Surface enhanced demineralized bone graft material

Abstract

Described herein are compositions comprising a BMP-2 derived peptide conjugated to albumin for use in bone grafts. The composition may further include bone matrix, such as demineralized bone matrix (DBM).

Claims

1. A demineralized bone matrix (DBM) composition comprising: DBM; and a surface coating on the DBM of a macromolecular-peptide structure, wherein the macromolecular-peptide comprises a peptide conjugated to a protein at a ratio of about 10 peptides to 1 protein, and results in bone growth in vivo and mimics naturally occurring bone morphogenic protein (BMP)-2, wherein the macromolecular-peptide is the active ingredient, wherein the peptide is of SEQ ID NO:6, and wherein the protein is albumin.

2. The DBM composition of claim 1, wherein the peptide has Cysteine at the N-terminus to facilitate covalent conjugation.

3. The DBM composition of claim 2, wherein Cysteine residue is followed by a flexible spacer.

4. The DBM composition of claim 3, wherein the flexible spacer comprises one or more amino acid residues.

5. The DBM composition of claim 4, wherein the flexible spacer comprises the amino acid glycine.

6. The DBM composition of claim 1, wherein the peptide is amidated at the C-terminus for protection.

7. The DBM composition of claim 1, wherein the peptide concentration is from 10 micrograms to 100 milligrams per cubic centimeter.

8. The DBM composition of claim 1, having an average particle size ranging from 10 microns to 10 mm.

9. The DBM composition of claim 1, wherein the composition comprises fibers ranging from 10 microns in diameter to 4 mm in diameter.

10. The DBM composition of claim 1, wherein the composition comprises fibers with lengths from 20 microns to 200 mm.

11. The DBM composition of claim 1, wherein the composition comprises fibers and particles.

12. The DBM composition of claim 1, further comprising one or more of bone graft/scaffold material selected from allograft or autograft bone; cancellous bone; untreated DBM; bone marrow aspirate; a synthetic calcium phosphate; blood or blood-derived concentrates; gelatin; PEG; pluronic; glycerol; and carboxymethyl cellulose (CMC).

13. The DBM composition of claim 1, wherein the percentage, by weight, of DBM in the composition is 5% to 99.5% of a total weight of the composition.

14. The DBM composition of claim 1, further comprising a biodegradable polymer.

15. The DBM composition of claim 1, further comprising one or more growth factors.

16. The DBM composition of claim 15, wherein the growth factor is recombinant BMP-2.

17. The DBM composition of claim 1, further comprising one or more of stem cells, platelet rich plasma (PRP) blood, calcium phosphates, calcium sulfates, calcium carbonate, synthetic growth factors, natural growth factors, collagen, and fibrin.

18. The DBM composition of claim 1, wherein the composition comprises a collagen sponge.

19. The DBM composition of claim 1, wherein the DBM is derived from an animal or human.

20. The DBM composition of claim 19, wherein the DBM is derived from a canine.

21. The DBM composition of claim 19, wherein the DBM is derived from a human.

22. A method of manufacturing the DBM composition of claim 1, comprising: conjugating a plurality of peptides of SEQ ID NO:6 to albumin at a ratio of about 10 peptides to 1 albumin to create macromolecular-peptides; combining the macromolecular peptides with a DBM; coating the surface of the DBM with the macromolecular-peptides; and thereby creating a bone repair composition for supporting bone growth in vivo that mimics naturally occurring BMP.

23. A method of repairing a damaged bone or a defect in a bone of a subject, the method comprising: applying the DBM of claim 1 to the bone of the subject.

24. The method of claim 23, wherein the subject is a human.

25. The method of claim 23, wherein the subject is a mammal.

26. The method of claim 23, wherein the subject is a canine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee

(2) FIG. 1 depicts the basic peptide design FIG. 2 depicts the albumin-peptide complex. Schematic diagram of the Albumin-peptide complex based on the globular nature of the Albumin molecule.

(3) FIG. 3 depicts the albumin-peptide-cell complex. Schematic diagram of Albumin-peptide complex interacting with BMP Receptors on a single cell. P: peptide, R1: Type I receptor, R2: Type II receptor.

