CERAMIC COMPOSITIONS AND METHODS OF USE

SEQUENCE LISTING

The application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Dec. 3, 2020, is named 50222-709_601_SL.txt and is 285,147 bytes in size.

BACKGROUND

Three-dimensional (3D) printing is a manufacturing process of making three dimensional solid objects from a digital file. In the additive process of 3D printing an object is created by laying down successive layers of material until the desired object is created, with up to micrometer accuracy. Paired with computer-aided design (CAD) software, 3D printing enables production of complex functional shapes that can be easily customized compared with traditional manufacturing methods.

Surgically implanting scaffolds and/or other forms of graft materials to promote tissue regeneration is a useful technique if the implant can match the mechanical properties of the native tissue. Various materials can be used as ink in the fabrication of porous 3D-printed structures for implantation, including materials that mimic tissue and enable tissue regrowth. Inks with effective bioactive and mechanical properties are required for regeneration of native tissue, and if used in 3D printing can be customized and adopted for large tissue defect repair.

SUMMARY

The present disclosure provides ink formulations and methods for 3D printing scaffolds. Further provided are scaffolds that may be generated using such ink formulations and methods. The scaffolds may be coated with therapeutic agents, such as those that promote bone growth, and/or seeded with cells to generate devices for use in tissue replacement and grafting. For some devices, therapeutic agents may be tethered to the scaffold via a targeting moiety that interacts with a scaffold component, such as a ceramic material. Advantages of the materials and methods described herein include providing a 3D-printed, customizable implant, as well as more universal objects, such as strip, block, or cylindrical objects. As the implants are 3D-printed, precise control of the implant geometry is possible. Implants printed with these inks are thus suitable for many different therapies including long bone repair, spinal fusion, maxio-facial structures, etc. The different formulations of the inks produce materials that result in implantable devices with differing properties, including differing porosity and flexibility.

Various inks and scaffolds of the present disclosure are designed to enhance the accessibility of therapeutic agents and/or targeting moieties to scaffold components, such as ceramic materials like beta-tricalcium phosphate. For instance, ink formulations described in Examples 1-2.

Various inks and methods of the present disclosure are designed to improve the manufacturability of 3D printed scaffolds by converting the ink to 1.75 mm diameter filaments. In filament form (rather than syringe-based bioprinting ink), a larger quantity of material can be prepared, stored, and used continuously (e.g., 1 kg of filament wound around a spool) before needing to recharge material in the 3D printer. Additionally, fused filament fabrication (FFF) 3D printers generally have a larger available build volume than syringe-based bioprinters and can be printed at a faster nozzle speed, both of which improve manufacturing throughput. Also, there is a very wide variety of FFF printers on the market compared to a relatively small assortment of syringe-based bioprinters. For instance, ink formulations #5 and #6 were both fabricated into 1.75 mm diameter filaments material and demonstrated in a FFF 3D printer, as described further in Examples 1-2.

Various inks and scaffolds of the present disclosure are designed to optimize bioresorption characteristics of the scaffolds. For instance, the copolymers used in ink formulation #2 (caprolactone/glycolide copolymer (95:5)), ink formulation #3 (caprolactone/glycolide copolymer (90:10)), and ink formulation #4 (poly(D,L-lactide-co-glycolide) copolymer (50:50)) have faster resorption rates than polycaprolactone. The resorption rates vary from slowest to fastest as: polycaprolactone, polycaprolactone/polyglycolide copolymer (95:5), polycaprolactone/glycolide copolymer (90:10), polydioxanone/L-lactide copolymer (90:10), poly(D,L-lactide-co-glycolide) copolymer (50:50).

In one aspect, provided herein is a three-dimensional structure comprising about 50% to about 90% by weight ceramic and about 10% to about 50% by weight polymer, wherein the structure has a plurality of micropores. In some embodiments, the micropores have an average pore size of about 1 micron to about 500 microns, or about 50 microns to about 250 microns, or about 150 microns in diameter. In some embodiments, the micropores provide additional surface area to the structure for contact with a therapeutic agent. In some embodiments, micropores are formed after removal of one or more pore formers from the structure. In some embodiments, the pore former comprises a second polymer, a sugar, or a salt, or a combination thereof. In some embodiments, micropores are formed by release of carbon dioxide from a blowing agent present during manufacture of the structure. In some embodiments, the blowing agent comprises sodium bicarbonate.

In another aspect, provided herein is a three-dimensional structure comprising about 50% to about 90% by weight ceramic and about 10% to about 50% by weight polymer, wherein the structure has an open porosity of about 15% to about 50%.

In some embodiments of a structure provided herein, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, orvanadate, or a combination thereof. In some embodiments of a structure provided herein, the polymer comprises polycaprolactone, caprolactone/glycolide copolymer, poly(D,L-lactide-co-glycolide) copolymer, or dioxanone/L-lactide copolymer, or a combination thereof. In some embodiments of a structure provided herein, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, orvanadate, or a combination thereof, and the polymer comprises polycaprolactone, caprolactone/glycolide copolymer, poly(D,L-lactide-co-glycolide) copolymer, or dioxanone/L-lactide copolymer, or a combination thereof.

In some embodiments of a structure provided herein, the polymer comprises polycaprolactone and the ceramic comprises calcium phosphate. In some embodiments, the structure comprises about 65% to about 85% by weight calcium phosphate and about 15% to about 35% by weight polycaprolactone. In some embodiments, provided is a structure comprising about 65% to about 85% by weight calcium phosphate and about 15% to about 35% by weight polycaprolactone.

In some embodiments of a structure provided herein, the polymer comprises caprolactone/glycolide copolymer and the ceramic comprises calcium phosphate. In some embodiments, the structure comprises about 65% to about 85% by weight calcium phosphate and about 15% to about 35% by weight caprolactone/glycolide copolymer. In some embodiments, provided is a structure comprising about 65% to about 85% by weight calcium phosphate and about 15% to about 35% by weight caprolactone/glycolide copolymer.

In some embodiments of a structure provided herein, the polymer comprises poly(D,L-lactide-co-glycolide) copolymer or dioxanone/L-lactide copolymer, and the ceramic comprises calcium phosphate. In some embodiments, the structure comprises about 65% to about 85% by weight calcium phosphate and about 15% to about 35% by weight poly(D,L-lactide-co-glycolide) copolymer or dioxanone/L-lactide copolymer. In some embodiments, provided is a structure comprising about 65% to about 85% by weight calcium phosphate and about 15% to about 35% by weight poly(D,L-lactide-co-glycolide) copolymer or dioxanone/L-lactide copolymer.

In some embodiments of a structure provided herein, the structure is formed from an ink comprising the ceramic, the polymer, and a sacrificial pore former. In some embodiments of a structure provided herein, the structure is formed from a filament using an extrusion based three dimensional printing method.

Further provided is a device comprising a structure provided herein, and a therapeutic agent. In some embodiments, the therapeutic agent comprises a bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting moiety, and the targeting moiety is non-covalently bound to the three-dimensional structure. In some embodiments, the targeting moiety comprises a polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences of Tables 2-3. In some embodiments, the therapeutic agent comprises a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 433-441.

In one aspect, provided herein is a device comprising: a therapeutic agent and a printed three-dimensional structure, the printed three-dimensional structure comprising about 50% to about 90% by weight ceramic and about 10% to about 50% by weight polymer. In some embodiments, the therapeutic agent is non-covalently bound to the printed three-dimensional structure. In some embodiments, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or a combination thereof. In some embodiments, the ceramic comprises beta-tricalcium phosphate (β-TCP). In some embodiments, the structure comprises about 70% to about 80% by weight ceramic. In some embodiments, the structure comprises about 75% by weight ceramic. In some embodiments, the polymer comprises a polyester. In some embodiments, the polymer comprises polycaprolactone (PCL), polyglycolide, lactide, or polydioxanone (PDS), or a combination thereof. In some embodiments, the polymer comprises polycaprolactone (PCL). In some embodiments, the polymer comprises polyglycolide. In some embodiments, the polymer comprises polydioxanone (PDS). In some embodiments, the polymer comprises a lactide. In some embodiments, the lactide comprises L-lactide. In some embodiments, the lactide comprises poly(D,L-lactide). In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer comprises polycaprolactone (PCL) and polyglycolide. In some embodiments, the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide. In some embodiments, the copolymer comprises poly(D,L-lactide-co-glycolide) copolymer. In some embodiments, the copolymer comprises about 40-60 percent by mole poly(D,L-lactide) and about 40-60 percent by mole polyglycolide. In some embodiments, the copolymer comprises PDS-glycolide copolymer. In some embodiments, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole glycolide. In some embodiments, the copolymer comprises PDS-L-lactide copolymer. In some embodiments, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole L-lactide. In some embodiments, the structure comprises about 15% to about 25% by weight polymer. In some embodiments, the structure comprises about 25% by weight polymer. In some embodiments, the printed three-dimensional structure is formed from an ink comprising about 30% to about 70% by weight the ceramic, about 10% to about 30% by weight the polymer, and optionally one or more additional agents. In some embodiments, the ink comprises about 50% to about 70% by weight the ceramic. In some embodiments, the ink comprises ab out 60% by weight the ceramic. In some embodiments, the ink comprises ab out 15% to about 25% by weight the ceramic. In some embodiments, the ink comprises about 20% by weight the ceramic. In some embodiments, the ink comprises the one or more additional agents. In some embodiments, the ink comprises about 1% to about 30% by weight the one or more additional agents. In some embodiments, the one or more additional agents comprises an additional polymer, particulate, or blowing agent, or a combination thereof. In some embodiments, the one or more additional agents comprises the additional polymer. In some embodiments, the additional polymer comprises polyethylene glycol. In some embodiments, the additional polymer is present in the ink at about 10% to about 30% by weight. In some embodiments, the one or more additional agents comprises the particulate. In some embodiments, the particulate comprises sodium chloride, calcium chloride, sucrose, trehalose (e.g., a, a trehalose dihydrate), or mannitol (e.g., D-mannitol), or a combination thereof. In some embodiments, the particulate is present in the ink at about 1% to about 10% by weight. In some embodiments, the one or more additional agents comprises the blowing agent. In some embodiments, the blowing agent comprises baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and/or azodicarbonamide. In some embodiments, the particulate is present in the ink at about 5% to about 20% by weight. In some embodiments, the therapeutic agent comprises a mammalian growth factor or a functional portion thereof. In some embodiments, the therapeutic agent comprises one or more polypeptides selected from Table 1, or a functional portion thereof. In some embodiments, the therapeutic agent comprises a bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting moiety, and the targeting moiety is non-covalently bound to the printed three-dimensional structure. In some embodiments, the targeting moiety is bound to the printed three-dimensional structure with an affinity of ab out 1 pM to about 100 μM. In some embodiments, the targeting moiety comprises a polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences of Tables 2-3. In some embodiments, the targeting moiety comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences selected from a sequence from Tables 2-3. In some embodiments, the therapeutic agent comprises a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 433-441.

Further provided is a method of treating a condition in a subject in need thereof, the method comprising administering to the subject the device. In some embodiments, the condition comprises a bone defect, cartilage defect, soft tissue defect, tendon defect, fascia defect, ligament defect, organ defect, osteotendinous tissue defect, dermal defect, osteochondral defect, osteoporosis, avascular necrosis, or congenital skeletal malformation, or a combination thereof. In some embodiments, the method comprises spinal fusion. In some embodiments, the spinal fusion comprises posterior lumbar fusion (PLF) and/or interbody fusion. In some embodiments, the method comprises bone repair, dental repair, craniomaxillofacial repair, ankle fusion, kyphoplasty, osteoplasty, scaphoid fracture repair, tendeno-osseous repair, costal reconstruction, subchondral bone repair, cartilage repair, or surgical implantation of the three-dimensional structure or device, or a combination thereof.

Further provided is a formulation comprising about 30% to about 70% by weight ceramic, about 10% to about 30% by weight polymer, and optionally one or more additional agents. In some embodiments, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or a combination thereof. In some embodiments, the ceramic comprises beta-tricalcium phosphate (β-TCP). In some embodiments, the ink comprises about 50% to about 70% by weight the ceramic. In some embodiments, the ink comprises about 60% by weight the ceramic. In some embodiments, the polymer comprises a polyester. In some embodiments, the polymer comprises polycaprolactone (PCL), polyglycolide, lactide, or polydioxanone (PDS), or a combination thereof. In some embodiments, the polymer comprises polycaprolactone (PCL). In some embodiments, the polymer comprises polyglycolide. In some embodiments, the polymer comprises polydioxanone (PDS). In some embodiments, the polymer comprises a lactide. In some embodiments, the lactide comprises L-lactide. In some embodiments, the lactide comprises poly(D,L-lactide). In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer comprises polycaprolactone (PCL) and polyglycolide. In some embodiments, the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide. In some embodiments, the copolymer comprises poly(D,L-lactide-co-glycolide) copolymer. In some embodiments, the copolymer comprises about 40-60 percent by mole poly(D,L-lactide) and about 40-60 percent by mole polyglycolide. In some embodiments, the copolymer comprises PDS-glycolide copolymer. In some embodiments, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole glycolide. In some embodiments, the copolymer comprises PDS-L-lactide copolymer. In some embodiments, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole L-lactide. In some embodiments, the ink comprises the one or more additional agents. In some embodiments, the ink comprises about 1% to about 30% by weight the one or more additional agents. In some embodiments, the one or more additional agents comprises an additional polymer, particulate, or blowing agent, or a combination thereof. In some embodiments, the one or more additional agents comprises the additional polymer. In some embodiments, the additional polymer comprises polyethylene glycol. In some embodiments, the additional polymer is present in the ink at about 10% to about 30% by weight. In some embodiments, the one or more additional agents comprises the particulate. In some embodiments, the particulate comprises sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α trehalose dihydrate), or mannitol (e.g., D-mannitol), or a combination thereof. In some embodiments, the particulate is present in the ink at about 1% to about 10% by weight. In some embodiments, the one or more additional agents comprises the blowing agent. In some embodiments, the blowing agent comprises baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and/or azodicarbonamide. In some embodiments, the particulate is present in the ink at about 5% to about 20% by weight.

Further provided is a filament comprising the formulation. In some embodiments, the filament has a diameter of about 1.5 mm to about 2 mm.

Further provided is a method of preparing a three-dimensional structure, the method comprising using the formation in a three-dimensional printing method.

Further provided is a three-dimensional structure prepared using the ink formulation. In some embodiments, the structure comprises about 50% to about 90% by weight ceramic. In some embodiments, the structure comprises about 50% to about 90% by weight tricalcium phosphate. In some embodiments, the structure comprises about 10% to about 50% by weight polymer. In some embodiments, the polymer comprises a polyester. In some embodiments, the polymer comprises polycaprolactone (PCL), polyglycolide, lactide, or polydioxanone (PDS), or a combination thereof. In some embodiments, the polymer comprises polycaprolactone (PCL). In some embodiments, the polymer comprises polyglycolide. In some embodiments, the polymer comprises polydioxanone (PDS). In some embodiments, the polymer comprises a lactide. In some embodiments, the lactide comprises L-lactide. In some embodiments, the lactide comprises poly(D,L-lactide). In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer comprises polycaprolactone (PCL) and polyglycolide. In some embodiments, the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide. In some embodiments, the copolymer comprises poly(D,L-lactide-co-glycolide) copolymer. In some embodiments, the copolymer comprises about 40-60 percent by mole poly(D,L-lactide) and about 40-60 percent by mole polyglycolide. In some embodiments, the copolymer comprises PDS-glycolide copolymer. In some embodiments, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole glycolide. In some embodiments, the copolymer comprises PDS-L-lactide copolymer. In some embodiments, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole L-lactide. In some embodiments, the structure comprises about 15% to about 25% by weight polymer. In some embodiments, the structure comprises about 25% by weight polymer.

Further provided is a three-dimensional structure comprising about 50% to about 90% by weight ceramic, and about 10% to about 30% polymer. In some embodiments, the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or a combination thereof. In some embodiments, the ceramic comprises beta-tricalcium phosphate (β-TCP). In some embodiments, the structure comprises ab out 65% to ab out 85% by weight ceramic. In some embodiments, the structure comprises about 70% to about 80% by weight ceramic. In some embodiments, the structure comprises about 75% by weight ceramic. In some embodiments, the polymer comprises a polyester. In some embodiments, the polymer comprises polycaprolactone (PCL), polyglycolide, lactide, or polydioxanone (PDS), or a combination thereof. In some embodiments, the polymer comprises polycaprolactone (PCL). In some embodiments, the polymer comprises polyglycolide. In some embodiments, the polymer comprises polydioxanone (PDS). In some embodiments, the polymer comprises a lactide. In some embodiments, the lactide comprises L-lactide. In some embodiments, the lactide comprises poly(D,L-lactide). In some embodiments, the polymer comprises a copolymer. In some embodiments, the copolymer comprises polycaprolactone (PCL) and polyglycolide. In some embodiments, the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide. In some embodiments, the copolymer comprises poly(D,L-lactide-co-glycolide) copolymer. In some embodiments, the copolymer comprises about 40-60 percent by mole poly(D,L-lactide) and about 40-60 percent by mole polyglycolide. In some embodiments, the copolymer comprises PDS-glycolide copolymer. In some embodiments, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole glycolide. In some embodiments, the copolymer comprises PDS-L-lactide copolymer. In some embodiments, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole L-lactide. In some embodiments, the structure comprises about 15% to about 25% by weight polymer. In some embodiments, the structure comprises about 25% by weight polymer. In some embodiments, the structure is prepared by a three-dimensional printing method.

Further provided is a method of manufacturing the three-dimensional structure, the method comprising depositing an ink formulation in a three-dimensional form. In some embodiments, the ink formulation comprises the ink formulation herein.

Further provided is a method of treating a condition in a subject in need thereof, the method comprising delivering to an organ or tissue of the subject the structure.

Further provided is a method of treating a condition in a subject in need thereof, the method comprising delivering to an organ or tissue of the subject the structure manufactured by a method herein.

In some embodiments, the condition comprises a bone defect, cartilage defect, soft tissue defect, tendon defect, fascia defect, ligament defect, organ defect, osteotendinous tissue defect, dermal defect, osteochondral defect, osteoporosis, avascular necrosis, or congenital skeletal malformation, or a combination thereof. In some embodiments, the method comprises spinal fusion. In some embodiments, the spinal fusion comprises posterior lumbar fusion (PLF) and/or interbody fusion. In some embodiments, the method comprises bone repair, dental repair, craniomaxillofacial repair, ankle fusion, kyphoplasty, osteoplasty, scaphoid fracture repair, tendeno-osseous repair, costal reconstruction, sub chondral b one rep air, cartilage repair, or surgical implantation of the three-dimensional structure or device, or a combination thereof. In some embodiments, the method further comprises treating the subject with a therapeutic agent.

Further provided is a method of delivering a therapeutic agent to a subject in need thereof, the method comprising delivering to an organ or tissue of the subject a device comprising a therapeutic agent and the structure.

In some embodiments, the therapeutic agent comprises a mammalian growth factor or a functional portion thereof. In some embodiments, the therapeutic agent comprises one or more polypeptides selected from Table 1, or a functional portion thereof. In some embodiments, the therapeutic agent comprises a bone morphogenetic protein (BMP). In some embodiments, the therapeutic agent comprises a targeting moiety that non-covalently binds to the structure. In some embodiments, the targeting moiety binds to the printed three-dimensional structure with an affinity of about 1 pM to about 100 μm. In some embodiments, the targeting moiety comprises a polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences of Tables 5-6. In some embodiments, the targeting moiety comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences selected from the sequences of Tables 5-6. In some embodiments, the therapeutic agent comprises or is part of a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 433-441.

In some embodiments of a device and/or structure herein, the structure has a density of about 1 g/cm3 to about 2 g/cm3. In some embodiments of a device and/or structure herein, the structure has an open porosity of about 15% to about 50%. In some embodiments of a device and/or structure herein, the structure of any one of claims 91-139, wherein the structure has a strut diameter of about 300 μm to about 600 μm.

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #1 as outlined in Example 2. FIG. 1A and FIG. 1C are SEM images of the surface of the object at increasing magnifications. FIG. 1B is an SEM image of the side of the object.

FIGS. 2A-2C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #2 as outlined in Example 2. FIG. 2A and FIG. 2C are SEM images of the surface of the object at increasing magnifications. FIG. 2B is an SEM image of the side of the object.

FIGS. 3A-3C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #3 as outlined in Example 2. FIG. 3A and FIG. 3C are SEM images of the surface of the object at increasing magnifications. FIG. 3B is an SEM image of the side of the object.

FIGS. 4A-4C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #4 as outlined in Example 2. FIG. 4A and FIG. 4C are SEM images of the surface of the object at increasing magnifications. FIG. 4B is an SEM image of the side of the object.

FIGS. 5A-5C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #5 as outlined in Example 2. FIG. 5A and FIG. 5C are SEM images of the surface of the object at increasing magnifications. FIG. 5B is an SEM image of the side of the object.

FIGS. 6A-6C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #6 as outlined in Example 2. FIG. 6A and FIG. 6C are SEM images of the surface of the object at increasing magnifications. FIG. 6B is an SEM image of the side of the object.

FIGS. 7A-7C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #7 as outlined in Example 2. FIG. 7A and FIG. 7C are SEM images of the surface of the object at increasing magnifications. FIG. 7B is an SEM image of the side of the object.

FIGS. 8A-8C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #8 as outlined in Example 2. FIG. 8A and FIG. 8C are SEM images of the surface of the object at increasing magnifications. FIG. 8B is an SEM image of the side of the object.

FIGS. 9A-9C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #9 as outlined in Example 2. FIG. 9A and FIG. 9C are SEM images of the surface of the object at increasing magnifications. FIG. 9B is an SEM image of the side of the object.

FIGS. 10A-10C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #10 as outlined in Example 2. FIG. 10A and FIG. 10C are SEM images of the surface of the object at increasing magnifications. FIG. 10B is an SEM image of the side of the object.

FIGS. 11A-11C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #11 as outlined in Example 2. FIG. 11A and FIG. 11C are SEM images of the surface of the object at increasing magnifications. FIG. 11B is an SEM image of the side of the object.

FIGS. 12A-12C are images from scanning electron microscopy (SEM images) of an example 3D printed object made with ink formulation #12 as outlined in Example 2. FIG. 12A and FIG. 12C are SEM images of the surface of the object at increasing magnifications. FIG. 12B is an SEM image of the side of the object.

DETAILED DESCRIPTION

Various formulations and structures are provided herein. The structures may be coated with a tetherable protein (for example, a growth factor) for a desired therapeutic effect, such as promotion of bone growth after implantation of the tethered structure.

Formulations

In one aspect, provided herein are formulations for fabrication of 3D-printed structures. As a non-limiting example, the formulations include a ceramic material such as calcium phosphate (e.g., tricalcium phosphate, beta tricalcium phosphate, alpha tricalcium phosphate), hydroxyapatite, fluorapatite, bone (e.g., demineralized bone), glasses (bioglasses) such as silicates, vanadates, and related ceramic minerals, or chelated divalent metal ions, or a combination thereof. In some embodiments, the ceramic material comprises beta-tricalcium phosphate (β-TCP). In some embodiments, the formulation is about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70 percent ceramic by weight of the formulation. For instance, the formulation is about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% ceramic by weight. In some embodiments, the ceramic is β-TCP. In some embodiments, the β-TCP is introduced into the formulation as a powder. In some embodiments, the formulation comprises one or more additional components. Non-limiting examples of additional components include water, polymer (including copolymer), antifoaming agent, dispersing agent, solvent, particulate, blowing agent, and plasticizer.

