Patent Application
20180071433
The present invention relates to a bone graft comprising a tannin-hydroxyapatite resin scaffold, osteocompetent stem cells, and a growth medium, and methods of use.
The “background” description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description which may not otherwise qualify as prior art at the time of filing, are neither expressly or impliedly admitted as prior art against the present invention.
Tannin and its derivatives can be used in various applications [U.S. Pat. No. 9,302,413; WO2014117946 A1; Lacoste, C., et al., Pine tannin-based rigid foams: Mechanical and thermal properties, Industrial Crops and Products, 2013, 43, 245; WO2013010668 A1; US20150361240; US20150274921; US20150259460; U.S. Pat. No. 6,043,350; US20140193322; US20140158927; US20060194918; Szczurek, A., et al., The use of tannin to prepare carbon gels. Parts I: Carbon aerogels, Carbon, 2011, 49, 2773; Szczurek, A., et al., The use of tannin to prepare carbon gels. Parts II: Carbon aerogels, Carbon, 2011, 49, 2785; Amaral-Labat, G., et al., Pore structure and electrochemical performances of tannin-based carbon cryogels, Biomass Bioenergy, 2012, 39, 274; Grishechko, L. I., et al., New tannin-lignin aerogels, Industrial Crops and Products, 2013, 41, 347—each incorporated herein by reference in its entirety], because their mechanical properties can vary in a wide range, depending on their method of preparation. Among others materials, tannin and tannin compounds have been approved for human use and have been accepted as totally non-toxic and environmentally friendly by the European commission REACH program and European Food Safety Authority (EFSA) [Ajinomoto OmniChem, Impact of REACh on Ajinomoto OmniChem's Natural Specialties, http://www.natural-specialities.com/PDF/Applications/REACh%20compliance%20OmniChem%20Natural%20Specialties%20v3.0.pdf, 2009; European Food Safety Authority, Scientific Opinion on the safety and efficacy of tannic acid when used as feed flavouring for all animal species, EFSA Journal, 2014, 12, 3828—each incorporated herein by reference in its entirety]. Tannins can be very soft materials such as the organic resin used as a “formaldehyde-free cornstarch-tannin adhesive” [Moubarik, A., et al., Preparation and Mechanical Characterization of Particleboard made from Maritime Pine and Glued with Bio-Adhesives based on Cornstarch and Tannins, Maderas Ciencia y Technologia, 2010, 12, 189; Basso, M. C., et al., A New Approach to Environmentally Friendly Protein Plastics and Foams, Bioresources, 2015, 10, 8014—each incorporated herein by reference in its entirety], or very hard materials such as grinding discs [Lagel, M. C., et al., Cutting and grinding wheels for angle grinders with a bio-resin matrix solid grinding wheels, Industrial Crops and Products, 2015, 67, 264—incorporated herein by reference in its entirety] and automobile brake pads [Lagel, M. C., et al., Automotive brake pads made with a bio resin matrix, Industrial Crops and Products, 2015, 85, 372—incorporated herein by reference in its entirety].
The need for bone tissue substitutes for dental, craniofacial, and orthopedic reconstructions is rapidly increasing due to rapid global population growth and extension of life expectancy, with the number of elderly people (+65 years) estimated to be about 2 billion by 2050 [Clegg, A. et al., Frailty in elderly people, The Lancet, 2013, 381, 752—incorporated herein by reference in its entirety]. Available options to treat bone deficiencies are based on transplantation of bone grafts or implantation of alloplastic materials [Hing, K. A., Bone repair in the twenty-first century: biology, chemistry or engineering?, Philos Trans A Math Phys Eng Sci., 2004, 362, 2821—incorporated herein by reference in its entirety], which can restore tissue integrity and functionality but fail to provide optimal therapeutic solutions in several clinical cases, such as in situations characterized by extensive tissue loss, poor bone quality, or otherwise compromised regenerative capacity [Oreffo, R. O., et al., Future potentials for using osteogenic stem cells and biomaterials in orthopedics, Bone, 1999, 25, 5S—incorporated herein by reference in its entirety]. Conversely, engineering bone substitutes by culturing osteocompetent cells onto compliant biomaterials offers the possibility to grow unlimited amounts of tissue products with enhanced regenerative potential and broader clinical use [de Peppo G. M., et al., Engineering bone tissue substitutes from human induced pluripotent stem cells, Proc Natl Acad Sci USA. 2013, 110, 8680—incorporated herein by reference in its entirety].
Interfacing osteocompotent stem cells onto porous tannin spray-dried powder (PTSDP) resin scaffolds holds the potential to enhance the healing properties of these materials. However, currently available PTSDP scaffolds lack macroporosity, a critical feature that allows cell infiltration, communication, and growth. Scaffold porosity can also facilitate bone ingrowth and remodeling, therefore maximizing the therapeutic potential of tissue-engineered products. Specifically in bone engineering, porosity is important for developing biomaterial scaffolds that mimic the architecture of the native bone tissue.
In view of the forgoing, one objective of the present disclosure is to provide a bone graft comprising a tannin-hydroxyapatite resin scaffold, osteocompetent stem cells, and a growth medium, and methods of use.
According to a first aspect, the present disclosure relates to a biocompatible bone graft which includes a porous scaffold structure comprising a tannin-hydroxyapatite resin which has a 17-80 vol % porosity, a pore diameter of 40-300 μm, and a compressive strength of 0.15-1.90 MPa; a population of osteocompetent stem cells obtained from a mammalian donor; and a growth medium.
In one embodiment the tannin-hydroxyapatite resin of the biocompatible bone graft comprises 50-90 wt % tannin and 10-50 wt % hydroxyapatite.
In one embodiment the osteocompetent stem cells within the biocompatible bone graft are derived from a sample of bone marrow, periosteum, dermal fibroblasts, or adipose tissue.
In another embodiment at least 50% of the osteocompetent stem cells express CD13, CD29, CD44, CD90, or CD105.
In another related embodiment, the biocompatible bone graft also comprises a growth factor. In a related embodiment, that growth factor is β-glycerophosphate, dexamethasone, ascorbic acid, transforming growth factor β (TGF-β1), fibroblast growth factor (FGF), active vitamin D, a bone morphogenic protein (BMP), and/or parathyroid hormone.
In another embodiment, osteoinductive biomolecules are adsorbed or chemically-linked on the surface of the porous scaffold structure.
In one embodiment the porous scaffold structure is made with an additional biodegradable polymer of poly lactic-co-glycolic acid, poly lactic acid, poly glycolic acid, polyanhydride, poly(ortho)ester, polyurethane, poly(butyric acid), poly(valeric acid), polycaprolactone, poly(lactide-co-caprolactone), or poly(trimethylene carbonate).
In one embodiment, the biocompatible bone graft also comprises a first portion of bone on one side of the porous scaffold structure, and a second portion on another side of the porous scaffold structure, where both portions of bone comprise a bone wound site in a patient.
In a related embodiment, the biocompatible bone graft in the bone wound site further comprises mineralized osseous tissue supported by the porous scaffold structure and connecting both portions of bone.
In another related embodiment, the patient is the mammalian donor of the stem cells.
In another related embodiment, the biocompatible bone graft also comprises a prosthesis.
In another related embodiment, the biocompatible bone graft also comprises a second biocompatible bone graft in the wound site and which has a different size, shape, and/or composition.
According to a second aspect, the present disclosure relates to a method of monitoring the growth of the osteocompetent stem cells in the biocompatible bone graft for up to 16 weeks by X-ray imaging, MRI, or ultrasonography.
In another embodiment of the method, the growth of the osteocompetent stem cells in the biocompatible bone graft is monitored up to 16 weeks by the expression level of one of the following osteogenic differentiation genes: RUNX2, COL1A1, ALPL, OPN, or PDGFRB.
According to a third aspect, the present disclosure relates to a method of screening a bone disease drug by adding the drug to the biocompatible bone graft, growing the osteocompetent stem cells in the presence of the drug to form new bone tissue on the bone graft, measuring the porosity, density, and/or compressive strength of the drug-treated bone graft, and comparing it to a substantially similar bone graft that has not been treated with the drug.
In one embodiment, the bone disease drug is denosumab, teriparatide, alendronate, risedronate, ibandronate, zoledronic acid, teriparatide, strontium ranelate, aluminium chlorohydrate, or odanacatib.