(4) FIG. 4 depicts human BMP-2 chain sequence, with contiguous region highlighted.

(5) FIG. 5 depicts the albumin-collagen complex (Albumin: globular shape dimer molecules colored in blue and green).

(6) FIG. 6 is a graph of in-vitro Osteoinductivity (OI) scores based on ALP measurements.

(7) FIG. 7 depicts radiographs from a canine maxillary plate cyst case, specifically a case of radicular/periapical cyst with maxillary plate resorption XSS 208; filled with AAPC graft and covered with membrane.

(8) FIG. 8 depicts radiographs from a canine mandibular fracture case. Specifically, a case of bilateral mandibular fixation; separation of the rostral segment (fight trauma); Mandibular reconstruction; AAPC graft in alveoli. Additional AAPC graft was added across fracture at 3 days because patient returned with traumatic re-opening of repair.

(9) FIG. 9 depicts radiographs from a canine non-union case, specifically, a case of femoral fracture repair (neglected non-union) debrided and grafted with AACP.

(10) FIG. 10. depicts radiographs from a feline comminuted fracture repair, specifically, a case of comminuted fracture repair filled with the AAPC graft.

(11) FIG. 11. depicts time to heal (TTH) comparisons to rhBMP2 for Long Bone, Mandibular Defect, and Sinus Augmentation studies.

DETAILED DESCRIPTION

(12) The peptide sequence is derived from Bone Morphogenetic Protein-2. The sequence is modified on the N terminal to allow for conjugation to Albumin (recombinant form). Cysteine residue (C) is added on the peptide's N-terminal to allow for covalent conjugation on the Albumin's Lysine residues (FIG. 1). The activated Albumin has reactive maleimide groups on its surface available for conjugation with a sulfhydryl containing peptide to form stable thioether bonds. Briefly, Albumin is reacted in isolation with a crosslinker to allow the NHS-ester end of Sulfo-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) to attach at several available primary amines (side chain of lysine) on the protein surface. This reaction creates maleimide-activated Albumin, namely Albumin molecules that are labeled with several sulfhydryl-reactive maleimide groups.

(13) The Cysteine residue is followed by three Glycine (G) residues that work as a flexible spacer. The spacer is followed by the BMP-2-derived sequence (FIG. 1). This spacer is critical to allow for free movement of the peptide so that it can bind the receptor with a high affinity. Each Albumin molecule was engineered to carry at least 10 peptides (FIG. 2).

(14) The surface of the bone allograft material (DBM and allograft bone chips) was coated with the Albumin-peptide complex. Effective doses can range from 100 microgram per CC to 100 milligram per CC, or about 0.1-100×10{circumflex over ( )}5 molecules per mm.sup.2.

Design Logic

(15) The ideal design should have a peptide that can induce BMP receptor signaling, and can naturally immobilize/localize on the graft surface. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. The dimer nature (i.e. multivalence) of naturally occurring BMPs is capable of receptor oligomerization.

(16) Physiologically, BMP interactions with extracellular matrix (ECM) components facilitate localized and spatially regulated signaling; therefore, we reasoned that the lack of ECM binding in the current, clinically used forms of BMP-2 (such as INFUSE) could underlie the limited translation.

(17) Affinity for Receptor Binding:

(18) We analyzed the three dimensional structure of human BMP-2 and its receptor complex (2GOO) (BMP-2 dimer in complex with Type I and Type II dimers; at rcsb.org/pdb/explore/explore.do?structureld=2goo). The protein-protein interface was analyzed using a PDBePISA server (ebi.ac.uk/pdbe/pisa/). The objective was to find a contiguous region that has bonds (hydrogen bonds or salt bridges) with both types of the receptors. There was only one contiguous region (70-109) that has this unique advantage (Table 1, Table 2, and FIG. 4). We therefore designed sequences derived from this region that can bind Type I and Type II receptors and induce the needed oligomerization when presented correctly