In some embodiments, the formulation comprises one or more polymers, e.g., about 1, 2, 3, 4, or 5 polymers. Non-limiting examples of polymers include poly(ethylene oxide), poly(propylene oxide), polyethylene glycol (PEG), and polyester. In some embodiments, the formulation comprises a polymer that is about 5-30 percent by weight of the formulation. In some embodiments, the formulation is about 20-60 percent total polymer by weight. For instance, the total polymer includes two or more polymers in the formulation, where the total percentage of polymers in the formulation is about 20-60 percent of the weight of the formulation. In some embodiments, the formulation is about 30 to about 50 percent total polymer by weight. As non-limiting examples, the formulation is ab out 35-45 percent total polymer by weight. In an example, the polymer comprises a poloxamer. Poloxamers are block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). A non-limiting example of a poloxamer is poloxamer 407, such as Pluronic® F-127. In some cases, the formulation comprises about 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 percent poloxamer 407 by weight. As another example, the polymer comprises polyethylene glycol (PEG). In some cases, the formulation comprises about 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight PEG. For instance, the formulation comprises about 5-30, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 percent by weight PEG. In some embodiments, there is a first PEG with a first molecular weight, and a second PEG with a second molecular weight. In some embodiments, PEG can have a molecular weight from 500 g/mol to 25,000 g/mol. In some cases, the molecular weight of PEG is about 500 g/mol, about 1,000 g/mol, about 1,500 g/mol, about 20.00 g/mol, about 2,500 g/mol, about 3,000 g/mol, about 3,500 g/mol, about 4,000 g/mol, about 4,500 g/mol, about 5,000 g/mol, about 5,500 g/mol, about 6,000 g/mol, about 6,500 g/mol, about 7,000 g/mol, about 7,500 g/mol, about 8,000 g/mol, about 8,500 g/mol, about 9,000 g/mol, about 9,500 g/mol, about 10,000 g/mol, about 10,500 g/mol, about 11,000 g/mol, about 11,500 g/mol, about 12,000 g/mol, about 12,500 g/mol, about 13,000 g/mol, about 13,500 g/mol, about 14,000 g/mol, about 14,500 g/mol, about 15,000 g/mol, about 15,500 g/mol, about 16,000 g/mol, about 16,500 g/mol, about 17,000 g/mol, about 17,500 g/mol, about 18,000 g/mol, about 18,500 g/mol, about 19,000 g/mol, about 19,500 g/mol, about 20,000 g/mol, about 20,500 g/mol, about 21,000 g/mol, about 21,500 g/mol, about 22,000 g/mol, about 22,500 g/mol, about 23,000 g/mol, about 23,500 g/mol, about 24,000 g/mol, about 24,500 g/mol, or about 25,000 g/mol. In a nonlimiting example embodiment, the formulation comprises PEG having a molecular weight of 1,500 g/mol. In a further nonlimiting example embodiment, the formulation comprises PEG having a molecular weight of 8,000 g/mol. In a further nonlimiting example embodiment, the formulation comprises PEG having a molecular weight of 20,000 g/mol. In a nonlimiting example embodiment, the formulation comprises a first PEG at about 15 percent by weight and a second PEG at about 15 percent by weight. As another example, the polymer comprises polydioxanone (PDS). In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight PDS. For instance, the formulation comprises about 15-25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 percent by weight PDS. As another example, the polymer comprises a polyester. In some embodiments, the polyester comprises a biodegradable polyester such as polycaprolactone (PCL). In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight PCL. For instance, the formulation comprises about 15-25, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 percent by weight PCL. In some cases, the formulation comprises PCL having a molecular weight of 50,000 g/mol. In some embodiments, the polyester comprises a polyglycolide or poly(glycolic acid) (PGA). In some cases, the formulation comprises about 0.5-20, 0.5-18, 0.5-16, 0.5-14, 0.5-12, 0.5-10, 0.5-8, 0.5-6, 0.5-4, 0.5-2, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 percent by weight PGA. For instance, the formulation comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 percent by weight PGA. In some cases, the PGA has a molecular weight of about 38,000-54,000. In some embodiments, the polyester comprises a polylactide, such as poly(D, L-lactide). In some cases, the formulation comprises about 0.5-20, 0.5-18, 0.5-16, 0.5-14, 0.5-12, 0.5-10, 0.5-8, 0.5-6, 0.5-4, 0.5-2, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 percent by weight polylactide. For instance, the formulation comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 percent by weight polylactide.

In some embodiments, the polymer comprises a copolymer. In some cases, the copolymer comprises polyglycolide. In some cases, the copolymer comprises PCL and polyglycolide. For instance, the copolymer comprises about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 percent molar PCL, and about 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 percent molar polyglycolide. In some cases, the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide. In some cases, the copolymer comprises PDS and polyglycolide. For instance, the copolymer comprises about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 percent molar PDS, and about 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 percent molar polyglycolide. In some cases, the copolymer comprises about 90-95 percent by mole PDS and ab out 5-10 percent by mole polyglycolide. In some cases, the copolymer comprises PDS-glycolide copolymer. For instance, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, 25-30, or 20 percent by weight PDS-glycolide copolymer. In some cases, the copolymer comprises poly(D, L-lactide-co-glycolide) copolymer. For instance, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, 25-30, or 20 percent by weight poly(D, L-lactide-co-glycolide) copolymer. In some cases, the copolymer comprises lactide (e.g., poly(D, L-lactide)) and polyglycolide. For instance, the copolymer comprises about 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-65, 40-60, 40-55, 40-50, 40-45, 45-65, 45-60, 45-55, 45-50, 50-65, 50-60, 50-55, 55-65, 55-60, 60-65 percent molar lactide (e.g., poly(D, L-lactide)), and about 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-65, 40-60, 40-55, 40-50, 40-45, 45-65, 45-60, 45-55, 45-50, 50-65, 50-60, 50-55, 55-65, 55-60, 60-65 percent molar glycolide. In some cases, the copolymer comprises poly(D, L-lactide-co-glycolide) copolymer. For instance, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, 25-30, or 20 percent by weight poly(D, L-lactide-co-glycolide) copolymer. In some cases, the copolymer comprises lactide (e.g., L-lactide) and PDS. For instance, the copolymer comprises about 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-65, 40-60, 40-55, 40-50, 40-45, 45-65, 45-60, 45-55, 45-50, 50-65, 50-60, 50-55, 55-65, 55-60, 60-65 percent molar PDS, and about 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-65, 40-60, 40-55, 40-50, 40-45, 45-65, 45-60, 45-55, 45-50, 50-65, 50-60, 50-55, 55-65, 55-60, 60-65 percent molar lactide (e.g., L-lactide). In some cases, the copolymer comprises PDS-L-lactide copolymer. For instance, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, 25-30, or 20 percent by weight PDS-L-lactide copolymer. In some cases, the copolymer comprises dioxanone. In some cases, the copolymer comprises dioxanone and lactide (e.g., L-lactide). For instance, the copolymer comprises about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 percent molar dioxanone, and about 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 percent molar lactide (e.g., L-lactide). In some cases, the copolymer comprises about 85-95 percent by mole dioxanone and about 5-15 percent by mole lactide (e.g., L-lactide).

In some instances, the copolymer has a faster resorption rate than a single polymer. For instance, the copolymers used in example embodiments of ink formulation #2 (polycaprolactone/polyglycolide copolymer (95:5)), ink formulation #3 (polycaprolactone/polyglycolide copolymer (90:10)), and ink formulation #4 (poly(D,L-lactide-co-glycolide) copolymer (50:50)) have faster resorption rates than polycaprolactone. The resorption rates vary from slowest to fastest as: polycaprolactone, polycaprolactone/polyglycolide copolymer (95:5), polycaprolactone/glycolide copolymer (90:10), polydioxanone/L-lactide copolymer (90:10), poly(D,L-lactide-co-glycolide) copolymer (50:50).

In some embodiments, the formulation comprises two or more polymers. In some embodiments, the formulation is about 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight of a first polymer, and about 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight of a second polymer. In non-limiting examples, the formulation is about 10-30 percent by weight of a first polymer, and about 10-30 percent by weight of a second polymer, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 percent by weight of the first polymer, and about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 percent by weight of the second polymer. In some cases, the first and/or second polymer comprises PEG. In some cases, the first polymer comprises PCL and the second polymer comprises PEG. In some cases, the first polymer comprises PD S and the second polymer comprises PEG. In some cases, the first polymer comprises a copolymer and the second polymer comprises PEG. The copolymer may comprise PCL and polyglycolide (e.g., 95 mol % polycaprolactone, 5 mol % polyglycolide; 90 mol % polycaprolactone, 10 mol % polyglycolide). The copolymer may comprise polylactide (e.g., poly(D,L-lactide) and polyglycolide (e.g., 50 mol % poly(D,L-lactide), 50 mol % polyglycolide). The copolymer may comprise PDS-glycolide copolymer (e.g., 90 mol % PDS, 10 mol % polyglycolide). The copolymer may comprise PDS-L-lactide copolymer (e.g., 90 mol % PDS, 10 mol % L-lactide).

In some embodiments, the formulation comprises one or more particulates. The particulate may be a pore former. The particulate may be water soluble. The particulate may comprise a salt and/or sugar. Non-limiting examples of particulates include sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α trehalose dihydrate), and mannitol (e.g., D-mannitol). In some cases, the particulate comprises sucrose. In some embodiments, the formulation comprises about 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, or 5-6 percent by weight particulate. In some cases, the formulation comprises about 1-10%, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% particulate. In some embodiments, the particulate or pore former has an average size of about 1 micron to about 500 microns in diameter. For instance, about 1 micron to about 450 microns, about 1 micron to about 400 microns, about 1 micron to about 350 microns, about 1 micron to about 300 microns, about 1 micron to about 250 microns, about 1 micron to about 200 microns, about 1 micron to about 150 microns, about 50 microns to about 500 microns, about 50 microns to about 450 microns, about 50 microns to about 400 microns, about 50 microns to about 350 microns, about 50 microns to about 300 microns, about 50 microns to about 250 microns, about 50 microns to about 200 microns, about 50 microns to about 150 microns, about 100 microns to about 500 microns, about 100 microns to about 450 microns, about 100 microns to about 400 microns, about 100 microns to about 350 microns, about 100 microns to about 300 microns, about 100 microns to about 250 microns, about 100 microns to about 200 microns, about 100 microns to about 150 microns, about 150 microns to about 500 microns, about 150 microns to about 450 microns, about 150 microns to about 400 microns, about 150 microns to about 350 microns, about 150 microns to about 300 microns, about 150 microns to about 250 microns, or ab out 150 microns to ab out 200 microns in diameter. In some cases, the particular or pore former has an average size of about 50 microns to about 250 microns, about 60 microns to about 240 microns, about 70 microns to about 230 microns, about 80 microns to about 220 microns, or about 90 microns to about 210 microns in diameter. In some embodiments, the particulate of pore former has an average size of about 100 microns to about 200 microns, e.g., about 110 microns to about 190 microns, about 120 microns to about 180 microns, about 130 microns to about 170 microns, about 140 microns to about 160 microns, or about 100 microns, about 110 microns, about 120 microns, about 130 microns, about 140 microns, about 150 microns, about 160 microns, about 170 microns, about 180 microns, about 190 microns, or about 200 microns in diameter. In some embodiments, the particulate or pore former has an average size of about 150 microns in diameter. In some embodiments, upon removal of the particulate or pore former, a structure formed from the formulation has micropores that provide additional surface area to the structure for contact with a therapeutic agent as compared to a structure formed with a formulation lacking the particulate or pore former. In some embodiments, the micropores of the structure have an average pore size of about 1 micron to about 500 microns, or about 50 microns to about 250 microns, or about 150 microns in diameter.

In some embodiments, the formulation comprises one or more blowing agents. In some embodiments, the blowing agent comprises about 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-20, 10-18, 10-16, 10-14, 10-12, 5-15, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 percent by weight blowing agent. In some cases, the blowing agent releases carbon dioxide base during printing to create a foamed structure that can increase porosity of the 3D printed structure. Non-limiting examples of blowing agents include baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and azodicarbonamide. In some cases, the blowing agent comprises sodium bicarbonate. In some cases, the formulation comprises ab out 5-15, or ab out 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 percent by weight sodium bicarbonate. In some embodiments, the blowing agent provides for micropores in the structure having an average diameter of about 1 micron to about 500 microns, or about 50 microns to about 250 microns, or about 150 microns.

In some embodiments, a formulation comprises a ceramic material (e.g., β-TCP) and a polymer. Polymers include PEO, PPO, PDS, PEG, polyester, copolymers, or a combination thereof. In some embodiments, the formulation is about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70 percent ceramic material (e.g., β-TCP) by weight, e.g., about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% ceramic material (e.g., β-TCP) by weight. In some cases, the formulation comprises about 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 percent poloxamer 407 by weight. In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight PEG. In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight PCL. In some cases, the formulation comprises about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight PDS. In some embodiments, the formulation further comprises an antifoaming agent. In some embodiments, the formulation further comprises a dispersing agent. In some embodiments, the formulation further comprises a solvent. In some embodiments, the formulation further comprises a plasticizer. In some embodiments, the formulation further comprises a particulate. In some cases, the formulation further comprises a blowing agent.

In some embodiments, a formulation comprises a ceramic material (e.g., β-TCP) and a particulate. The particulate may be water soluble. Non-limiting examples of particulates include salts and sugars, e.g., sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α trehalose dihydrate), and mannitol (e.g., D-mannitol). In some embodiments, the formulation is about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70 percent ceramic material (e.g., (3-TCP) by weight, e.g., about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% ceramic material (e.g., (3-TCP) by weight. In some embodiments, the formulation comprises about 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, or 5-6 percent by weight particulate. In some embodiments, the formulation further comprises water. In some embodiments, the formulation further comprises a polymer. In some embodiments, the formulation further comprises an antifoaming agent. In some embodiments, the formulation further comprises a dispersing agent. In some embodiments, the formulation further comprises a solvent. In some embodiments, the formulation further comprises a plasticizer. In some cases, the formulation further comprises a blowing agent.

In some embodiments, a formulation comprises a ceramic material (e.g., β-TCP) and a blowing agent. Non-limiting examples of blowing agents include baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and azodicarbonamide. The blowing agent may comprise sodium bicarbonate. In some embodiments, the formulation is about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 55-70, 55-65, 55-60, 60-70, 60-65, or 65-70 percent ceramic material (e.g., β-TCP) by weight, e.g., about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% ceramic material (e.g., β-TCP) by weight. In some embodiments, the blowing agent comprises about 5-20, 5-18, 5-16, 5-15, 5-14, 5-12, 5-10, 5-8, 5-6, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-20, 10-18, 10-16, 10-14, 10-12, 5-15, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 percent by weight blowing agent. In some cases, the formulation comprises about 5-15, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 percent by weight sodium bicarbonate. In some embodiments, the formulation further comprises water. In some embodiments, the formulation further comprises a polymer. In some embodiments, the formulation further comprises an antifoaming agent. In some embodiments, the formulation further comprises a dispersing agent. In some embodiments, the formulation further comprises a solvent. In some embodiments, the formulation further comprises a plasticizer. In some embodiments, the formulation further comprises a particulate.

In another aspect, a formulation comprises a ceramic material and one or more polymers. In some embodiments, the formulation comprises about 30% to about 70% a ceramic material (e.g., β-TCP). For instance, the formulation comprises about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 60-70, 60-65, 65-70, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 percent by weight a ceramic material (e.g., β-TCP). In some embodiments, the formulation comprises a first polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight first polymer. The first polymer may be polycaprolactone (PCL). The first polymer may be polydioxanone (PDS). In some embodiments, the formulation comprises a second polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight second polymer. The second polymer may be polyethylene glycol (PEG). In a non-limiting embodiment, the formulation comprises about 30-70% by weight ceramic, about 10-30% by weight a first polymer, and about 10-30% by weight a second polymer. For example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight PCL, and about 10-30% by weight PEG. As another example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight PDS, and about 10-30% by weight PEG.

In some further embodiments, the formulation comprises a particulate. The particulate may be water soluble. In some cases, the particulate comprises sucrose. In some embodiments, the formulation comprises about 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, or 5-6 percent by weight particulate. For example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight PCL, about 10-30% by weight PEG, and about 1-10% by weight particulate.

In some further embodiments, the formulation comprises a blowing agent. In some cases, the blowing agent comprises sodium bicarbonate. In some embodiments, the formulation comprises about 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 8-20, 8-18, 8-16, 8-14, 8-12, 8-10, 10-20, 10-18, 10-16, 10-14, 10-12, 5-15, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 percent by weight blowing agent. For example, the formulation may comprise ab out 30-70% by weight β-TCP, ab out 10-30% by weight PCL, ab out 10-30% by weight PEG, and about 5-20% by weight blowing agent.

In some further embodiments, the polymer of the formulation is a copolymer, such as a PCL and polyglycolide copolymer. For instance, the copolymer comprises about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 percent molar PCL, and about 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 percent molar polyglycolide. In some cases, the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide. In an example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight PCL and polyglycolide copolymer (e.g., 95 mol % polycaprolactone, 5 mol % polyglycolide), and about 10-30% by weight PEG. In an example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight PCL and polyglycolide copolymer (e.g., 90 mol % polycaprolactone, 10 mol % polyglycolide), and about 10-30% by weight PEG.

In some further embodiments, the polymer of the formulation is a copolymer, such as a PDS and polyglycolide copolymer. For instance, the copolymer comprises about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 percent molar PDS, and about 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, or 8-10 percent molar polyglycolide. In some cases, the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole polyglycolide. In an example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight PDS and polyglycolide copolymer (e.g., 90 mol % PDS, 10 mol % polyglycolide), and about 10-30% by weight PEG.

In some further embodiments, the polymer of the formulation is a copolymer, such as a PDS and lactide copolymer. For instance, the copolymer comprises about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 percent molar PDS, and about 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, orb-10 percent molar lactide. In some cases, the copolymer comprises ab out 90-95 percent by mole PDS and about 5-10 percent by mole lactide. In an example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight PDS and lactide copolymer (e.g., 90 mol % PDS, 10 mol % lactide), and about 10-30% by weight PEG.

In another aspect, a formulation comprises a ceramic material and one or more polymers. In some embodiments, the formulation comprises about 30% to about 70% a ceramic material (e.g., β-TCP). For instance, the formulation comprises about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 60-70, 60-65, 65-70, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 percent by weight a ceramic material (e.g., β-TCP). In some embodiments, the formulation comprises a first polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight first polymer. The first polymer may be a copolymer. In some embodiments, the copolymer comprises a lactide (e.g., poly(D, L-lactide)) and polyglycolide. In some embodiments, the copolymer comprises poly(D, L-lactide-co-glycolide) copolymer. In some embodiments, the copolymer comprises about 50 mol % poly(D, L-lactide) and ab out 50 mol % polyglycolide. In some embodiments, the formulation comprises a second polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight second polymer. The second polymer may be polyethylene glycol (PEG). In a non-limiting embodiment, the formulation comprises about 30-70% by weight ceramic, about 10-30% by weight a first polymer, and about 10-30% by weight a second polymer. For example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight copolymer, and about 10-30% by weight PEG.

In some further embodiments, a formulation comprises a ceramic material and one or more polymers. In some embodiments, the formulation comprises about 30% to about 70% a ceramic material (e.g., β-TCP). For instance, the formulation comprises about 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 30-35, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-70, 40-65, 40-60, 40-55, 40-50, 40-45, 45-70, 45-65, 45-60, 45-55, 45-50, 50-70, 50-65, 50-60, 50-55, 60-70, 60-65, 65-70, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 percent by weight a ceramic material (e.g., β-TCP). In some embodiments, the formulation comprises a first polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight first polymer. The first polymer may be a copolymer, such as a dioxanone and lactide (e.g., L-lactide) copolymer. For instance, the copolymer comprises about 80-99, 80-98, 80-97, 80-96, 80-95, 80-94, 80-93, 80-92, 80-91, 80-90, 80-89, 80-88, 80-87, 80-86, 80-85, 85-99, 85-98, 85-97, 85-96, 85-95, 85-94, 85-93, 85-92, 85-91, 85-90, 90-99, 90-98, 90-97, 90-96, 90-95, 90-94, 90-93, 90-92, 90-91, 95-99, 95-98, 95-97, or 95-96 percent molar dioxanone, and about 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 4-20, 4-18, 4-16, 4-14, 4-12, 4-10, 4-8, 4-6, 5-20, 5-18, 5-16, 5-14, 5-12, 5-10, 5-8, 5-6, 8-20, 8-18, 8-16, 8-14, 8-12, orb-10 percent molar lactide. In some cases, the copolymer comprises about 90-95 percent by mole dioxanone and about 5-10 percent by mole lactide. In some embodiments, the formulation comprises a second polymer, e.g., about 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 percent by weight second polymer. The second polymer may be polyethylene glycol (PEG). In a non-limiting embodiment, the formulation comprises about 30-70% by weight ceramic, about 10-30% by weight a first polymer, and about 10-30% by weight a second polymer. For example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight copolymer, and about 10-30% by weight PEG In an example, the formulation may comprise about 30-70% by weight β-TCP, about 10-30% by weight PDS and lactide copolymer (e.g., 90 mol % dioxanone, 10 mol % L-lactide), and about 10-30% by weight PEG.

In one aspect, the formulation has a low viscosity that may be useful during manufacture for extruding through a small diameter nozzle. The nozzle may have a diameter of about 240 μm to about 500 μm or about 280 to about 450 μm, e.g., about 240, 260, 280,300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 μm. In a nonlimiting example embodiment, the formulation is melt-mixed in a dual asymmetric centrifugal mixer to create a homogenous liquid ink.

In one aspect, the formulation has a higher viscosity that may be useful during manufacture by forming the formulation into a filament. The filament may then be used for fused filament fabrication.

In one aspect, the formulation is in the form of a filament. For instance, as further described in Example 2, ink formulations were prepared into filaments for use in 3D printing structures on a fused filament fabrication (FFF) 3D printer. In some embodiments, the filament formulation has a diameter of about 1 to about 3 mm, or about 1 to about 2.75 mm, about 1 to about 2.5 mm, about 1 to about 2.25 mm, about 1 to about 2 mm, about 1 to about 1.75 mm, about 1 to about 1.5 mm, about 1.25 to about 3 mm, about 1.25 to about 2.75 mm, about 1.25 to about 2.5 mm, about 1.25 to about 2.25 mm, about 1.25 to about 2 mm, about 1.25 to about 1.75 mm, about 1.25 to about 1.5 mm, about 1.5 to about 3 mm, about 1.5 to about 2.75 mm, about 1.5 to about 2.5 mm, about 1.5 to about 2.25 mm, about 1.5 to about 2 mm, about 1.5 to about 1.75 mm, about 1.75 to about 3 mm, about 1.75 to about 2.75 mm, about 1.75 to about 2.5 mm, about 1.75 to about 2.25 mm, about 1.75 to about 2 mm, about 2 to about 3 mm, about 2 to about 2.75 mm, about 2 to about 2.5 mm, or about 2 to about 2.25 mm. As a non-limiting example, the filament formulation has a diameter of about 1.5 mm to about 2 mm, or about 1.5 mm, about 1.75 mm, or about 2 mm.

In one aspect, the formulation is in the form of a pellet. For instance, as further described in example 2, ink formulations were prepared into pellets for use in 3D structures. In some embodiments, the pellets can be made into filaments. In some embodiments, pellets can be made into powders. In some embodiments, pellets have a length of about 1 to about 6 mm, or about 1 to about 5.5 mm, about 1 to about 5 mm, about 1 to about 4.5 mm, about 1 to about 4 mm, about 1 to about 3.5 mm, about 1 to about 3 mm, about 1 to about 2.5 mm, about 1 to about 2 mm, about 1 to about 1.5 mm, about 1.5 to about 6 mm, about 1.5 to about 5.5 mm, about 1.5 to about 5 mm, ab out 1.5 to ab out 4.5 mm, ab out 1.5 to about 4 mm, ab out 1.5 to ab out 3.5 mm, ab out 1.5 to about 3 mm, about 1.5 to about 2.5 mm, about 2 to about 6 mm, about 2 to about 5.5 mm, about 2 to about 5 mm, about 2 to about 4.5 mm, about 2 to about 4 mm, about 2 to about 3.5 mm, about 2 to about 3 mm, about 2 to about 2.5 mm, about 2.5 to about 6 mm, about 2.5 to about 5.5 mm, about 2.5 to about 5 mm, about 2.5 to about 4.5 mm, about 2.5 to about 4 mm, about 2.5 to about 3.5 mm, about 2.5 to about 3 mm, about 3 to about 6 mm, about 3 to about 5.5 mm, about 3 to about 5 mm, about 3 to about 4.5 mm, about 3 to about 4 mm, about 3 to about 3.5 mm, about 3.5 to about 6 mm, about 3.5 to about 5.5 mm, about 3.5 to about 5 mm, about 3.5 to about 4.5 mm, about 3.5 to about 4 mm, about 4 to about 6 mm, about 4 to about 5.5 mm, about 4 to about 5 mm, about 4 to about 4.5 mm, about 4.5 to about 6 mm, about 4.5 to about 5.5 mm, about 4.5 to about 5 mm, about 5 to about 6 mm, about 5 to about 5.5 mm, or about 5.5 to about 6 mm. As a non-limiting example embodiment, the pellets have a length of about 2.5 mm to about 4.5 mm, or about 2.5 mm, about 3 mm, about 3.5 mm, about 4 mm, or about 4.5 mm. In some embodiments, a pellet encompasses a variety of different shapes including spears, rods, granules, blocks, particles, and particles of any suitable shape.

