Implantable engineered bone scaffolds with auto-graft-like properties (referred to as implantable scaffolds) may be surgically implanted into the tissue(s) of a subject, such as a human or animal. Implantable scaffolds often fail because the implant fails to integrate with the surrounding bone tissues.
The long-term success of dental implants is dependent upon sustained osseointegration. Without sufficient bone to support the implant placement, loosening will occur, increasing the risk of biomechanical overload and/or implant fracture, which often require implant removal and re-implantation.
Embodiments disclosed relate to implantable scaffolds including at least one integration aid and methods of making and using the same. In an embodiment, an implantable scaffold is disclosed. The implantable scaffold includes a fluoridated apatite structure sized and shaped for implantation in an animal. The fluoridated apatite structure defines a plurality of pores. The implantable scaffold also includes at least one integration aid including at least one of stromal vascular fraction adhered to the fluoridated apatite structure or at least one metal substitute substituted into the fluoridated apatite structure, the at least one metal substitute including one or more of zinc, silver, or iron.
In an embodiment, a method of making an implantable scaffold is disclosed. The method includes providing fluoridated apatite particles, sintering the fluoridated apatite particles at a sintering temperature of at least 950° C. to form a fluoridated apatite structure, and introducing at least one integration aid into the fluoridated apatite structure. The at least one integration aid includes at least one of stromal vascular fraction adhered to at least a portion of the fluoridated apatite structure or at least one metal substitute substituted into the fluoridated apatite structure, the at least one metal substitute including one or more of zinc, iron, or silver.
In an embodiment, a method of using an implantable scaffold. The method includes providing an implantable scaffold. The implantable scaffold includes a fluoridated apatite structure sized and shaped for implantation in an animal. The fluoridated apatite structure defining a plurality of pores. The implantable scaffold also includes at least one integration aid including at least one of stromal vascular fraction adhered to at least a portion of the fluoridated apatite structure or at least one metal substitute substituted into the fluoridated apatite structure, the at least one metal substitute including one or more of zinc, iron, or silver. The method also includes implanting the implantable scaffold in a subject.
Features from any of the disclosed embodiments may be used in combination with one another, without limitation. In addition, other features and advantages of the present disclosure will become apparent to those of ordinary skill in the art through consideration of the following detailed description and the accompanying drawings.
The drawings illustrate several embodiments of the invention, wherein identical reference numerals refer to identical or similar elements or features in different views or embodiments shown in the drawings.
Embodiments disclosed relate to implantable scaffolds including at least one integration aid and methods of making and using the same. An example, implant includes a fluoridated apatite structure sized and shaped for implantation in an animal (e.g., a human). The implantable scaffold also includes at least one integration aid configured to improve osseointegration with the implantable scaffolds. In an embodiment, the integration aid includes stromal vascular fraction adhered to (e.g., disposed in and/or on) the fluoridated apatite structure. It has been found that stromal vascular fraction facilitates bone regrowth and integration of the implantable scaffold with the native bone tissue of the subject. In an embodiment, the integration aid includes metal substitute (e.g., zinc, iron, or silver) substituted into the fluoridated apatite structure. It has been found that the metal substitute substituted into the fluoridated apatite structure provided antibacterial properties to the fluoridated apatite structure thereby minimizing bacteria on the fluoridated apatite structure that may impede new bone deposition and promotes cell differentiation (e.g., direct stem cells to osteogenic lineage) relative to an implantable scaffold that only includes FA.
An ideal engineered bone substitute should replicate the beneficial qualities of autograft bone, including structural support, a reliable source of osteogenic cells, and the capacity to generate osteogenic signals. One biomaterial that has been used clinically as a structural and biological bone graft substitute is synthetic hydroxyapatite [Ca10(PO4)6(OH)2] (“HA”). The HA scaffold has the advantage of possessing a similar chemical structure of the mineral component of native bone tissue stoichiometrically-making the HA biocompatible. HA substitutes, however lack necessary mechanical strengths to be an adequate replacement to autograft. In the hope of improving the mechanical and degradation properties of apatite-based scaffolds, the crystallinity of the HA may be improved by fluoridation and heat treatment. The resultant, fully fluoridated apatite particles may be similar to HA but has enhanced properties in terms of biocompatibility, strength, in vivo stability, and cell adhesion. The fluoridated apatite particles have also been shown to produce comparable mechanical strengths (4.17 to 13.5 MPa), upon sintering at high temperatures, to human cancellous bone tissue.
The fluoridated apatites (e.g., fluoridated apatite particles and fluoridated apatite structures) disclosed herein include one or more of fluorohydroxyapatite (“FHA”) or fluorapatite (“FA”) that is sintered at a sintering temperature selected to provide a desired surface morphology for the scaffold. FHA (Ca10(PO4)6Fy(OH)2-y) and FA (Ca10(PO4)6F2) are partially and fully fluoridated forms of apatite (e.g., HA). The scaffolds disclosed herein including fluoridated apatite demonstrate excellent adhesion to tissue cells relative to implants or scaffolds formed from other materials such as HA. The scaffolds disclosed herein may be used as bone grafts, such as a bone substitute or carrier for the same. Proper integration of the bone with the implant surface is important for maintaining a stable interface and preserving implant integrity.
The fluoridated apatite structure of the scaffolds herein are made of nonbiological materials (e.g., materials foreign to the body's internal environment) that may be used as bone grafts. Conventional bone grafts may include autografts or allografts. Autografts have no immunogenic response, but it has a limited supply. Decellularized allografts harvested from cadaveric sources have the advantage of being osteoconductive and osteogenic; however, they can be associated with risk of infectious disease immunogenicity, host rejection, and accelerated graft resorption. Allografts have no autologous cells and require cells to migrate in, which takes time, during which time large portion of grafts may resorb and lose strength and structure, which may cause failure of the allografts. Bone substitutes have been developed in response to the shortcomings of autografts and allografts. Bone substitutes have focused on providing the necessary matrix to support bone-ingrowth/ongrowth and integration by providing a biocompatible, bioresorbable, and porous scaffold made from materials such as HA, collagen, and biodegradable synthetic materials.
Conventional scaffolds that have integrated extracellular matrix proteins or growth factors, typically BMPs, have certain limitations, such as the uncontrolled release of growth factors, which leads to the formation of bone in undesirable areas of the body (e.g., ectopic bone formation). In contrast, the autograft-like living bone substitutes of the scaffolds disclosed herein may incorporate cell types that the patient's own body already utilizes in response to bone loss or physical insult. As the scaffold material (e.g., FA and FHA) is naturally resorbed within the body over time, it is replaced with new bone tissues that may be stimulated within the porous structure by the addition of the integration aids disclosed herein. Moreover, the use of fluoridated apatite structures results in a relatively slower resorption rate than in conventional bone grafts, thereby mitigating the undesirable premature release of growth factors described above.
The scaffolds disclosed herein are sized and shaped for implantation in an animal (e.g., human). For example, the scaffolds disclosed herein can be custom fabricated (e.g., 3-D printed) to fit the precise size and shape of a defect a patient presents with. The scaffolds disclosed herein eliminate requirements for autograft donor sites, a second surgery to harvest grafts, pain associated with graft removal, and prolonged recovery time. The scaffolds disclosed herein may also be sterile, off-the-shelf products that may be opened in the operating room by the surgical team such as immediately prior to implantation.