(19) TABLE-US-00001 TABLE 1 Hydrogen bonds between Type I receptor and BMP-2. Chains E and B are Type I receptors. Chains A and D are the ligands (BMP-2). Distance (Dist) is reported in Angstrom (A). Yellow highlights demonstrate the residues of interest within BMP-2 chains Receptor I Dist. [Å] Ligand E:GLN 86[NE2] 2.98 D:LEU 51[O] E:THR 55[OG1] 2.56 D:ASP 53[OD2] E:GLN 94[N] 2.81 D:SER 69[O] E:ARG 97[NH2] 2.99 D:SER 69[OG] E:GLN 86[OE1] 2.63 D:LEU 51[N] E:CYS 77[O] 2.93 D:ASP 53[N] E:HIS 43[O] 3.14 D:HIS 54[NE2] E:GLU 81[OE1] 3.43 D:ASN 59[ND2] E:GLN 94[OE1] 3.73 D:SER 72[N] E:GLN 94[OE1] 3.39 D:SER 72[OG] B:SER 90[OG] 2.48 A:VAL 26[O] B:LYS 92[NZ] 3.08 A:SER 24[O] B:ASP 84[OD1] 2.68 A:TYR 103[OH] B:ASP 84[OD1] 3.33 A:TYR 91[OH] B:ASP 89[O] 2.81 A:TRP 28[NE1] E:SER 90[OG] 2.60 D:VAL 26[O] E:LYS 92[NZ] 3.07 D:SER 24[O] E:TYR 80[OH] 3.43 D:LYS 101[NZ] E:ASP 84[OD2] 2.83 D:LYS 101[NZ] E:ASP 84[OD2] 2.67 D:TYR 103[OH] E:ASP 84[OD2] 3.19 D:TYR 91[OH] E:ASP 89[O] 2.87 D:TRP 28[NE1]

(20) TABLE-US-00002 TABLE 2 Hydrogen bonds between Type II receptor and the ligand (BMP-2). C and F are the Type II receptor chains. A and D are the ligand (BMP-2) chains. Distance (Dist) is reported in Angstrom (A). Receptor II Dist. [Å] Ligand C:LYS 37[NZ] 3.13 A:GLU 109[OE2] C:LEU 61[N] 2.79 A:SER 88[OG] F:LYS 37[NZ] 3.32 D:SER 85[OG] F:LYS 37[NZ] 2.92 D:GLU 109[OE1] F:LYS 56[NZ] 2.59 D:VAL 98[O] F:LEU 61[ N] 2.88 D:SER 88[OG]

(21) We further modeled one of the selected peptides and generated three dimensional complexes of the peptide and the receptors (Type I and Type II). The data demonstrated that the selected sequence has high and specific affinity to bind both receptors (Table 3).

(22) TABLE-US-00003 TABLE 3 Protein-protein interface data of the peptide-receptor complexes. P-value less than 0.5 indicates specific binding between the two structures. Receptor II Receptor 1 Surface A 640 669.8 Energy −9.8 −5.6 P-value 0.313 0.310 HB 4 8 SB 0 4
Receptors Oligomerization and Signaling:

(23) Multivalent binding allows for receptors' oligomerization which is needed to initiate receptor signaling (FIG. 3). We therefore conjugated multiple peptides per Albumin molecule (for example about 10 peptides per single Albumin molecule). Successful conjugation at this ratio was confirmed through the appropriate laboratory methods, using for example without limitation, high performance liquid chromatography (HPLC). Albumin was selected for various reasons including, but not limited to, the following: Large number of Lysine residues (about 62 per molecule; see jbc.org/content/239/3/850.full.pdf): This advantage increases the probability for successful conjugation at the desired ratio (e.g., 10 peptides per single Albumin molecule) and allows for multivalent binding between the ligand and the receptor. Globular shape: The globular nature of Albumin is a great advantage in this design because it allows for close proximity between the peptide ligands. Close proximity aids successful receptor oligomerization and signaling (Sugio, S.; Kashima, A.; Mochizuki, S.; Noda, M.; Kobayashi, K. (1 Jun. 1999). “Crystal structure of human serum albumin at 2.5 A resolution”. Protein Engineering Design and Selection. 12 (6): 439-446). The combination of close proximity between the peptide ligands as well as multi-valent design make this macromolecule comparable to the naturally occurring growth factor ligand. Albumin's high molecular weight (about 65 Kilo Dalton before conjugation): High molecular weight may increase the effectiveness of ligand receptor interaction and/or prolong the interaction duration. Additionally adding a large molecule like Albumin on the N-terminal may help the stabilization of the three dimensional structure of the peptide so that it can mimic the parent molecule. Hydrophobic domains: Albumin has many hydrophobic domains which may facilitate the interaction/adhesion with cell surface Albumin is a clinically safe carrier