In one aspect, the formulation is in the form of a powder. In some embodiments, the powder could be produced from a pellet. In a nonlimiting example embodiment, components of the formulation are melt-mixed into homogenous ink, and cryomilled to form powder. In a nonlimiting example embodiment, components of the formulation are dissolved in a solvent-based slurry and spray dried to form a powder. In some embodiments, powders are used in selective laser sintering.

3D Printed Structures

In another aspect, provided herein are 3D printed structures. The structures may be prepared using a formulation and/or method of manufacture described herein.

In some embodiments a three-dimensional structure has micropores. The micropores may be formed after removal of a particulate or pore former. The micropores may be formed by a use of a blowing agent during formulation. In some embodiments, the micropores provide additional surface area to the structure for contact with a therapeutic agent as compared to a structure lacking micropores. In a non-limiting example, the therapeutic agent comprises a targeting moiety that is non-covalently bound to a ceramic material of the structure. In some embodiments, the micropores have an average diameter of about 1 micron to ab out 500 microns. For instance, about 1 micron to about 450 microns, about 1 micron to about 400 microns, about 1 micron to about 350 microns, about 1 micron to about 300 microns, about 1 micron to about 250 microns, about 1 micron to about 200 microns, about 1 micron to about 150 microns, about 50 microns to about 500 microns, about 50 microns to about 450 microns, about 50 microns to about 400 microns, about 50 microns to about 350 microns, about 50 microns to about 300 microns, about 50 microns to about 250 microns, about 50 microns to about 200 microns, about 50 microns to about 150 microns, about 100 microns to about 500 microns, about 100 microns to about 450 microns, about 100 microns to about 400 microns, about 100 microns to about 350 microns, about 100 microns to about 300 microns, about 100 microns to about 250 microns, about 100 microns to about 200 microns, about 100 microns to about 150 microns, about 150 microns to about 500 microns, about 150 microns to about 450 microns, about 150 microns to about 400 microns, about 150 microns to about 350 microns, about 150 microns to about 300 microns, about 150 microns to about 250 microns, or about 150 microns to about 200 microns in diameter. In some cases, the micropores have an average diameter of about 50 microns to about 250 microns, about 60 microns to about 240 microns, about 70 microns to about 230 microns, about 80 microns to about 220 microns, or about 90 microns to about 210 microns. In some embodiments, the micropores an average diameter of about 100 microns to about 200 microns, e.g., about 110 microns to about 190 microns, about 120 microns to about 180 microns, about 130 microns to about 170 microns, about 140 microns to about 160 microns, or about 100 microns, about 110 microns, about 120 microns, about 130 microns, about 140 microns, ab out 150 microns, about 160 microns, ab out 170 microns, about 180 microns, about 190 microns, or about 200 microns. In some embodiments, the micropores have an average diameter of about 150 microns.

In some embodiments a three-dimensional structure has a density of ab out 1 g/cm³to about 3 g/cm³. In some embodiments a three-dimensional structure has a density of about 1 g/cm³to about 2 g/cm³. (e.g., about 1, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2 g/cm³or any value therebetween).

In some embodiments a three-dimensional structure has an open porosity of about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 20% to about 50%, about 20% to about 45%, about 25% to about 40%, about 25% to about 50%, about 25% to about 45%, or about 25% to about 40%. In some embodiments, the open porosity is about 25% to about 40%, e.g., about 25%, 30%, 35%, or 40%, or any value therebetween).

In some embodiments a three-dimensional structure has a strut diameter of about 300 nm to about 600 nm, about 325 nm to about 600 nm, about 350 nm to about 600 nm, about 375 nm to about 600 nm, about 400 nm to about 600 nm, about 425 nm to about 600 nm, about 450 nm to about 600 nm, about 475 nm to about 600 nm, about 500 nm to about 600 nm, about 525 nm to about 600 nm, about 550 nm to about 600 nm, about 300 nm to about 575 nm, about 325 nm to about 575 nm, about 350 nm to about 575 nm, about 375 nm to about 575 nm, about 400 nm to about 575 nm, about 425 nm to about 575 nm, about 450 nm to about 575 nm, about 475 nm to about 575 nm, about 500 nm to about 575 nm, about 525 nm to about 575 nm, about 550 nm to about 575 nm, about 300 nm to about 550 nm, about 325 nm to about 550 nm, about 350 nm to about 550 nm, about 375 nm to about 550 nm, about 400 nm to about 550 nm, about 425 nm to about 550 nm, about 450 nm to about 550 nm, about 475 nm to about 550 nm, about 500 nm to about 550 nm, about 525 nm to about 550 nm, about 300 nm to about 525 nm, about 325 nm to about 525 nm, about 350 nm to about 525 nm, about 375 nm to about 525 nm, about 400 nm to about 525 nm, about 425 nm to about 525 nm, about 450 nm to about 525 nm, about 475 nm to about 525 nm, about 500 nm to about 525 nm, about 300 nm to about 500 nm, about 325 nm to about 500 nm, about 350 nm to about 500 nm, about 375 nm to about 500 nm, about 400 nm to about 500 nm, about 425 nm to about 500 nm, about 450 nm to about 500 nm, about 475 nm to about 500 nm, about 300 nm to about 475 nm, about 325 nm to about 475 nm, about 350 nm to about 475 nm, about 375 nm to about 475 nm, about 400 nm to about 475 nm, about 425 nm to about 475 nm, about 450 nm to about 475 nm, ab out 300 nm to about 450 nm, about 325 nm to about 450 nm, about 350 nm to about 450 nm, about 375 nm to about 450 nm, about 400 nm to about 450 nm, about 425 nm to about 450 nm, about 300 nm to about 400 nm, about 325 nm to about 400 nm, about 350 nm to about 400 nm, or about 375 nm to about 400 nm.

In some embodiments, the structure comprises a ceramic material such as a calcium phosphate. In some embodiments, the structure comprises about 50-100, 50-95, 50-90, 50-85, 50-80, 50-75, 50-70, 50-65, 50-60, 50-55, 55-100, 55-95, 55-90, 55-85, 55-80, 55-75, 55-70, 55-65, 55-60, 60-100, 60-95, 60-90, 60-85, 60-80, 60-75, 60-70, 60-65, 65-100, 65-95, 65-90, 65-85, 65-80, 65-75, 65-70, 70-100, 70-95, 70-90, 70-85, 70-80, 70-75, 75-100, 75-95, 75-90, 75-85, 75-80, 80-100, 80-95, 80-90, 80-85, 85-100, 85-95, 85-90, 90-100, 90-95, 95-100, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent ceramic material. In some cases, the ceramic material is calcium phosphate, such as beta-tricalcium phosphate ((3-TCP).

The structure may be manufactured using 3D printing from an ink comprising about 30-70% by weight β-TCP powder, about 10-30% by weight first polymer, and about 10-30% by weight second polymer. In some cases, the first polymer comprises PCL. In some cases, the first polymer comprises PDS. In some cases, the second polymer comprises PEG. In some cases, the ink further comprises about 1-10% by weight particulate (e.g., sucrose). In some cases, the ink further comprises about 5-20% blowing agent (e.g., sodium bicarbonate).

In some embodiments the three-dimensional structure has a density of about 1 g/cm³to about 2 g/cm³or about 1.25 g/cm³to about 1.75 g/cm³. In some embodiments the three-dimensional structure has an open porosity of about 20% to about 40%, about 25% to about 35%, e.g., about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. In some embodiments a three-dimensional structure has a strut diameter of about 400 μm to about 500 μm, about 400 μm to about 450 μm, or about 425 μm to about 450 μm.

In some embodiments the three-dimensional structure has a density of about 1 g/cm³to about 2 g/cm³or about 1 g/cm³to about 1.5 g/cm³. In some embodiments the three-dimensional structure has an open porosity of about 30% to about 50%, about 35% to about 45%, e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments a three-dimensional structure has a strut diameter of about 325 μm to about 425 μm, about 350 μm to about 400 μm, or about 360 μm to about 390 μm.

In some embodiments the three-dimensional structure has a density of about 1 g/cm³to about 2 g/cm³or about 1 g/cm³to about 1.5 g/cm³. In some embodiments the three-dimensional structure has an open porosity of about 30% to about 50%, about 35% to about 45%, e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments a three-dimensional structure has a strut diameter of about 350 μm to about 450 μm, about 350 μm to about 400 μm, or about 380 μm to about 405 μm.

In a non-limiting example, a structure has about 50-90% ceramic material such as β-TCP. In some cases, the structure has about 50, 55, 60, 65, 70, 75, 80, 85, or 90% ceramic material such as β-TCP. In some embodiments, the structure has about 10-50% copolymer such as polycaprolactone/polyglycolide copolymer (PCL/PGA, e.g., 90:10, 95:5), poly(D,L-lactide-co-glycolide) copolymer (PLGA, e.g., 50:50), PDS-glycolide copolymer (PDS/PGA, e.g., 90:10), PDS-L-lactide copolymer (PDS/PLA, e.g., 90:10), or Dioxanone/L-lactide copolymer (e.g., 90:10). In some cases, the structure has about 10, 15, 20, 25, 30, 35, 40, 45, or 50% polymer such as PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide. Example structures include those having: about 85-90% ceramic (e.g., β-TCP) and about 10-15% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 80-85% ceramic (e.g., β-TCP) and about 15-20% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 75-80% ceramic (e.g., β-TCP) and about 20-25% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 70-75% ceramic (e.g., β-TCP) and about 25-30% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 65-70% ceramic (e.g., β-TCP) and about 30-35% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 60-65% ceramic (e.g., β-TCP) and ab out 35-40% polymer (e.g., PCL/PGA, PD S/PGA, PD S/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 55-60% ceramic (e.g., β-TCP) and about 40-45% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 50-55% ceramic (e.g., β-TCP) and about 45-50% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 90% ceramic (e.g., β-TCP) and about 10% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 89% ceramic (e.g., β-TCP) and about 11% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 88% ceramic (e.g., β-TCP) and about 12% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 87% ceramic (e.g., β-TCP) and about 13% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 86% ceramic (e.g., β-TCP) and about 14% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 85% ceramic (e.g., β-TCP) and about 15% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 84% ceramic (e.g., β-TCP) and about 16% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 83% ceramic (e.g., β-TCP) and about 17% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 82% ceramic (e.g., β-TCP) and about 18% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 81% ceramic (e.g., β-TCP) and about 19% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 80% ceramic (e.g., β-TCP) and about 20% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 79% ceramic (e.g., β-TCP) and about 21% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 78% ceramic (e.g., β-TCP) and about 22% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 77% ceramic (e.g., β-TCP) and about 23% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 76% ceramic (e.g., β-TCP) and about 24% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 75% ceramic (e.g., β-TCP) and about 25% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 74% ceramic (e.g., β-TCP) and about 26% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 73% ceramic (e.g., β-TCP) and about 27% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 72% ceramic (e.g., β-TCP) and about 28% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 71% ceramic (e.g., β-TCP) and about 29% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 70% ceramic (e.g., β-TCP) and about 30% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 69% ceramic (e.g., β-TCP) and about 31% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 68% ceramic (e.g., β-TCP) and about 32% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 67% ceramic (e.g., β-TCP) and about 33% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 66% ceramic (e.g., β-TCP) and about 34% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 65% ceramic (e.g., β-TCP) and about 35% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 64% ceramic (e.g., β-TCP) and about 36% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 63% ceramic (e.g., β-TCP) and about 37% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 62% ceramic (e.g., β-TCP) and about 38% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 61% ceramic (e.g., β-TCP) and about 39% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 60% ceramic (e.g., β-TCP) and about 40% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 59% ceramic (e.g., β-TCP) and about 41% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 58% ceramic (e.g., β-TCP) and about 42% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 57% ceramic (e.g., β-TCP) and about 43% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 56% ceramic (e.g., β-TCP) and about 44% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 55% ceramic (e.g., β-TCP) and about 45% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 54% ceramic (e.g., β-TCP) and about 46% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 53% ceramic (e.g., β-TCP) and about 47% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 52% ceramic (e.g., β-TCP) and about 48% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, about 51% ceramic (e.g., β-TCP) and about 49% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight, and about 50% ceramic (e.g., β-TCP) and about 50% polymer (e.g., PCL/PGA, PDS/PGA, PDS/PLA, PLGA, or Dioxanone/L-lactide) by weight.

In some embodiments the three-dimensional structure has a density of about 1 g/cm³to about 2 g/cm³or about 1.25 g/cm³to about 1.75 g/cm³. In some embodiments the three-dimensional structure has an open porosity of about 15% to about 35%, about 20% to about 30%, e.g., about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. In some embodiments a three-dimensional structure has a strut diameter of ab out 350 μm to about 450 μm, about 375 μm to about 425 μm, or about 385 μm to about 415 μm.

In some embodiments the three-dimensional structure has a density of about 1 g/cm³to about 2 g/cm³or about 1.5 g/cm³to about 2 g/cm³. In some embodiments the three-dimensional structure has an open porosity of about 15% to about 35%, about 20% to about 30%, e.g., about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. In some embodiments a three-dimensional structure has a strut diameter of about 500 μm to about 600 μm, about 550 μm to about 600 μm, or about 555 μm to about 585 μm.

In some embodiments the three-dimensional structure has a density of about 1 g/cm³to about 2 g/cm³or about 1.25 g/cm³to about 1.75 g/cm³. In some embodiments the three-dimensional structure has an open porosity of about 20% to about 40%, about 25% to about 35%, e.g., about 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. In some embodiments a three-dimensional structure has a strut diameter of ab out 400 μm to about 500 μm, about 450 μm to about 500 μm, or about 450 μm to about 475 μm.

In some embodiments, the compositions of ink formulations herein are varied to optimize specific surface area. The surface area may be optimized for combination with a certain therapeutic agent. For example, the structure has a surface area of about 0.2-2 m²/g for combination with a BMP protein (e.g., tBMP-2). In some embodiments, the surface area of a structure herein is about 0.2-2, 0.2-1.8, 0.2-1.6, 0.2-1.4, 0.2-1.2, 0.2-1, 0.2-0.8, 0.2-0.6, 0.2-0.4, 0.4-2, 0.4-1.8, 0.4-1.6, 0.4-1.4, 0.4-1.2, 0.4-1, 0.4-0.8, 0.4-0.6, 0.6-2, 0.6-1.8, 0.6-1.6, 0.6-1.4, 0.6-1.2, 0.6-1, 0.6-0.8, 0.8-2, 0.8-1.8, 0.8-1.6, 0.8-1.4, 0.8-1.2, 0.8-1, 1-2, 1-1.8, 1-1.6, 1-1.4, 1-1.2, 1.2-2, 1.2-1.8, 1.2-1.6, 1.2-1.4, 1.4-2, 1.4-1.8, 1.4-1.6, 1.6-2, 1.6-1.8, or 1.8-2 m²/g. In some embodiments, the surface area is calculated by Brunauer-Emmett-Teller (BET) by gas physisorption.

In some embodiments, the compositions of ink formulations herein are varied to optimize resorption rate of one or more materials of the scaffold. For instance, the polymers are selected based on resorption rate. The resorption rates vary from slowest to fastest as: polycaprolactone, polycaprolactone/polyglycolide copolymer (95:5), polycaprolactone/glycolide copolymer (90:10), polydioxanone/L-lactide copolymer (90:10), poly(D,L-lactide-co-glycolide) copolymer (50:50).

Methods of Manufacture

In another aspect, provided are methods of manufacturing a structure using 3D printing techniques.

In some embodiments, the method comprises syringe-based melt extrusion bioprinting Example inks for this method may be low in viscosity for extrusion of the ink through a narrow nozzle. Non-limiting example methods of manufacturing using this method are described in Example 2, for instance, with regard to printing ink formulations #1, #2, #3, #4.

In one aspect, the method is an extrusion based method comprising a 3D printing method that extrudes the material out of a nozzle.

In some embodiments, the extrusion based method encompasses bioprinting (syringe-based pneumatic printing) or Fused Granular Fabrication (FGF) where pellets of feedstock are fed from a hopper into a mini-screw extrusion head which melts and pushes the material out of a fine nozzle. In some embodiments, the inks are formed into small (e.g., 2-5 mm) pellets or granules.

In some embodiments, the method comprises fused filament fabrication (FFF). Example inks for this method may be formed into filaments for printing on FFF 3D printers. Non-limiting example methods of manufacturing using this method are described in Example 2, for instance, with regard to printing ink formulations #5 and #6.

In some embodiments, the method comprises pelletized fused deposition modeling. Fused deposition modeling (FDM) is an additive manufacturing process. Three dimensional objects are formed through extrusion and deposition of individual layers of thermoplastic materials. FDM involves the melt extrusion of filament materials through a heated nozzle and deposition as thin solid layers on a platform. A thermoplastic polymer material is fed into a temperature-controlled FDM extrusion head and it is heated to a semi-liquid state. Afterward, the FDM extrusion head extrudes and deposits the material in ultra-thin layers onto a base with precision. The material solidifies, laminating to the preceding layer. In this way, parts are fabricated in layers, where each layer is built by extruding a small bead of material, called a road, in a particular pattern, such that the layer is covered with the adjacent roads. After each layer is completed, extrusion head height is increased and sub sequent layers are built to construct the part. Usually, FDM is used to fabricate solid models. In order to fabricate porous structures, raster fill gaps have a positive value which is applied to impart a channel within a build layer. Arranged in a regular manner, the channels are interconnected even in three dimensions. Layer by layer fabrication allows design of a pore morphology which varies across a scaffold structure.

In some embodiments, the method comprises selective laser sintering (SLS). Selective laser sintering (SLS) is a process wherein a dispenser deposits layers of powdered material into a target area. There is a laser control mechanism that typically includes a computer with the article design stored on it. The laser control mechanism modulates and moves a laser beam to selectively irradiate the powder layer within defined boundaries of the design, melting the powder on which the laser beam falls. This is done to selectively sinter sequential powder layers. The method produces a completed article comprised of a plurality of layers sintered together.

In some embodiments, after 3D printing, the resulting subject is soaked in water to dissolve certain components of the ink, e.g., PEG, particulate (e.g., pore forming agent, sucrose), blowing agent (e.g., sodium bicarbonate), or a combination thereof. The structure may then be dried, sterilized, treated with a therapeutic as described elsewhere herein, or a combination thereof.

Any of the 3D-printed structures described herein can be coated with a tetherable protein (for example, tBMP2). Following completion of the structures using any of the methods discussed herein, the structures can be washed in an acidic sodium acetate buffer. This can be one, two, or more washes. The washing can then be followed by a two-hour incubation of the structures in sodium acetate buffer that contains a 1 mg/mL concentration of tBMP2 protein. The tetherable tBMP2 binds to the β-TCP surface of the implantable structures in a monolayer.

In further embodiments, the ink formulations discussed herein can include a light-sensitive resin that is mixed with the ceramic powder for digital light processing (DLP), an additive manufacturing technique that is faster than robocasting or melt extrusion. Components in a photosensitive, ceramic-filled resin for DLP 3D printing of bone implants typically include ceramic powder (e.g., β-TCP, hydroxyapatite, bioglass, typically <10 μm particle size), one or more crosslinking acrylates or methacrylates (e.g., polyethylene glycol diacrylate, polycaprolactone methacrylate), a plasticizer to reduce resin viscosity (e.g., water), a dispersant to promote breakdown of powder agglomerates (e.g., Darvan® 821-A), photoinitiator to initiate the photocrosslinking reaction (e.g., Lithium phenyl-2,4,6-trimethylbenzoylphosphinate), and a photoabsorber to retain high x-y resolution (e.g., tartrazine). Once resin formulations are prepared by asymmetric centrifugal mixing of the components, the ink is exposed layer by layer to a DLP image, causing the lighted pixels to selectively solidify when the resin encounters the light Once the implantable structure has been built up layer by layer, it can be thermally processed to burn out the included polymer and densify the ceramic (e.g., a polyethylene glycol diacrylate-containing resin), or left as-is, resulting in a flexible ceramic/polymer composite implant (e.g., a polycaprolactone methacrylate-containing resin).

Devices

In another aspect, provided are devices and kits comprising a 3D printed structure described herein and a therapeutic agent. In some embodiments, a device comprises the therapeutic agent connected to, dispersed within, or otherwise combined with the 3D printed structure. As used herein, a therapeutic agent is inclusive of a plurality of therapeutic agents, such as 2, 3, 4, or 5 therapeutic agents.

Therapeutic Agents

In some embodiments, the therapeutic agent comprises a mammalian growth factor or a functional portion thereof. Mammalian growth factors can be osteoinductive molecules that are capable of initiating and enhancing the bone repair process. A functional portion of the mammalian growth factor is a region that has a therapeutic effect. For instance, a functional portion of a mammalian growth factor is osteoinductive. As another example, a functional portion of a mammalian growth factor is capable of initiating and/or enhancing bone repair. A functional portion of a mammalian growth factor may have osteogenic activity.

Non-limiting examples of mammalian growth factors are described herein. In some instances, the mammalian growth factor comprises: epidermal growth factor (EGF), platelet derived growth factor (PDGF), insulin like growth factor (IGF-1), fibroblast growth factor (FGF), fibroblast growth factor 2 (FGF2), fibroblast growth factor 18 (FGF18), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), transforming growth factor beta 1 (TGF-β1), transforming growth factor beta 3 (TGF-β3), osteogenic protein 1 (OP-1), osteogenic protein 2 (OP-2), osteogenic protein 3 (OP-3), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 3 (BMP-3), bone morphogenetic protein 4 (BMP-4), bone morphogenetic protein 5 (BMP-5), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), bone morphogenetic protein (BMP-9), bone morphogenetic protein 10 (BMP-10), bone morphogenetic protein 11 (BMP-11), bone morphogenetic protein 12 (BMP-12), bone morphogenetic protein 13 (BMP-13), bone morphogenetic protein 15 (BMP-15), dentin phosphoprotein (DPP), vegetal related growth factor (Vgr), growth differentiation factor 1 (GDF-1), growth differentiation factor 3 (GDF-3), growth differentiation factor 5 (GDF-5), growth differentiation factor 6 (GDF-6), growth differentiation factor 7 (GDF-7), growth differentiation factor 8 (GDF8), growth differentiation factor 11 (GDF11), growth differentiation factor 15 (GDF15), vascular endothelial growth factor (VEGF), hyaluronic acid binding protein (HABP), and collagen binding protein (CBP), fibroblast growth factor 18 (FGF-18), keratinocyte growth factor (KGF), tumor necrosis factor alpha (TNFα), tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), wnt family member 1 (WNT1), wnt family member 2 (WNT2), wnt family member 2B (WNT2B), wnt family m emb er 3 (WNT3), wnt family member 3 A (WNT3 A), wnt family member 4 (WNT4), wnt family member 5A (WNT5A), wnt family member 5B (WNT5B), wnt family member 6 (WNT6), wnt family member 7A (WNT7A), wnt family member 7B (WNT7B), wnt family member 8A (WNT8A), wnt family member 8B (WNT8B), wnt family member 9A (WNT9A), wnt family member 9B (WNT9B), wnt family member 10A (WNT10A), wnt family member 10B (WNT10B), wnt family member 11 (WNT11), or wnt family member 16 (WNT16), or a mature peptide or functional portion thereof.