Autograft-like porous bone scaffolds are described herein. They may be fabricated with a mineral matrix (e.g., fluoridated apatite structures) and integration aids (e.g., agents configured to cause or otherwise facilitate de novo osseous tissue repair and regeneration). As the scaffold is naturally resorbed over time, it is replaced with an influx of new bone formation, due in part to the integration aids. Accordingly, the scaffolds disclosed herein are particularly useful in the orthopedic, plastic surgery, and dental fields as customizable scaffold material to repair instances of bone loss, defects, and trauma.
As previously discussed, the implantable scaffolds disclosed herein include at least one integration aid. The integration aids disclosed herein (e.g., stromal vascular fraction and the metal substitute) provide additional properties to the fluoridated apatite structure that facilitate integration of the implantable scaffold. For example, it is currently believed that the implantable scaffolds disclosed herein that include one or both of the integration aids disclosed herein may perform similar to autografts.
In an embodiment, the integration aids include a stromal vascular fraction. The stromal vascular fraction may be deposited in the pores of the fluoridated apatite structure such that at least some of the pores are at least partially occupied by the stromal vascular fraction. The stromal vascular fraction may also be deposited on other surfaces of the fluoridated apatite structure. It has been found that stromal vascular fraction may have the capacity to regenerate osseous tissue to a level comparable with autograft bone. Stromal vascular fraction includes a combination of cell types. In an example, the stromal vascular fraction may include adipose-derived stem cells (“ADSC”) along with other cell types. In an example, the stromal vascular fraction includes progenitor stem cells since such cells can differentiate into multiple lineages that can be differentiated on the osteogenic lineage. In an example, the stromal vascular fraction may include perivascular cells, leukocytes, endothelial cells, fibroblasts, progenitor stem cells, ADSC, other adipose cells, or combinations thereof. In an embodiment, the stromal vascular fraction may include minimally processed stromal vascular fraction (i.e., stromal vascular fraction provided in an extraction process that involves minimal manipulation of the fat source of respective patients)
The stromal vascular fraction may also represent an improvement over implantable scaffolds that include growth factors and other stem cells instead of stromal vascular fraction. For example, growth factors like bone morphogenetic proteins (“BMP”) have been shown to promote bone growth at injury sites and differentiate stem cells into a bone lineage. However, BMP has a disadvantage that their bioavailability may decrease over time and may have a short half-life. In another example, BMP and osteoblasts require the implant scaffold to exhibit rough surfaces to maximize their benefit. ADSC express bone lineage markers on the implant scaffold but the timing of the expression is dependent upon the type of material forming the implantable scaffold. However, stromal vascular fraction may not have at least some of these issues associated with osteoblasts, and ADSC, for example, because the stromal vascular fraction may include a plurality of cell types that will contribute to neo-vascularization. Further, it has been found that stromal vascular fraction significantly encourages stem cell differentiation towards osteogenic lineage on FA surfaces.
The autologous adipose-derived stromal vascular fraction (i.e., the stromal vascular fraction is derived from tissue of the subject that receives the bone graft) can be obtained at the time of the surgery from the patient. Adipose-derived stromal vascular fraction is an ideal stem cell source in a surgical setting as stromal vascular fraction can be extracted from local adipose tissues and administered with bone scaffolds. For example, the stromal vascular fraction may be obtained from the fat tissue (e.g., from liposuction) of the subject that receives the bone scaffold. The adipose-derived stromal vascular fraction may generate osseous tissue better than other stem cell types. In other words, the adipose-derived stromal vascular fraction may cause the implantable scaffold to behave more like an autograft than other stem cell populations. In an embodiment, the stromal vascular fraction may be formed from (i.e., isolated from) the breakdown of adipose tissue either by enzymatic or mechanical techniques. In other words, stromal vascular fraction may be obtained from the subject through minimal manipulation of the adipose tissue and the stromal vascular fraction can be isolated in the same operative setting as the reconstruction of the bone.
In an embodiment, the implantable scaffolds may include about 1×102 or more stromal vascular fraction cells, such as about 1×103 or more, about 1×104 or more, about 1×105 or more, about 1×106 or more, about 1×107 or more, about 1×108 or more stromal vascular fraction cells/cm2. The amount of stromal vascular fraction cells may depend on the size of the implantable scaffold and/or the number of stromal vascular fraction cells removed from the patient.
In an embodiment, the integration aid includes at least one metal substitute substituted (e.g., doped) into the fluoridated apatite structure. The metal substitute may include zinc, iron, silver, any other biocidal metal, or any other suitable metal. It has been found that the presence of the metal substitute in the fluoridated apatite structure improves the antimicrobial properties of the implantable scaffold without increasing the cell toxicity of the implantable scaffold. Further, it has been found that the presence of the metal substitute in the fluoridated apatite structure increases cell differentiation after implantation compared to non-metal substituted fluoridated apatite structures. In a particular example, the metal substitute includes zinc because natural zinc substituted HA/FHA is present in the bone and enamel of human teeth. It has also been found that zinc has a stimulatory effect on cells.
The metal substitute may replace some of the calcium in the fluoridated apatite structure. For example, when the fluoridated apatite structure includes FA, the chemical formula of the FA may be FA Ca(10-X)(PO4)6F2ZX, where Z the metal substitute. When the fluoridated apatite structure includes FHA, the chemical formula may be Ca(10-X)(PO4)6Fy(OH)2-yZX, where Z is the metal substitute. X in either chemical formula may be selected such that 0.25 molar % to about 15 molar % of the calcium is replaced with the metal substitute, such as in ranges of about 0.25 molar % to about 0.75 molar %, about 0.5 molar % to about 1 molar %, about 0.75 molar % to about 1.5 molar %, about 1 molar % to about 2 molar %, about 1.5 molar % to about 2.5 molar %, about 2 molar % to about 3 molar %, about 2.5 molar % to about 3.5 molar %, about 3 molar % to about 4 molar %, about 3.5 molar % to about 4.5 molar %, about 4 molar % to about 5 molar %, about 4.5 molar % to about 6 molar %, about 5 molar % to about 7 molar %, about 6 molar % to about 8 molar %, about 7 molar % to about 9 molar %, about 8 molar % to about 10 molar %, about 9 molar % to about 12 molar %, or about 10 molar % to about 15 molar %. The metal substitute may replace the calcium in the FA and FHA during the synthesis of the FA and FHA and/or during the formation of the fluoridated apatite structure. It is noted that the chemical formulas of the FA and FHA including the metal substitute may be slightly different from the chemical formulas provided above because the substitution of the metal substitute into the FA and FHA may slightly change the stoichiometry of the FA and FHA. It is noted that the molar % of calcium that is replaced with the metal substitute may be selected to be less than 5 molar % to prevent secondary phase formations in the hydroxyapatites.
The implantable scaffolds disclosed herein that include the metal substitute represent an improvement over “non-metal substituted implantable scaffolds.” Examples of non-metal substituted implantable scaffolds include decellularized cadaveric bone tissues, synthetic polymeric-based engineered bone grafts. Such non-metal substituted implantable structures exhibit no natural antimicrobial properties. Thus, the non-metal substituted implantable scaffolds might not be able to limit infection of the contaminated implantation site which, in turn, prevents osseous tissue ingrowth and integration and results in failed bone implants. To remedy this situation, the non-metal substituted implantable scaffolds may be cleaned prior to implantation but, due to the porosity thereof, it may be difficult to completely sterilize the implantable scaffold prior to implantation. The non-metal substituted implantable scaffolds may also have an antimicrobial agent disposed in the pores thereof but the antimicrobial agent may limit the volume available for other agents (e.g., stromal vascular fraction) and may inhibit integration of the non-metal substituted implantable scaffolds into the tissue. However, it has been found that the implantable scaffolds that include the metal substitute exhibit antimicrobial properties that at least inhibit contamination of the implantation site. Further, it has been found that the antimicrobial properties of the implantable scaffolds that include metal substitute exhibit enhanced cell differentiation and do not increase the cell toxicity of the implantable scaffold.
The scaffold 100 serves as a structure that provides mechanical strength, a substrate for bone growth. The bulk structure of the scaffold 100 is formed of fluoridated apatite such as one or more of FA or FHA (e.g., FA or FHA including the metal substitute). The fluoridated apatite structure of the scaffold 100 may be a porous sponge-like (though substantially rigid) structure. The fluoridated apatite structure of the scaffold 100 may be framework, block, rod, plug, wedge, or any other structure formed from a plurality of fluoridated apatite particles and has a plurality of pores therein. The bulk structure of the scaffold may be shaped to fit into a selected space or cavity, within a subject's body. At least some of the pores in the bulk structure of the scaffold may be formed by casting fluoridated apatite material in an investment material and removing the investment material, such as by one or more of dissolution, combusting, heating, machining, lasing, or any other suitable technique. At least some of the pores in the scaffold 100 (e.g., in the microstructure) may be due to the crystalline nature of the fluoridated apatite of the scaffold 100. In an embodiment, as shown in
The scaffold 100 defines a plurality of surfaces that can bond to bone or other tissues and may provide a substrate through which dopants may be delivered to the implantation site. The scaffold 100 may include one or more void spaces 130 therein. The void spaces 130 may be pores. In some embodiments, the void spaces 130 may include pores or chambers formed (e.g., molded, machined, dissolved, etc.) in the bulk structure of the scaffold 100.
The scaffold 100 may include at least one integration aid. For example, one or more surfaces of the scaffold 100 may have the stromal vascular fraction 135 disposed thereon or disposed in the void space (e.g., pores) of the scaffold 100. The one or more void spaces 130 may be at least partially filled with the stromal vascular fraction 135 configured to promote bone growth that are distinct from the stromal vascular fraction. Alternatively or additionally, the void spaces 130 may be at least partially filled with dopants. Suitable dopants may include collagen, keratose, differentiation promoters (e.g., BMP-2), platelet-rich plasma, stem cells (e.g., ADSCs), demineralized bone matrix, and the like.
The scaffold 100 may be formed in any shape (e.g., size and dimensions) for implantation into the tissues (e.g., hard and/or soft tissues) of a subject. For example, the scaffold 100 may be sized and shaped to form a post, a screw, a joint, a socket, a ball, or any other bone structure. In embodiments, the scaffold 100 may be disposed on or sized and shaped to host percutaneous implant such as percutaneous osseointegrated (OI) prosthetics, dental implants, orthopedic implants, or the like.
The fluoridated apatite in the scaffold 100 provides a medium for preferential attachment of tissue cells (e.g., osteoblast cells, epithelial cells, etc.) to the scaffold 100. The scaffold 100 includes fluoridated apatite material such as, FHA, FA, or combinations thereof. For example, the scaffold 100 may consist of or consist essentially of FA, FHA, one or more integration aids, one or more optional dopants 135, or combinations of any of the foregoing. In embodiments, the scaffold 100 may consist of or consist essentially of FA and one or more integration aids. FA has proven to be particularly effective at adhering to the osteoblast cells. FA in the scaffold 100 promotes tissue adhesion which provides a substrate for new bone growth, and which reduces or eliminates gaps along the surfaces of the scaffold, thereby reducing or eliminating infections at the wound site 112 (e.g., implant site). The scaffolds disclosed herein increase tissue bonding and growth at the surface of the scaffold. The scaffolds disclosed herein reduce or eliminate downgrowth of osteoblast cells, epithelial cells, or other local cells along the scaffold surface relative to conventional scaffolds (e.g., scaffolds that do not have the fluoridated apatite structure disclosed herein). Osteoblast cells showed great affinity for fluoridated apatite surfaces that were sintered at 1050° C. to 1250° C. when compared to HA and titanium surfaces.
The material that forms the fluoridated apatite structure may include FA and/or FHA particles that are pressed into a green body. For example, to form the green body, the material that forms the fluoridated apatite structure may be disposed in a mold and be pressed (i.e., have a compressive load applied thereto). The compressive load may be about 25 MPa or greater, about 30 MPa or greater, about 45 MPa or greater, about 60 MPa or greater, about 75 MPa or greater, about 90 MPa or greater, about 105 MPa or greater, about 120 MPa or greater, or in ranges of about 30 MPa to about 60 MPa, about 45 MPa to about 75 MPa, about 60 MPa to about 90 MPa, about 75 MPa to about 105 MPa, or about 90 MPa or greater. It has been unexpectedly found that the compressive load used to form the green body affects the antimicrobial properties of the fluoridated apatite structure, for example, when the fluoridated apatite structure includes the metal substitute. In particular, it has been found that compressive loads above 25 MPa improves the antimicrobial properties of the fluoridated apatite structure and, further, that increasing the compressive load above 25 MPa further improves the antimicrobial properties of the fluoridated apatite structure.
The material that forms the fluoridated apatite structure of the scaffolds 100 disclosed herein includes fluoridated apatite that has been sintered at a temperature between about 950° C. and about 1,350° C., or more particularly between about 1,050° C. and about 1,250° C., or even more particularly about 1,100° C. and about 1,200° C. In a particular example, the fluoridated apatite structure of the scaffolds 100 may be sintered in air for 3 hours at 1250° C. with a heating and cooling rate of 2° C./minute, starting and finishing at room temperature. The inventors currently believe that fluoridated apatite sintered in the temperature range(s) disclosed above agglomerate to form a plurality of bonded agglomerations of fluoridated apatite that have a size, shape, and zeta potential that encourage adhesion between tissue cells and the fluoridated apatite (e.g., FA) in the scaffold.
In an unsintered state, fluoridated apatite (FA and/or FHA) particles exhibits a substantially rod-like or needle-like crystal structure. During sintering, the subject fluoridated apatite crystals agglomerate and exhibit various bulk structures and surface morphologies. For example, through sintering, the fluoridated apatite particles may be formed into agglomerates exhibiting greater three dimensional characteristics, such as substantially granular shapes (e.g., prismatic, pseudo-prismatic, rounded, spherical, semi-spherical, ellipsoid, or irregularly rounded shapes). The as-sintered fluoridated apatite particles may be substantially devoid of the rod-like or needle-like fluoridated apatite of the unsintered fluoridated apatite particles. The average volume of an average sintered fluoridated apatite agglomerate may be at least ten times the average volume of the average unsintered fluoridated apatite particles. The resulting sintered fluoridated apatite particles (e.g., agglomerates) exhibit an overall smoother surface morphology than the unsintered fluoridated apatite particles.
Bulk fluoridated apatite particles may be a coherent mass of agglomerations provided in a specific form, such as grains of the scaffold. Bulk fluoridated apatite particles may be formed by sintering a mass of fluoridated apatite particles and then grinding, crushing, or otherwise breaking the resulting sintered bulk body into smaller bulk particles. The smaller bulk particles may be sized, such as using a sieve, to provide a plurality of particles having a substantially homogenous average particle size. The bulk particle size (e.g., a coherent mass of agglomerations provided in a granular form) of the bulk fluoridated apatite particles disclosed herein may be at least about 5 μm, such as about 30 μm to 300 μm, about 60 μm to 200 μm, about 65 μm to 150 μm, about 60 μm to 120 μm, about 120 μm to 200 μm, or less than about 300 μm. The particles may be sieved to obtain the desired particle size.
The porosity of a scaffold of the sintered fluoridated apatite particles is also different than the porosity of a scaffold of the unsintered fluoridated apatite particles. For example, the bulk structure of the sintered fluoridated apatite particles exhibits less porosity than the unsintered fluoridated apatite particles. This is believed to be due to the agglomerates densifying (e.g., self-organizing or building into naturally fitting structures) during sintering, thereby providing less pore space therebetween than the unsintered particles.
Fluoridated apatite maintains a relatively strong mechanical strength, even after sintering. For example, fabricated heat-treated FA and FHA scaffolds, which showed enhanced osteoblast cellular adhesion and proliferation properties when compared to HA surfaces treated at the same temperatures, also showed compressive strengths of 100-200 MPa, which is similar to cortical bone (170-193 MPa cortical bone; 7-10 MPa cancellous bone).
The inventors currently believe that the porosity and surface morphology of the sintered FA particles increase adhesion to tissue cells (e.g., endothelial cells, osteoblasts, fibroblasts, etc.). The charge of the fluoridated apatite material also contributes to increased tissue adhesion. For example, the surface charge of the fluoridated apatite material is believed to increase differentiation of cells at the interface therebetween. The FA is more electronegative than FHA and HA. As shown below, experiments have demonstrated that sintered FA promotes adhesion to osteoblasts, and to a much higher degree than sintered FHA and HA.
The surface charge of the scaffold material may be measured as the zeta potential. In some cases, the zeta potential of FA is more than double the zeta potential of FHA or HA sintered under the same conditions. The zeta potential of the fluoridated apatite scaffolds disclosed herein may be less than (e.g., have a greater negative value than) about −10 mV, such as about −10 mV to −80 mV, about −20 mV to −65 mV, about −26 mV to −80 mV, about −26 mV to −65 mV, about −40 mV to −80 mV, less than about −26 mV, less than about −35 mV, or less than about −40 mV. The inventors currently believe that the electronegativity of the fluorine atoms in the fluoridated apatite drive the zeta potential lower and stimulate cell adhesion, such as by causing differentiation. The zeta potential of may be determined, for example, using a Massively Parallel Phase Analysis Light Scattering (MP-PALS) spectrometer.
Various techniques may be used to manufacture the implantable scaffolds disclosed herein.
Block 210 of providing fluoridated apatite particles may include providing FA particles, FHA particles, or combinations of the foregoing. Providing fluoridated apatite particles may include providing a plurality of fluoridated apatite particles, such as FA, FHA, or combinations thereof. In an embodiment, providing fluoridated apatite particles includes providing fluoridated apatite particles that include at least one metal substitute. The plurality of fluoridated apatite particles may exhibit any of the average fluoridated apatite particle sizes disclosed herein. The fluoridated apatite particles may exhibit any of the bulk fluoridated apatite particle sizes disclosed herein (e.g., 60 μm to 200 μm).
In some embodiments, providing fluoridated apatite particles may include forming the fluoridated apatite particles, such as FA particles, FHA particles, FA and/or FHA particles including the metal substitute, or mixtures thereof. In some embodiments, a precipitation (e.g., continuous aqueous precipitation) method may be used to synthesize the fluoridated apatite particles. In an example, the precipitation method may include substituting at least some of the calcium with the metal substitute. In an example, forming the fluoridated apatite particles includes pre-sintering the fluoridated apatite particles.
In a particular example, forming the fluoridated apatite particles includes forming fluoridate apatite particles having zinc substituted therein. In such an example, forming the fluoridated apatites particles may include using a calcium source (e.g., Ca(NO3)2.H2O), a zinc source (e.g., Zn(NO3)2), a phosphate source (e.g., Na2HPO4), and a fluorine source (e.g., NaF). To synthesize the fluoridated apatite particles, the calcium, zinc, phosphate, and fluorine sources may be mixed at boiling point in stoichiometric ion solutions of Ca2+, PO43−, and F− (CA2+/PO43−/F−=5:3:1 molar ratio). For x molar % zinc-fluoridated apatite particles syntheses, x molar % Ca2+ cation solution may be replaced by x molar % Zn2+ ions to maintain the molar ratios of the reaction to be (Ca2+ Zn2+)/PO3−/F−=5:3:1. It is noted that this specific method of forming the fluoridated apatite particles may include a metal substitute source other than or in addition to zinc.
In some embodiments, providing fluoridated apatite particles may include forming the plurality of fluoridated apatite particles into a coherent body. The coherent body may consist of or consist essentially of FA, FHA, one or more stromal vascular fraction, one or more dopants, or combinations of any of the foregoing. In some embodiments, additional materials may be present in the coherent body, such as a ceramic, metal, polymer, etc. Forming the plurality of fluoridated apatite particles into a coherent body may include pressing, rolling, molding, casting (e.g., foam casting), adhering, three-dimensional printing, or otherwise forming an at least partially bonded body or mass of fluoridated apatite particles. In an example, forming the plurality of fluoridated apatite particles into a coherent body includes pressing the fluoridated apatite particles at any of the compressive loads disclosed above. In an example, the coherent body may be created by forming a slurry having fluoridated apatite particles and a sacrificial structural material. The slurry may be dried, cooled, or reacted to harden into the coherent body. The coherent body includes a solid or semi-solid structure containing fluoridated apatite particles and the sacrificial structural material (e.g., investment material). The coherent body may be frozen or compressed to form a green state part that remains intact as a solid unitary structure. The sacrificial structural material may be selected to harden at a desired temperature or condition (800° C.-1300° C.), to provide a selected porosity, and/or to be removable from the coherent body (e.g., plurality of at least partially bonded fluoridated apatite particles) via one or more of combustion, melting, dissolution, vacuum, or any other technique for removing an investment material. For example, the sacrificial structural material may be a polymer, a salt, a ceramic, or the like, composed to dissolve or otherwise dissociate in selected conditions. The sacrificial structural material may be removed prior to, after, or concurrently with sintering the fluoridated apatite particles using any of the sintering techniques disclosed herein.
In an example, forming the plurality of fluoridated apatite particles into a coherent body includes foam-casting the particles. In such an example, aqueous slurries may be made. The slurries may include binders (e.g., 1 wt % polyvinyl alcohol and 1 wt % polyethylene glycol), a dispersant (e.g., 1 wt % Dynol 604), and distilled water. The slurries may infiltrate and fill a correctly sized foam template and dried under vacuum. The resultant green bodies may then be heated (e.g., sintered) to remove the foam template.
In some embodiments, the porous scaffoldings may be fabricated out of a fluoridated apatite slurry. The fluoridated apatite (e.g., FA) slurry may be used as an investment material or casting material. For example, scaffolds may be prepared using polymeric sponges as investment material or a mold, which are then infiltrated with the fluoridated apatite slurry containing monomers and initiators for rapid gelation via in situ polymerization. This gel sponge processing technique integrates gel-casting with polymer sponge methods. Next, the polymeric sponge can be removed (e.g., burned off at elevated temperatures (e.g., 1,050° C. to 1,250° C.)) and the remaining coherent body (e.g., fluoridated apatite scaffold) may be cleaned with distilled water.
In some embodiments, providing fluoridated apatite particles may include forming the fluoridated apatite particles into a predetermined shape. For example, fluoridated apatite scaffolding with pre-determined shapes (e.g., flat, tubular, or cubic) and porosities. Fluoridated apatite particles, DI water, and a binder (e.g., acrylamide/methylenebysacrylamide) may be mixed such as in a ball-mill and then, one or more of an initiator (e.g., Tetramethylenediamine), binder (e.g., carboxymethyl cellulose or Polyvinyl alcohol), dispersant, surfactant, or excess DI water may be added and mixed for duration (e.g., 12 hours). This slurry may be cured under vacuum, and sequentially poured over an infiltrated into a shaped polyether sponge as a frame for obtaining the desired shape, size, and porosity. The infiltrated sponge may be put under vacuum and a catalyst (e.g., ammonium persulfate) solution may be applied for facilitating polymerization. The sponges may be placed inside a nitrogen chamber to avoid surface contamination, which may prevent the polymerization process. After drying at room temperature, samples may be sintered to form the sintered body as disclosed in more detail below (e.g., at 1250° C. at a heating rate of 5° C./min).
Further methods of forming a coherent body of fluoridated apatite particles may include mixing the fluoridated apatite particles with polymers, slip casting, freeze-casting, sol-gel formation, foaming, polymer replication, solid freeform fabrication, three-dimensional printing, or the like.
The fluoridated apatite particles in the coherent body may be further subjected to sintering at a predetermined temperature. Block 220 of sintering the fluoridated apatite particles to form a sintered body may include sintering the fluoridated apatite particles prior to, contemporaneously with, or after providing the fluoridated apatite particles. The sintered body may have a denser bulk structure than the unsintered coherent body. The porosity of the sintered body may exhibit less porosity than the unsintered coherent body. Sintering the fluoridated apatite particles may include sintering the coherent body. Sintering the fluoridated apatite particles may include sintering a coherent body of fluoridated apatite particles that have been previously sintered. The sintered body may consist of or consist essentially of FA, FHA, one or more dopants, or combinations of any of the foregoing.
The fluoridated apatite particles may be sintered as one or both of a loose powder or in the cohesive body (e.g., polymer sponge impregnated with FA particles or pressed pellet of subject FA particles). Sintering the fluoridated apatite particles may include heating the fluoridated apatite particles to a temperature of at least about 950° C., such as about 950° C. to about 1,350° C., about 1,050° C. to about 1,250° C., about 1,050° C. to about 1,150° C., about 1,150° C. to about 1,250° C., at least 1,050° C., at least about 1,150° C., less than about 1,300° C., or less than about 1,250° C. The heating (e.g., sintering) may be carried out for at least 1 minute, such as about 1 minute to about 24 hours, about 1 hour to about 18 hours, about 2 hours to about 12 hours, about 4 hours to about 10 hours, about 20 minutes to about 4 hours, about 30 minutes to about 3 hours, about 1 hour to about 10 hours, about 8 hours to about 16 hours, at least about 2 hours, less than about 24 hours, or less than about 12 hours. The above-noted sintering times may be hold times at the sintering temperature. For example, a plurality of fluoridated apatite particles may be placed in a sintering oven that is ramped up to the sintering temperature at a selected rate (e.g., about 5° C./min., about 7° C./min., about 10° C./min., about 5° C./min. to 15° C./min, or about 1° C./min or more), maintains the sintering temperature for the selected duration, and ramps back down to the ambient temperature at a selected rate (e.g., any of the rates disclosed above). The sintering temperatures within the ranges disclosed herein do not alter the chemical composition of the fluoridated apatites disclosed herein. Sintering may be carried out in an inert environment or an ambient environment.
Sintering the fluoridated apatite particles may include heating the fluoridated apatite particles in an inert atmosphere (e.g., N2 or Argon), in a vacuum, in an open or oxidizing atmosphere (e.g., in the presence of oxygen, carbon dioxide, N2, etc.), or combinations of any of the foregoing.
Sintering the fluoridated apatite particles to form a sintered body may include sintering the fluoridated apatite particles (e.g., coherent body) at a temperature sufficient to burn out any investment or mold material such as a polymer, so that substantially only the fluoridated apatite or other selected materials desired for implantation remain. In such embodiments, the as-cast fluoridated apatite particles (e.g., coherent body) and the material the fluoridated apatite particles were cast in (e.g., polymer sponge or matrix material) may be subjected to sintering.
The sintered fluoridated apatite particles may exhibit the surface morphology, porosity, zeta potential, average particle size, or any other characteristics of any of the sintered fluoridated apatite particles disclosed herein. For example, the sintered body may include sintered fluoridated apatite particles having a spherical, semi-spherical, prismatic, pseudo-prismatic, ellipsoid, or irregularly rounded shape and are devoid of rod-like or needle-like fluoridated apatite particles.
The sintered fluoridated apatite particles are densified via the sintering process while the polymer material is combusted or melts out of the coherent body. The shrinkage of the fluoridated apatite particles during sintering is reproducible (about 15% upon sintering). Accordingly, the techniques disclosed herein provide the ability to custom make scaffoldings for the desired shapes and sizes to fit the clinical needs of grafts for reconstructive surgeries in plastic, orthopedic and dental surgeries. Unfortunately, tensile properties of pure apatite ceramics are limited. For example, unsintered apatite ceramics exhibit a hardness value about of 5.1 GPa and fracture toughness value of about 2.0 MPa·m1/2. When compared to the fracture toughness of human bone (about 12 MPa·m1/2), apatite's toughness is relatively poor. Thus, the fluoridated apatite scaffolds disclosed herein may be used as heavy-loaded implants after sintering to improve strength.
The scaffolds disclosed herein (e.g., FA scaffolds) may exhibit at least 10% porosity, such as 30% to 70% porosity. Accordingly, the scaffolds may provide a ready delivery means for one or more dopants. Such scaffolds may be used as bone fillers, such as for dental applications or the like.
Returning to
In some embodiments, forming the scaffold may be carried out substantially simultaneously with sintering the coherent body to form a sintered body. For example, the forming the scaffold and sintering the coherent body may both be carried out via sintering. In such embodiments, the coherent body may be provided in a size and shape such that the sintered body may be the scaffold (e.g., implantable size and shape). Providing such a shape may be provided or formed by one or more of molding, grinding, cutting, lapping, etc.
In an embodiment, when the integration aid includes a stromal vascular fraction, the method 200 may include disposing the stromal vascular fraction in or on the fluoridated apatite particles, coherent body, sintered body, or scaffold with the stromal vascular fraction. In an example, the method 200 may include obtaining adipose tissue from the subject and isolating the stromal vascular fraction from the adipose tissue. The stromal vascular fraction obtained from the adipose tissue may then be disposed in or on the fluoridated apatite structure. In a particular example, the method 200 may include obtaining adipose tissue from the subject and implanting the implantable scaffold in the subject during the same procedure. In an example, the method 200 may include providing non-adipose stromal vascular fraction and disposing the non-adipose stromal vascular fraction in or on the fluoridated apatite structure. In an example, the stromal vascular fraction may be disposed in or on the fluoridated apatite structure after sintering the fluoridated apatite structure since sintering the stromal vascular fraction may damage the stromal vascular fraction.
In an example, the stromal vascular fraction may be provided in a liquid form exhibiting a viscosity sufficient to allow the cells to be disposed in and/or on the fluoridated apatite structure. In an example, disposing the stromal vascular fraction in and/or on the fluoridated apatite structure may include a solution containing the stromal vascular fraction (i.e., diluted stromal vascular fraction) cells into or onto the coherent body, sintered body, or scaffold. For example, the stromal vascular fraction may be suspended, dispersed, or dissolved in a liquid medium which is applied to the coherent body, sintered body, or scaffold, such as via immersing, spraying, pipetting, aliquoting, pouring, or any other liquid application technique. In embodiments, a scaffold may be wetted and loaded with a suspension of stromal vascular fraction.
The method 200 may include disposing sufficient quantities of stem cells within the stromal vascular fraction that can stimulate bone growth, cell differentiation, or vascularization. For example, the stromal vascular fraction may be present in and/or on the fluoridated apatite structure in cell densities 102 cells/ml to 1010 cells/ml per unit area, such as in ranges of 102 cells/ml to 104 cells/ml, 103 cells/ml to 105 cells/ml, 104 cells/ml to 106 cells/ml, 105 cells/ml to 107 cells/ml, 106 cells/ml to 108 cells/ml, 107 cells/ml to 109 cells/ml, or 108 cells/ml to 1010 cells/ml. The amount of cells present in stromal vascular fraction of the implantable scaffold may be selected based on the size of the implantation site and the size of the implantable scaffold.
The method 200 may include doping the fluoridated apatite particles, coherent body, sintered body, or scaffold with one or more dopants, such as any of the dopants disclosed herein. Doping the fluoridated apatite particles may include mixing one or more dopants into the fluoridated apatite particles prior to, contemporaneously with, or after providing the fluoridated apatite particles or forming the coherent body of fluoridated apatite particles. For example, doping the fluoridated apatite particles may include adding one or more dopants to the plurality of fluoridated apatite particles prior to forming the coherent body, or coating at least a portion of the coherent body with one or more dopants after forming the coherent body. Each of the one or more dopants may be present in amounts composed to stimulate bone growth, cell differentiation, or soft tissue growth, such as at least 1 nanogram (ng), 10 micrograms (g) to 10 milligrams (mg), about m to 1 mg, 50 μg to 500 μg, or less than 1 mg.
Combinations of dopants may be utilized to provide controlled release of one or more dopants in vivo. For example, ADSCs may be used with BMP-2 and a hydrogel such as keratose, where keratose is relatively stable in vivo to allow for controlled release of dopants disposed therein. For example, the keratose may be applied as a coating over the scaffold, where upon degradation of the keratose, the dopants there beneath are released. For example, the keratose may dissolve to release growth factors such as BMP-2. In some examples, the dopants may be present in a layered systems where multiple layers of dopants are each disposed beneath a layer of hydrogel such as keratose. Accordingly, time released benefits may be realized using the scaffolds disclosed herein.
Doping the coherent body, sintered body, or scaffold may include applying a solution containing the one or more dopants into or onto the coherent body, sintered body, or scaffold. For example, one or more of the dopants may be suspended, dispersed, or dissolved in a liquid medium which is applied to the coherent body, sintered body, or scaffold, such as via immersing, spraying, pipetting, aliquoting, or any other liquid application technique. In embodiments, BMP-2 may be dispersed in a keratose hydrogel, which may be poured over a scaffold and allowed to incubate. Similarly, a scaffold may be wetted and loaded with a suspension of ADSCs.
In some examples, doping the scaffold may include disposing the scaffold in the tissue of an implantee, such as soft tissue to deposit autologous tissues, growth factors, etc. in the scaffold prior to final implantation in a bone.
The block 320 of providing an implantable scaffold including fluoridated apatite structure and one or more integration aids may include providing any of the scaffolds disclosed herein. Providing the implantable scaffold may include providing an implantable scaffold having fluoridated apatite structure sintered at any of the temperatures disclosed herein (about 950° C. to about 1350° C. or about 1050° C. to about 1250° C.), having any of the zeta values disclosed herein, having any of the surface morphologies disclosed herein, or any of the properties of sintered fluoridated apatite particles disclosed herein. The implantable scaffold may consist of or consist essentially of FA, FHA, one or more integration aids, or combinations of any of the foregoing. The implantable scaffold may be sized and shaped for at least partial bone replacement, an osseointegrated implant, a dental implant, or the like. Providing the implantable scaffold may include making at least a portion of the implantable scaffold, such as by using any of the techniques for making scaffolds disclosed herein. For example, making at least a portion of the implantable scaffold may include isolating stromal vascular fraction from adipose tissue.
The block 320 of implanting the implantable scaffold in a subject may include implanting the implantable scaffold into the tissue of a subject, such as into the skin, bone, or other tissues of a subject. For example, implanting the implantable scaffold in a subject may include positioning the implantable scaffold within a pocket in a bone of a subject, such as in a jaw, hip, vertebrae, femur, etc. For example, an osseointegrated scaffold may be inserted into bone whereby the fluoridated apatite structure contacts one or both of the bone or soft tissue of the subject. Implanting the implantable scaffold in a subject may include surgically implanting the scaffold into the tissue of a subject. Implanting the scaffold in a subject may include closing the implantation site, such as by suturing.
Implanting the scaffold may include one or more of sizing or shaping the implantable scaffold as disclosed herein.
The method 300 may include preparing an implantation site such as by removing tissue (e.g., bone) from an implantation site in a patient. For example, preparing an implantation size may include removing at least some bone to form a pocket in a bone. In such embodiments, the scaffold may be shaped and sized to fit in the pocket. Preparing the implantation site may also include removing tissue and then isolating stromal vascular fraction from the removed tissue.
Preparing the implantation site may include cleaning the implantation site, such as cleaning the interface between the bone of the subject and the scaffold. Such cleaning may include washing with water or another fluid (e.g., iodine, soap, alcohol, etc.).
The method 300 may include implanting the implantable scaffold in soft tissue prior to implanting the implantable scaffold in bone, such as to allow the one or more dopants to produce autograft cells. The implantable scaffold may be disposed in the soft tissue for at least a week, such as 1 week to 3 months.
In a particular example, the method 300 may include anesthetizing the subject until intubated. The skin around the incision may be shaved, prepared, and draped for sterile surgery. Initially, skin and subcutaneous incisions may be made. Adipose tissue in this region may be collected and placed in a sterile lactated Ringer solution. The collected adipose tissue may be weighed, rinsed with lactated Ringer solution, suspended in collagenase solution for a time period (e.g., 30 minutes), centrifuged, and then the stromal vascular fraction may be collected. The viable cells in the stromal vascular fraction may then be determined using a small sample of the stromal vascular fraction (e.g., staining with DAPI and counting the number of nucleated cells with an automated cell counter). The implantable scaffold may then be placed in a concentrated solution of the stromal vascular fraction (e.g., 1×107 stromal vascular fraction cells in a Ringer solution) for a define time period sufficient to allow adherence of cells on the implantable scaffold. The implantable scaffold may then at least a portion fill a bone defect. After placing the implantable scaffold, the tissue cut during the incision may then be closed.
Further examples of implantable structures, methods of making implantable structures, and methods of using implantable structures that may be used in any of the embodiments disclosed herein are disclosed in U.S. patent application Ser. No. 17/420,579 filed on Jul. 2, 2021 and U.S. patent application Ser. No. 17/420,589 filed on Jul. 2, 2021, the disclosure of each of which is incorporated herein, in its entirety, by this reference.
The following working examples provide further detail in connection with the specific embodiments described above.
Several implantable scaffolds formed from HA and FHA were manufactured.
Implantable scaffolds including FA were fabricated using a foam-casting technique and then sintered at a range of temperatures between 850° C. and 1450 C°.
An ideal engineered bone implant should replicate the beneficial qualities of autograft bone, including structural support, a reliable source of osteogenic cells, and the capacity to generate osteogenic signals. Since apatites are known for their osteoconductivity and, as presented above, higher sintering temperatures result in mechanically robust scaffolds, it is vital to include at least one integration aid to make them an effective autograft-like scaffold. As stated above, the integration aid may include stromal vascular fraction. To confirm the effectiveness of fluoridated apatite structure to guide the differentiation of stromal vascular fraction, in vitro studies were carried out. The stromal vascular fraction is separated from adipose (fat) tissue. Stromal vascular fraction comprised pervascular cells, leukocytes, endothelial cells, other adipose cells, fibroblasts, and progenitor stem cells. The stromal vascular fraction was extracted from the subject's own body and administered to effect therapeutic results. As stromal vascular fraction can differentiate into multiple lineages, it was important to access the fluoridated apatite structure ability to differentiate stromal vascular fraction to the osteogenic pathway.
First, it was determined the impact that HA, FA, FHA, and sintering temperatures have on the viability of cells within stromal vascular fraction and fluoridated apatites' ability to differentiate into the osteoblast lineage. For this, small HA and fluoridated apatite structure disks were prepared from the raw powder. These disks were sintered at pre-selected temperatures. Post-sintering, a scanning electron image was used to confirm the micro-scaled topographical features of the surface of the small HA and fluoridated apatite structures. A known density (1,300 cells/cm2) of passage 3-5 ADSCs (RASMD-01001, Santa Clara, Calif.) were incubated on the disks at 37° C. in a 5% CO2 incubator for 2 and 10 days. Cells were mechanically detached from the surface at 2 and 10 days, post-seeding, and subjected to cell viability assays (alamarBlue® assay), immunohistochemistry, and RT-PCR analysis, data is compared to cells plated onto a cell culture dish (cell drop controls).
At two days post-seeding, it was found that were relatively more viable cells on the HA 1150° C. surface (1.9±0.09) compared to all other groups, suggesting stromal vascular fraction either preferentially proliferated or selectively adhered to a greater extent on this surface (p<0.05; data not given). Additionally, the cell drop control group had statistically fewer viable cells than the HA, FA, and FHA apatite disks sintered at 1250° C. By 10 days post-seeding, there were no statistical differences between the cell drop control group and the different apatite surfaces (p>0.05), indicating all of these surfaces support the cell adhesion and growth.
For assessing these surfaces' ability to promote differentiation, total RNAs were extracted using standard techniques. After confirming the quality, RNAs were reverse transcribed. Gene expression was then quantified using real-time PCR with gene-specific primers for runt-related transcription factor 2 (Runx2; RN01512298_m1; NM_001278483.1) and secreted phosphoprotein 1 (SPP1, also known as osteopontin; RN00681031_m1, Thermo Fisher ID; NM_012881.2, NCBI Reference Sequence). All values were normalized to the housekeeping gene 18s ribosomal RNA (Hs999999901_s1; X03205.1, GenBank) and data reported as 2{circumflex over ( )}ΔΔct and calculated relative to the cell drop control. To confirm the mRNA expression data, ADSCs seeded on surfaces for 2 or 10 days were fixed in 10% formalin and then incubated with a primary antibody for osteocalcin or osteopontin. The samples were then incubated with fluorescently labeled secondary antibodies for osteocalcin and osteopontin and were imaged using a confocal microscope at 10× magnification.
RUNX2, a transcription factor associated with the early differentiation of stem cells into pre-osteoblast cell lineage, was expressed at lower levels in ADSCs plated on HA1150° C. (p<0.01) and at equivalent levels to the cell drop control on HA1250° C. This data are supported by the expression of SPP1—a marker of late-stage osteoblast differentiation which was undetectable in cells plated on HA sintered at 1150° C. and equivalent when compared to the cell drop control plated on HA1250° C.
Next, SVF cells were harvested and characterized from humans and rats. For determining the composition of SVFs from rats, the abdominal fat from 5 male Lewis rats were gathered and weighed. These fat samples were then digested with collagenases (1 and 11) for one hour at 37° C., the red blood cells lysed, and the remaining cells were suspended in media. After lysing the red blood cells, finally detached cells were washed and suspended in media. A small sample of the cell suspension was stained with DAPI and evaluated along with AccuCount beads by fluorescent activated cell sorting (FACS) to determine the concentration of viable cells within our samples. On average, 8500±2300 cells/gram of fat were obtained. This experiment was repeated for human fat, which was harvested using the approved protocol (IRB:1094 Molecular Classification of Cancer). A higher concentration of cells was obtained from human tissues (125,000±28,000 cells/gram of fat). In order to quantify the number of ADSC cells within the SVF portion, these cells were labeled with CD45, CD31, CD34, CD 73, and CD 90, stained for DAPI, and then sorted using FACS. Here, we defined stem cells as viable cells, negative for CD45 and CD31, and positive for CD34, CD 73, and CD 90. On average, ˜68±4% cells were alive within our extracted suspension and of those live cells, about 22% were stem cells (i.e., ADSC). It is important to note that all samples had a similar amount of viable cells post-processing. However, the number of ADSC cells differed from human fat sample to sample.
Next, a transcriptome study was undertaken to compare the differential expression of transcriptomes of the stromal vascular fraction cells grown on sintered FA surfaces versus cell culture plate control. For this, the stromal vascular fraction cells isolated from human fat were seeded on cell culture plate wells (control) and FA disks at a seeding density of 80,000 cells/1.9 cm2. Growth media was selected instead of differentiating media in order to limit the media-induced differentiation of cells. After culturing for 6 days, samples were pooled, and scRNA-sequencing was performed at the University of Utah's genomics core. CellRanger and Seurat analysis packages were used for sequence alignment, quality control, feature quantification, and clustering, while SingleR was used for cell-type annotation based on the cell.
Based on the single-cell sequencing data, another cell-culture study was conducted to look at cell differentiation at a later time point, i.e., 11 days compared to 6 days post-seeding. As previously described, human adipose tissue was harvested and processed, and the stromal vascular fraction was characterized using FACS. Then equal numbers of stromal vascular fraction/area were seeded onto FA, HA, and cell culture plates, with the apatites sintered at 1150° C. The cells were cultured for 11 days and stained for osteoblast markers, osteopontin, and osteocalcin immunohistochemically. They were then imaged using confocal microscopy.
Thirty male Wistar rats were used for testing the efficacy of the scaffold to allow deposition of new bone. The rats were divided into four groups of six animals. Each animal received one of the following treatments (n=6): Group 1—defect left untreated (negative control), Group 2—defect filled with autograft bone (gold standard), Group 3—defect filled with HA scaffold, Group 4—defect filled with FA scaffold, and Group 5—defect filled with FA and 1·106 of stromal vascular fraction cells. During a sterile surgery, a longitudinal incision was made on the lateral surface of the patellar tendon to expose the femoral condyle. A bone defect was then created in the center of the condyle and parallel to the long of the femur using a drill/needle and saline flush (approximately 2 mm wide and 4 mm long). The drilled cavity was flushed with saline. Animals were treated as assigned. The incision was closed with sutures. Every two weeks, all animals were subjected to in situ micro-CT scans to monitor the progress of bone regeneration. All animals remained in the study for 12 weeks post-surgery without any adverse events. At necropsy, the scaffolding and the surrounding tissues were harvested and subjected to histological analyses and micro-CT imaging. The volume data that was calculated from the micro-CT scans showed that new bone formed preferentially around and in-between the spaces occupied by the FA granules, while the defect-only remained unfilled.
An FA powder and three zinc-substituted fluorapatites (0.5% Zn-FA, 1.0% Zn-FA, and 2.0% Zn-FA) powders were synthesized using a chemical co-precipitation technique. It is noted that the percentages used in this section refers to molar percent. Each of the powders were characterized using inductively coupled plasma mass spectrometry (ICP-MS) to determine the phosphate, zinc, and calcium contents of the powders, an x-ray diffraction technique was used to quantify the crystallography of the powders, and a fluoride probe to determine the fluoride content of the powders. Table 1 shows the molar weight of calcium and zinc in the synthesized powders and the calculated ICP-MS as percent molar substitution of zinc within the apatite crystal structure.
In a first set, the powders were compressed into 10 mm disks using a compressive load of about 31 MPa, about 62 MPa, and 94 MPa (i.e., a compressive force of 1,000 kg, 2,000 kg, and 3,000 kg, respectively). Initially, compression pressures were also altered to produce uniform surface micro-features. Compressed and sintered disks were imaged under a scanning electron microscope to document the surface microstructures. In the second set, the powders were formed into scaffolds using a foam-casting technique.
Each of the disks were sintered at 1100° C., 1150° C., and 1200° C.
Phosphate, zinc, and calcium content was determined with ICP-MS. ICP-MS showed a slightly lower Ca/P ratio at 1.42, compared to the expected value of stoichiometric FA, which is 1.67. The Ca/P ratio for 0.5, 1.0, and 2.0% Zn-FA was also lower than the expected value. The zinc substitution values for these powders were found to be at the expected substitution values. The lower Ca/P ratio may be attributed to the technique used for the synthesis,
Cell viability studies were carried out using ADSCs maintained in growth media supplemented with 10% heat-inactivated fetal bovine serum, and incubated for 2 or 10 days on a control, FA scaffold sintered at 1150° C., and 2.0% Zn-FA scaffold sintered at 1150° C. All experiments were conducted with cells between passages 3-5. ADSCs were plated onto the different surfaces and then evaluated for cell viability with alamarBlue®. All data are reported as mean±SD and relative to cells grown on a cell culture plate (n=4/disks/group/assay). Group differences in ADSC viability and were evaluated by ANOVA, followed by a Tukey's post hoc test (JMP, SAS Institute). Significance was set at p<0.05. After 2 and 10 days of incubation, no significant difference in cell viability (p>0.05) was seen between the FA disks and the 2.0 molar % Zn-FA disks. In other words, the data showed that sintered, 2.0 molar % Zn-FA disks had no difference in cell toxicity when compared to an FA disks. ADSC adhesion assays showed that there were no differences between FA and 2% Zn-FA in cell adhesion numbers after 2 and 10 days of incubation.
Initial trials showed that the compressive loads used to form the disks affected the surface morphology and the microstructures which, in turn, affected the number of bacteria colonies that were adhered to the disks. For example, it was found that increasing the compressive loading used to form the disks significant decreased (e.g., by a 1 to 2 log-fold decrease) the bacterial adhesion on the disks. The disks pressed with higher compressive loads had smaller microstructural features and the least numbers of bacterial adhesion compared to disks pressed with lower compressive loads. This observation was made independent of sintering temperature. Furthermore, the biofilm data indicated the least amount of bacteria adhered to the disks sintered at 1150° C. when compared to other temperatures studied after a 48 hour period of incubation in the biofilm reactor. This sintering temperature was then used for fabricating porous scaffolding for in vivo testing. It was also found that adding zinc to the FA crystal structure decreased bacterial adhesions with the 2.0% Zn-FA substitution resulting in better antimicrobial surface. In particular, the 2.0 molar % Zn-FA disks have a 1 to 2-log fold reduction is bacterial adhesion when compared to the FA disks.
Another study included evaluating the abilities of zinc substituted apatite to limit bacterial adhesion in a highly contaminated environment following ASTM standard protocol (ASTM E3161-18) with Staphylococcus aureus (ATCC 6538; S. aureus). For this study, 10 mm disks were fabricated by compressing a known weight (0.3 g) of apatites and then sintered at 1100, 1150, or 1200° C. Some of the disks were then processed to quantify bacteria on the surface and others for SEM imaging.
A foam-casting technique was used to fabricate scaffolds with aqueous slurries of Zn-FA powder, which was formulated using distilled water, polyvinyl alcohol, and polyethylene glycol as binders. The foam-casting templates were dried and sintered at 1150° C. Resultant porous implantable scaffolds were cleaned sterilized, and implanted in a rat femoral intra-condyle bond defect model for twelve weeks (n=6/group). The groups were 1) empty defect left untreated (negative control), 2) defect filled with autograft (current gold standard, positive control), 3) defect filled with FA scaffold, and 4) defect filled with 2% Zn-FA scaffold. Every two weeks, all animals were subjected to in situ micro-CT scans to monitor the progress of boned regeneration.
The data obtained from the zinc-substituted FA disks shows that by substituting zinc into the apatite crystallite structure, the antimicrobial properties of FA can be improved without forgoing its osteogenic abilities. It is believed that such substitutions can impart protection during the lifetime of the bone scaffolds themselves.
While various aspects and embodiments have been disclosed herein, other aspects and embodiments are contemplated. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting.
This application claims priority to U.S. Provisional Patent Application No. 63/239,749 filed on Sep. 1, 2021 and U.S. Provisional Patent Application No. 63/300,742 filed on Jan. 19, 2022, the disclosure of each of which is incorporated herein, in its entirety, by this reference
Number | Date | Country | |
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63239749 | Sep 2021 | US | |
63300742 | Jan 2022 | US |