According to a fourth aspect, the present disclosure relates to a method of making a porous scaffold structure starting with mixing porous tannin powder particles, dried polyethylene glycol particles, and hydroxyapatite together to make a powder mixture; mixing the powder mixture with an organic acid and a formaldehyde solution to form a liquid mixture; incubating the liquid mixture to form a set scaffold; and heating the set scaffold to remove the polyethylene glycol particles to form the porous scaffold structure.
In another embodiment of the method, a biodegradable polymer is also added to the powder mixture. In a further embodiment, the biodegradable polymer is poly lactic-co-glycolic acid, poly lactic acid, poly glycolic acid, polyanhydride, poly(ortho)ester, polyurethane, poly(butyric acid), poly(valeric acid), polycaprolactone, poly(lactide-co-caprolactone), or poly(trimethylene carbonate).
The foregoing paragraphs have been provided by way of general introduction, and are not intended to limit the scope of the following claims. The described embodiments, together with further advantages, will be best understood by reference to the following detailed description taken in conjunction with the accompanying drawings.
A more complete appreciation of the disclosure and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
FIG. IJ is an SEM image of a decellularized bone scaffold.
Embodiments of the present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the disclosure are shown.
The present disclosure will be better understood with reference to the following terms and meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.
As used herein, the term “biocompatible” denotes being biologically compatible by not producing a toxic, injurious, or immunological response when in physical contact with a living cell or tissue.
As used herein, the term “porosity” is the percentage of void volume of a material's bulk volume that can be reached from the exterior surfaces of the material. In the current disclosure, macroporosity, mesoporosity, and microporosity all refer to porosity. As defined here, a macroporous material contains pores with an average diameter greater than 50 nm; a mesoporous material contains pores with an average diameter of 2 nm-50 nm, and a microporous material contains pores with an average diameter less than 2 nm.
According to a first aspect, the present disclosure relates to a biocompatible bone graft which includes a porous scaffold structure comprising a tannin-hydroxyapatite resin, a population of osteocompetent stem cells obtained from a mammalian donor, and a growth medium.
The tannin-hydroxyapatite resin of the porous scaffold structure may have a porosity of 5-90 vol %, preferably 10-95 vol %, more preferably 17-80 vol %; a pore diameter of 10-700 μm, preferably 25-500 μm, more preferably 40-300 μm; and a compressive strength of 0.05-3.00 MPa, preferably 0.01-2.50 MPa, more preferably 0.15-1.90 MPa. The tannin-hydroxyapatite resin of the porous scaffold structure may have a bulk density of 0.10-0.50 g/cm3, preferably 0.20-0.40 g/cm3, more preferably 0.27-0.35 g/cm3.
The porous scaffold structure may be formed in the shape of a cuboid, spheroid, ovoid, ellipsoid, or other irregular shape, and with ends that may be rounded, squared, tapered, beveled, conical, concave, convex, scalloped, angular, or in some other form. Additionally, the porous scaffold structure may be formed in one shape and then filed, drilled, sanded, cut, or grinded into another shape. The optimal shape and dimensions for the scaffold structure as a bone graft depend on the size and location of the bone wound site, and the presence of other materials, such as an orthopedic implant. The longest dimension of the porous scaffold structure may range from 4-20 mm, preferably 5-12 mm, more preferably 6-10 mm. The shortest dimension of the porous scaffold structure may range from 1-10 mm, preferably 1-8 mm, more preferably 1-5 mm. In one embodiment, the porous scaffold structure has a cylindrical shape with an 8 mm diameter and a 2 mm height.
The tannin-hydroxyapatite resin is made of a tannin extract chemically cross-linked in a mixture that also contains hydroxyapatite. This creates a matrix of cross-linked tannin molecules in which hydroxyapatite is adsorbed and embedded throughout.
In one embodiment, the hydroxyapatite is dispersed within the tannin matrix, and no chemical bonding is present therebetween.
Here, tannin may be extracted from mimosa, acacia, wattle, quebracho, pine, spruce, fir, tanoak, hemlock, mangrove, gambier, oak, birch, maple, eucalyptus, tara, and/or catechu trees, or mixtures thereof, and may come from the leaf, bud, seed, root, bark, trunk, and/or stem tissue. Preferably the tannin is extracted from the bark of mimosa, quebracho, or wattle trees.
Tannin refers to a group of plant-based oligomeric polyphenolic compounds. The tannin extract used for the resin may include a mixture of condensed, flavonoid tannin molecules and hydrolyzable tannin molecules. Condensed, flavonoid tannin molecules are based on flavon-3-ol monomers (1) and generally contain 3-6 hydroxyl groups per monomeric unit. Examples of common condensed tannin molecules include, but are not limited to, those based on prorobinetinidin (II), profisetinidin (III), procyanidin (IV), and prodelphinidin (V) flavonoid units and are connected through bonds at C4, C6, and/or C8. The major tannin constituent in pine bark extract may be procyanidin (IV) with an average degree of polymerization between 6 and 7, with units linked through C4 and C8. The major tannin constituent in mimosa tannin may be profisetinidin (III) and prorobinetinidin (II), with an average degree of polymerization between 4 and 5, with units linked through C4 and C6. Hydrolyzable tannin molecules may contain a saccharide and may be a type of gallotannin or ellagitannin, which are oligomers of gallic acid (galloyl) units (VI) connected through ester or C—C bonds, respectively. One example of a common gallotannin is tannic acid, with one polymeric form shown in formula VII. Gallotannins may contain 2-12 galloyl groups per molecule, with each galloyl group having 2-3 hydroxyl groups and one ester group. It is further possible to have complex tannins in which a condensed, flavonoid tannin is bound to a gallotannin and/or an ellagitannin.
In a preferred embodiment, the tannin is a prorobinetinidin oligomer of molecular weight 0.5-3.5 kDa and is extracted from mimosa tree bark. In another embodiment, condensed polyflavonoid tannins with a molecular weight greater than 3.5 kDa may be used in the resin. In an alternative embodiment, monomeric flavoinoid units and condensed polyflavonoid tannins with a molecular weight less than 1 kDa may be used in the tannin resin. In an alternative embodiment, the tannin used for the tannin resin may contain a mixture of both condensed tannins (containing flavan-3-ol units), hydrolyzable tannins (containing galloyl units), and complex tannins (containing both flavan-3-ol and galloyl units). In this mixture, the molar ratio of total galloyl units to total flavan-3-ol units may range from 4:1-1:100, preferably 1:1-1:100, more preferably 1:4-1:100.
In one embodiment, tannin from an aqueous tannin extract may be chemically modified prior to cross-linking, which may change the mechanical, chemical, and/or biological properties of the tannin resin. In another embodiment, the chemical modification changes the reaction conditions needed to form the tannin resin. The hydroxyl sites of tannin molecules may be methylated, carboxymethylated, acylated, alkoxylated, substituted with ammonia, reacted with isocyanates, and/or etherified. Other parts of tannin molecules may be sulfonated, hydrolyzed, condensed, halogenated, or reacted with furfuryl alcohol. In addition, tannin may be esterified with fatty acids including, but not limited to, oleic acid, linoleic acid, elaidic acid, erucic acid, gondoic acid, paullinic acid, γ-linolenic acid, α-linolenic acid, and/or stearidonic acid.
In one embodiment, the tannin is functionalized fully or partially with reactive functional groups to chemically bind the hydroxyapatite within the resin. This may be accomplished with a reagent that may crosslink the hydroxyl groups of the tannin with the hydroxyl groups of the hydroxyapatite. Such a reagent may be a diisocyanate such as methylene diphenyl diisocyanate (MDI), toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), and/or hexamethylene diisocyanate (HDI).
An aqueous tannin extract may be dried to create a tannin powder for use in the tannin-hydroxyapatite resin. The powder may be formed by spray drying, freeze drying, air drying, or vacuum drying the extract. Preferably, the powder is formed by spray drying. The formed powder may comprise 60-95 wt %, preferably 70-90 wt %, more preferably 78-85 wt % flavonoid tannins, with the remaining portion of the dried residue comprising hemicellulose fragments, hydrolyzable tannins, complex tannins, amino acids, imino acids, and/or water. It is possible that the extract may be purified prior to drying in order to enrich the concentration of flavonoid tannins. The dried tannin powder particles may have an average largest dimension of 150 μm or smaller, preferably 75 μm or smaller, more preferably 50 μm or smaller.