In some embodiments, the mammalian growth factor is a human growth factor. Non-limiting examples of human growth factors and mature peptides and/or functional portions thereof are provided in Table 1. In some embodiments, the mammalian growth factor comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to any of the sequences in Table 1 or any secreted human growth factor, and has osteogenic activity. In some embodiments, the amino acids in a mammalian growth factor that are conserved between different species are likely important for osteogenic activity and may not be mutated, while amino acids in a mammalian growth factor that are not conserved between different species are not likely important for osteogenic activity and may be mutated.

In some embodiments, the mammalian growth factor comprises BMP-2. In some embodiments, the mammalian growth factor is a mature peptide of BMP-2 (e.g., does not comprise a signal sequence). In some embodiments, the mammalian growth factor comprises a functional portion of BMP-2. In some embodiments, the functional portion of BMP-2 comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to: QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNS TNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 454). In some embodiments, the mammalian growth factor comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 454. In some embodiments, the mammalian growth factor comprises a sequence at least about 90% identical to SEQ ID NO: 454. In some embodiments, the mammalian growth factor comprises SEQ ID NO: 454.

In some embodiments, the mammalian growth factor is a non-human mammalian growth factor. The non-human mammalian growth factor may be homologous to a human growth factor, such as one or more of the human growth factors of Table 1. In some embodiments, a non-human mammalian growth factor is homologous to a human growth factor if the non-human mammalian growth factor is at least ab out 80% identical to the human mammalian growth factor as determined using the NCBI Blast alignment algorithm as of the date of this filing. In some cases, the coverage is at least about 90%. In some embodiments, a non-human mammalian growth factor is homologous to a human growth factor if the non-human mammalian growth factor is at least about 80% positive as compared to the human mammalian growth factor as determined using the NCBI Blast alignment algorithm as of the date of this filing. In some cases, the coverage is at least about 90%. In some embodiments, a non-human mammalian growth factor is homologous to a human growth factor if the non-human mammalian growth factor aligned with the human growth factor using the NCBI Blast as of the date of this filing has an E value of less than about 1E-40, at least about 1E-50, 1E-60, 1E-70, or 1E-10, with a query cover of at least about 90%.

TABLE 1

Therapeutic Growth Factors

SEQ ID

Name
Protein Sequence
NO:

EGF
NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLK
442

WWELR

PDGF
EEAEIPREVIERLARSQIHSIRDLQRLLEIDSVGSEDSLDTSLRAHGVHATK
443

HVPEKRPLPIRRKR

IGF-1
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFR
444

SCDLRRLEMYCAPLKPAKSA

FGF
FNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAES
445

VGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISK

KHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD

FGF2
PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPH
446

IKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLES

NNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS

FGF18
EENVDFRIHVENQTRARDDVSRKQLRLYQLYSRTSGKHIQVLGRRISARG
447

EDGDKYAQLLVETDTFGSQVRIKGKETEFYLCMNRKGKLVGKPDGTSKE

CVFIEKVLENNYTALMSAKYSGWYVGFTKKGRPRKGPKTRENQQDVHF

MKRYPKGQPELQKPFKYTTVTKRSRRIRPTHPA

TGF-α
ENSTSPLSADPPVAAAVVSHFNDCPDSHTQFCFHGTCRFLVQEDKPACVC
448

HSGYVGARCEHADLLAVVAASQKKQAITALVVVSIVALAVLIITCVLIHC

CQVRKHCEWCRALICRHEKPSALLKGRTACCHSETVV

TGF-α
VVSHFNDCPDSHTQFCFHGTCRFLVQEDKPACVCHSGYVGARCEHADLL
449

A

TGF-ß1
ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCP
450

YIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKV

EQLSNMIVRSCKCS

TGF-ß3
ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCP
451

YLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVE

QLSNMVVKSCKCS

OP-2
AVRPLRRRQPKKSNELPQANRLPGIFDDVHGSHGRQVCRRHELYVSFQD
452

(BMP-8)
LGWLDWVIAPQGYSAYYCEGECSFPLDSCMNATNHAILQSLVHLMMPD

AVPKACCAPTKLSATSVLYYDSSNNVILRKHRNMVVKACGCH

BMP8A
AVRPLRRRQPKKSNELPQANRLPGIFDDVRGSHGRQVCRRHELYVSFQD
453

LGWLDWVIAPQGYSAYYCEGECSFPLDSCMNATNHAILQSLVHLMKPN

AVPKACCAPTKLSATSVLYYDSSNNVILRKHRNMVVKACGCH

BMP-2
QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECP
454

FPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKV

VLKNYQDMVVEGCGCR

BMP-3
QWIEPRNCARRYLKVDFADIGWSEWIISPKSFDAYYCSGACQFPMPKSLK
455

PSNHATIQSIVRAVGVVPGIPEPCCVPEKMSSLSILFFDENKNVVLKVYPN

MTVESCACR

BMP-4
SPKHHSQRARKKNKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGD
456

CPFPLADHLNSTNHAIVQTLVNSVNSSIPKACCVPTELSAISMLYLDEYDK

VVLKNYQEMVVEGCGCR

BMP-5
AANKRKNQNRNKSSSHQDSSRMSSVGDYNTSEQKQACKKHELYVSFRD
457

LGWQDWIIAPEGYAAFYCDGECSFPLNAHMNATNHAIVQTLVHLMFPD

HVPKPCCAPTKLNAISVLYFDDSSNVILKKYRNMVVRSCGCH

BMP-6/
SASSRRRQQSRNRSTQSQDVARVSSASDYNSSELKTACRKHELYVSFQDL
458

VGR
GWQDWIIAPKGYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEY

VPKPCCAPTKLNAISVLYFDDNSNVILKKYRNMVVRACGCH

BMP-7/
STGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRD
459

OP-1
LGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPET

VPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH

BMP-9
SAGAGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGCFFPLADDVT
460

PTKHAIVQTLVHLKFPTKVGKACCVPTKLSPISVLYKDDMGVPTLKYHY

EGMSVAECGCR

BMP-10
NAKGNYCKRTPLYIDFKEIGWDSWIIAPPGYEAYECRGVCNYPLAEHLTP
461

TKHAIIQALVHLKNSQKASKACCVPTKLEPISILYLDKGVVTYKFKYEGM

AVSECGCR

BMP-11/
NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEY
462

GDF-11
MFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKI

PGMVVDRCGCS

BMP-12
TALAGTRTAQGSGGGAGRGHGRRGRSRCSRKPLHVDFKELGWDDWIIA
463

PLDYEAYHCEGLCDFPLRSHLEPTNHAIIQTLLNSMAPDAAPASCCVPAR

LSPISILYIDAANNVVYKQYEDMVVEACGCR

BMP-13/
TAFASRHGKRHGKKSRLRCSKKPLHVNFKELGWDDWIIAPLEYEAYHCE
464

GDF-6
GVCDFPLRSHLEPTNHAIIQTLMNSMDPGSTPPSCCVPTKLTPISILYIDAG

NNVVYKQYEDMVVESCGCR

BMP-15
QADGISAEVTASSSKHSGPENNQCSLHPFQISFRQLGWDHWIIAPPFYTPN
465

YCKGTCLRVLRDGLNSPNHAIIQNLINQLVDQSVPRPSCVPYKYVPISVL

MIEANGSILYKEYEGMIAESCTCR

DPP isoform
IPVPQSKPLERHVEKSMNLHLLARSNVSVQDELNASGTIKESGVLVHEGD
466

1
RGRQENTQDGHKGEGNGSKWAEVGGKSFSTYSTLANEEGNIEGWNGDT

GKAETYGHDGIHGKEENITANGIQGQVSIIDNAGATNRSNINGNTDKNTQ

NGDVGDAGHNEDVAVVQEDGPQVAGSNNSTDNEDEIIENSCRNEGNTSE

ITPQINSKRNGTKEAEVTPGTGEDAGLDNSDGSPSGNGADEDEDEGSGDD

EDEEAGNGKDSSNNSKGQEGQDHGKEDDHDSSIGQNSDSKEYYDPEGKE

DPHNEVDGDKTSKSEENSAGIPEDNGSQRIEDTQKLNHRESKRVENRITK

ESETHAVGKSQDKGIEIKGPSSGNRNITKEVGKGNEGKEDKGQHGMILG

KGNVKTQGEVVNIEGPGQKSEPGNKVGHSNTGSDSNSDGYDSYDFDDKS

MQG

DPP isoform
IPVPQSKPLERHVEKSMNLHLLARSNVSVQDELNASGTIKESGVLVHEGD
467

2
RGRQENTQDGHKGEGNGSKWAEVGGKSFSTYSTLANEEGNIEGWNGDT

GKAETYGHDGIHGKEENITANGIQGQVSIIDNAGATNRSNTNGNTDKNTQ

NGDVGDAGHNEDVAVVQEDGPQVAGSNNSTDNEDEIIENSCRNEGNTSE

ITPQINSKRNGTKEAEVTPGTGEDAGLDNSDGSPSGNGADEDEDEGSGDD

EDEEAGNGKDSSNNSKGQEGQDHGKEDDHDSSIGQNSDSKEYYDPEGKE

DPHNEVDGDKTSKSEENSAGIPEDNGSQRIEDTQKLNHRESKRVENRITK

ESETHAVGKSQDKGIEIKGPSSGNRNITKEVGKGNEGKEDKGQHGMILG

KGNVKTQGEVVNIEGPGQKSEPGNKVGHSNTGSDSNSDGYDSYDFDDKS

MQGDDPNSSDESNGNDDANSESDNNSSSRGDASYNSDESKDNGNGSDS

KGAEDDDSDSTSDTNNSDSNGNGNNGNDDNDKSDSGKGKSDSSDSDSS

DSSNSSDSSDSSDSDSSDSNSSSDSDSSDSDSSDSSDSDSSDSSNSSDSSDSS

DSSDSSDSSDSSDSKSDSSKSESDSSDSDSKSDSSDSNSSDSSDNSDSSDSS

NSSNSSDSSDSSDSSDSSSSSDSSNSSDSSDSSDSSNSSESSDSSDSSDSDSS

DSSDSSNSNSSDSDSSNSSDSSDSSNSSDSSDSSDSSNSSDSSDSSDSSNSSD

SSDSSDSSDSSDSSNSSDSNDSSNSSDSSDSSNSSDSSNSSDSSDSSDSSDSD

SSNSSDSSNSSDSSDSSNSSDSSDSSDSSDGSDSDSSNRSDSSNSSDSSDSSD

SSNSSDSSDSSDSNESSNSSDSSDSSNSSDSDSSDSSNSSDSSDSSNSSDSSE

SSNSSDNSNSSDSSNSSDSSDSSDSSNSSDSSNSSDSSNSSDSSDSNSSDSSD

SSNSSDSSDSSDSSDSSDSSDSSNSSDSSDSSDSSDSSNSSDSSNSSDSSNSS

DSSDSSDSSDSSDSSDSSDSSDSSNSSDSSDSSDSSDSSDSSDSSDSSDSSES

SDSSDSSNSSDSSDSSDSSDSSDSSDSSDSSDSSDSSNSSDSSDSSDSSDSSD

SSNSSDSSDSSESSDSSDSSDSSDSSDSSDSSDSSDSSDSSNSSDSSDSSDSS

DSSDSSDSSDSSDSSDSSDSSDSSDSSDSSDSSDSSDSSDSNESSDSSDSSDS

SDSSNSSDSSDSSDSSDSTSDSNDESDSQSKSGNGNNNGSDSDSDSEGSDS

NHSTSDD

DPP isoform
DDPNSSDESNGNDDANSESDNNSSSRGDASYNSDESKDNGNGSDSKGAE
468

3
DDDSDSTSDTNNSDSNGNGNNGNDDNDKSDSGKGKSDSSDSDSSDSSNS

SDSSDSSDSDSSDSNSSSDSDSSDSDSSDSSDSDSSDSSNSSDSSDSSDSSDS

SDSSDSSDSKSDSSKSESDSSDSDSKSDSSDSNSSDSSDNSDSSDSSNSSNS

SDSSDSSDSSDSSSSSDSSNSSDSSDSSDSSNSSESSDSSDSSDSDSSDSSDSS

NSNSSDSDSSNSSDSSDSSNSSDSSDSSDSSNSSDSSDSSDSSNSSDSSDSSD

SSDSSDSSNSSDSNDSSNSSDSSDSSNSSDSSNSSDSSDSSDSSDSDSSNSSD

SSNSSDSSDSSNSSDSSDSSDSSDGSDSDSSNRSDSSNSSDSSDSSDSSNSSD

SSDSSDSNESSNSSDSSDSSNSSDSDSSDSSNSSDSSDSSNSSDSSESSNSSD

NSNSSDSSNSSDSSDSSDSSNSSDSSNSSDSSNSSDSSDSNSSDSSDSSNSSD

SSDSSDSSDSSDSSDSSNSSDSSDSSDSSDSSNSSDSSNSSDSSNSSDSSDSS

DSSDSSDSSDSSDSSDSSNSSDSSDSSDSSDSSDSSDSSDSSDSSESSDSSDS

SNSSDSSDSSDSSDSSDSSDSSDSSDSSDSSNSSDSSDSSDSSDSSDSSNSSD

SSDSSESSDSSDSSDSSDSSDSSDSSDSSDSSDSSNSSDSSDSSDSSDSSDSS

DSSDSSDSSDSSDSSDSSDSSDSSDSSDSSDSSDSNESSDSSDSSDSSDSSNS

SDSSDSSDSSDSTSDSNDESDSQSKSGNGNNNGSDSDSDSEGSDSNHSTSD

D

GDF-1
DAEPVLGGGPGGACRARRLYVSFREVGWHRWVIAPRGFLANYCQGQCA
469

LPVALSGSGGPPALNHAVLRALMHAAAPGAADLPCCVPARLSPISVLFFD

NSDNVVLRQYEDMVVDECGCR

GDF-3
AAIPVPKLSCKNLCHRHQLFINFRDLGWHKWIIAPKGFMANYCHGECPFS
470

LTISLNSSNYAFMQALMHAVDPEIPQAVCIPTKLSPISMLYQDNNDNVILR

HYEDMVVDECGCG

GDF-5
APLATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHC
471

EGLCEFPLRSHLEPTNHAVIQTLMNSMDPESTPPTCCVPTRLSPISILFIDSA

NNVVYKQYEDMVVESCGCR

GDF8
DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEF
472

VFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIP

AMVVDRCGCS

GDF15
ARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIG
473

ACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG

VSLQTYDDLLAKDCHCI

VEGF
APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPS
474

CVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHN

KCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCL

MPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNE

RTCRCDKPRR

HABP
FSLMSLLESLDPDWTPDQYDYSYEDYNQEENTSSTLTHAENPDWYYTED
475

Isoform 1
QADPCQPNPCEHGGDCLVHGSTFTCSCLAPFSGNKCQKVQNTCKDNPCG

RGQCLITQSPPYYRCVCKHPYTGPSCSQVVPVCRPNPCQNGATCSRHKRR

SKFTCACPDQFKGKFCEIGSDDCYVGDGYSYRGKMNRTVNQHACLYWN

SHLLLQENYNMFMEDAETHGIGEHNFCRNPDADEKPWCFIKVINDKVK

WEYCDVSACSAQDVAYPEESPTEPSTKLPGFDSCGKTEIAERKIKR

HABP
IYGGFKSTAGKHPWQASLQSSLPLTISMPQGHFCGGALIHPCWVLTAAHC
477

Isoform 2
TDIKTRHLKVVLGDQDLKKEEFHEQSFRVEKIFKYSHYNERDEIPHNDIAL

LKLKPVDGHCALESKYVKTVCLPDGSFPSGSECHISGWGVTETGKGSRQ

LLDAKVKLIANTLCNSRQLYDHMIDDSMICAGNLQKPGQDTCQGDSGGP

LTCEKDGTYYVYGIVSWGLECGKRPGVYTQVTKFLNWIKATIKSESGF

CBP
AEVKKPAAAAAPGTAEKLSPKAATLAERSAGLAFSLYQAMAKDQAVEN
479

ILVSPVVVASSLGLVSLGGKATTASQAKAVLSAEQLRDEEVHAGLGELLR

SLSNSTARNVTWKLGSRLYGPSSVSFADDFVRSSKQHYNCEHSKINFRDK

RSALQSINEWAAQTTDGKLPEVTKDVERTDGALLVNAMFFKPHWDEKF

HHKMVDNRGFMVTRSYTVGVMMMHRTGLYNYYDDEKEKLQIVEMPL

AHKLSSLIILMPHHVEPLERLEKLLTKEQLKIWMGKMQKKAVAISLPKGV

VEVTHDLQKHLAGLGLTEAIDKNKADLSRMSGKKDLYLASVFHATAFEL

DTDGNPFDQDIYGREELRSPKLFYADHPFIFLVRDTQSGSLLFIGRLVRPK

GDKMRDEL

KGF
CNDMTPEQMATNVNCSSPERHTRSYDYMEGGDIRVRRLFCRTQWYLRID
480

KRGKVKGTQEMKNNYNIMEIRTVAVGIVAIKGVESEFYLAMNKEGKLY

AKKECNEDCNFKELILENHYNTYASAKWTHNGGEMFVALNQKGIPVRG

KKTKKEQKTAHFLPMAIT

TNFα
GPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLN
481

RRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTI

SRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGD

RLSAEINRPDYLDFAESGQVYFGIIAL

TRAIL
TNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLR
482

QLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTL

SSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQ

TYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAE

YGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG

WNT1
ANSSGRWWGIVNVASSTNLLTDSKSLQLVLEPSLQLLSRKQRRLIRQNPG
483

ILHSVSGGLQSAVRECKWQFRNRRWNCPTAPGPHLFGKIVNRGCRETAFI

FAITSAGVTHSVARSCSEGSIESCTCDYRRRGPGGPDWHWGGCSDNIDFG

RLFGREFVDSGEKGRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSG

SCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNRASRAELLR

LEPEDPAHKPPSPHDLVYFEKSPNFCTYSGRLGTAGTAGRACNSSSPALD

GCELLCCGRGHRTRTQRVTERCNCTFHWCCHVSCRNCTHTRVLHECL

WNT2
SWWYMRATGGSSRVMCDNVPGLVSSQRQLCHRHPDVMRAISQGVAEW
484

TAECQHQFRQHRWNCNTLDRDHSLFGRVLLRSSRESAFVYAISSAGVVF

AITRACSQGEVKSCSCDPKKMGSAKDSKGIFDWGGCSDNIDYGIKFARAF

VDAKERKGKDARALMNLHNNRAGRKAVKRFLKQECKCHGVSGSCTLR

TCWLAMADFRKTGDYLWRKYNGAIQVVMNQDGTGFTVANERFKKPTK

NDLVYFENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGY

DTSHVTRMTKCGCKFHWCCAVRCQDCLEALDVHTCKAPKNADWTTAT

WNT2B
SWWYIGALGARVICDNIPGLVSRQRQLCQRYPDIMRSVGEGAREWIREC
485

QHQFRHHRWNCTTLDRDHTVFGRVMLRSSREAAFVYAISSAGVVHAITR

ACSQGELSVCSCDPYTRGRHHDQRGDFDWGGCSDNIHYGVRFAKAFVD

AKEKRLKDARALMNLHNNRCGRTAVRRFLKLECKCHGVSGSCTLRTCW

RALSDFRRTGDYLRRRYDGAVQVMATQDGANFTAARQGYRRATRTDL

VYFDNSPDYCVLDKAAGSLGTAGRVCSKTSKGTDGCEIMCCGRGYDTT

RVTRVTQCECKFHWCCAVRCKECRNTVDVHTCKAPKKAEWLDQT

WNT3
GYPIWWSLALGQQYTSLGSQPLLCGSIPGLVPKQLRFCRNYIEIMPSVAEG
486

VKLGIQECQHQFRGRRWNCTTIDDSLAIFGPVLDKATRESAFVHAIASAG

VAFAVTRSCAEGTSTICGCDSHHKGPPGEGWKWGGCSEDADFGVLVSRE

FADARENRPDARSAMNKHNNEAGRTTILDHMHLKCKCHGLSGSCEVKT

CWWAQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRAKYSLFK

PPTERDLVYYENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGR

GHNTRTEKRKEKCHCIFHWCCYVSCQECIRIYDVHTCK

WNT3A
SYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMPSVAEG
487

IKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAG

VAFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSRE

FADARENRPDARSAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKT

CWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESRGWVETLRPRYTYFK

VPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGR

GHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCK

WNT4
SNWLYLAKLSSVGSISEEETCEKLKGLIQRQVQMCKRNLEVMDSVRRGA
488

QLAIEECQYQFRNRRWNCSTLDSLPVFGKVVTQGTREAAFVYAISSAGV

AFAVTRACSSGELEKCGCDRTVHGVSPQGFQWSGCSDNIAYGVAFSQSF

VDVRERSKGASSSRALMNLHNNEAGRKAILTHMRVECKCHGVSGSCEV

KTCWRAVPPFRQVGHALKEKFDGATEVEPRRVGSSRALVPRNAQFKPHT

DEDLVYLEPSPDFCEQDMRSGVLGTRGRTCNKTSKAIDGCELLCCGRGF

HTAQVELAERCSCKFHWCCFVKCRQCQRLVELHTCR

WNT5A
IIGAQPLCSQLAGLSQGQKKLCHLYQDHMQYIGEGAKTGIKECQYQFRH
489

RRWNCSTVDNTSVFGRVMQIGSRETAFTYAVSAAGVVNAMSRACREGE

LSTCGCSRAARPKDLPRDWLWGGCGDNIDYGYRFAKEFVDARERERIHA

KGSYESARILMNLHNNEAGRRTVYNLADVACKCHGVSGSCSLKTCWLQ

LADFRKVGDALKEKYDSAAAMRLNSRGKLVQVNSRFNSPTTQDLVYIDP

SPDYCVRNESTGSLGTQGRLCNKTSEGMDGCELMCCGRGYDQFKTVQT

ERCHCKFHWCCYVKCKKCTEIVDQFVCK

WNT5B
QLLTDANSWWSLALNPVQRPEMFIIGAQPVCSQLPGLSPGQRKLCQLYQ
490

EHMAYIGEGAKTGIKECQHQFRQRRWNCSTADNASVFGRVMQIGSRETA

FTHAVSAAGVVNAISRACREGELSTCGCSRTARPKDLPRDWLWGGCGD

NVEYGYRFAKEFVDAREREKNFAKGSEEQGRVLMNLQNNEAGRRAVY

KMADVACKCHGVSGSCSLKTCWLQLAEFRKVGDRLKEKYDSAAAMRV

TRKGRLELVNSRFTQPTPEDLVYVDPSPDYCLRNESTGSLGTQGRLCNKT

SEGMDGCELMCCGRGYNQFKSVQVERCHCKFHWCCFVRCKKCTEIVDQ

YICK

WNT6
LWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPEVVAELARGARLG
491

VRECQFQFRFRRWNCSSHSKAFGRILQQDIRETAFVFAITAAGASHAVTQ

ACSMGELLQCGCQAPRGRAPPRPSGLPGTPGPPGPAGSPEGSAAWEWGG

CGDDVDFGDEKSRLFMDARHKRGRGDIRALVQLHNNEAGRLAVRSHTR

TECKCHGLSGSCALRTCWQKLPPFREVGARLLERFHGASRVMGTNDGK

ALLPAVRTLKPPGRADLLYAADSPDFCAPNRRTGSPGTRGRACNSSAPDL

SGCDLLCCGRGHRQESVQLEENCLCRFHWCCVVQCHRCRVRKELSLCL

WNT7A
LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRW
492

NCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCG

CDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM

NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLK

DKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCE

EDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNC

KFHWCCYVKCNTCSERTEMYTCK

WNT7B
ALSSVVALGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQ
493

FRFGRWNCSALGEKTVFGQELRVGSREAAFTYAITAAGVAHAVTAACSQ

GNLSNCGCDREKQGYYNQAEGWKWGGCSADVRYGIDFSRRFVDAREIK

KNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKF

REVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVY

IEKSPNYCEEDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYT

KVWQCNCKFHWCCFVKCNTCSERTEVFTCK

WNT8A
VNNFLITGPKAYLTYTTSVALGAQSGIEECKFQFAWERWNCPENALQLST
494

HNRLRSATRETSFIHAISSAGVMYIITKNCSMGDFENCGCDGSNNGKTGG

HGWIWGGCSDNVEFGERISKLFVDSLEKGKDARALMNLHNNRAGRLAV

RATMKRTCKCHGISGSCSIQTCWLQLAEFREMGDYLKAKYDQALKIEMD

KRQLRAGNSAEGHWVPAEAFLPSAEAELIFLEESPDYCTCNSSLGIYGTE

GRECLQNSHNTSRWERRSCGRLCTECGLQVEERKTEVISSCNCKFQWCC

TVKCDQCRHVVSKYYCARSPGSAQSLGKGSA

WNT8B
WSVNNFLMTGPKAYLIYSSSVAAGAQSGIEECKYQFAWDRWNCPERAL
495

QLSSHGGLRSANRETAFVHAISSAGVMYTLTRNCSLGDFDNCGCDDSRN

GQLGGQGWLWGGCSDNVGFGEAISKQFVDALETGQDARAAMNLHNNE

AGRKAVKGTMKRTCKCHGVSGSCTTQTCWLQLPEFREVGAHLKEKYHA

ALKVDLLQGAGNSAAGRGAIADTFRSISTRELVHLEDSPDYCLENKTLGL

LGTEGRECLRRGRALGRWERRSCRRLCGDCGLAVEERRAETVSSCNCKF

HWCCAVRCEQCRRRVTKYFCSRAERPRGGAAHKPGRKP

WNT9A
YFGLTGSEPLTILPLTLEPEAAAQAHYKACDRLKLERKQRRMCRRDPGV
496

AETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFKETAFLYAI

SSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSS

KFVKEFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCT

VRTCWRQLAPFHEVGKHLKHKYETALKVGSTTNEAAGEAGAISPPRGRA

SGAGGSDPLPRTPELVHLDDSPSFCLAGRFSPGTAGRRCHREKNCESICCG

RGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEVYTCKG

WNT9B
SYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKLSRRQKQLCRRE
497

PGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRMGLLKRGFKETAFLY

AVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLK

YSTKFLSNFLGSKRGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVS

GSCAVRTCWKQLSPFRETGQVLKLRYDSAVKVSSATNEALGRLELWAP

ARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGTAGRVCSREASCSSL

CCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH

WNT10A
MPRSAPNDILDLRLPPEPVLNANTVCLTLPGLSRRQMEVCVRHPDVAASA
498

IQGIQIAIHECQHQFRDQRWNCSSLETRNKIPYESPIFSRGFRESAFAYAIA

AAGVVHAVSNACALGKLKACGCDASRRGDEEAFRRKLHRLQLDALQRG

KGLSHGVPEHPALPTASPGLQDSWEWGGCSPDMGFGERFSKDFLDSREP

HRDIHARMRLHNNRVGRQAVMENMRRKCKCHGTSGSCQLKTCWQVTP

EFRTVGALLRSRFHRATLIRPHNRNGGQLEPGPAGAPSPAPGAPGPRRRA

SPADLVYFEKSPDFCEREPRLDSAGTVGRLCNKSSAGSDGCGSMCCGRG

HNILRQTRSERCHCRFHWCCFVVCEECRITEWVSVCK

WNT10B
NEILGLKLPGEPPLTANTVCLTLSGLSKRQLGLCLRNPDVTASALQGLHIA
499

VHECQHQLRDQRWNCSALEGGGRLPHHSAILKRGFRESAFSFSMLAAGV

MHAVATACSLGKLVSCGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHS

LPSPGPGSSPSPGPQDTWEWGGCNHDMDFGEKFSRDFLDSREAPRDIQAR

MRIHNNRVGRQVVTENLKRKCKCHGTSGSCQFKTCWRAAPEFRAVGAA

LRERLGRAIFIDTHNRNSGAFQPRLRPRRLSGELVYFEKSPDFCERDPTMG

SPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTRVERCHCRFHWCCY

VLCDECKVTEWVNVCK

WNT11
IKWLALSKTPSALALNQTQHCKQLEGLVSAQVQLCRSNLELMHTVVHA
500

AREVMKACRRAFADMRWNCSSIELAPNYLLDLERGTRESAFVYALSAA

AISHAIARACTSGDLPGCSCGPVPGEPPGPGNRWGGCADNLSYGLLMGA

KFSDAPMKVKKTGSQANKLMRLHNSEVGRQALRASLEMKCKCHGVSGS

CSIRTCWKGLQELQDVAADLKTRYLSATKVVHRPMGTRKHLVPKDLDIR

PVKDSELVYLQSSPDFCMKNEKVGSHGTQDRQCNKTSNGSDSCDLMCC

GRGYNPYTDRVVERCHCKYHWCCYVTCRRCERTVERYVCK

WNT16
NWMWLGIASFGVPEKLGCANLPLNSRQKELCKRKPYLLPSIREGARLGIQ
501

ECGSQFRHERWNCMITAAATTAPMGASPLFGYELSSGTKETAFIYAVMA

AGLVHSVTRSCSAGNMTECSCDTTLQNGGSASEGWHWGGCSDDVQYG

MWFSRKFLDFPIGNTTGKENKVLLAMNLHNNEAGRQAVAKLMSVDCRC

HGVSGSCAVKTCWKTMSSFEKIGHLLKDKYENSIQISDKTKRKMRRREK

DQRKIPIHKDDLLYVNKSPNYCVEDKKLGIPGTQGRECNRTSEGADGCNL

LCCGRGYNTHVVRHVERCECKFIWCCYVRCRRCESMTDVHTCK

Targeting Moieties

In some embodiments, the device or kit comprises a targeting moiety that tethers the therapeutic agent to the structure. In some embodiments, the targeting moiety is connected to the therapeutic agent, and the moiety non-covalently binds to the structure. As a non-limiting example, the targeting moiety is covalently connected to the therapeutic agent via a peptide bond. For instance, targeting moiety comprises a targeting peptide, and the targeting peptide is linked to the therapeutic agent via a peptide bond.

In some embodiments, the targeting moiety has an affinity for the structure, or a component of the structure, e.g., to a ceramic material of the structure such as calcium phosphate. In some embodiments, the dissociation constant (KD) for binding between the targeting moiety and the structure or component thereof is: (i) at least about 1 fM, at least about 10 fM, at least about 100 fM, or at least about 1 pM; and (ii) less than about 100 uM, less than about 90 uM, less than about 80 uM, less than about 70 uM, less than about 60 uM, less than about 50 uM, less than about 40 uM, less than about 30 uM, less than about 20 uM, less than about 10 uM, less than about 5 uM, less than about 1 uM, or less than about 100 pM. For example, the targeting moiety may bind to beta-tricalcium phosphate with an affinity of about 100 fM to about 100 uM, about 1 pM to about 100 μM, about 10 pM to about 100 μM, about 100 pM to about 100 μM, or about 1 μM to about 100 μM.

In some embodiments, the targeting moiety comprises one or more targeting peptides that each bind to the structure. In some embodiments, the targeting peptide binds to the ceramic material of the structure. For example, the targeting peptide binds to calcium phosphate (e.g., tricalcium phosphate, beta tricalcium phosphate, alpha tricalcium phosphate), hydroxyapatite, fluorapatite, bone (e.g., demineralized bone), glasses (bioglasses) such as silicates, vanadates, and related ceramic minerals, or chelated divalent metal ions, or a combination thereof. In some embodiments, the targeting peptide comprises two or more targeting peptides. In some embodiments, two or more targeting peptides is no more than about 50, 45, 40, 35, 30, 25, 20, 15, or 10 targeting peptides. In some embodiments, two or more targeting peptides is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 targeting peptides. In some embodiments, two or more targeting peptides is about 2 to about 10 targeting peptides. In some embodiments, two or more targeting peptides is about 5 targeting peptides.

In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 2. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 3. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 4. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 5. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 6. In some embodiments, the targeting peptide comprises a sequence at least ab out 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 7. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 8. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 9. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 10. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 11. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 12. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 13. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 14. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 15. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 16. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 17. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 18. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 19. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 20. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 21. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 22. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 23. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 24. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 25. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 26. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 27. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 28. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 29. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 30. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 31. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 32. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 33. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 34. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 35. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 36. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 37. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 38. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 39. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 40. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 41. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 42. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 43. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 44. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 45. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 46. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 47. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 48. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 49. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 50. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 51. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 52. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 53. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 54. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 55. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 56. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 57. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 58. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 59. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 60. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 61.

TABLE 2

Targeting Peptides

SEQ

ID

Sequence
NO:

LLADTTHHRPWT
1

VIGESTHHRPWS
2

LIADSTHHSPWT
3

ILAESTHHKPWT
4

ILAETTHHRPWS
5

IIGESSHHKPFT
6

GLGDTTHHRPWG
7

VLGDTTHHKPWT
8

IVADSTHHRPWT
9

STADTSHHRPS
10

TSGGESTHHRPS
11

TSGGESSHHKPS
12

TGSGDSSHHRPS
13

GSSGESTHHKPST
14

VGADSTHHRPVT
15

GAADTTHHRPVT
16

AGADTTHHRPVT
17

GGADTTHHRPAT
18

GGADTTHHRPGT
19

LLADTTHHRPWTVIGESTHHRPWS
20

LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFT
21

LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWT
22

LLADTTHHRPWTILAESTHHKPWT
23

LLADTTHHRPWTILAESTHHKPWTLLADTTHHRPWTILAESTHHKPWTLLADTTHHRPW
24

T

LLADTTHHRPWTGLGDTTHHRPWG
25

LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWT
26

LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRP
27

WT

LLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRPWTGLGDTTHHRPWGLLADTTHHRP
28

WTGLGDTTHHRPWGLLADTTHHRPWT

STADTSHHRPSTSGGESTHHRPSTSGGESSHHKPSTGSGDSSHHRPSGSSGESTHHKPST
29

VGADSTHHRPVTGAADTTHHRPVTAGADTTHHRPVTGGADTTHHRPATGGADTTHHRP
30

GT

STADTSHHRPSLLADTTHHRPWTTSGGESTHHRPSVGADSTHHRPVTTSGGESSHHKPSG
31

AADTTHHRPVTTGSGDSSHHRPSGSSGESTHHKPSTGGADTTHHRPAT

AAADTTHHRPWT
32

AAADTTHHRPWTAAADTTHHRPWTAAADTTHHRPWTAAADTTHHRPWTAAADTTHH
33

RPWT

LLADAAHHRPWTLLADAAHHRPWTLLADAAHHRPWTLLADAAHHRPWTLLADAAHH
34

RPWT

LLADTTAARPWTLLADTTAARPWTLLADTTAARPWTLLADTTAARPWTLLADTTAARP
35

WT

LLADTTHHRPWTLLADTTHHRPWT
36

LLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWT
37

LLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWTLLADTTHHRPWTLLADTTHHRP
38

WT

STSGSTVIGESTHHRPWSLIADSTHHSPWTILAESTHHKPWTILAETTHHRPWSIIGESSHH
39

KPFTGLGDTTHHRPWGVLGDTTHHKPWTIVADSTHHRPWTGQVLPTTTPSSPSTTSGS

LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWG
40

VIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWT
41

(X1)(X2), wherein X1 comprises SEQ ID NO: 1 and X2 comprises one or more of SEQ ID NOS:
42

1-41.

(X1)(X2), wherein X1 comprises SEQ ID NO: 2 and X2 comprises one or more of SEQ ID NOS:
43

1-41.

(X1)(X2), wherein X1 comprises SEQ ID NO: 4 and X2 comprises one or more of SEQ ID NOS:
44

1-41.

(X1)(X2), wherein X1 comprises SEQ ID NO: 6 and X2 comprises one or more of SEQ ID NOS:
45

1-41.

(X1)(X2), wherein X1 comprises SEQ ID NO: 7 and X2 comprises one or more of SEQ ID NOS:
46

1-41.

(X1)(X2), wherein X1 comprises SEQ ID NO: 1 and X2 comprises one or more of SEQ ID NOS:
47

2, 4, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 2 and X2 comprises one or more of SEQ ID NOS:
48

1,4, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 4 and X2 comprises one or more of SEQ ID NOS:
49

1, 2, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 6 and X2 comprises one or more of SEQ ID NOS:
50

1, 4, 2, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 7 and X2 comprises one or more of SEQ ID NOS:
51

1, 4, 6, or 2.

(X1)(X2), wherein X1 comprises SEQ ID NO: 1 and X2 comprises two or more of SEQ ID NOS:
52

2, 4, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 2 and X2 comprises two or more of SEQ ID NOS:
53

1, 4, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 4 and X2 comprises two or more of SEQ ID NOS:
54

1, 2, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 6 and X2 comprises two or more of SEQ ID NOS:
55

1, 4, 2, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 7 and X2 comprises two or more of SEQ ID NOS:
56

1, 4, 6, or 2.

(X1)(X2), wherein X1 comprises SEQ ID NO: 1 and X2 comprises three or more of SEQ ID
57

NOS: 2, 4, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 2 and X2 comprises three or more of SEQ ID
58

NOS: 1, 4, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 4 and X2 comprises three or more of SEQ ID
59

NOS: 1, 2, 6, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 6 and X2 comprises three or more of SEQ ID
60

NOS: 1, 4, 2, or 7.

(X1)(X2), wherein X1 comprises SEQ ID NO: 7 and X2 comprises three or more of SEQ ID
61

NOS: 1, 4, 6, or 2.

In some embodiments, a targeting peptide comprises one or more sequences of Table 2. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of Table 2.

TABLE 3

Additional Targeting Peptides

SEQ ID

Sequence
NO

ACAPLMFSQC
62

ACHASLKHRC
63

ACLSTKTNIC
64

ACTTPSKHQC
65

AHFSPNLLLGG
66

AHSLKSITNHGL
67

AKQTVPV
68

AKTLMPSPFPRT
69

AMSQTMTAAIEK
70

ANPPLSL
71

ANPYHRH
72

APLSLSL
73

APYHPTIPASVHGGGK
74

ASAVGSLSIRWX
75

ASGPTNV
76

ASHNPKL
77

ASWVDSRQPSAA
78

ATFSPPL
79

ATWSHHLSSAGL
80

ATWSHHLSSAGLGGGS
81

CAHLSPHKC
82

CDIPWRNEC
83

CDPLRQHSC
84

CDSLGHWLC
85

CDYTTRHSC
86

CHGTLNPEC
87

CHHNLSWEC
88

CHIWTLASC
89

CHNTFSPRC
90

CIPLHASLC
91

CITTTSLSC
92

CKLTTCKDC
93

CKNHTTFWC
94

CLKLLSRSC
95

CLLKAHPSC
96

CLNQLKQAC
97

CLSTKTNIC
98

CMNFPSPHC
99

CNYPTLKSC
100

CPQLTVGQHRT
101

CPQSPTYTC
102

CPSSAIHTC
103

CPTSTARIC
104

CQASSFPSC
105

CQPYFWYRC
106

CQTLTPSIC
107

CSKLGHLWC
108

CSKTPERIX
109

CSNNNRMTC
110

CSPILSLSC
111

CSPTNFTRC
112

CSRPAMNVC
113

CSTKAYPNC
114

CSTSSCGSC
115

CSYWGHRDC
116

CTAHDANAC
117

CTANSEKTC
118

CTHPKASMC
119

CTKTINGKC
120

CTNMQSPLC
121

CTPFTKLPC
122

CTPTTDSIC
123

CTQQNGHPC
124

CTTPSKHQC
125

CTYNVAKPC
126

DKLHRLA
127

DLNYFTLSSKRE
128

DLPPTLHTTGSP
129

DMRQQRS
130

DQYWGLR
131

DSSNPIFWRPSS
132

DSSNPIFWRPSSGGGS
133

EFLGVPASLVNP
134

EPNHTRF
135

EPRRAVAAL
136

EPRRAVAEL
137

EPRREVAEL
138

EPRREVCEL
139

ESDLTHALHWLG
140

ESLKSIS
141

ETRTQLL
142

ETVCASS
143

ETYARPL
144

ETYQQPL
145

EVHSTDRYRSIP
146

FGLQPTGDIARR
147

FSMDDPERVRSP
148

FSPLHTSTYRPS
149

FTLPTIR
150

FVNLLGQ
151

GDFNSGHHTTTR
152

GGGAAAA
153

GIHVPWMPPVAF
154

GIHVPWMPPVAFGGGS
155

GPSNNLPWSNTP
156

GSAGLKYPLYKS
157

GSCPPKK
158

GSLFKAL
159

GTQTPQP
160

GTSRLFS
161

GVHKHFYSRWLG
162

HAPLTRSPAPNL
163

HAPVQPN
164

HGSLTTLXRYEP
165

HHFHLPKLRPPV
166

HHQRSPA
167

HHTWDTRIWQAF
168

HMLAQTF
169

HNVTTRTQRLMP
170

HPTTPIHMPNF
171

HQFISPEPFLIS
172

HQFPXSNLVWKP
173

HQWDHKY
174

HRDPXSXPSAXRP
175

HRLGHMS
176

HSACHASLKHRC
177

HSACKLTTCKDG
178

HSACLSTKTNIC
179

HSMPHMGTYLLT
180

HSTGPTR
181

HTLLSTT
182

HYPTVNF
183

IAHVPETRLAQM
184

IFSMGTALARPL
185

IGYPVLP
186

INFQFLKPSTTR
187

INKHPQQVSTLL
188

IQHQAKT
189

ISPSHSQAQADL
191

KAFDKHG
192

KATITGM
193

KEIPPIPLLAPS
194

KEIPPIPLLAPSGGGS
195

KIPKACCVPTELSAISMLYL
196

KIPKASSVPTELSAIATLYL
197

AAAAEPRRAVAAL

KIPKASSVPTELSAISTLYL
198

KIPKASSVPTELSAISTLYL
199

AAAAEPRRAVAAL

KIPKASSVPTELSAISTLYL
200

AAAAEPRRAVAEL

KIPKASSVPTELSAISTLYL
201

AAAAEPRREVAEL

KIPKASSVPTELSAISTLYL
202

AAAAXPRRXVAXL

KIPKASSVPTELSAISTLYL
203

XPRRXVAXL

KLHASLA
204

KLSAWSF
205

KLTWQELYQLKYKGI
206

KLTWQELYQLKYKGIGGG
207

AAAAEPRREVAEL

KMNHMPN
208

KPMQFVH
209

KTSSWAN
210

LASTTHV
211

LDYPIPQTVLHH
212

LFAAVPSTQFFR
213

LGFDPTSTRFYT
214

LGPGKAF
215

LKPFSGA
216

LLADTTHHRPWP
217

LLADTTHHRPWT
218

LLADTTHHRPWTGGGS
219

LLPLKFK
220

LPFQPPI
221

LPLTPLP
222

LPRDLHATPQQI
223

LPSIHNL
224

LPWAPNLPDSTA
225

LPWTEPSFWRTP
226

LPWTEPSFWRTPGGGS
227

LQKSPSL
228

LQPSQPQRFAPT
229

LRAFPSLPHTVT
230

LSAPMEY
231

LSKNPLL
232

LSLRASAATDFQ
233

LSPPMQLQPTYS
234

LTPTMFNMHGVL
235

LTQTLQY
236

MHNVSDSNDSAI
237

MKVHERS
238

MPQTLVLPRSLL
239

MQFTPAPSPSDH
240

MTSQTLR
241

MYPLPAP
242

NERQMEL
243

NFAMNLR
244

NITQLGS
245

NKPLSTL
246

NNVSQKWQQRLI
247

NNVSQKWQQRLIGGGS
248

NPDHPDIPQDVHGGGK
249

NPMIMNQ
250

NPQMQRS
251

NPRSQAT
252

NPYAPTIPQSVAGGGK
253

NPYHPTIPQSVH
254

NPYHPTIPQSVHGGGK
255

NSMIAHNKTRMH
256

NSMIAHNKTRMHGGGS
257

NSSMLGMLPSSF
258

NTSSSQGTQRLG
259

NTTTDIPSPSQF
260

NYPTLKS
261

NYSHLRVKLPTP
262

NYSHLRVKLPTPGGGS
263

PAKQKAH
264

PDIPLSR
265

PGQWPSSLTLYK
266

PHNPGKL
267

PIDAFFD
268

PLTQPSH
269

PPKDSRG
270

PPNMARA
271

PSMKHWR
272

PTNKPHT
273

PTTMTRW
274

PTTWGHL
275

PXGPXGPXGPXGPXGPXA
276

PXGPXGPXGPXGPXGPXG

QHNFRGASSSAP
277

QIPQMRILHPYG
278

QIQKPPRTPPSL
279

QLTQTMWKDTTL
280

QNLPPERYSEAT
281

QNPRQIY
282

QNYLLPK
283

QPGLWPS
284

QRSWTLDSALSM
285

QRSWTLDSALSMGGGS
286

QSLSFAGPPAWQ
287

QSSYNPI
288

QTHARHQ
289

QTHSSLW
290

QTTMTPLWPSFS
291

RCMSEVISFNCP
292

RHTLPLH
293

RPHTITN
294

RSPYYNKWSSKF
295

RTPLQPLEDFRP
296

SAGHIHEAHRPL
297

SAISDHRAHRSH
298

SAKGRAD
299

SAKKVFS
300

SASGTPS
301

SEPTYWRPNMSG
302

SFAPDIKYPVPS
303

SFQSMSLMTLVV
304

SFWHHHSPRSPL
305

SGHQLLLNKMPN
306

SGHQLLLNKMPNGGGS
307

SIFAHQTPTHKN
308

SIPKMIPTESLL
309

SIPSHSIHSAKA
310

SIRTSMNPPNLL
311

SKTSSTS
312

SLLTPWL
313

SLPHYIDNPFRQ
314

SLSKANILHLYG
315

SLVTADASFTPS
316

SMAAKSS
317

SMVYGNRLPSAL
318

SMYDTHS
319

SPEMKPR
320

SPNFSWLPLGTT
321

SPNLPWSKLSAY
322

SPNNPRE
323

SPNNTRE
324

SPSLMARSSPYW
325

SQHSTQD
326

SQTLPYSNAPSP
327

SRTGAHH
328

SSHHHRH
329

SSPPRVY
330

SSSMAKM
331

SSTLKTFFGFPD
332

SSTLKTFFGFPDGGGS
333

SSTQAHPFAPQL
334

SSTQVQHTLLQT
335

SSVPGRP
336

SSYEYHA
337

STLASMR
338

STPNSYSLPQAR
339

STQAHPW
340

STSAKHW
341

STVVMQPPPRPA
342

SVFLPTRHSPDL
343

SVQTRPLFHSHF
344

SVSVGMKPSPRP
345

SVSVGMNAESXA
346

SVSVGMNAESYG
347

SVSVGTEAESXA
348

SWPLYSRDSGLG
349

SYIDSMVPSTQT
350

SYKTTDSDTSPL
351

SYSQMDPPRSLPGGGS
352

TAAASNLRAVPP
353

TAPLSHPPRPGA
354

TDHPPKA
355

TGLAKTA
356

TGLLPNSSGAGI
357

TGPPSRQPAPLH
358

TGPTSLS
359

THPVVFEDERLF
360

TIHSKPA
361

TKDWLPS
362

TLAFQTA
363

TLAPTFR
364

TLDKYTRLLSRY
365

TLGLPML
366

TLLRTQV
367

TLMTTPP
368

TLPSPLALLTVH
369

TLQRMGQ
370

TLSNGHRYLELL
371

TMGFTAPRFPHY
372

TMRNPITSLISV
373

TMRNPITSLISVGGGS
374

TMTNMAK
375

TPLSYLKGLVTV
376

TPLTSPSLVRPQ
377

TPSPKLLQVFQA
378

TPSTGLGMSPAV
379

TPVYSLKLGPWP
380

TQTWPQSSSHGL
381

TRFYDSL
382

TRLVPSRYYHHP
383

TSPIPQMRTVPP
384

TTKNFNK
385

TTLSPRT
386

TTNSSMTMQLQR
387

TTTLPVQPTLRN
388

TTTWTTTARWPL
389

TTYNSPP
390

TVAQMPPHWQLT
391

TVLGTFP
392

TWNSNSTQYGNR
393

TWTLPAMHPRPA
394

VHLTHGQ
395

VHPRPSL
396

VHTSLLQKHPLP
397

VLPNIYMTLSA
398

VMDFASPAHVLP
399

VNQEYWFFPRRP
400

VPPISXTFLFXSTXS
401

VPPLHPALSRXN
402

VSPFLSPTPLLF
403

VSRLGTPSMHPS
404

VVKSNGE
405

VYSSPLSQLPR
406

WLPPRTQ
407

WPANKLSTKSMY
408

WPFNHFPWWNVP
409

WPTYLNPSSLKA
410

WSAHIVPYSHKP
411

WWPNSLNWVPRP
412

WYPNHLA
413

XITXGAY
414

XPRRAVAAL
415

XPRRAVAXL
416

XPRRXVAXL
417

XXFPLXG
418

YATQHNWRLKHE
419

YCPMRLCTDC
420

YELQMPLTLPLN
421

YEPAAAE
422

YGKGFSPYFHVT
423

YPHYSLPGSSTL
424

YPIMSHTCCHGV
425

YPKALRN
426

YPSLLKMQPQFS
427

YQPRPFVTTSPM
428

YSAPLARSNVVM
429

YTRLSHNPYTLS
430

YTTHVLPFAPSS
431

YTWQTIREQYEM
432

In some embodiments, a targeting peptide comprises one or more sequences of Table 3. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of Table 3.