(24) In conclusion, the conjugation to Albumin was specifically engineered to mimic the natural ligand receptor interaction mode.

(25) Extracellular Matrix Immobilization/Localization:

(26) Albumin has another great advantage because it has a high binding affinity to collagen which is a major component of demineralized bone matrix (DBM).

(27) We developed a three dimensional model for the Albumin-collagen complex (FIG. 5). We then analyzed the protein-protein interface. Data demonstrated that Albumin has a high affinity to collagen that includes a large interface area and high energy: surface area of about 2213 Å.sup.2; energy of −22.5 ΔiG kcal/mol; with 4 hydrogen bonds; and 1 salt bridge.

(28) This natural immobilization amplifies the effectiveness of ligand-receptor binding which translates into higher levels of osteoinductivity. Amplified effectiveness could be attributed to prolonged ligand-receptor binding and the lack of or reduced receptor internalization. Prolonged signaling is an advantage when compared to soluble recombinant growth factors such as INFUSE (soluble rhBMP) which has a short half-life.

(29) Natural immobilization confers on this design several safety advantages over soluble forms of growth factors. Soluble factors can easily escape the graft site and cause unwanted outcomes such as hetero-topic ossification, radiculopathy, or seroma and swelling.

(30) Preparation of a Bone Graft Containing the DBM/Peptide Complex:

(31) The albumin/peptide complex (APC) can be applied to a collagen graft (such as a collagen sponge made from bovine tendon) or preferably to DBM of animal or human origin. The APC-modified DBM or collagen can be used “as is” as a bone graft. It can be mixed with any other bone graft material either in the manufacturing plant or at the time of surgery. Such materials can include, for example, unmodified DBM, unmodified collagen, allograft or autograft bone; blood, PRP, stem cells, calcium phosphates, calcium sulfates, calcium carbonate, and natural or synthetic growth factors. The physical properties can be changed to a putty or an injectable by the addition of suitable viscosity modifiers, for example polymers such as Pluronic, CMC (carboxymethyl cellulose), PVA (poly vinyl alcohol); glycerol, gelatin, collagen. These materials can be added individually or in combination.

(32) The modified DBM/collagen may also be incorporated into a complex with one or more biodegradable polymers, for example Poly(lactic acid)/Poly(glycolic acid) (PLA/PGA), in order to make a potentially load bearing graft.

(33) The modified DBM/collagen is preferably aseptically processed to preserve the maximum amount of activity. It is possible, however, to terminally sterilize the modified graft product with the understanding that this process may reduce the biological activity, for example without limitation by exposure to e-beam or gamma-radiation in the presence of dry ice.

(34) List of Peptide Sequences

(35) A-BMP-Derived Peptide/Linear/Synthetic

(36) TABLE-US-00004 Seq ID No. 1:  CGGGVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVE Seq ID No. 2:  CGGGKIPKASSVPTELSAISTLYL Seq ID No. 3: CGGGSAISMLYLDENEKVVLKNYQDMVVE Seq ID No. 4:  CGGGKIPKAAAVPTELSAISMLYLDENEKVVLKNYQDMVVE Seq ID No. 5:  CGGGKIPKAAAVPTELSAISTLYL Seq ID No. 6:  CGGGKIPKAGSVPTELSAISTLYL Seq ID No. 7:  CGGGKIPKASGVPTELSAISTLYL Seq ID No. 8:  CGGGKIPKAGGVPTELSAISMLYLDENEKVVLKNYQDMVVE Seq ID No. 9:  CGGGKIPKAGSVPTELSAISMLYLDENEKVVLKNYQDMVVE Seq ID No. 10:  CGGGKIPKASGVPTELSAISMLYLDENEKVVLKNYQDMVVE Seq ID No. 11:  CGGGVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVE Seq ID No. 12:  CGGGKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVE
Sequence of B-Human Serum Albumin/Recombinant