Hydroxyapatite and a dry porogen are mixed with the tannin powder to form a powder mixture. As used herein, a “porogen” is a non-reacting substance added to a liquid settable material in order to reserve the space that forms pores. The mass ratio of hydroxyapatite to tannin may be in the range of 1:10-2:1, preferably 1:8-1:1, more preferably 1:5-1:3. The mass ratio of the dry porogen to tannin may be in the range of 4:1 to 1:2, preferably 3:1 to 1:1.5, more preferably 2.5:1 to 1:1. Hydroxyapatite within the resin may support the growth and differentiation of the osteocompetent stem cells, as well as supply a starting mineralization source. In an envisioned embodiment, other inorganic materials could be used in place of, or in addition to, hydroxyapatite. These materials include tri-calcium phosphate, fluorapatite, carbonated hydroxyapatite, coralline hydroxyapatite, mineralized bone matrix, demineralized bone matrix, chlorapatite, bone meal powder, calcium sulfate, and bioactive glass (Bioglass). In an envisioned embodiment, hydroxyapatite is used with tri-calcium phosphate at a mass ratio ranging from 1:10-10:1, preferably 1:5-5:1, more preferably 1:4-4:1 hydroxyapatite to tri-calcium phosphate.
In one embodiment the porous scaffold structure is made with an additional biodegradable polymer of gelatin, lignin, poly(2-hydroxyethyl methacrylate), polyhydroxobutyrate, hyaluronic acid, a polysaccharide such as starch, dextran, sodium alginate, carboxymethyl cellulose (CMC) or hydroxypropyl methylcellulose (HPMC), chitosan, glycosaminoglycan, poly lactic-co-glycolic acid, poly lactic acid, poly glycolic acid, polyanhydride, poly(ortho)ester, polyurethane, poly(butyric acid), poly(valeric acid), polycaprolactone, poly(lactide-co-caprolactone), or poly(trimethylene carbonate). Any mixtures of these biodegradable polymers may also be used.
Preferably, the biodegradable polymer is poly lactic-co-glycolic acid, poly lactic acid, poly glycolic acid, polyanhydride, poly(ortho)ester, polyurethane, poly(butyric acid), poly(valeric acid), polycaprolactone, poly(lactide-co-caprolactone), or poly(trimethylene carbonate). In one preferred embodiment, the biodegradable polymer is poly lactic-co-glycolic acid.
Following the setting of the tannin-hydroxyapatite resin, the porogen is removed by dissolution, melting, evaporation, and/or sublimation, leaving a solid porous scaffold. Porogens that can be used in the tannin-hydroxyapatite resin include salt crystals, such as NaCl, MgCl2, CaCl2, NH4Cl, NH4PO4, or NH4CO3; polysaccharides such as amylose, cellulose, cellulose derivatives, chitin, chitosan, dextran, glycogen, inulin, glycosaminoglycans, pectin, pullulan, starch, starch derivatives, or xanthan; monosaccharides such as glucose, ribose, fructose, or triose; saccharide oligomers; polymers including, but not limited to the aforementioned biodegradable polymers; waxes such as animal waxes, vegetable waxes, mineral waxes, petroleum waxes, synthetic waxes or mixtures thereof; and surfactants such as cationic surfactants (based on quaternary ammonium cations), anionic surfactants (based on sulfate, sulfonate, or carboxylate anions), zwitterionic (amphoteric) surfactants, or non-ionic surfactants. Particles of these porogens may comprise a mixture of different porogen compounds and can be added to the powder mixture, or they can be added to the liquid resin prior to setting. Following the formation of the solid scaffold, the porogens can be removed by dissolving in different solutions and/or heating the scaffold. The porogen particles may have an average largest dimension of 50-800, preferably 75-700, more preferably 100-600 μm. Preferably the porogen comprises dry, solid particles of polyethylene glycol with the polyethylene glycol having an average molecular weight of 1,000-100,000 Da, preferably 5,000-80,000 Da, more preferably 10,000-40,000 Da. It may be possible that the porogen creates pores in the resin by adsorbing to the surface of the dried tannin particles, which limits the amount of tannin available to react. Following the removal of the porogen, in one embodiment, the porous scaffold structure consists of the tannin-hydroxyapatite resin.
A crystal growth inhibitor may be added to the powder mixture to prevent the formation of hydroxyapatite crystals. Such inhibitors may be 1-hydroxyethylidene 1,1-diphosphonic acid (HEDP), nitrilotri (methylene phosphonic acid) (NTMP), ethylenediamine tetra(methylene phosphonic acid) (EDTMP), citric acid, disodium pyrophosphate, or tetrasodium pyrophosphate. More than one inhibitor can be used at once; preferably the inhibitors used are citric acid and disodium pyrophosphate. The combined mass of the crystal growth inhibitors added to the powder mixture may make up 0.5-5% total mass, preferably 1-4% total mass, more preferably 2.5-3.5% total mass of the powder mixture. In an embodiment using two crystal growth inhibitors, the mole ratio between them may range from 10:1-1:10, preferably 5:1-1:5, more preferably 3:1-1:3. In a preferred embodiment, disodium pyrophosphate is added to compose 0.1-3.5 wt %, preferably 0.5-2 wt %, more preferably 0.8-1.5 wt % of the powder mixture, and citric acid is added to compose 1-5 wt %, preferably 2-4 wt %, more preferably 2.2-3 wt % of the powder mixture.
The powder mixture is mixed with a volume of 0.10-0.40 mL, preferably 0.15-0.35 mL, more preferably 0.20-0.30 mL of an organic acid per gram of the powder mixture and a formaldehyde solution to form a liquid mixture. The concentration of organic acid added to the powder may range from 0.1-1 M, preferably 0.3-0.8 M, more preferably 0.4-0.6 M. The organic acid may be formic, acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and/or isethionic acid; preferably the organic acid is citric acid.
The formaldehyde solution may be added to make up 1-20 wt %, preferably 2-18 wt %, more preferably 5-15 wt % of the liquid mixture. The formaldehyde solution may contain 20-38 wt %, preferably 30-38 wt %, more preferably 35-38 wt % formaldehyde. The formaldehyde solution may also include an alcohol at a weight concentration of 5-15%, preferably 7-13%, more preferably 8-12% to prevent formaldehyde polymerization. This alcohol may be ethanol, methanol, t-butyl alcohol, 1-propanol, or isopropanol; preferably the alcohol is methanol. In a preferred embodiment, the formaldehyde solution contains 37 wt % formaldehyde and 10% methanol, and is otherwise known as formalin.
In alternative embodiments, different cross-linking reagents may be used instead of, or in addition to, formaldehyde to create the tannin resin. For example, a hydrolyzable tannin may be used with phenol and formaldehyde to create a tannin resin. In another embodiment, furfuryl alcohol, succinaldehyde, glutaraldehyde, malondialdehyde, phthalaldehyde and/or glyoxal may be used as crosslinking reagents. In addition, tannin could be polymerized into a polyurethane with an isocyanate reagent such as methylene diphenyl diisocyanate (MDI), toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), and/or hexamethylene diisocyanate (HDI). An ethoxylated amine may also be used in the polyurethane-forming reaction. In another example, a tannin-based epoxy resin may be formed by reacting hydrolyzable or condensed tannins with epoxides such as diglycidyl ether, polyglycidyl ether, and/or epichlorohydrin.
The liquid mixture is mixed vigorously for 20 s-5 min, preferably 30 s-2 min, more preferably 30 s-1 min, and then transferred to molds. The molds may be made of any non-reactive substance, but preferably the molds comprise a flexible material such as silicone, rubber, or a plastic. The mixing may be accomplished with a blender, a turbula, a vortex mixer, a general laboratory mixer, or by manually shaking. Preferably, the mixing is accomplished with a turbula. After 3-10 min, preferably after 4-8 min, more preferably after 5-7 min of transferring the samples, the molds may be completely submerged in a saline buffer to expose the samples to the buffer. Alternatively, the saline buffer may be added to the mold to completely cover the sample. The saline buffer may be phosphate buffered saline (PBS), tris buffered saline (TBS), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS) buffered saline, or Dulbecco's phosphate buffered saline (DPBS). Preferably, the buffer is DPBS with calcium. The saline buffer may be supplemented with a soluble calcium salt, such as CaCl2, to a concentration of 0.5-1.3 mM, preferably 0.7-1.1 mM, more preferably 0.8-1.0 mM Ca2+ in solution. The samples are incubated in the saline buffer for 12-48 h, preferably 18-36 h, more preferably 20-30 h and at a temperature of 30-50° C., preferably 34-42° C., more preferably 35-40° C. to allow for complete setting of the tannin-hydroxyapatite resin.