Additional targeting peptides useful in the present disclosure include any one of SEQ ID NO: 1 to SEQ ID NO: 558 of U.S. Pat. No. 7,572,766. In some embodiments, the targeting peptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 558 of U.S. Pat. No. 7,572,766.

In some embodiments, the device or kit comprises a chimeric polypeptide comprising the targeting peptide and a targeting moiety. In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 433 (ASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPL YVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKA CCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 434 (VIPIGSLLADTTHEIRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTH HKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLK SSC KRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNS KIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 435 (LLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPW TASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPL YVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKA CCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 436 (VIGESTHHRPWSIIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGG SEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWND WIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISML YLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 437 (IIGESSHHKPFTGLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGA GTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAF YCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVL KNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 438 (GLGDTTHHRPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGAST GGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLA DHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEG CGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 439 (ILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQR KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQT LVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR). In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 440 ((X)QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHL NSTNHAIVQTLVNSVNSKIPKACCVPIELSAISMLYLDENEKVVLKNYQDMVVEGCGC R), wherein X comprises a targeting peptide and optionally a linker. For example, the targeting peptide comprises one or more of SEQ ID NOS: 1-41. In some cases, the chimeric polypeptide comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 441 ((X)ASGAGGSEGGGSEGGTSGATGAGTSTSGGGASTGGGTGQAKHKQRKRLKSSCKRH PLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIP KACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR), wherein X comprises a targeting peptide and optionally a linker. For example, the targeting peptide comprises one or more of SEQ ID NOS: 1-41.

In some embodiments, a therapeutic agent is not connected to a structure using a targeting moiety. For example, the therapeutic agent may interact with the structure via non-covalent b onds. The therapeutic agent may be connected to a structure by hydrogen bonding, ionic bonding hydrophobic interactions, or van der Waals forces. The therapeutic agent may also be connected to a structure using covalent bonds. Examples of methods for connecting using covalent bonds includes chemical linkers and spacers that are used for modifying active groups within proteins such as amines, thiols and carbohydrates.

In some embodiments, provided is a device comprising a structure that is seeded with cells. Non-limiting examples of cells include osteocytes and other bone cells, chondrocytes, and meniscal cells. In some instances, the cells can be added to the completed implantable structures.

Device Manufacture

Further provided herein are methods of manufacturing a device comprising a structure and a therapeutic agent. Some methods comprise: (a) providing a first solution of a therapeutic agent (e.g., a chimeric polypeptide comprising the therapeutic agent and a targeting moiety), (b) providing a 3D structure, and (c) combining (a) and (b). In some embodiments, the therapeutic agent is present in the first solution at a concentration of about 0.25-1.5, 0.25-1.25, 0.25-1, 0.25-0.75, 0.25-0.5, 0.5-1.5, 0.5-1.25, 0.5-1, 0.5-0.75, 0.75-1.5, 0.75-1.25, 0.75-1, 1-1.5, 1-1.25, 1.25-1.5, 0.25, 0.5, 1, 1.25, or 1.5 mg/mL. In some methods, step (c) comprises incubating the first solution and structure for about 10-240, 10-180, 10-120, 20-240, 20-180, 20-120, 30-240, 30-180, 30-120, 40-240, 40-180, 40-120, 50-240, 50-180, 50-120, 60-240, 60-180, 60-120, 70-240, 70-180, 70-120, 80-240, 80-180, 80-120, 90-240, 90-180, 90-120, 100-240, 100-180, 100-120, 110-240, 110-180, 110-120, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 minutes. In some methods, step (c) comprises incubating the first solution and structure with movement, such as rotation and/or shaker (e.g., using a plate shaker). In some cases, the first solution comprises a buffer. For example, the buffer comprises sodium acetate and acetic acid. In some cases, the first solution has a pH from about 4 to about 5, or about 4, 4.1, 4.15, 4.2, 4.25, 4.3, 4.35, 4.4, 4.45, 4.5, 4.45, 4.6, 4.65, 4.7, 4.75, 4.8, 4.85, 4.9, 4.95, or 5. In some cases, the first solution comprises a salt. For example, the first solution comprises sodium chloride, such as about 50-150, 50-140, 50-130, 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-150, 60-140, 60-130, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-150, 80-140, 80-130, 80-120, 80-110, 80-100, 80-90, 90-150, 90-140, 90-130, 90-120, 90-110, 90-100, 100-150, 100-140, 100-130, 100-120, 100-110, 110-150, 110-140, 110-130, 110-120, 120-150, 120-140, 120-130, 130-150, 130-140, or 140-150. In some embodiments, the first solution comprises 10 mM sodium acetate, 7 mM acetic acid, 100 mM NaCl, pH=4.75. In some embodiments, the method further comprises (d) washing the 3D structure of step (c) with a second solution, such as phosphate buffered saline (PBS). In some embodiments, the method further comprises drying the 3D structure of step (c) or step (d).

In some embodiments, the mass of the therapeutic agent (e.g., a therapeutic agent alone or a therapeutic agent connected to a targeting moiety) per cubic centimeter of the structure in a device is between about 0.05 and 50 (mg/cc), e.g., about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/cc or any number therebetween. For example, the therapeutic agent is about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mg per cubic centimeter device. One method of measuring the amount of therapeutic peptide bound to the structure includes: (1) measuring the mass of therapeutic peptide input in the first solution, (2) measuring the mass of the therapeutic agent remaining in the first solution after combination with and removal from the structure, (3) measuring the mass of the therapeutic agent in the second solution if a wash step is included, (4) summing (2) and (3); and subtracting the sum of (4) from (1).

Methods of Treatment

In another aspect, provided are methods of treating a subject with a structure herein. In some methods, the subject is treated with a device comprising a therapeutic peptide and the structure. In some instances, the subject has a bone fracture or a bone defect. In some instances, the subject requires a vertebral fusion of the spine. In some instances, the subject has a cartilage tear or cartilage defect. In some instances, the subject has cartilage loss.

In some embodiments, the subject is suffering from a defect in bone, cartilage, soft tissue, tendon, fascia, ligament, organ, osteotendinous tissue, dermal, or osteochondral, or a combination of one or more of the aforementioned defects. In some embodiments, a defect is a lack of bone, cartilage, soft tissue, tendon, fascia, ligament, organ, osteotendinous tissue, dermal, or osteochondral, or a combination of one or more of the aforementioned defects. In some embodiments, a defect in the subject arises from trauma. In some embodiments, a defect in the subject arises due to a congenital condition. In some embodiments, a defect in the subject arises due to an acquired condition. In some embodiments, a defect refers to the absence, loss, and/or break in a tissue and/or organ of the body. In some embodiments, a “bone defect” refers to the absence or loss (e.g., partial loss) of bone at an anatomical location in a subject where it would otherwise be present in a control healthy subject. A bone defect may be the result of an infection (e.g., osteomyelitis), a tumor, a trauma, or an adverse event of a treatment. A bone defect may also affect the muscles, soft tissue, tendons, or joints surrounding the bone defect and cause injury. In some embodiments, a bone defect includes damage to a soft tissue. In some embodiments, a “cartilage defect” refers to the absence or loss (e.g., partial loss) of cartilage at an anatomical location in a subject where it would otherwise be present in a control healthy subject. A cartilage defect may be the result of disease, osteochondritis, osteonecrosis, or trauma. For example, a cartilage defect may affect the knee joint.

Non-limiting examples of conditions suitable for treatment with a structure or device described herein include osteoarthritis, disc degeneration, congenital defect, spinal stenosis, spondylolisthesis, spondylosis, bone fracture, scoliosis, kyphosis, spinal fusion (PLF, and interbody fusions), trauma repair of bone, dental repair, craniomaxillofacial repair, ankle fusion, kyphoplasty, balloon osteoplasty, scaphoid facture repair, tendeno-osseous repair, osteoporosis, avascular necrosis, congenital skeletal malformations, costal reconstruction, subchondral bone repair, cartilage repair (e.g., at low doses), or trauma, or a combination thereof. BMP2 is also involved in hair follicle development, therefore the methods may comprise treatment to hair follicles. The trauma may be to the bone, cartilage, soft tissue, tendon, fascia, ligament, organ, osteotendinous tissue, or dermal tissue, or osteochondral tissue. In some embodiments, the method is to treat an osteochondral injury.

The methods of treatment may comprise spinal fusion. In some embodiments, spinal fusion is a surgical technique to join two or more vertebrae. In some embodiments, the spinal fusion comprises PLF. In some embodiments, the spinal fusion comprises interbody fusions.

Provided herein are methods of promoting bone or cartilage formation in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the structures or devices described herein. Some embodiments of these methods can further include first selecting a subject in need of bone or cartilage formation. In some embodiments, the structure or device is administered to the subject proximal to the desired site of bone or cartilage formation in the subject.

Also provided herein are methods of replacing and/or repairing bone or cartilage in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the structure or devices described herein. Some embodiments of these methods can further include first selecting a subject in need of bone replacement, bone repair, cartilage replacement, or cartilage repair. In some embodiments, the structure or device is administered to the subject proximal to the desired site of bone or cartilage replacement or repair in the subject.

Also provided herein are methods of treating a bone fracture or bone loss in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the structure or devices described herein. Some embodiments of these methods can further include first selecting a subject having a bone fracture or bone loss. In some embodiments, the structure or device is administered to the subject proximal to the bone fracture or the site of bone loss in the subject.

Also provided herein are methods of repairing soft tissue in a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the structure or devices described herein. Some embodiments of these methods can further include first selecting a subject having a bone fracture or bone loss. In some embodiments, the composition is administered to the subject proximal to the bone fracture or the site of bone loss in the subject.

Also provided herein are methods of localized delivery of a therapeutic to a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the structure or devices described herein. Some embodiments of these methods can further include first selecting a subject having a bone fracture or bone loss. In some embodiments, the structure or device is administered to the subject proximal to the bone fracture or the site of bone loss in the subject.

Methods of determining the efficacy of treatment of a bone fracture or bone loss in a subject are known in the art and include, e.g., imaging techniques (e.g., magnetic resonance imaging, X-ray, or computed tomography).

Methods of detecting bone or cartilage formation, or replacement or repair of bone or cartilage in a subject are also known in the art and include, e.g., imaging techniques (e.g., magnetic resonance imaging, X-ray, or computed tomography).

Suitable animal models for treatment of a bone fraction or bone loss, bone or cartilage formation, or bone or cartilage replacement or repair are known in the art. Non-limiting examples of such animal models are described in the Examples and in, e.g., Drosse et al., Tissue Engineering Part C 14(1):79-88, 2008; Histing et al., Bone 49:591-599, 2011; and Poser et al., Hindawi Publishing Corporation, BioMed Research International; Article ID 348635, 2014.

As used herein, a method of treatment comprises administering to the subject a structure or device herein. In some embodiments, administration comprises implanting a polypeptide or composition herein.

In some embodiments, a polypeptide and/or composition herein comprising BMP-2 is administered to the subject. In some embodiments, the BMP2 comprises a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 454. In some embodiments, the BMP-2 is administered to induce formation of bone in the subject. In some embodiments, the BMP-2 is administered to induce formation of cartilage. In some embodiments, the BMP-2 is administered in a spinal fusion.

The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. The singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. To the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

In some embodiments, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value.

The term “subject” as used herein refers to any mammal. A subject therefore refers to, for example, mice, rats, dogs, cats, horses, cows, pigs, guinea pigs, rats, humans, monkeys, and the like. When the subject is a human, the subject may be referred to herein as a patient. In some embodiments, the subject or “subject in need of treatment” may be a canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), ovine, bovine, porcine, caprine, primate, e.g., a simian (e.g., a monkey (e.g., marmoset, baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon), a human, or a rodent (e.g., a mouse, a guinea pig, a hamster, or a rat). In some embodiments, the subject or “subject in need of treatment” may be a non-human mammal, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, lapine, porcine, canine, or primate animals) may be employed.

In some embodiments, the term “therapeutically effective amount” refers to an amount of a polypeptide or composition effective to “treat” a disease, condition or disorder in a subject. In some cases, therapeutically effective amount of the polypeptide or composition reduces the severity of symptoms of the disease, condition or disorder. In some instances, the disease, condition or disorder comprises a defect in an organ or tissue.

In some embodiments, “affinity” refers to the strength of the sum total of non-covalent interactions between a β-TCP binding sequence (or a chimeric polypeptide or polypeptide comprising a β-TCP binding sequence) and its binding partner (e.g., β-TCP). Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U. S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

Each of the embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Additionally, while specific formulations for the inks are described, variations of the specific quantities of each ink ingredient are possible. Accordingly, other embodiments are within the scope of the following claims.

Example Embodiments

- 1. A device comprising: a therapeutic agent and a printed three-dimensional structure, the printed three-dimensional structure comprising about 50% to about 90% by weight ceramic and about 10% to about 50% by weight polymer.
- 2. The device of embodiment 1, wherein the therapeutic agent is non-covalently bound to the printed three-dimensional structure.
- 3. The device of embodiment 1 or embodiment 2, wherein the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or a combination thereof.
- 4. The device of any one of embodiments 1-3, wherein the ceramic comprises beta-tricalcium phosphate (β-TCP).
- 5. The device of any one of embodiments 1-4, wherein the structure comprises about 70% to about 80% by weight ceramic.
- 6. The device of embodiment 5, wherein the structure comprises about 75% by weight ceramic.
- 7. The device of any one of embodiments 1-6, wherein the polymer comprises a polyester.
- 8. The device of any one of embodiments 1-7, wherein the polymer comprises polycaprolactone (PCL), polyglycolide, lactide, or polydioxanone (PDS), or a combination thereof.
- 9. The device of any one of embodiments 1-8, wherein the polymer comprises polycaprolactone (PCL).
- 10. The device of any one of embodiments 1-9, wherein the polymer comprises polyglycolide.
- 11. The device of any one of embodiments 1-10, wherein the polymer comprises polydioxanone (PDS).
- 12. The device of any one of embodiments 1-11, wherein the polymer comprises a lactide.
- 13. The device of embodiment 12, wherein the lactide comprises L-lactide.
- 14. The device of embodiment 12 or embodiment 13, wherein the lactide comprises poly(D,L-lactide).
- 15. The device of any one of embodiments 1-14, wherein the polymer comprises a copolymer.
- 16. The device of embodiment 15, wherein the copolymer comprises polycaprolactone (PCL) and polyglycolide.
- 17. The device of embodiment 16, wherein the copolymer comprises ab out 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide.
- 18. The device of any one of embodiments 15-17, wherein the copolymer comprises poly(D,L-lactide-co-glycolide) copolymer.
- 19. The device of embodiment 18, wherein the copolymer comprises ab out 40-60 percent by mole poly(D,L-lactide) and about 40-60 percent by mole polyglycolide.
- 20. The device of any one of embodiments 15-19, wherein the copolymer comprises PDS-glycolide copolymer.
- 21. The device of embodiment 20, wherein the copolymer comprises ab out 90-95 percent by mole PDS and about 5-10 percent by mole glycolide.
- 22. The device of any one of embodiments 15-21, wherein the copolymer comprises PDS-L-lactide copolymer.
- 23. The device of embodiment 20, wherein the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole L-lactide.
- 24. The device of any one of embodiments 1-23, wherein the structure comprises about 15% to about 25% by weight polymer.
- 25. The device of embodiment 24, wherein the structure comprises about 25% by weight polymer.
- 26. The device of any one of embodiments 1-25, wherein the printed three-dimensional structure is formed from an ink comprising about 30% to about 70% by weight the ceramic, about 10% to about 30% by weight the polymer, and optionally one or more additional agents.
- 27. The device of embodiment 26, wherein the ink comprises about 50% to about 70% by weight the ceramic.
- 28. The device of embodiment 27, wherein the ink comprises about 60% by weight the ceramic.
- 29. The device of any one of embodiments 26-28, wherein the ink comprises about 15% to about 25% by weight the ceramic.
- 30. The device of embodiment 29, wherein the ink comprises about 20% by weight the ceramic.
- 31. The device of any one of embodiments 26-30, wherein the ink comprises the one or more additional agents.
- 32. The device of any one of embodiments 26-31, wherein the ink comprises about 1% to about 30% by weight the one or more additional agents.
- 33. The device of any one of embodiments 26-32, wherein the one or more additional agents comprises an additional polymer, particulate, or blowing agent, or a combination thereof 34. The device of embodiment 33, wherein the one or more additional agents comprises the additional polymer.
- 35. The device of embodiment 33 or embodiment 34, wherein the additional polymer comprises a first polyethylene glycol and optionally a second polyethylene glycol wherein if present the second polyethylene glycol has a different molecular weight than the first polyethyleneglycol.
- 36. The device of any one of embodiments 33-35, wherein the additional polymer is present in the ink at about 10% to about 30% by weight.
- 37. The device of any one of embodiments 33-36, wherein the one or more additional agents comprises the particulate.
- 38. The device of any one of embodiments 33-37, wherein the particulate comprises sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α trehalose dihydrate), or mannitol (e.g., D-mannitol), or a combination thereof.
- 39. The device of any one of embodiments 33-38, wherein the particulate is present in the ink at about 1% to about 10% by weight.
- 40. The device of any one of embodiments 33-39, wherein the one or more additional agents comprises the blowing agent.
- 41. The device of any one of embodiments 33-40, wherein the blowing agent comprises baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and/or azodicarbonamide.
- 42. The device of any one of embodiments 33-41, wherein the particulate is present in the ink at about 5% to about 20% by weight.
- 43. The device of any one of embodiments 1-42, wherein the therapeutic agent comprises a mammalian growth factor or a functional portion thereof.
- 44. The device of any one of embodiments 1-43, wherein the therapeutic agent comprises one or more polypeptides selected from Table 1, or a functional portion thereof.
- 45. The device of any one of embodiments 1-44, wherein the therapeutic agent comprises a bone morphogenetic protein (BMP).
- 46. The device of any one of embodiments 1-45, wherein the therapeutic agent comprises a targeting moiety, and the targeting moiety is non-covalently bound to the printed three-dimensional structure.
- 47. The device of embodiment 46, wherein the targeting moiety is bound to the printed three-dimensional structure with an affinity of about 1 pM to about 100 μm.
- 48. The device of embodiment 46 or embodiment 47, wherein the targeting moiety comprises a polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences of Tables 2-3.
- 49. The device of embodiment 46 or embodiment 47, wherein the targeting moiety comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences selected from a sequence from Tables 2-3.
- 50. The device of any one of embodiments 1-49, wherein the therapeutic agent comprises a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 433-441.
- 51. A method of treating a condition in a subject in need thereof, the method comprising administering to the subject the device of any one of embodiments 1-50.
- 52. The method of embodiment 51, wherein the condition comprises a bone defect, cartilage defect, soft tissue defect, tendon defect, fascia defect, ligament defect, organ defect, osteotendinous tissue defect, dermal defect, osteochondral defect, osteoporosis, avascular necrosis, or congenital skeletal malformation, or a combination thereof.
- 53. The method of embodiment 51 or embodiment 52, wherein the method comprises spinal fusion.
- 54. The method of embodiment 53, wherein the spinal fusion comprises posterior lumbar fusion (PLF) and/or interbody fusion.
- 55. The method of embodiment 51 or embodiment 52, wherein the method comprises bone repair, dental repair, craniomaxillofacial repair, ankle fusion, kyphoplasty, osteoplasty, scaphoid fracture repair, tendeno-osseous repair, costal reconstruction, subchondral bone repair, cartilage repair, or surgical implantation of the three-dimensional structure or device, or a combination thereof.
- 56. A formulation comprising about 30% to about 70% by weight ceramic, about 10% to about 30% by weight polymer, and optionally one or more additional agents.
- 57. The formulation of embodiment 56, wherein the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or a combination thereof
- 58. The formulation of embodiment 56 or embodiment 57, wherein the ceramic comprises beta-tricalcium phosphate (β-TCP).
- 59. The formulation any one of embodiments 56-58, wherein the ink comprises about 50% to about 70% by weight the ceramic.
- 60. The formulation of embodiment 59, wherein the ink comprises about 60% by weight the ceramic.
- 61. The formulation of any one of embodiments 56-60, wherein the polymer comprises a polyester.
- 62. The formulation of any one of embodiments 56-61, wherein the polymer comprises polycaprolactone (PCL), polyglycolide, lactide, or polydioxanone (PDS), or a combination thereof.
- 63. The formulation of any one of embodiments 56-62, wherein the polymer comprises polycaprolactone (PCL).
- 64. The formulation of any one of embodiments 56-63, wherein the polymer comprises polyglycolide.
- 65. The formulation of any one of embodiments 56-64, wherein the polymer comprises polydioxanone (PDS).
- 66. The formulation of any one of embodiments 56-65, wherein the polymer comprises a lactide.
- 67. The formulation of any one of embodiments 56-66, wherein the lactide comprises L-lactide.
- 68. The formulation of any one of embodiments 56-67, wherein the lactide comprises poly(D,L-lactide).
- 69. The formulation of any one of embodiments 56-68, wherein the polymer comprises a copolymer.
- 70. The formulation of embodiment 69, wherein the copolymer comprises polycaprolactone (PCL) and polyglycolide.
- 71. The formulation of embodiment 70, wherein the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide.
- 72. The formulation of any one of embodiments 69-71, wherein the copolymer comprises poly(D,L-lactide-co-glycolide) copolymer.
- 73. The formulation of any one of embodiments 69-72, wherein the copolymer comprises about 40-60 percent by mole poly(D,L-lactide) and about 40-60 percent by mole polyglycolide.
- 74. The formulation of any one of embodiments 69-73, wherein the copolymer comprises PDS-glycolide copolymer.
- 75. The formulation of embodiment 74, wherein the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole glycolide.
- 76. The formulation of any one of embodiments 69-75, wherein the copolymer comprises PDS-L-lactide copolymer.
- 77. The formulation of embodiment 76, wherein the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole L-lactide.
- 78. The formulation of any one of embodiments 56-77, wherein the ink comprises the one or more additional agents.
- 79. The formulation of any one of embodiments 56-78, wherein the ink comprises about 1% to about 30% by weight the one or more additional agents.
- 80. The formulation of any one of embodiments 56-79, wherein the one or more additional agents comprises an additional polymer, particulate, or blowing agent, or a combination thereof.
- 81. The formulation of embodiment 80, wherein the one or more additional agents comprises the additional polymer.
- 82. The formulation of embodiment 80 or embodiment 81, wherein the additional polymer comprises a first polyethylene glycol and optionally a second polyethylene glycol wherein if present the second polyethylene glycol has a different molecular weight than the first polyethylene glycol.
- 83. The formulation of any one of embodiments 80-82, wherein the additional polymer is present in the ink at about 10% to about 30% by weight.
- 84. The formulation of any one of embodiments 80-83, wherein the one or more additional agents comprises the particulate.
- 85. The formulation of any one of embodiments 80-84, wherein the particulate comprises sodium chloride, calcium chloride, sucrose, trehalose (e.g., α,α trehalose dihydrate), or mannitol (e.g., D-mannitol), or a combination thereof.
- 86. The formulation of any one of embodiments 80-85, wherein the particulate is present in the ink at about 1% to about 10% by weight.
- 87. The formulation of any one of embodiments 80-86, wherein the one or more additional agents comprises the blowing agent.
- 88. The formulation of any one of embodiments 80-87, wherein the blowing agent comprises baking powder (e.g., monocalcium phosphate, sodium bicarbonate, corn starch) and/or azodicarbonamide.
- 89. The formulation of any one of embodiments 80-88, wherein the particulate is present in the ink at about 5% to about 20% by weight.
- 90. A method of preparing a three-dimensional structure, the method comprising using the formation of any one of embodiments 56-89 in a three-dimensional printing method.
- 91. A three-dimensional structure prepared using the ink formulation of any one of embodiments 56-89.
- 92. The structure of embodiment 91, comprising about 50% to about 90% by weight ceramic.
- 93. The structure of embodiment 91, comprising about 50% to about 90% by weight tricalcium phosphate.
- 94. The structure of any one of embodiments 91-93, comprising about 10% to about 50% by weight polymer.
- 95. The structure of embodiment 94, wherein the polymer comprises a polyester.
- 96. The structure of embodiment 94 or embodiment 95, wherein the polymer comprises polycaprolactone (PCL), polyglycolide, lactide, or polydioxanone (PDS), or a combination thereof.
- 97. The structure of any one of embodiments 94-96, wherein the polymer comprises polycaprolactone (PCL).
- 98. The structure of any one of embodiments 94-97, wherein the polymer comprises polyglycolide.
- 99. The structure of any one of embodiments 94-98, wherein the polymer comprises polydioxanone (PDS).
- 100. The structure of any one of embodiments 94-99, wherein the polymer comprises a lactide.
- 101. The structure of embodiment 100, wherein the lactide comprises L-lactide.
- 102. The structure of embodiment 100 or embodiment 101, wherein the lactide comprises poly(D,L-lactide).
- 103. The structure of any one of embodiments 94-102, wherein the polymer comprises a copolymer.
- 104. The structure of embodiment 103, wherein the copolymer comprises polycaprolactone (PCL) and polyglycolide.
- 105. The structure of embodiment 104, wherein the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide.
- 106. The structure of any one of embodiments 103-105, wherein the copolymer comprises poly(D,L-lactide-co-glycolide) copolymer.
- 107. The structure of any one of embodiments 103-106, wherein the copolymer comprises about 40-60 percent by mole poly(D,L-lactide) and about 40-60 percent by mole polyglycolide.
- 108. The structure of any one of embodiments 103-107, wherein the copolymer comprises PDS-glycolide copolymer.
- 109. The structure of embodiment 108, wherein the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole glycolide.
- 110. The structure of any one of embodiments 103-109, wherein the copolymer comprises PDS-L-lactide copolymer.
- 111. The structure of embodiment 110, wherein the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole L-lactide.
- 112. The structure of any one of embodiments 94-111, wherein the structure comprises about 15% to about 25% by weight polymer.
- 113. The structure of embodiment 112, wherein the structure comprises about 25% by weight polymer.
- 114. A three-dimensional structure comprising about 50% to about 90% by weight ceramic, and about 10% to about 30% polymer.
- 115. The structure of embodiment 114, wherein the ceramic comprises calcium phosphate, hydroxyapatite, fluorapatite, bone, silicate, or vanadate, or a combination thereof.
- 116. The structure of embodiment 114 or embodiment 115, wherein the ceramic comprises beta-tricalcium phosphate (β-TCP).
- 117. The structure of any one of embodiments 114-116, comprising about 65% to about 85% by weight ceramic.
- 118. The structure of embodiment 117, comprising about 70% to about 80% by weight ceramic.
- 119. The structure of embodiment 118, comprising about 75% by weight ceramic.
- 120. The structure of any one of embodiments 114-119, wherein the polymer comprises a polyester.
- 121. The structure of any one of embodiments 114-120, wherein the polymer comprises polycaprolactone (PCL), polyglycolide, lactide, or polydioxanone (PDS), or a combination thereof.
- 122. The structure of any one of embodiments 114-121, wherein the polymer comprises polycaprolactone (PCL).
- 123. The structure of any one of embodiments 114-122, wherein the polymer comprises polyglycolide.
- 124. The structure of any one of embodiments 114-123, wherein the polymer comprises polydioxanone (PDS).
- 125. The structure of any one of embodiments 114-124, wherein the polymer comprises a lactide.
- 126. The structure of embodiment 125, wherein the lactide comprises L-lactide.
- 127. The structure of embodiment 125 or embodiment 126, wherein the lactide comprises poly(D,L-lactide).
- 128. The structure of any one of embodiments 114-127, wherein the polymer comprises a copolymer.
- 129. The structure of embodiment 128, wherein the copolymer comprises polycaprolactone (PCL) and polyglycolide.
- 130. The structure of embodiment 129, wherein the copolymer comprises about 90-95 percent by mole PCL and about 5-10 percent by mole polyglycolide.
- 131. The structure of any one of embodiments 128-130, wherein the copolymer comprises poly(D,L-lactide-co-glycolide) copolymer.
- 132. The structure of embodiment 131, wherein the copolymer comprises about 40-60 percent by mole poly(D,L-lactide) and about 40-60 percent by mole polyglycolide.
- 133. The structure of any one of embodiments 128-132, wherein the copolymer comprises PDS-glycolide copolymer.
- 134. The structure of embodiment 133, wherein the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole glycolide.
- 135. The structure of any one of embodiments 128-134, wherein the copolymer comprises PDS-L-lactide copolymer.
- 136. The structure of embodiment 135, wherein the copolymer comprises about 90-95 percent by mole PDS and about 5-10 percent by mole L-lactide.
- 137. The structure of any one of embodiments 114-136, wherein the structure comprises about 15% to about 25% by weight polymer.
- 138. The structure of embodiment 137, wherein the structure comprises about 25% by weight polymer.
- 139. The structure of any one of embodiments 91-138, prepared by a three-dimensional printing method.
- 140. A method of manufacturing the three-dimensional structure of any one of embodiments 91-139, the method comprising depositing an ink formulation in a three-dimensional form.
- 141. The method of embodiment 140, wherein the ink formulation comprises the ink formulation of any one of embodiments 56-89.
- 142. A method of treating a condition in a subject in need thereof, the method comprising delivering to an organ or tissue of the subject the structure of any one of embodiments 91-139.
- 143. A method of treating a condition in a subject in need thereof, the method comprising delivering to an organ or tissue of the subject the structure manufactured by the method of any one of embodiments 90, 140 or 141.
- 144. The method of embodiment 142 or embodiment 143, wherein the condition comprises a bone defect, cartilage defect, soft tissue defect, tendon defect, fascia defect, ligament defect, organ defect, osteotendinous tissue defect, dermal defect, osteochondral defect, osteoporosis, avascular necrosis, or congenital skeletal malformation, or a combination thereof.
- 145. The method of any one of embodiments 142-144, wherein the method comprises spinal fusion.
- 146. The method of embodiment 145, wherein the spinal fusion comprises posterior lumbar fusion (PLF) and/or interbody fusion.
- 147. The method of embodiment 142 or embodiment 143, wherein the method comprises bone repair, dental repair, craniomaxillofacial repair, ankle fusion, kyphoplasty, osteoplasty, scaphoid fracture repair, tendeno-osseous repair, costal reconstruction, subchondral bone repair, cartilage repair, or surgical implantation of the three-dimensional structure or device, or a combination thereof.
- 148. The method of any one of embodiments 142-147, further comprising treating the subject with a therapeutic agent.
- 149. A method of delivering a therapeutic agent to a subject in need thereof, the method comprising delivering to an organ or tissue of the subject a device comprising a therapeutic agent and the structure of any one of embodiments 91-139.
- 150. The method of embodiment 148 or embodiment 149, wherein the therapeutic agent comprises a mammalian growth factor or a functional portion thereof
- 151. The method of any one of embodiments 148-150, wherein the therapeutic agent comprises one or more polypeptides selected from Table 1, or a functional portion thereof.
- 152. The method of any one of embodiments 148-151, wherein the therapeutic agent comprises a bone morphogenetic protein (BMP).
- 153. The method of any one of embodiments 148-152, wherein the therapeutic agent comprises a targeting moiety that non-covalently binds to the structure.
- 154. The method of embodiment 153, wherein the targeting moiety binds to the printed three-dimensional structure with an affinity of about 1 pM to about 100 μm.
- 155. The method of embodiment 153 or embodiment 154, wherein the targeting moiety comprises a polypeptide at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of the sequences of Tables 5-6.
- 156. The method of embodiment 153 or embodiment 154, wherein the targeting moiety comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences selected from the sequences of Tables 5-6.
- 157. The method of any one of embodiments 148-156, wherein the therapeutic agent comprises or is part of a chimeric polypeptide comprising a sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 433-441.
- 158. The device of any one of embodiments 1-50, the method of any one of embodiments 51-55, 90, 140-157, or the structure of any one of embodiments 91-139, wherein the structure has a density of about 1 g/cm³to about 2 g/cm³.
- 159. The device of any one of embodiments 1-50, the method of any one of embodiments 51-55, 90, 140-157, or the structure of any one of embodiments 91-139, wherein the structure has an open porosity of about 15% to about 50%.
- 160. The device of any one of embodiments 1-50, the method of any one of embodiments 51-55, 90, 140-157, or the structure of any one of embodiments 91-139, wherein the structure has a strut diameter of about 300 μm to about 600 μm.
- 161. A filament comprising the formulation of any one of embodiments 56-89.
- 162. The filament of embodiment 161, having a diameter of about 1.5 mm to about 2 mm.
- 163. A pellet comprising the formulation of any one of embodiments 56-89.
- 164. The pellet of embodiment 163, having a length of about 3 mm to about 4 mm.

EXAMPLES
Example 1: Ink Formulations and 3D Printed Scaffolds

Ink Formulation and Scaffold #1:

This ink is also converted into a filament form and utilized to prepare a 3D printed scaffold using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol and sucrose pore formers. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight polycaprolactone.

This ink is also converted into pellets (e.g., a filament is formed into small pellets) and fed from a hopper into a mini-screw extrusion head which melts and pushes the material out of a fine nozzle.

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 4

Example ink formulation #1

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
57
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing, binding

sites for targeting moiety

Poly-
19
50,000 MW,
Flexible polymer binder for

caprolactone

fine powder,
β-TCP powder; not water

Tmelt = 60 C.
soluble

Polyethylene
19
1500 MW, flake,
Water soluble polymer with

Glycol

Tmelt = 60 C.
low melt viscosity for good

extrusion

Sucrose
5
Fine powder
Water soluble particulate,

sacrificial pore former

Ink Formulation #2

This ink is also converted into a filament form and utilized to prepare a 3D printed scaffold using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight caprolactone/glycolide copolymer (95:5).

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 5

Example ink formulation #2

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing,

binding sites for

therapeutic

Caprolactone/
20
95 mol %
Flexible polymer binder

Glycolide

polycaprolactone,
for β-TCP powder; not

copolymer

5 mol %
water soluble; faster

polyglycolide,
bioresorption compared to

pellets, Tmelt =
polycaprolactone

52-62 C.

Polyethylene
20
1500 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

for good extrusion

Ink Formulation #3

This ink is also converted into a filament form and utilized to prepare a 3D printed scaffold using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight caprolactone/glycolide copolymer (90:10).

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 6

Example ink formulation #3

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing,

binding sites for

therapeutic

Caprolactone/
20
90 mol %
Flexible polymer binder

Glycolide

polycaprolactone,
for β-TCP powder; not

copolymer

10 mol %
water soluble; fast

polyglycolide,
bioresorption compared to

chips
polycaprolactone

Polyethylene
20
1500 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

for good extrusion

Ink Formulation #4

This ink is also converted into a filament form and utilized to prepare a 3D printed scaffold using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight poly(D,L-lactide-co-glycolide) copolymer (50:50).

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 7

Example ink formulation #4

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing,

binding sites for

therapeutic

poly(D,L-
20
50 mol % poly(D,L-
Flexible polymer binder

lactide-co-

lactide), 50 mol %
for β-TCP powder; not

glycolide)

polyglycolide,
water soluble; fast

copolymer

chunks, 38,000-54,000
bioresorption compared

MW, Tg = 46-50 C.
to polycaprolactone

Polyethylene
20
1500 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

for good extrusion

Ink Formulation #5

This 3D printing ink material is a flexible, polymer-ceramic composite material containing β-TCP that may be tethered to a therapeutic agent, such as tBMP2. This formulation contains a sacrificial pore former (water soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. This ink is a moderate viscosity formulation that was formed into a 1.75 mm filament for 3D printing on a RepRap style FFF 3D printers (e.g. Prusa i3 MK3 S 3D printer) with a 400 μm diameter nozzle. The higher molecular weight polyethylene glycol (8000 MW for FFF 3D printing vs. 1500 MW for syringe-based bioprinting) results in a higher viscosity material which aids in extrusion of 1.75 mm diameter filaments. After 3D printing was complete, the resulting scaffold was soaked in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold #5 contains 75% by weight β-TCP powder, and 25% by weight polycaprolactone.

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 8

Example ink formulation #5

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried
Spherical β-TCP Powder

powder,
for good extrudability

10-38 micron
during 3D printing,

particle size
binding sites for

therapeutic

Polycaprolactone
20
50,000 MW, fine
Flexible polymer binder

powder,
for β-TCP powder; not

Tmelt = 60 C.
water soluble

Polyethylene
20
8,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

for good extrusion

Ink Formulation #6

This 3D printing ink material is a flexible, polymer-ceramic composite material containing β-TCP that may be tethered to a therapeutic agent, such as tBMP2. This formulation contains a sacrificial pore former (water soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. This ink also contains a blowing agent (sodium bicarbonate) which thermally decomposes and releases CO2 gas during 3D printing to create a foamed structure, thus increasing the porosity of the 3D printed scaffold. This ink is a moderate viscosity formulation that was formed into a 1.75 mm filament for 3D printing on a RepRap style FFF 3D printers (e.g. Prusa i3 MK3S 3D printer) with a 400 μm diameter nozzle. The higher molecular weight polyethylene glycol (8000 MW for FFF 3D printing vs. 1500 MW for syringe-based bioprinting) results in a higher viscosity material which aids in extrusion of 1.75 mm diameter filaments. After 3D printing was complete, the resulting scaffold was soaked in water to dissolve the sacrificial polyethylene glycol pore former and sodium carbonate by-product from the sodium bicarbonate thermal decomposition. The resulting scaffold #6 contains 75% by weight β-TCP powder, and 25% by weight polycaprolactone.

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 9

Example ink formulation #6

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
54
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing,

binding sites for

therapeutic

Polycaprolactone
18
50,000 MW, fine
Flexible polymer binder

powder,
for β-TCP powder; not

Tmelt = 60 C.
water soluble

Polyethylene
18
8,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

for good extrusion

Sodium
10
Fine powder,
Blowing agent

Bicarbonate

thermal

decomposition

temperature 80 C.

Ink Formulation #7

This ink is also converted into a filament form and utilized to prepare a 3D printed scaffold using a fused filament fabrication (FFF or FDM) 3D printer. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight dioxanone/L-lactide copolymer (90:10).

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 10

Example ink formulation #7

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing,

binding sites for

therapeutic

Dioxanone/
20
90 mol %
Flexible polymer binder

L-lactide

polydioxanone,
for β-TCP powder; not

copolymer

10 mol % poly-L-
water soluble; faster

lactide, white
bioresorption compared

chips,
to polycaprolactone

Tmelt = ~110 C.

Polyethylene
20
1500 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

for good extrusion

Ink Formulation #8

This 3D printing ink material is a flexible, polymer-ceramic composite material containing β-TCP that may be tethered to a therapeutic agent, such as tBMP2. This formulation contains a sacrificial pore former (water soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. This ink is a moderate viscosity formulation that can be formed into a 1.75 mm filament for 3D printing on a RepRap style FFF 3D printers (e.g. Prusa i3 MK3 S 3D printer) with a 400 μm diameter nozzle. The higher molecular weight blend of polyethylene glycol (8000 MW and 20000 MW for FFF 3D printing vs. 1500 MW for syringe-based bioprinting) results in a higher viscosity material which aids in extrusion of 1.75 mm diameter filaments. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight polycaprolactone.

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 11

Example ink formulation #8

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried
Spherical β-TCP Powder

powder,
for good extrudability

10-38 micron
during 3D printing,

particle size
binding sites for

therapeutic

Polycaprolactone
20
50,000 MW, fine
Flexible polymer binder

powder,
for β-TCP powder; not

Tmelt = 60 C.
water soluble

Polyethylene
10
8,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

Polyethylene
10
20,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with moderate melt

viscosity

Ink Formulation #9

This 3D printing ink material is a flexible, polymer-ceramic composite material containing β-TCP that may be tethered to a therapeutic agent, such as tBMP2. This formulation contains two sacrificial pore formers (water soluble polyethylene glycol and water soluble glucose) to expose more β-TCP surface area for agent binding. This ink is a moderate viscosity formulation that can be formed into a 1.75 mm filament for 3D printing on a RepRap style FFF 3D printers (e.g. Prusa i3 MK3 S 3D printer) with a 400 μm diameter nozzle. The higher molecular weight blend of polyethylene glycol (8000 MW and 20000 MW for FFF 3D printing vs. 1500 MW for syringe-based bioprinting) results in a higher viscosity material which aids in extrusion of 1.75 mm diameter filaments. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol and sucrose pore formers. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight polycaprolactone.

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 12

Example ink formulation #9

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing,

binding sites for

therapeutic

Polycaprolactone
20
50,000 MW, fine
Flexible polymer binder

powder,
for β-TCP powder; not

Tmelt = 60 C.
water soluble

Polyethylene
10
8,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

Polyethylene
10
20,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with moderate melt

viscosity

Sucrose
5
Fine powder
Water soluble particulate,

sacrificial pore former

Ink Formulation #10

This 3D printing ink material is a flexible, polymer-ceramic composite material containing β-TCP that may be tether to a therapeutic agent, such a tBMP2. This formulation contains a sacrificial pore former (water soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. This formulation employs a 95 mol % caprolactone 5 mol % glycolide copolymer for faster bioresorption characteristics compared to polycaprolactone. This ink is a moderate viscosity formulation that can be formed into a 1.75 mm filament for 3D printing on a RepRap style FFF 3D printers (e.g. Prusa i3 MK3 S 3D printer) with a 400 μm diameter nozzle. The higher molecular weight blend of polyethylene glycol (8000 MW and 20000 MW for FFF 3D printing vs. 1500 MW for syringe-based bioprinting) results in a higher viscosity material which aids in extrusion of 1.75 mm diameter filaments. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight caprolactone/glycolide copolymer (95:5).

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 13

Example ink formulation #10

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing,

binding sites for

therapeutic

Caprolactone/
20
95 mol %
Flexible polymer binder

Glycolide

polycaprolactone,
for β-TCP powder; not

copolymer

5 mol %
water soluble; faster

(95:5)

polyglycolide,
bioresorption compared

pellets, Tmelt =
to polycaprolactone

52-62 C.

Polyethylene
10
8,000 MW, flake,
Water soluble polymer with

Glycol

Tmelt = 60 C.
low melt viscosity

Polyethylene
10
20,000 MW, flake,
Water soluble polymer with

Glycol

Tmelt = 60 C.
moderate melt viscosity

Ink Formulation #11

This 3D printing ink material is a flexible, polymer-ceramic composite material containing β-TCP that may be tether to a therapeutic agent, such as tBMP2. This formulation contains a sacrificial pore former (water soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. This formulation employs a 90 mol % caprolactone 10 mol % glycolide copolymer for fast bioresorption characteristics compared to polycaprolactone. This ink is a moderate viscosity formulation that can be formed into a 1.75 mm filament for 3D printing on a RepRap style FFF 3D printers (e.g. Prusa i3 MK3 S 3D printer) with a 400 μm diameter nozzle. The higher molecular weight blend of polyethylene glycol (8000 MW and 20000 MW for FFF 3D printing vs. 1500 MW for syringe-based bioprinting) results in a higher viscosity material which aids in extrusion of 1.75 mm diameter filaments. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene glycol pore former. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight caprolactone/glycolide copolymer (90:10).

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 14

Example ink formulation #11

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried powder,
Spherical β-TCP Powder

10-38 micron
for good extrudability

particle size
during 3D printing,

binding sites for

therapeutic

Caprolactone/
20
95 mol %
Flexible polymer binder

Glycolide

polycaprolactone,
for β-TCP powder; not

copolymer

5 mol %
water soluble; fast

(90:10)

polyglycolide, chips
bioresorption compared

to polycaprolactone

Polyethylene
10
8,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

Polyethylene
10
20,000 MW, flake,
Water soluble polymer with

Glycol

Tmelt = 60 C.
moderate melt viscosity

Ink Formulation #12

This 3D printing ink material is a flexible, polymer-ceramic composite material containing β-TCP that may be tethered to a therapeutic agent, such as tBMP2. This formulation contains a sacrificial pore former (water soluble polyethylene glycol) to expose more β-TCP surface area for agent binding. This formulation employs a 50 mol %:50 mol % poly(D,L-lactide-co-glycolide) copolymer for fast bioresorption characteristics compared to polycaprolactone. This ink is a moderate viscosity formulation that can be formed into a 1.75 mm filament for 3D printing on a RepRap style FFF 3D printers (e.g. Prusa i3 MK3 S 3D printer) with a 400 μm diameter nozzle. The higher molecular weight blend of polyethylene glycol (8000 MW and 20000 MW for FFF 3D printing vs. 1500 MW for syringe-based bioprinting) results in a higher viscosity material which aids in extrusion of 1.75 mm diameter filaments. After 3D printing is complete, the resulting scaffold is soaked in water to dissolve the sacrificial polyethylene gly col pore former. The resulting scaffold contains 75% by weight β-TCP powder, and 25% by weight poly(D,L-lactide-co-glycolide) copolymer (50:50).

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 15

Example ink formulation #12

Wt %
Component

Component
in ink
characteristics
Purpose

β-TCP Powder
60
Spray dried
Spherical β-TCP Powder

powder,
for good extrudability

10-38 micron
during 3D printing,

particle size
binding sites for

therapeutic.

poly(D,L-
20
50 mol % poly(D,L-
Flexible polymer binder

lactide-co-

lactide), 50 mol %
for β-TCP powder; not

glycolide)

polyglycolide,
water soluble; fast

copolymer

chunks, 38,000-
bioresorption compared to

54,000 MW,
polycaprolactone

Tg = 46-50 C.

Polyethylene
10
8,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with low melt viscosity

Polyethylene
10
20,000 MW, flake,
Water soluble polymer

Glycol

Tmelt = 60 C.
with moderate melt

viscosity

Ink Formulation #13

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 16

Example ink formulation #13

Component
Wt % in ink

β-TCP Powder
50-80

Polydioxanone (PDS)
10-30

Polyethylene Glycol
10-30

Ink Formulation #14

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 17

Example ink formulation #14

Component
Wt % in ink

β-TCP Powder
50-80

PDS-Glycolide Copolymer (90:10)
10-30

Polyethylene Glycol
10-30

Ink Formulation #15

This ink is also cryomilled into a fine powder. A laser heats the powder into the desired configuration using the SLS method.

TABLE 18

Example ink formulation #15

Component
Wt % in ink

β-TCP Powder
50-80

PDS-L-Lactide Copolymer (90:10)
10-30

Polyethylene Glycol
10-30

Example 2: Ink Preparation and Scaffold Manufacture by 3D Printing

Ink Formulation and Scaffold #1

Method: To make 5.3 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of polycaprolactone powder, 1.87 g of polyethylene glycol flake and 0.49 g sucrose were added to a glass mixing container. The glass jar was placed in a dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. The mixer was mixed for 5 min at high intensity (3500 rpm). During mixing, the internal friction causes the polycaprolactone and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder and sucrose powder into the molten polymer blend. The blended ink was allowed to cool for 10-15 min, then mixed for 5 more minutes at 3500 rpm. The mixing/cooling process was repeated for a total of four 5 min mixes at 3500 rpm. After the fourth mix at 3500 rpm, the ink charge was poured out on a glass plate and two spatulas were used to form into a roughly 1 cm diameter×6 cm long cylinder. While ink was still semi-molten, it was cut into several ˜1-2 cm long pieces with straight razor.

3D Printing: solid polymer/β-TCP pieces were transferred to a 5 cc stainless steel syringe (for use in Allevi 3 Bioprinter). Extruder CORE printing head was heated to 135° C. and allowed to dwell for approximately 30 min to ensure melting of the ink. The ink was printed with 400 micron I.D. conical metallic Luer lock tip using 70 psi pressure and 7 mm/s nozzle velocity. The scaffold was 3D printed on painter's tape applied to a smooth glass or polymer surface, such as a glass microscope slide, larger glass plate, or 96 well plate lid.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol and sucrose from the printed material, thus creating a porous and flexible β-TCP/polycaprolactone composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent such as tBMP2 protein. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 1A-1C.

3D printing is also performed using a FFF 3D printer. The ink #1 is extruded into filaments and the filaments are loaded in a FFF 3D printer to generate a 3D printed scaffold. The scaffold is processed using the post processing method outlined above.

Ink Formulation and Scaffold #2

Method: To make a 5 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of 95:5 caprolactone/glycolide copolymer pellets, and 1.87 g of polyethylene glycol flake were added to a glass mixing container. The glass jar was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. The mixture was mixed for 5 min at high intensity (3500 rpm). During mixing, the internal friction causes the 95:5 caprolactone/glycolide copolymer and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The blended ink was allowed to cool for 10-15 min, and then mixed for 5 more minutes at 3500 rpm. The mixing/cooling process was repeated for a total of four 5 min mixes at 3500 rpm. After the fourth mix at 3500 rpm, the ink charge was poured out on a glass plate and two spatulas were used to form into a roughly 1 cm diameter×6 cm long cylinder. While ink was still semi-molten, it was cut into several ˜1-2 cm long pieces with straight razor.

3D Printing: The solid polymer/β-TCP pieces were transferred to a 5 cc stainless steel syringe (for use in Allevi 3 Bioprinter). Extruder CORE printing head was heated to 130° C. and allowed to dwell for approximately 30 min to ensure melting of the ink. Ink was printed with 320 micron I.D. conical metallic Luer lock tip using 80 psi pressure and 6 mm/s nozzle velocity. Scaffolds were 3D printed on painter's tape applied to a smooth glass or polymer surface, such as a glass microscope slide, larger glass plate, or 96 well plate lid.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material, thus creating a porous and flexible β-TCP/95:5 caprolactone/glycolide copolymer composite. The scaffolds were dried for at least twelve hours to ensure residual water evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 2A-2C.

3D printing is also performed using a FFF 3D printer. The ink #2 is extruded into filaments and the filaments are loaded in a FFF 3D printer to generate a 3D printed scaffold. The scaffold is processed using the post processing method outlined above.

Ink Formulation and Scaffold #3

Method: To make a 5 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of 90:10 caprolactone/glycolide copolymer chips, and 1.87 g of polyethylene glycol flake were added to a glass mixing container. The mixture was placed in a glass jar in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. It was then mixed for 5 min at high intensity (3500 rpm). During mixing, the internal friction causes the 90:10 caprolactone/glycolide copolymer and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The blended ink was allowed to cool for 10-15 min and then mixed for 5 more minutes at 3500 rpm. The mixing/cooling process was repeated for a total of four 5 min mixes at 3500 rpm. After the fourth mix at 3500 rpm, the ink charge was poured out on a glass plate and two spatulas were used to form into a roughly 1 cm diameter×6 cm long cylinder. While the ink was still semi-molten, it was cut into several ˜1-2 cm long pieces with straight razor.

3D Printing: the solid polymer/β-TCP pieces were transferred to a 5 cc stainless steel syringe (for use in Allevi 3 Bioprinter). Extruder CORE printing head was heated to 130° C. and allowed to dwell for approximately 30 min to ensure melting of the ink. Ink was printed with 320 micron I.D. conical metallic Luer lock tip using 45 psi pressure and 7 mm/s nozzle velocity. Scaffolds were be 3D printed on painter's tape applied to a smooth glass or polymer surface, such as a glass microscope slide, larger glass plate, or 96 well plate lid.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material, thus creating a porous and flexible β-TCP/90:10 caprolactone/glycolide copolymer composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 3A-3C.

3D printing is also performed using a FFF 3D printer. The ink #3 is extruded into filaments and the filaments are loaded in a FFF 3D printer to generate a 3D printed scaffold. The scaffold is processed using the post processing method outlined above.

Ink Formulation and Scaffold #4

Method: To make a 2.5 cc batch of ink, 2.8 g of β-TCP powder, 0.94 g of 50:50 poly(D,L-lactide-co-glycolide) copolymer chunks, and 0.94 g of polyethylene glycol flake were added to a glass mixing container. The glass jar was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. It was mixed for 5 min at high intensity (3500 rpm). During mixing the internal friction causes the 50:50 poly(D,L-lactide-co-glycolide) copolymer and polyethylene glycol to flow, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The blended ink was allowed to cool for 10-15 min and then mixed for 5 more minutes at 3500 rpm. The mixing/cooling process was repeated for a total of four 5 min mixes at 3500 rpm. After the fourth mix at 3500 rpm, the ink charge was poured out on a glass plate and two spatulas were used to form into a roughly 1 cm diameter×3 cm long cylinder. While ink was still semi-molten, it was cut into several ˜1-2 cm long pieces with straight razor.

3D Printing: The solid polymer/β-TCP pieces were transferred to a 5 cc stainless steel syringe (for use in Allevi 3 Bioprinter). Extruder CORE printing head was heated to 85° C. and allowed to dwell for approximately 30 min to ensure melting of the ink. Ink was printed with a 400 micron I.D. conical metallic Luer lock tip using 60 psi pressure and 7 mm/s nozzle velocity. Scaffolds were 3D printed on painter's tape applied to a smooth glass or polymer surface, such as a glass microscope slide, larger glass plate, or 96 well plate lid.

Post Processing: 3D printed structures are soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material, thus creating a porous and flexible β-TCP/50:50 poly(D,L-lactide-co-glycolide) composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 4A-4C.

3D printing is also performed using a FFF 3D printer. The ink #4 is extruded into filaments and the filaments are loaded in a FFF 3D printer to generate a 3D printed scaffold. The scaffold is processed using the post processing method outlined above.

Ink Formulation and Scaffold #5

Method: To make a 5 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of polycaprolactone powder, and 1.87 g of polyethylene glycol flake were added to a glass mixing container. The glass jar was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mix at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. It was mixed for 5 min at high intensity (3500 rpm). During mixing, the internal friction causes the polycaprolactone and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flatten with a spatula to approximately 3 mm thick layer for cooling. After cooling, shears were used to cut into approximately 3-4 mm pellets. This was repeated for two additional 5 cc batches to create a total 15 cc of pellets for filament extrusion.

Filament Fabrication: 15 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A 3× length extended melt filter nozzle (with filter screens removed) with 1.75 mm diameter hole size was used. A cooling fan was placed near the extrusion nozzle to speed up solidification of the extruded filament. The 1.75 mm diameter filament was extruded at extrusion temperature to 62° C. and speed at ½ on the analog dial (approximately 1 cm/second extrusion speed).

3D Printing: The 1.75 mm filament was loaded in a Prusa i3 MK3 S 3D printer. This filament material was printed using a 400 micron brass nozzle, 105° C. extruder temperature, and 15 mm/s print speed.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material, thus creating a porous and flexible β-TCP/polycaprolactone composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 5A-5C.

Ink Formulation and Scaffold #6

Method: To make a 5.5 cc batch of ink, 5.6 g of β-TCP powder, 1.87 g of polycaprolactone powder, 1.87 g of polyethylene glycol flake, and 1.04 g of sodium bicarbonate were added to a glass mixing container. The glass jar was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. It was mixed for 2 min at high intensity (3500 rpm). During mixing the internal friction causes the polycaprolactone and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder and sodium bicarbonate powder into the molten polymer blend. The molten ink was poured onto a glass plate and flattened with a spatula to approximately 3 mm thick layer for cooling. After cooling, shears were used to cut into approximately 3-4 mm pellets. The was repeated for two additional 5.5 cc batches to create a total 16.5 cc of pellets for filament extrusion.

Filament Fabrication: 16.5 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A 3× length extended melt filter nozzle (with filter screens removed) with 1.75 mm diameter hole size was used. A cooling fan was placed near the extrusion nozzle to speed up solidification of the extruded filament. The 1.75 mm diameter filament was extruded at extrusion temperature to 62° C. and speed at ½ on the analog dial (approximately 1 cm/second extrusion speed).

3D Printing: The 1.75 mm filament was loaded in a Prusa i3 MK3 S 3D printer. This filament material was printed using a 400 micron brass nozzle, 155° C. extruder temperature, and 15 mm/s print speed.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol and sodium carbonate (by-product of the sodium bicarbonate thermal decomposition) from the foamy printed material, thus creating a porous and flexible (3-TCP/polycaprolactone composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 6A-6C.

Ink Formulation and Scaffold #7

Method: To make a 10 cc batch of ink, 11.2 g of β-TCP powder, 3.74 g of Dioxanone/L-lactide (90:10) copolymer chips, and 3.74 g of polyethylene glycol flake were added to a glass mixing container. The glass jar was placed in a dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. The glass jar was transferred to a hot plate and heated until it reached 185° C. (measured with IR thermometer). Next, the glass jar was immediately transferred back to the dual asymmetric centrifugal mixer and mixed for 5 min at high intensity (3500 rpm). Liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. Next, the glass jar was transferred back to the hotplate and the temperature was increased to 185° C. Upon reaching the temperature, the glass jar was immediately transferred back to the dual asymmetric centrifugal mixer and mixed for 5 more minutes at 3500 rpm. The mixing/heating process was repeated for a total of four 5 min mixes at 3500 rpm. After the fourth mix at 3500 rpm, the ink charge was poured out on a glass plate and two spatulas were used to form into a roughly 1 cm diameter×6 cm long cylinder. While ink was still semi-molten, it was cut into several ˜1-2 cm long pieces with straight razor.

3D Printing: solid polymer/β-TCP pieces were transferred to a 5 cc stainless steel syringe (for use in Allevi 3 Bioprinter). Extruder CORE printing head was heated to 110° C. and allowed to dwell for approximately 30 min to ensure melting of the ink. The ink was printed with 400 micron I.D. conical metallic Luer lock tip using 15 psi pressure and 10 mm/s nozzle velocity. The scaffold was 3D printed on painter's tape applied to a smooth glass or polymer surface, such as a glass microscope slide, larger glass plate, or 96 well plate lid.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material. The process created a porous and flexible β-TCP/90:10 dioxanone-L-lactide copolymer composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent such as tBMP2 protein. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 7A-7C.

3D printing is also performed using a FFF 3D printer. The ink #7 is extruded into filaments and the filaments are loaded in a FFF 3D printer to generate a 3D printed scaffold. The scaffold is processed using the post processing method outlined above.

Ink Formulation and Scaffold #8

Method: To make a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of polycaprolactone powder, 3 g of polyethylene glycol (8,000 MW) flake and 3 g of polyethylene glycol (20,000 MW) flake were added to a teflon mixing container. The teflon container was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. Next, the teflon container was mixed for 2.5 min at high intensity (3500 rpm). During mixing, the internal friction causes the polycaprolactone and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flatten with a spatula to approximately 3 mm thick layer for cooling. It was cooled for 10-15 min. The mixture was returned to the teflon container and mixed for 2.5 more minutes at a high intensity (3500 rpm). The mixing/cooling process was repeated for a total of four 2.5 min mixes at 3500 rpm. After the ink was cooled after the fourth and final mix, shears were used to cut it into approximately 3-4 mm pellets.

Filament Fabrication: 16 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A 3× length extended melt filter nozzle (with filter screens removed) with 1.75 mm diameter hole size was used. A cooling fan was placed near the extrusion nozzle to speed up solidification of the extruded filament. The 1.75 mm diameter filament was extruded at extrusion temperature to 62° C. and speed at ½ on the analog dial (approximately 1 cm/second extrusion speed).

3D Printing: The 1.75 mm filament was loaded in a Prusa i3 MK3S 3D printer. This filament material was printed using a 400 micron brass nozzle, 105° C. extruder temperature, and 15 mm/s print speed.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material, thus creating a porous and flexible β-TCP/polycaprolactone composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 8A-8C.

Ink Formulation and Scaffold #9

Method: To make a 16.3 cc batch of ink, 17.1 g of β-TCP powder, 5.7 g of polycaprolactone powder, 2.85 g of polyethyleneglycol (8,000 MW) flake, 2.85 g of polyethylene glycol (20,000 MW) flake, and 1.5 g of sucrose were added to a teflon mixing container. The teflon container was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. Next, the teflon container was mixed for 2 min at high intensity (3500 rpm). During mixing, the internal friction causes the polycaprolactone and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flatten with a spatula to approximately 3 mm thick layer for cooling. It was cooled for 10-15 min. The mixture was returned to the teflon container and mixed for 2 more minutes at a high intensity (3500 rpm). The mixing/cooling process was repeated for a total of four 2 min mixes at 3500 rpm. After the ink was cooled after the fourth and final mix, shears were used to cut it into approximately 3-4 mm pellets.

Filament Fabrication: 16.3 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A 3× length extended melt filter nozzle (with filter screens removed) with 1.75 mm diameter hole size was used. A cooling fan was placed near the extrusion nozzle to speed up solidification of the extruded filament. The 1.75 mm diameter filament was extruded at extrusion temperature to 62° C. and speed at ½ on the analog dial (approximately 1 cm/second extrusion speed).

3D Printing: The 1.75 mm filament was loaded in a Prusa i3 MK3S 3D printer. This filament material was printed using a 400 micron brass nozzle, 140° C. extruder temperature, and 10 mm/s print speed.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol and sucrose from the printed material, thus creating a porous and flexible β-TCP/polycaprolactone composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 9A-9C.

Ink Formulation and Scaffold #10

Method: To make a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of caprolactone/glycolide copolymer (95:5) pellets, 3 g of polyethylene glycol (8,000 MW) flake and 3 g of polyethyleneglycol (20,000 MW) flake were added to a teflon mixing container. The teflon container was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. Next, the teflon container was mixed for 2.5 min at high intensity (3500 rpm). During mixing, the internal friction causes the caprolactone/glycolide copolymer (95:5) and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flatten with a spatula to approximately 3 mm thick layer for cooling. It was cooled for 10-15 min. The mixture was returned to the teflon container and mixed for 4 more minutes at a high intensity (3500 rpm). The mixing/cooling process was repeated for a total of one 2.5 minute mix and three 4 min mixes at 3500 rpm. After the ink was cooled after the fourth and final mix, shears were used to cut it into approximately 3-4 mm pellets.

3D Printing: The 1.75 mm filament was loaded in a Prusa i3 MK3S 3D printer. This filament material was printed using a 400 micron brass nozzle, 150° C. extruder temperature, and 10 mm/s print speed.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material, thus creating a porous and flexible β-TCP/caprolactone/glycolide copolymer (95:5) composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 10A-10C.

Ink Formulation and Scaffold #11

Method: To make a 16 cc batch of ink, 18 g of β-TCP powder, 6 g of caprolactone/glycolide copolymer (90:10) chips, 3 g of polyethylene glycol (8,000 MW) flake and 3 g of polyethyleneglycol (20,000 MW) flake were added to a teflon mixing container. The teflon container was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. Next, the teflon container was mixed for 2 min at high intensity (3500 rpm). During mixing, the internal friction causes the caprolactone/glycolide copolymer (90:10) and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flatten with a spatula to approximately 3 mm thick layer for cooling. It was cooled for 10-15 min. The mixture was returned to the teflon container and mixed for 5 more minutes at a high intensity (3500 rpm). The mixing/cooling process was repeated for a total of one 2 minute mix and three 5 min mixes at 3500 rpm. After the ink was cooled after the fourth and final mix, shears were used to cut it into approximately 3-4 mm pellets.

3D Printing: The 1.75 mm filament was loaded in a Prusa i3 MK3S 3D printer. This filament material was printed using a 400 micron brass nozzle, 150° C. extruder temperature, print bed temperature of 40° C., and 10 mm/s print speed.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material, thus creating a porous and flexible β-TCP/caprolactone/glycolide copolymer (90:10) composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold b eforebinding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 11A-11C.

Ink Formulation and Scaffold #12

Method: To make a 8 cc batch of ink, 9 g of β-TCP powder, 3 g of Poly(D,L-lactide-co-glycolide) copolymer (50:50) chunks, 1.5 g of polyethylene glycol (8,000 MW) flake, and 1.5 g of polyethylene glycol (20,000 MW) flake were added to a teflon mixing container. The teflon container was placed in dual asymmetric centrifugal mixer (FlackTek Speedmixer) and mixed at low intensity (300 rpm) for 2 min to homogenize the powder blend before high rpm mixing. Next, the teflon container was mixed for 2 min at high intensity (3500 rpm). During mixing, the internal friction causes the Poly(D,L-lactide-co-glycolide) copolymer (50:50) and polyethylene glycol to melt, changing the ink to a viscous molten liquid. This liquid phase mixing facilitates intimate dispersion of the β-TCP powder into the molten polymer blend. The molten ink was poured onto a glass plate and flatten with a spatula to approximately 3 mm thick layer for cooling. It was cooled for 10-15 min. The mixture was returned to the teflon container and mixed for 2.5 more minutes at a high intensity (3500 rpm). The mixing/cooling process was repeated for a total of one 2 minute mix and two 2.5 min mixes at 3500 rpm. After the ink was cooled after the third and final mix, shears were used to cut it into approximately 3-4 mm pellets.

Filament Fabrication: 8 cc of 3-4 mm pellets were loaded into the hopper of a filament extruder (Filabot EX2 Filament Extruder). A 3× length extended melt filter nozzle (with filter screens removed) with 1.75 mm diameter hole size was used. A cooling fan was placed near the extrusion nozzle to speed up solidification of the extruded filament. The 1.75 mm diameter filament was extruded at extrusion temperature to 62° C. and speed at ½ on the analog dial (approximately 1 cm/second extrusion speed).

3D Printing: The 1.75 mm filament was loaded in a Prusa i3 MK3S 3D printer. This filament material was printed using a 400 micron brass nozzle, 140° C. extruder temperature, and 10 mm/s print speed.

Post Processing: 3D printed structures were soaked overnight in distilled water to dissolve the polyethylene glycol from the printed material, thus creating a porous and flexible β-TCP/Poly(D,L-lactide-co-glycolide) copolymer (50:50) composite. Scaffolds were dried for at least twelve hours to ensure residual water has evaporated from the porous scaffold before binding with a therapeutic agent like tBMP2. Scaffolds were sterilized by soaking for 2-4 hours in a 70% ethanol solution and allowed to dry in biosafety cabinet for approximately 12 hours. Images of the scaffold are shown in FIGS. 12A-12C.

Ink Formulation and Scaffold #13

Ink #13 is printed using a syringe-based melt extrusion printing method, for example, using methods as described for printing inks #1-#4. Ink #13 is printed using fused filament fabrication 3D printing, for example, using methods as described for printing ink #5 and ink #6.

Ink Formulation and Scaffold #14 Ink #14 is printed using a syringe-based melt extrusion printing method, for example, using methods as described for printing inks #1-#4. Ink #14 is printed using fused filament fabrication 3D printing, for example, using methods as described for printing ink #5 and ink #6.

Ink Formulation and Scaffold #15 Ink #9 is printed using a syringe-based melt extrusion printing method, for example, using methods as described for printing inks #1-#4. Ink #15 is printed using fused filament fabrication 3D printing, for example, using methods as described for printing ink #5 and ink #6.

Structure Properties

Physical properties of the scaffolds #1-#6 were determined and are outlined in Table 19.

TABLE 19

Properties of Examples Scaffolds

Ink
Density (g/cm³)
Open Porosity (%)
Strut Diameter (μm)

Ink #1
1.45
30
434

Ink #2
1.56
23
397

Ink #3
1.70
25
569

Ink #4
1.49
32
464

Ink #5
1.23
39
378

Ink #6
1.22
39
392

Ink #7
1.50
33
499

Ink #8
1.22
37
359

Ink #9
1.23
37
351

Ink #10
1.28
37
374

Ink #11
1.45
29
335

Ink #12
1.30
37
364

The structures of this example are tested using Brunauer-Emmett-Teller (BET) surface area analysis by gas physisorption.

A compression test is also performed on the structures.

Example 3: Therapeutic Agent

A chimeric polypeptide comprising the BMP therapeutic peptide connected to five b eta-tricalcium phosphate binding peptides was expressed and purified using standard expression and purification methods. The chimeric polypeptide is referred to as tBMP-2 and has the following sequence:

(SEQ ID NO: 434)

MPIGSLLADTTHHRPWTVIGESTHHRPWSIIGESSHHKPFTGLGDTTHH

RPWGILAESTHHKPWTASGAGGSEGGGSEGGTSGATGAGTSTSGGGAST

GGGTGQAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCH

GECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLD

ENEKVVLKNYQDMVVEGCGCR.

Example 4: Device Manufacture

The 3D printed structures are combined with the tBMP-2 therapeutic agent to create a device. 0.75 mg/mL tBMP-2 binding solution (10 mM sodium acetate, 7 mM acetic acid, 100 mM NaCl, pH=4.75) is prepared and sterilized with 0.22 μm filter. In biosafety cabinet, tBMP-2 binding solution is added to a sterile polypropylene tube with sterile pipette such that a ratio of 15 mg of tBMP-2 per g of 3D printed scaffold is achieved. A sterile scaffold is added to binding solution with sterile tweezers, then the polypropylene tube is closed and wrapped with para film. The tube is placed on a LabLine Instruments Titer Plate Shaker, set at speed 6, and shaken for 2 hours. In a biosafety cabinet, the scaffold+tBMP-2 is removed with sterile tweezers and placed in a different sterile polypropylene tube containing sterile PBS (same volume as the tBMP-2 binding solution). The lid is closed, wrapped with parafilm, and returned to the Titer Plate Shaker to shake for 5 minutes at speed 6. The tube is opened in the biosafety cabinet, the scaffold+tBMP-2 is removed with sterile tweezers, and placed in a sterile petri dish. The scaffold+tBMP-2 is allowed to dry overnight in the biosafety cabinet, resulting in the tBMP-2 device.

The mass of tBMP-2 remaining in the binding solution and mass of tBMP-2 in the PBS wash solution is measured using A280 absorbance measurements. The sum of these masses is calculated and then subtracted from the initial mass of tBMP-2 in binding solution to arrive at the mass of tBMP-2 which remains bound to the 3D printed scaffold.

Example 5: Animal Models

Demonstrating the utility of the embodiments described herein is done by demonstrating bone regeneration in an animal model (e.g., rats, rabbits, goats, pigs, dogs, sheep) and assessing through μCT imaging and histological analysis. Indications can include lumbar spinal fusion (a 3D printed insert for spinal fusion cage), posterolateral (PLF) spine fusion (3D printed scaffold that spans transverse processes), tibial segmental defects (a 3D printed scaffold based on patient CT data), and/or alveolar ridge augmentation (a 3D printed thin barrier membrane).

CERAMIC COMPOSITIONS AND METHODS OF USE

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