(37) TABLE-US-00005 Seq ID No. 13: MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKAL VLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHT LFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPR LVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKF GERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEM PADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVV LLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQ NCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSK CCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLV NRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTA LVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKK LVAASQAALGL

(38) It is well known in the art that some modifications and changes can be made in the structure of a peptide, such as those described herein, without substantially altering the structure or sequence of that peptide, and still obtain a biologically active equivalent. In one aspect, disclosed compositions, peptides, and proteins may possess amino acid sequences that differ from the sequences disclosed herein (especially SEQ ID NOs: 1-14). In some embodiments, the amino acids may be naturally occurring or synthetic amino acids well known to those of skill in the art. In some embodiments, these differences may be accomplished by conservative amino acid substitutions of the disclosed sequences. As used herein, the term “conservative amino acid substitutions” refers to the substitution of one amino acid for another at a given location in the protein, where the substitution can be made without substantial loss of the relevant function (e.g. growth factor activity, receptor binding, etc.). In making such changes, substitutions of like amino acid residues can be made by one of skill in the art on the basis of relative similarity of side-chain substituents, for example, size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the protein by routine testing.

(39) In some embodiments, the disclosed peptides and proteins may have sequence identity greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, and 65%.

(40) Previous attempts to use BMP derived peptides suffered from several shortcomings (mentioned above) that made it difficult for them to mimic the natural growth factor behavior. These shortcomings made it difficult to obtain reliable and reproducible effects.

(41) In many embodiments, the disclosed compositions may be useful in procedures involving various bone and/or soft tissue grafting, for example wherein there may be an exposed collagen surface that may be intended to contact and/or bind bone. For example, bone, ligament, and tendon grafts may be performed with the disclosed composition. In many embodiments, failure of a procedure may be caused by the ends of the tendon not attaching properly to bone, and the disclosed compositions are useful in allowing such tissues to attach to bone.

EXAMPLES

Example 1; Preparation of the Graft Material

(42) Materials and Methods:

(43) All production operations are carried out aseptically in an ISO 6 (class 1000) clean room. DBM is made from canine cortical bone sourced from cadavers. For the study examples detailed below in this document, the source bone was canine bone. The bone is cleaned of all soft tissue, the cancellous ends are removed, and the bone is broken down into shards from which the marrow and other cellular elements are removed by purging with multiple water washes and exposure to other reagents such as antibiotics, alcohol and/or hydrogen peroxide. The bone is ground and sieved to the desired particle size (for example: 125-710 microns), then demineralized with a 0.6N HCl solution resulting in a residual calcium of <8%. After demineralization, the DBM is buffered with phosphate buffered saline, rinsed with water and either packaged directly for lyophilization or stored frozen until it can be packaged for lyophilization. Cancellous bone chips (which may be later mixed with DBM) are made from available cancellous bone (e.g., metaphyseal bone from the ends of the shafts used to make DBM). The marrow elements are removed from coarsely ground cancellous bone with multiple water washes and reagents as described above. The cleaned cancellous chips are then ground and sieved to the desired size range which may be the same or different than the corresponding DBM particles. The chips are either packaged directly for lyophilization or stored frozen until they can be packaged for lyophilization. If the allograft carrier is to be a mixture of DBM and cancellous bone chips they may be mixed prior to or after lyophilization. Typically, though non-exclusively, DBM and cancellous chips are mixed in an approximate 50/50 ratio by volume. All bone is stored lyophilized until treated with the peptide.