Then the samples are removed from the molds, optionally polished with a coated abrasive, such as sandpaper, and incubated in a saline buffer, such as those listed previously, for 24-96 h, preferably 36-60 h, more preferably 44-52 h, and at a temperature of 50-95° C., preferably 55-80° C., more preferably 65-75° C., to melt and leach the polyethylene glycol porogen particles, leaving a porous scaffold. It is conceivable that the minimal time and temperature required for porogen leaching may depend on the thickness and density of the resin sample. In an envisioned embodiment with a different type of porogen, the leaching step may not be done in an aqueous environment but in a solvent or a dry oven. In another envisioned embodiment, porosity may be introduced by a foam blowing agent, such as pentane, isopentane, cyclopentane, diethyl ether, and/or supercritical CO2, instead of a porogen. In another embodiment, instead of a non-reactive porogen, a reactive substance could be mixed into the resin to produce gas bubbles in the structure. In yet another embodiment, porosity may be introduced to the scaffold structure by omitting a porogen substance and instead using larger spray-dried tannin particles in the powder mixture. These larger spray-dried tannin particles may have an average largest dimension of at least 100 μm, preferably at least 125 μm, more preferably at least 150 μm.
Depending on the concentration and type of porogen used, a small subset of pores might not be interconnected, meaning that their interior surface does not connect with the exterior surface of the scaffold structure. Preferably at least 95% of the pores are interconnected, more preferably at least 98%, even more preferably at least 99%.
Following the porogen leaching step of the tannin-hydroxyapatite resin, the sample may be dried for 10-48 h, preferably 12-24 h, more preferably 14-20 h at a temperature of 50-95° C., preferably 55-80° C., more preferably 65-75° C. Following the drying, the sample may be sterilized for cell culture by autoclaving at 150° C. for 30 min, however, other autoclave cycles or sterilization methods may be used, such as gamma ray sterilization. In one embodiment, the scaffolds are submerged in an aqueous solution of at least 70 vol %, at least 80 vol %, at least 90 vol % alcohol (e.g. methanol, ethanol, isopropanol, etc.) overnight to sterilize. Following sterilization, aseptic techniques may be used to prevent contamination of the bone graft.
In another embodiment, osteoinductive biomolecules are adsorbed or chemically-linked on the surface of the porous scaffold structure before adding the cells. The biomolecules may be mixed directly into the liquid mixture so that they may be exposed on the scaffold surface. The biomolecules may instead be chemically linked to a polymer on the scaffold surface, or linked to a polymer that had been mixed into the liquid mixture while forming the tannin-hydroxyapatite resin. As used herein, the “surface” of the porous scaffold denotes connected interior and exterior surfaces. These biomolecules may assist in cell attachment, growth, or differentiation of the osteocompetent stem cells. Such biomolecules include collagen, laminin, nidogen-1, vitronectin, fibronectin, bone morphogenic protein, RGD tripeptide, and mixtures of biomolecules like those from bone marrow aspirate and Matrigel. Certain synthetic polymer coatings on the scaffold surface may also promote cell attachment and differentiation such as poly(methylmethacrylate) (PMMA), polycarbonate, and amine group-grafted polyethylene.
In the embodiment where the porous scaffold structure is part of a biocompatible bone graft, the scaffold structure may also be seeded with osteocompetent stem cells. Osteocompetent stem cells are stem cells that can differentiate into osteoblast and osteoclast cells. These osteocompetent stem cells may be derived from mammalian stem cell sources such as embryonic or fetal tissue, placental tissue, umbilical cord tissue or blood, menstrual blood, muscle, synovial membrane tissue, bone marrow, periosteum, dermal tissue, or adipose tissue. Preferably, the osteocompetent stem cells are derived from bone marrow, periosteum, dermal fibroblasts, or adipose tissue. The osteocompetent stem cells for use in the bone graft may be obtained from a primary source or from a cell bank.
The mammalian source of the stem cells may be Homo sapiens sapiens, Pan troglodytes, Bos primigenius, Sus scrofa domesticus, Canis lupus familiaris, Felis calus, Rattus norvegicus, Mus musculus, or Equusferus caballus. Preferably, the mammalian source may be Homo sapiens sapiens.
In one embodiment, at least 50%, preferably at least 60%, more preferably at least 70% of the osteocompetent stem cells express CD13, CD29, CD44, CD49e, CD73, CD81, CD90, CD105 and/or CD151, which are biomarkers typical of primary mesenchymal stem cells. More preferably, the biomarkers are CD13, CD29, CD44, CD90, and/or CD105. In a related embodiment, at least 50%, preferably at least 60%, more preferably at least 70% of the osteocompetent stem cells show negative expression of OCT4, SOX2, TRA-1-60, CD31, CD34, and/or CD45, which means that the osteocompetent stem cells are not pluripotent nor hematopoietic. It is possible that a mixture of stem cells could be sorted by flow cytometry to choose osteocompetent stem cells based on the expression of the biomarkers.
Following collection of osteocompetent stem cells, the cells may be seeded onto the scaffold in the presence of a liquid growth medium to make the biocompatible bone graft. The liquid growth medium may be based on common formulations such as DMEM or DMEM:F12, or the medium may be a completely specialized formulation. The medium may include common supplements not specific to osteocompetent stem cells such as fetal bovine serum (FBS), ROCK inhibitor Y-27632, l-glutamine, sodium pyruvate, or antibiotics. To support the differentiation of the osteocompetent stem cells, the liquid growth medium may include additional supplements and growth factors including, but not limited to, platelet derived growth factor (PDGF), osteopontin, calcium, insulin-like growth factor (IGF-1), β-glycerophosphate, dexamethasone, ascorbic acid-2-phosphate, ascorbic acid, transforming growth factor β (TGF-β1), fibroblast growth factor (FGF), active vitamin D, a bone morphogenic protein (BMP), parathyroid hormone, or any combination thereof. Preferably, the supplement or growth factor is β-glycerophosphate, dexamethasone, ascorbic acid, transforming growth factor β (TGF-β1), fibroblast growth factor (FGF), active vitamin D, a bone morphogenic protein (BMP-2, BMP-4, or BMP-7), or parathyroid hormone. These growth factors may be added individually in a purified form to the liquid growth medium, or they may be added together with their biological source, such as platelet-rich plasma or bone marrow aspirate. In an envisioned embodiment, a growth factor or supplement may be adsorbed onto or chemically attached to the surface of the scaffold, as described previously with other biomolecules.
In a further embodiment, the cells prior to seeding may be grown within a 3D gel or other semi-solid, rather than a liquid growth medium.
Additionally, the composition of the growth medium may be altered at any point prior to implanting the bone graft. It is likely that in the steps from expanding the stem cell population in a flask to seeding the cells on the scaffold, different growth media may be used to supply different types and concentrations of nutrients to the cells.
Additionally, depending on the number of cells and the size of the scaffold, the cell population may need to be expanded prior to seeding on the scaffold. In some cases, the cells may be seeded on the scaffold, and then grown on the scaffold before implanting the bone graft. Cells may be added to one side of the scaffold if the scaffold is relatively small.
Additionally, for larger scaffolds, cells may be added to more than one side. Also, the scaffold may be intermittently rotated or flipped to encourage cell growth throughout the porous structure. The scaffold may be partially submerged in liquid growth medium or completely submerged. In the case of a partially submerged scaffold, liquid growth medium may periodically be added to the unsubmerged portion to keep the cells from drying out.
In one embodiment, a cryopreservative liquid medium may be added to the bone graft. The cryopreservative liquid medium may include FBS and a cryoprotectant such as glycerol and/or DMSO, or the medium may be a proprietary formulation available commercially. When stored at a temperature of −75 to −85° C., the bone graft may be preserved up to 5 years. When stored in a liquid nitrogen vapor phase, the bone graft may be preserved indefinitely.
The present disclosure also relates to a method of regenerating bone in a patient by implanting the aforementioned biocompatible bone graft within a bone wound site in a patient.
The bone wound site may be a defect resulting from injury such as a transverse fracture, a linear fracture, an oblique fracture, a spiral fracture, a greenstick fracture, or a comminuted fracture; from surgery; from an infection such as osteomyelitis; from malignancy, such as osteoma, osteoid osteoma, osteochondroma, osteoblastoma, osteoblastoma, enchondroma, aneurysmal bone cyst, giant cell tumor of the bone, chondrosarcoma, fibrus dysplasia, osteosarcoma, fibrosarcoma, or Ewing sarcoma; or from a developmental malformation such as a metaphyseal defect. The bone wound site may be in cancellous or cortical bone, or in a single bone wound site that comprises both. The bone wound site may also result from a previous bone graft that became infected or otherwise failed to heal.
In one embodiment of the method, the patient is the mammalian donor, wherein the bone graft is an autograft by definition. In a related embodiment, the bone graft is an allograft, in which the patient and the donor are different organisms, but the same species. In another related embodiment, the bone graft is a xenograft, in which the patient and the donor are different species.
In another embodiment, the bone graft is implanted with a prosthesis, such as a tooth, a knee, a shoulder, or a hip. The bone graft may also be implanted with an orthopedic implant to support an existing bone or joint, or to replace, in whole or in part, a missing bone or joint. Such orthopedic implants may comprise pins, rods, screws, nails, wires, and plates, and may be made of biocompatible metals such as titanium and related alloys, cobalt-based alloys, or stainless steel, or biocompatible polymeric materials such as carbon fiber, high density polyethylene, ultra-high molecular weight polyethylene (UHMWPE), polymethylmethacrylate (PMMA), or thermoplastic polyether ether ketone (PEEK).
In one embodiment of the method, a second bone graft is implanted into the implant site. This second bone graft may be identical to the first, or it may have a different size and/or composition. Where the implant site is shaped too irregularly or is too large for the insertion of a single bone graft, multiple, smaller bone grafts may be inserted. These smaller bone grafts may be shaped for specific locations in the bone wound sites and may require a certain order of insertion. In contrast, the bone grafts may be at a scale much smaller than the bone wound site in which case they can fill the bone wound site in any order or placement.
In addition, the porous scaffold could be broken into small pieces that are combined with osteocompetent stem cells and a growth medium to form a semi-solid bone graft paste. This paste could fill both regular and irregularly-shaped bone wounds as an alternative to implanting a solid scaffold structure. In this embodiment, a porogen may not be required in the tannin-hydroxyapatite resin, nor would the resin need to set in a mold. Depending on the viscosity, the bone graft paste may be injected into a bone wound site using a syringe. In a further embodiment, the bone graft paste could be used to support or surround a larger intact bone graft or other implant in the center of a bone wound site.
In one embodiment, the second bone graft comprises a macroporous hydroxyapatite-tannin resin but has a different composition, which may encourage bone development at different rates within the bone wound site. For example, the second bone graft may have a different porosity or a different composition of hydroxyapatite, polymers, tannins, and/or osteoinductive biomolecules. The second bone graft may have a different compressive strength or biodegrade a rate different than the first bone graft.
In another embodiment, the second bone graft is different than the first bone graft in that the second bone graft contains mineralized osseous tissue from a mammalian donor. This mineralized osseous tissue may be from cancellous and/or cortical bone, and may or may not contain cells. Preferably, this mineralized osseous tissue is fragmented into a paste as described previously. The donor of the osseous tissue may be a member of a different species than the patient, or may be a member of the same species as the patient, or may be the patient. In a related embodiment, a bone graft may be implanted with other living tissues such as cancellous bone marrow.
In one embodiment, a previously described growth factor may be added to the bone wound site when implanting the bone graft. Additionally, the growth factor may be administered by oral administration, intravenous administration, topical administration, intradermal administration, transdermal administration, subcutaneous administration, intramuscular administration, intralesional administration, intrapulmonal administration, or sublingual administration. The growth factor may be administered prior to, during, or after the grafting surgery, or at any combination of those times, and likely, more than one type of growth factor may be administered. The growth factor may also be administered with an analgesic or anti-inflammatory drug.
In one embodiment the tannin-hydroxyapatite resin of the biocompatible bone graft degrades into inert products that can be resorbed in a bone graft implant site. In that case, the growth and differentiation of the osteocompetent stem cells breaks down the scaffold structure into biocompatible products. Preferably, this process occurs as newly-developed osteoblasts lay down bone tissue, leading to the eventual replacement of the scaffold structure with mature bone tissue. It is conceivable that the hydroxyapatite embedded in the scaffold structure could be used by the osteoblasts in mineralizing new bone tissue. It is also possible that the tannin resin may be broken down into dissolved constituent polyphenolic molecules that are either metabolized by neighboring cells or are left in the extracellular space of the tissue.
According to a second aspect, the present disclosure relates to a method of monitoring the growth of the osteocompetent stem cells in the biocompatible bone graft up to 16 weeks by medical imaging, such as X-ray imaging, MRI, or ultrasonography. It is envisioned that the growth and bone formation within the bone graft create changes in density which can be detected by either of these imaging modes over a certain length of time. An increase in density may occur where osteoblast and osteoclast cells populate the scaffold and create new bone tissue. A decrease in density may occur where the scaffold is degraded. Likely, a single bone graft site may have both increases and decreases in density, occurring at different places and/or at different times. To monitor the growth of the osteocompetent stem cells, it is envisioned that two sets of images recorded with the same imaging mode but at different time intervals may be compared with one another. Likely, more than two sets of images may be compared, and for certain bone wound sites, longer healing times may warrant images recorded up to 6 months following the implant surgery. To ensure healing of the bone wound site, treatment may be modified depending on the progression of growth, such as the amount of activity allowed to the patient, the types of drugs administered, or the removal of sutures, casts, or braces. For example, if a drug is administered to support bone remineralization, the dosage may be decreased when a sufficient amount of new bone tissue has formed within the bone graft.
In another embodiment of the method, the growth of the osteocompetent stem cells are monitored up to 16 weeks by the expression level of one of the following osteogenic differentiation genes: BGLAP, BMP-2, BMP-4, BMP-6, BMP-7, SP7, RUNX2, COL1A1, ALPL, OPN, or PDGFRB. Preferably the gene is RUNX2, COL1A1, ALPL, OPN, or PDGFRB. Preferably, the expression level of the gene is inferred by measuring the concentration of its mRNA and comparing that to the mRNA concentration of a housekeeping gene, such as GAPDH. For these measurements, a tissue sample may be taken from the bone graft, preferably by a needle biopsy. Following routine protocol to extract and purify the mRNA, the concentration may be determined through quantitative RT-PCR. Preferably, more than one osteogenic differentiation gene is measured. To track the differentiation of the cells, the mRNA levels may be compared between two sets of measurements taken at different times. The first set of measurements may be prior to seeding the stem cells on the scaffold, or prior to implanting the bone graft. When the first set of measurements is taken prior to implanting the bone graft, the second set of measurements may be taken via biopsy at least one week, preferably at least 1 month, more preferably at least 2 months following the bone graft implant surgery. Given a larger than average bone wound site, it is likely that mRNA levels may depend on the location where the sample is taken. It is further envisioned that to improve growth conditions in vitro, the osteogenic differentiation genes may be measured of a bone graft that is not intended to be implanted.
According to a third aspect, the present disclosure relates to a method of screening a bone disease drug by adding the drug to the growth medium of the biocompatible bone graft, growing the osteocompetent stem cells in the presence of the drug to form new bone tissue on the bone graft, measuring the porosity, density, and/or compressive strength of the drug-treated bone graft, and comparing it to a substantially similar bone graft that has not been treated with the drug. Bone disease drugs that affect the growth of bone tissue may change measurable properties of the bone graft, and these properties may be measured ex vivo and compared to a similar untreated bone graft. As bone tissue is developed in the bone graft, the density may increase while the porosity decreases. The compressive strength may increase or decrease at different times, depending on the rate of resin scaffold degradation versus bone tissue growth and mineralization. It is envisioned that for implanted bone grafts, biopsies are taken to provide a representative composition of tissue and scaffold material. Bone graft samples obtained by biopsies or other means may be fixed with a formaldehyde solution prior to measurement in order to secure cells and tissue within the porous scaffold. Porosity and density may be characterized by drying the scaffold and measuring with gas displacement pycnometry, mercury intrusion porosimetry, or micro-CT (micro-computed tomography, or μCT). The compressive strength may be measured with an ultrasonic cement analyzer or a more general compressive strength tester.
In the case where the bone graft is implanted into a patient, the bone disease drug may be administered by any of the aforementioned methods for growth factors.
In one embodiment, the bone disease drug is ipriflavone, incadronic acid, pamidronic acid, clodronic acid, tiludronic acid, denosumab, teriparatide, alendronate, risedronate, ibandronate, zoledronic acid, teriparatide, strontium ranelate, aluminium chlorohydrate, or odanacatib. Preferably, the bone disease drug is denosumab, teriparatide, alendronate, risedronate, ibandronate, zoledronic acid, teriparatide, strontium ranelate, aluminium chlorohydrate, or odanacatib.
In a preferred embodiment, the growth of the new bone tissue within the drug-treated bone graft and the untreated bone graft occurs in vitro. In this embodiment, the two bone grafts can be compared side-by-side. It is also envisioned that several bone grafts could be grown in vitro to systematically test different drugs or growing conditions. Media supplements and growth factors, as listed previously, may also be used.
The examples below are intended to further illustrate protocols for preparing and characterizing the biocompatible bone graft, and uses thereof, and are not intended to limit the scope of the claims.
Porous tannin spray-dried powder (PTSDP) was mixed with hydroxyapatite (Ca10(PO4)6(OH)2) to form a powder mixture with a composition of 80 wt % PTSDP and 20 wt % hydroxyapatite, prepared as previously detailed [Amaral-Labat, G. et al., Highly mesoporous organic aerogels derived from soy and tannin, Green Chem., 2012, 14, 3099—incorporated herein by reference in its entirety]. Then, the powder mixture was sieved to include only 25-75 μm diameter particles. Polyethylene glycol (PEG; 20,000 MW, Sigma, St. Louis Mo.) was melted at 100° C., then solidified at room temperature and ground to a fine powder. The milled powder was sieved to defined size ranges of 100-400, 400-600, or 100-600 μm. The PEG powder at a certain size range was added directly to the tannin-hydroxyapatite powder at a weight ratio of 0.4, 0.6, 0.8 or 1 gram of PEG powder per gram of tannin-hydroxyapatite powder, and mixed together in a turbula for 30 min. Crystal growth inhibitors disodium dihydrogen pyrophosphate (1% by weight) and citric acid (2.4% by weight) were added directly to the powder, along with a formalin solution (10% by weight). The macroporous scaffolds were fabricated by addition of a citric acid solution (0.5M) to the powder at a ratio of 0.25 mL per gram of powder (irrespective of the amount of PEG). The mixture was shaken in a cap mixer for 1 minute and immediately manually transferred to silicone molds (8 mm diameter×3 mm thick) using a spatula. Within 5-7 min the molds were transferred to Dulbecco's phosphate buffered saline with calcium (DPBS-8662, Sigma) to allow for complete setting at 37° C. for 24 h.
Then, the samples were removed from the molds, polished with sandpaper to about 2 mm thickness, and transferred to fresh DPBS-8662 at 70° C. to melt and leach the PEG for 48 h. The absence of PEG was confirmed by differential scanning calorimetry (DSC) and X-ray diffraction analysis (XRD) as described below. Each sample was dried at 70° C. for 16 h, then sterilized in an autoclave at 150° C. for 30 min and used for characterization and cell culture. Ten scaffold groups were thus manufactured as listed in Table 1. Bone disks were prepared as previously described [de Peppo G. M., et al., Engineering bone tissue substitutes from human induced pluripotent stem cells, Proc Natl Acad Sci USA. 2013, 110, 8680—incorporated herein by reference in its entirety]. Plugs of trabecular bone (8 mm diameter) were drilled from the subchondral region of meta-carpal joints of calves (Green Village Packing, Green Village, N.J.). Soon after, plugs were cleansed under a high-pressure water stream to remove the bone marrow and then sequentially washed with a solution of ethylenediamine tetra-acetic acid (EDTA) (0.1%) in DPBS-8662, then EDTA (0.1%) in Tris (10 mM), then SDS (0.5%) in Tris (10 mM), followed by treatment with a solution of DNase and RNase in Tris buffer (10 mM) to remove cellular material. Decellularized bone plugs were thoroughly rinsed in DPBS-8662, freeze-dried, cut, and then polished to about 2 mm thickness. Each individual scaffold was weighed and measured to calculate the density, and those in the range of 0.27-0.35 g/cm3 were used for material characterization analysis and cell culture. For cell culture, scaffolds were selected and sterilized overnight in ethanol (70% by volume) and then conditioned in expansion medium overnight.
PSDTP and decellularized bone scaffolds were imaged on a Hitachi TM100 scanning electron microscope (Hitachi High Technologies America Inc, Schaumburg, Ill.). Three random fields, from three samples per group, were photographed using the following settings: 15 kV, 100× magnification, and 7 mm working distance.
X-ray diffraction and Rietveld analysis were used to assess the material composition after the manufacturing process. Briefly, the ground powder from autoclaved tannin-hydroxyapatite scaffolds was analyzed using a C8 advanced Bruker diffractometer (Bruker Corporation, Billerica, Mass.), theta-theta setup at 40 kV and 40 mA and over diffraction angles of 5-60°. The step length was 0.3 seconds per step, with 0.01 degrees per step and a rotation speed of 80 rotations per minute (rpm). Open source software Profex (http://profex.doebelin.org) was used to quantify the spectra via Rietveld refinement. The reported values are the average of 3 readings for each group. Complete removal of PEG from leached scaffolds was confirmed by the lack of PEG peaks at the angles of 19.23° and 23.24° for samples for each group.
For differential scanning calorimetry, cylinders 8 mm in diameter and 3 mm thickness were prepared for cell culture and during the leaching process samples were removed after 2.5, 24, 32, or 56 h of leaching, and dried for 2 h at 25° C. Three samples from each group were ground to a fine powder and 10-20 mg of powder was heated from 25° C. to 120° C., at 10° C. per minute, in a TA Q2000 DSC (TA Instruments, New Castle, Del.). Then, the total enthalpy change for each sample was calculated and compared to tannin-hydroxyapatite powders containing a known amount of PEG, and data were exported to graphing software Origin (OriginLab Corporation, Northampton, Mass.).
The porosity of tannin-hydroxyapatite and decellularized bone scaffolds was determined with the Acupyc 1330 helium pycnometry (Micrometrics, Norcross, Ga.). Six samples were ground to a fine powder with mortar and pestle and the skeletal density (ρs) of the powder determined by helium gas intrusion, using a 1 cm3 chamber at 19.5 pounds per square inch of pressure. Samples were first dried completely and the apparent density (ρa) was determined by measuring the physical dimensions of each specimen with calipers. Following Unosson et al., the total porosity (5%) of samples was determined according to the following equation [Unosson, J. E., et al., An evaluation of methods to determine the porosity of calcium phosphate cements, Biomed. Mater. Res. B Appl. Biomater., 2015, 103, 62—incorporated herein by reference in its entirety]:
The macroporosity was calculated for each group by substituting the average apparent density of the control group composites (no PEG) for the skeletal density in equation (1).
Micro-CT was used to assess the 3D microstructure of tannin-hydroxyapatite and decellularized bone scaffolds (Skyscan 1172, Bruker, Kontich, Belgium). Samples were acquired using: source voltage of 100 kV, a current of 100 μA, and an isotropic pixel size of 10 μm2. Reconstruction of cross sections was done using the NRecon software package (SkyScan, Bruker, Kontich, Belgium). The reconstructed images were binarized using an optimized global threshold and structural analyses were done in the CTAn software package (SkysScan, Bruker, 3 Kontich-Belgium). Three-dimensional reconstructions of the samples were obtained using CTvox (SkyScan, Bruker, Kontich, Belgium). Five samples were analyzed for each synthetic scaffold group, and 30 bone disks were imaged to account for high sample-to-sample variability associated with biological samples.
Tannin-hydroxyapatite resin scaffold samples were fabricated in molds 6 mm diameter×13 mm in length, allowed to set for 24 h and the top and bottom faces were polished to a final length of 12 mm. After subsequently leaching for 48 h at 70° C., 6 mm×12 mm cylinders were autoclaved. The dimensions were recorded with calipers, and the samples were compressed to failure on a Shimadzu autograph AGS-X at 1 mm/min. Analysis software TrapeziumX (Shimadzu, Tokyo, JP) was used to record and analyze data. Decellularized bone scaffolds were compressed to failure at 1 mm/minute. Each group contained 5 samples.
Following Hua et al., [H. Hua, et al., iPSC-derived β cells model diabetes due to glucokinase deficiency, J Clin Invest., 2013 July; 123, 3146—incorporated herein by reference in its entirety], human iPSC line 1013A was generated from dermal fibroblasts obtained from healthy individuals using sendai virus, according to the previously published protocols [Nagamura-Inoue, T. et al., Umbilical cord-derived mesenchymal stem cells: Their advantages and potential clinical utility, World J Stem Cells, 2014, 6, 195—incorporated herein by reference in its entirety] and expanded on mouse embryonic fibroblasts. Prior to mesenchymal differentiation, cells were evaluated for the expression of pluripotency markers. Briefly, cultures were fixed in 10 vol % formaldehyde solution, permeabilized using 0.1% Triton X-100 in DPBS-8662 (DPBS-8662T), blocked with 10% (by volume) donkey serum (Jackson ImmunoResearch Laboratories Inc, West Grove, Pa.) in DPBS-8662T and incubated overnight with primary antibodies (all from STEMGENT, Cambridge, Mass.) against Oct-4 (2.5 μg/mL; cat no. 09-0023), Sox2 (2.5 μg/mL; cat no. 09-0024), and TRA-1-60 (2.5 μg/mL; cat no. 09-0010). Alexa Fluor® secondary antibodies (all from Life Technologies, Waltham, Mass.) were used for detection (2 μg/mL; cat no. A31571, A11057 and A21206), and the nuclei were counterstained with Hoechst 33342 (1:1000 dilution; Life Technologies, Carlsbad, Calif.). Negative control samples were generated omitting the primary antibodies. Fluorescence images were taken with an Olympus IX71 mounted with a Q-Color 3 imaging camera and controlled by Olympus DP-BSW software. Images were processed with ImageJ (National Institutes of Health), and all fluorescence images are represented with pseudo-colors.
Confluent cultures of 1013A at passage 18 (P18) were then coaxed toward the mesenchymal lineage in the presence of induction medium as previously described [Nagamura-Inoue, T. et al., Umbilical cord-derived mesenchymal stem cells: Their advantages and potential clinical utility, World J Stem Cells, 2014, 6, 195—incorporated herein by reference in its entirety] and derived mesenchymal progenitors (1013A-MP) were expanded to P4 on EmbryoMax® gelatin (0.1%; Millipore, Billerica, Mass.)-coated plasticware in medium consisting of KnockOut Dulbecco's Modified Eagle's Medium (KO-DMEM) supplemented with 20% (by volume) HyClone fetal bovine serum (FBS; Thermo Scientific, Waltham, Mass.), 2 mM GlutaMax, 0.1 mM nonessential amino acids, 0.1 mM 3-mercaptoethanol, and 100 U/mL penicillin-streptomycin (all from Life Technologies).
For immunohistochemistry, cell suspensions (25×103 cells in 200 μL DPBS-8662) were spun using the Cytospin 4 (Thermo Scientific) at 1000 rpm for 5 min on SUPERFROST® microscope slides (Thermo Scientific), fixed with 4% PFA for 20 min at room temperature, washed in DPBS-8662, and immunostained for Oct-4, Sox-2, and TRA-1-60 as described before. Nuclei were counterstained with Hoechst 33342 (Sigma). Fluorescence images were taken with an Olympus 1X71 and processed with ImageJ (NIH). All fluorescent images are represented in pseudo-colors.
For flow cytometry analysis, cells were enzymatically detached with trypsin/EDTA (0.25%), filtered through a 70 μm cell strainer (BD Biosciences, Franklin Lakes, N.J.) to obtain a single cell suspension and centrifuged at 1000 rpm for 5 min. Cells were then re-suspended in staining buffer (5×105 cells in 200 μL) consisting of DPBS-8662 containing 0.5% (by volume) bovine serum albumin fraction V (Invitrogen), 100 U/mL penicillin-streptomycin (Invitrogen), 2 mM EDTA (Invitrogen), and 20 mM glucose (Sigma), and stained with a panel of primary antibodies: phycoerythrin-conjugated CD13 (cat. no. 555394), CD29 (cat. no. 555443), CD34 (cat. no. 555822), CD45 (cat. no. 560975), CD49e (cat. no. 555617), CD81 (cat. no. 555676), CD90 (cat. no. 555596), CD151 (cat. no. 556057), Alexa Fluor® 647-conjugated CD31 (cat. no. 558094), fluorescein isothiocyanate-conjugated CD73 (cat. no. 561254) (all from BD Pharmingen, San Diego, Calif.), and phycoerythrin-conjugated CD105 (cat. no. 12-1057-42; eBioscience, San Diego, Calif.). Cells were incubated on ice for 30 min in the dark, followed by washing with staining buffer, and analyzed immediately on the ARIA-IIu™ SOU Cell Sorter (BD Biosciences) configured with a 100 μm ceramic nozzle and operating at 20 psi sheath fluid pressure. Data were acquired and analyzed using the Diva 6.0 software (BD Biosciences). Positive expression of surface markers was determined using isotope controls. Gates were set using 1% in negative controls.
Cultures were screened for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel-CH). Briefly, culture media were centrifuged at 450×g for 5 min, the supernatants collected, and luminescence measured using the Synergy Mx microplate reader and Gen5 software (BioTek, Winooski, Vt.).
For cell seeding and culture on scaffolds, cells were detached at P4 using trypsin/EDTA (0.25%), counted, and suspended in expansion medium at a density of 30×106 cells/mL, and a 40 mL aliquot of the cell suspension was pipetted onto the blot-dried scaffolds in Costar® six-well ultra-low attachment plates (Corning, Corning, N.Y.). For tannin-hydroxyapatite scaffolds Group 1 and 6, two seeding protocols were used in the attempt to increase the yield of attached cells. In protocol 1, a 20 mL aliquot of cell suspension was added on one scaffold side and after 30 min the scaffolds were flipped and an additional 20 mL aliquot of cell suspension was added. Following 30 min, the scaffolds were flipped every 15 min for 2 h, and each time 5 μL of medium was added to avoid drying. In protocol 2, a 20 mL aliquot of cell suspension was added on one scaffold side and then incubated for 1.5 h. Every 15 min, 5 μL of medium was added to avoid drying. The scaffolds were then flipped, a 20 mL aliquot of cell suspension was added, and then the constructs (i.e. bone grafts) were incubated for an additional 1.5 h as described above. For decellularized bone scaffolds, the 40 mL aliquot was pipetted only on one side and every 15 min for 2 h, the scaffolds were flipped to facilitate uniform cell distribution, and 5 μL of medium was added to prevent the cells from drying out.
Following seeding, constructs were incubated overnight in expansion medium and the medium was collected. Non-adherent cells were counted using disposable Kova® Glasstic® slides (Kova International, Garden Grove, Calif.). The amount of attached cells per construct was also estimated 1 day after seeding using the PrestoBlue™ assay (Life Technologies™, Frederick, Md.) and following the manufacturer's instructions. Briefly, 700 μL of osteogenic medium containing 10% of PrestoBlue™ reagent (by volume) was added to each construct and samples were incubated at 37° C. for 2 h. Then, a 200 μL aliquot was transferred to a black, clear, flat bottom 96-well Falcon™ plate (BD, Franklin Lakes, N.J.), and the amount of viable cells determined by measuring the fluorescence intensity at 560/590 nm (excitation/emission) using the Synergy Mx microplate reader and Gen5 software 1.09 (BioTek). Readings were corrected for background fluorescence using expansion media as a control. The constructs were cultured in expansion medium for 3 days and then in osteogenic medium [DMEM (Life Technologies) supplemented with 10% (by volume) HyClone FBS (Thermo Scientific), 1 μM dexamethasone (Sigma), 10 μM β-glycerophosphate (Sigma), 50 μM ascorbic acid-2-phosphate (Sigma) and antibiotic-antimycotic (1×; Life Technologies)] for 7 weeks. All cultures were screened for mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza) as described above.
Cell attachment and survival was investigated via microscopic investigation using the LIVE/DEAD assay (Molecular Probes/Life Technologies). Briefly, constructs were washed in DPBS-8662 and incubated with a solution of calcein AM (2 μM) and ethidium bromide (4 μM) in DPBS-8662 for 1 h and imaged by epifluorescence and confocal microscopy in RPMI medium without red phenol (Lonza). Fluorescence images were taken with an Olympus IX71 as described above. In order to completely image each construct, mosaic pictures were assembled from sequential fluorescent images using ImageJ equipped with MosaicJ and TurboReg plugins (NIH). Confocal images were taken with the microscope Axiovert 200M microscope (Zeiss, Oberkochen, GE) mounted with an LSM 5 Pascal exciter using the LSM 5 Pascal software (Zeiss) under defined settings.
The osteogenic differentiation of 1013A-MP cells cultured on tannin-hydroxyapatite and decellularized bone scaffolds was explored via real-time PCR (RT-PCR) by measuring the expression of genes recognized to play a role during bone formation. After growing the cells for 3 or 7 weeks after seeding, 1 mL of TRIzol (Life Technologies) was added to each scaffold or decellularized bone. The cells were vortex-mixed 3 times (20 sec each) before adding 190 mL of chloroform for extraction. Following centrifugation at 12,000×g for 15 min at 4° C., the aqueous phase was collected, mixed with equal volume of 70% ethanol, and RNA extracted using the RNeasy Mini Kit (Qiagen, Venlo, Nebr.) as above. Purified RNA was quantified and reversely transcribed with random hexamers using the GoScript™ Reverse Transcription System (Promega, Madison, Wis.) according the manufacturer's protocol. RT-PCR was performed using the StepOnePlus PCR System cycler (Applied Biosystems, Waltham, Mass.) in a 20 μL volume reaction using the TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Life Technologies) composed of FAM dye-labeled TaqMan MGB probe and PCR primers for runt-related transcription factor 2 (RUNX2; Hs00231692_m1), collagen, type I, alpha 1 (COL1A1; Hs00164004_m1), alkaline phosphatase (ALPL; Hs01029144_m1), osteopontin (OPN; Hs00959010_m1), platelet-derived growth factor receptor beta (PDGFRB; Hs01019589_m1), and VIC dye-labeled TaqMan MGB probe and primers for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs02758991_g1). Cycling conditions were 95° C. for 10 min followed by 40 cycles of 95° C. for 15 s (denaturation) and 60° C. for 60 s (annealing and extension). The data were analyzed using the StepOne™ v2.2.2 software (Applied Biosystems), and the expression levels of the target genes were normalized to the expression level of GAPDH. The expression of genes of interest was expressed as the fold change over the expression at day zero, i.e. the day when culture in osteogenic media started.
Statistical analysis was conducted using the GraphPad Prism 6 version 6.0e (GraphPad Software Inc, La Jolla, Calif.). Student's t-test was used for single comparisons between two groups. One-way or two-way ANOVA was used for multiple comparisons with a Bonferroni post-hoc test when Gaussian distribution was confirmed using the D'Agostino-Pearson or Shapiro-Wilk normality test. All results are shown as means and ±standard deviations. A difference between the mean values for each group was considered statistically significant when the p value was less than 0.05.
All scaffolds display surface porosities with pore sizes<100 μm, resulting from water present between crystal grains when the tannin-hydroxyapatite resin sets. Scaffolds manufactured with PEG also display porosities with pore sizes>100 μm that vary with the content and size of PEG particles (
PEG is hydrophilic, readily absorbs water, and typically creates a hydration shell containing 1-4 molecules of water per subunit of PEG [Crupi, V. et al., Raman spectroscopic study of water in the poly(ethylene glycol) hydration shell, Journal of Molecular Structure, 1999, 381, 207—incorporated herein by reference in its entirety]. When the size of the PEG particles decreases, the total surface area will commensurately increase for each content category. Water should be getting out of the PTSDP to form a hydration shell on the surface of PEG particles, with smaller particles pulling more water from the reaction than larger ones. For any given PEG content, the use of larger PEG particle sizes (400-600 μm compared to 100-600 μm) reduced the total porosity, which is consistent with the theory that smaller particles pull more water from the reaction, thus increasing the scaffold porosity [Natarajan V., et al., Preparation and properties of tannic acid cross-linked collagen scaffold and its application in wound healing, J Biomed Maler Res B Appl Biomater, 2013, 101, 560—incorporated herein by reference in its entirety]. During the PTSDP dissolution/precipitation reaction, PEG can also directly interfere with the precipitation of dissolved PTSDP onto PEG particles. DSC and XRD analysis was used to verify the leaching of PEG particles from the tannin-hydroxyapatite scaffolds. Calorimetric results show complete leaching of PEG particles after 24 h (
In this study scaffold porosity was investigated via micro computed tomography μCT analysis and pycnometry. In
The overall macroporosity of all tannin-hydroxyapatite scaffolds is shown in
Interestingly, tannin-hydroxyapatite scaffolds manufactured without PEG particles show a compressive strength of 15.18±2.7 MPa, almost twice as much as decellularized bone scaffolds (6.3±1.14 MPa). The significant reduction in compressive strength observed for scaffolds manufactured using PEG particles compared to control samples (no PEG) indicates a relation between the scaffold macroporosity and mechanical properties. Regression analysis was used to estimate the correlation strength between scaffold porosity (as measured by pycnometry) and compressive strength (
Derivation and Characterization of iPSC-MPs
Mesenchymal progenitors were derived from the pluripotent stem cell line 1013A as previously described [de Peppo G. M., et al., Engineering bone tissue substitutes from human induced pluripotent stem cells, Proc Natl Acad Sci USA. 2013, 110, 8680—incorporated herein by reference in its entirety]. Colonies of 1013A cells display typical iPSC morphology and are positive for the pluripotency markers OCT4, SOX2, and TRA-1-60 as shown in FIG. 9A-
Attachment, Viability and Osteogenic Differentiation of iPSC-MPs
Scaffold features such as composition, porosity, shape, pore size, interconnectivity, and mechanical properties are all known to affect cell attachment, viability, and differentiation. In order to achieve a high yield of attached cells following seeding, two protocols were established and tested in this study. The number of attached cells dispersed across the entire surface of the scaffolds is much higher when using protocol 2 as shown in
However, no studies exist on the effects of macro PTSDP scaffolds on the osteogenic differentiation of human iPSC-MPs. Differentiation toward the osteogenic lineage is associated with the controlled up-regulation of specific genes that regulate the differentiation process and/or play a role in the formation of new tissue [Gowri, A. M., et al., Foetal stem cell derivation & characterization for osteogenic lineage, Indian J Med Res, 2013, 137, 308—incorporated herein by reference in its entirety]. Also, master transcription factors can control the expression of downstream genes in the early phase of osteogenic differentiation including COL1A1 and OPN.
Other important markers of osteogenic differentiation are the metalloenzyme ALPL, the tyrosine kinase PDGFRB, and RUNX2 [de Peppo G. M., et al., Engineering bone tissue substitutes from human induced pluripotent stem cells, Proc Natl Acad Sci U.S.A, 2013, 110, 8680; Gulseren, G., et al., Alkaline Phosphatase-Mimicking Peptide Nanofibers for Osteogenic Differentiation, Biomacromolecules 2015, 16, 2198—each incorporated herein by reference in its entirety]. Differentiation toward the osteogenic lineage was studied via real-time PCR as shown in
Instead of scaffolds based mainly on calcium phosphate, scaffolds based on porous tannin spray-dried powder (PTSDP) have been developed here for use in clinical settings as a bone graft and for a variety of orthopedic applications. Interfacing PTSDP scaffolds with bone-forming cells holds the potential to enhance their healing properties, and a few studies have recently reported such attempts using non-porous scaffold configurations. However, scaffold porosity is critical to enable tissue formation in vitro and facilitate bone replacement following implantation. In this invention, macroporous PTSDP scaffolds were developed with an interconnected porosity that mimics the bone micro-environment, with an objective to engineer bone grafts with enhanced healing properties. Scaffold porosity could be tuned by varying the total amount and particle size of the non-toxic PEG porogen. While PEG has been used as a porogen in hydrogel and polymeric scaffolds, this invention is the first report of ceramic scaffolds with PEG as a macroporous porogen tested in vitro. The results show that macro PTSDP scaffolds with defined structural parameters and mechanical properties can be fabricated using our engineering approach based on a moldable tannin-hydroxyapatite paste using PEG particles as a porogen. These macroporous scaffolds support cell attachment, viability, and osteogenic differentiation of iPSC-derived osteocompotent cells, thereby opening the possibility to grow functional bone grafts of clinical relevance for autologous dental and orthopedic applications. PTSDP scaffolds with an advantageous combination of porosity and mechanical properties could also be used to develop patient-specific bone grafts as experimental platforms to study bone development and pathology, screen new drugs, and test implant materials within a context that highly reflects the native tissue.