(44) Recombinant human serum Albumin was obtained from Albumin Biosciences (see albuminbio.com/products.php?id=4). Peptide manufacturing and conjugation were custom manufactured at the laboratories of Bio-Synthesis (at biosyn.com). Briefly, Albumin is reacted in isolation with a crosslinker to allow the NHS-ester end of Sulfo-SMCC to attach at several available primary amines (e.g., the side chain of lysine) on the protein's surface. This created maleimide-activated Albumin. Specific peptide was combined with activated Albumin and mixed, allowing specific, linking between the maleimide group (on Albumin) and the Cysteine residue at the peptide. Albumin-peptide complex solutions were prepared at different doses sterilized by filtration and then mixed with the bone graft (about 1 ml per 1 CC graft material) for two hours at room temperature. The mixture was then incubated at 0-10 degree Celsius overnight to allow for maximum binding. On day 2, the mixture was removed from refrigerated storage and packaged for aseptic freeze-drying. At the end of freeze drying cycles, the aseptically lyophilized bone graft was vacuum sealed in impervious packaging until ready for final packaging into individual doses. At aseptic final packaging, the vacuum-sealed packets were opened and the required doses were quickly dispensed into the appropriate packaging, vacuum sealed and suitable for presentation to a sterile field for single patient use. Microbial cultures, samples for osteoinductivity and other test samples were also obtained at various stages of processing to document progress and for quality assurance purposes. These final-packaged peptide-augmented grafts and representative samples were then stored at room temperature until needed for in vivo and in vitro studies.

Example 2; Testing the Graft Osteoinductivity In Vitro

(45) Materials and Methods:

(46) The graft material described in Example 1 was examined for effectiveness in vitro. Control (non-coated allograft) and investigational particles (Allograft coated with Albumin-peptide complex; AAPC) were tested for inductivity using a modified Alkaline Phosphatase Assay (ALP; see Han, B., Tang, B., & Nimni, M. E. (2003). Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix. Journal of orthopaedic research, 21(4), 648-654). Briefly, C2C12 cells were cultured on the particles for 72 hours in serum-free medium and then submitted to ALP assay. The ALP assay includes the steps of: washing particles with 1×PBS twice; lysis of cells with 200 μl homogenized buffer; freeze-thaw cycle three times; transfer 30 μL of sample lysate into 96-well plate; addition of 100 μL PnPP substrate solution; incubation at 37° C. for 30 min; and then reading the plate at 405 nm and recording data. The OI Score Calculation Formula is 01=(ALP test−ALP neg)/ALP neg. This in-vitro ALP method is based on a validated mode I for assessment of 01. It is based on the ability of inductive agents to transform mesenchymal stem cells into osteoblasts in serum-free medium. To establish an expected range for an OI score in this assay for a positive control, rhBMP-2 was added to the cells, and this was comparable to the expected range for BMP-2 in the published literature.

(47) Results:

(48) AAPC had significantly higher OI when compared to the allograft controls (p-values=0.000). The OI of AAPC was similar to the OI of positive control cultures (i.e., BMP-2 treated cells). See FIG. 6. The data show that these surface modifications can result in substantial increases in standard markers of OI in vitro. When cells were cultured in the dish but not on the particles, the OI signal was strongly attenuated (data not shown), indicating that the majority of the induced activity occurred when the cells were in close proximity to the signal on the surface of the particles. This indicates that the presently claimed design accelerates bone healing locally without the risk of signaling molecules leaving the site. See FIG. 6.

Example 3; Testing the Effectiveness and Safety In Vivo: Sinus Augmentation Cases

(49) Materials and Methods:

(50) The graft material described in Example 1 was further tested for effectiveness and safety in compassionate-use canine cases (with expected difficulties) in need of sinus or ridge augmentations. (These and in the in vivo examples described below were considered compassionate-use because in the user surgeon's estimation these were severe cases of bone loss, trauma, mal- or non-unions and other cases that came with significant expected difficulties for which other available options to preserve or restore function were not certain to produce successful results.) Standard surgical procedures were used at the discretion of the treating surgeons. The animals received one or more (0.5 CC) units of AAPC graft and were followed up until treatment outcomes had been determined. Follow up techniques included radiography and medical assessment by the treating physicians (see FIG. 7).

(51) Results:

(52) None of the cases had device-related events. All cases had accelerated and robust bone formation as early as two weeks. The quality of bone and soft tissue were judged to be excellent, and the success rate was 100%. Average clinical and radiographic healing was 12.2 weeks. A representative case is presented in FIG. 7.

Example 4; Maxillary Facial Reconstruction Cases

(53) Materials and Methods:

(54) The graft material described in Example 1 was further tested for effectiveness and safety in compassionate-use canine cases (with expected difficulties as described above) in animals with maxillary facial trauma. Standard surgical procedures were used at the discretion of the treating surgeons. The dogs received one or more (0.5 CC) units of AAPC graft and were followed up until treatment outcomes had been determined. Follow up techniques included radiography and medical assessment by the treating physicians.

(55) Results:

(56) A case of premaxilla reconstruction and two canine cases of mandibular fracture received AAPC graft due to expected clinical challenges. Despite the challenging conditions, successful healing signs were observed as early as 6 days post-op. Average time to heal (TTH) for the mandible cases was 10.5 weeks. See example in FIG. 8.

Example 5; Non-Union Cases

(57) Materials and Methods:

(58) The graft material described in Example 1 was further tested for effectiveness and safety in compassionate-use canine cases (with expected difficulties as described above) in animals with long bone non-unions. Standard surgical procedures were used at the discretion of the treating surgeons. The dogs received one or more (0.5 CC) units of AAPC graft and were followed up until treatment outcomes had been determined. Follow up techniques included radiography and medical assessment by the treating physicians.

(59) Results:

(60) Eight non-union canine cases received AAPC graft and were evaluated until completely healed. All cases had multiple risk factors that can seriously interfere with bone healing. The average TTH was 10.25 weeks. See example in FIG. 9.

Example 6: Other Compassionate Cases

(61) The graft material described in Example 1 was further tested for effectiveness and safety in compassionate-use canine cases (with expected difficulties as described above) in animals with miscellaneous injuries including acute fracture (1 case), comminuted fractures (3 cases), joint fusion, and TPLO revision (3 cases). Standard surgical procedures were used at the discretion of the treating surgeons. The dogs received one or more (0.5 CC) units of AAPC graft and were followed up until treatment outcomes had been determined. Follow up techniques included radiography and medical assessment by the treating physicians.

(62) Results:

(63) Average TTH are available in Table 5. A representative case of a comminuted fracture is seen in FIG. 10.

Example 7, Summary of Clinical Experience

(64) TABLE-US-00006 TABLE 4 High-level summary of Time-to-Heal (TTH) in various indications Time to Healing Procedure Number of (TTH, Mean), Acute fracture 1 12 Comminuted Fractures 3 8.3 Joint Fusion 1 6 Non-union 8 10.25 TPLO revision 3 15 Sinus augmentation 10 12.2 Maxillary reconstruction 1 5 Mandibular 2 10.5

(65) No device-related events were observed in any of the cases. The accelerated robust new bone formation was reported in all cases and the time needed for healing was not inferior to historical BMP-2 data from similar cases (Example 8). These preliminary data are very encouraging and suggest a potential alternative for autograft and BMP-2.

Example 8; Time to Heal (TTH); Comparison to rhBMP-2 (Historical Data in Animals)

(66) BMP's historical data were based on Cory B. et al., CVJ/VOL 53/JULY 2012. We compared TTH data using Bayesian simulation approach. Data are expressed as credible intervals of TTH. AAPC's TTH is short and not inferior to BMP's healing time FIG. 11 (below).

(67) While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the disclosed compositions and methods are capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present disclosure. Accordingly, the detailed description is to be regarded as illustrative in nature and not restrictive.

(68) All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each was included at its citation, in its entirety. In case of conflict between reference and specification, the present specification, including definitions, will control.

(69) Although the present disclosure has been described with a certain degree of particularity, it is understood the disclosure has been made by way of example, and changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims.