PEPTIDE-COATED CALCIUM PHOSPHATE PARTICLES

Abstract

The invention features methods and compositions for (i) controlling the amount of peptide bound to calcium phosphate particles; and (ii) for tightly binding peptide to the surface of calcium phosphate particles. The methods and compositions can be useful for preparing implants useful for promoting bone deposition at the site of implantation, and for repairing a variety of orthopedic conditions.

Claims

1.-4. (canceled)

5. A composition comprising hydroxyapatite particles coated with P-15 peptide, wherein the P-15 peptide is tightly bound to the surface of the hydroxyapatite particles and wherein the concentration of P-15 peptide bound to the surface of the hydroxyapatite particles is from 260 to 1,200 ng of P-15 per gram of hydroxyapatite particles.

6. The composition of claim 5, wherein said hydroxyapatite particles are anorganic bone mineral particles.

7.-8. (canceled)

9. A method for coating calcium phosphate particles with a peptide, said method comprising: (i) combining the calcium phosphate particles with a first aqueous salt solution having an osmolarity value greater than 290 mOsm and a pH of between 6.5 and 8.5 to produce pretreated calcium phosphate particles; (ii) combining the pretreated calcium phosphate particles with a second aqueous salt solution containing the peptide and having an osmolarity value greater than 290 mOsm and a pH of between 6.5 and 8.5 to produce coated calcium phosphate particles; and (iii) separating the coated calcium phosphate particles from the second aqueous salt solution.

10. A method for coating calcium phosphate particles with a peptide, said method comprising: (i) providing pretreated calcium phosphate particles prepared by combining the calcium phosphate particles with a first aqueous salt solution having an osmolarity value greater than 290 mOsm and a pH of between 6.5 and 8.5; (ii) combining the pretreated calcium phosphate particles with a second aqueous salt solution containing the peptide and having an osmolarity value greater than 290 mOsm and a pH of between 6.5 and 8.5 to produce coated calcium phosphate particles; and (iii) separating the coated calcium phosphate particles from the second aqueous salt solution.

11. The method of claim 9, wherein the first aqueous salt solution or the second aqueous salt solution comprise HEPES buffer, TAPS buffer, Bicine buffer, Tris buffer, TAPSO buffer, TES buffer, MOPS buffer, PIPES buffer, or phosphate buffer.

12. The method of claim 9, wherein the first aqueous salt solution or the second aqueous salt solution comprises from 300 to 600 mM NaCl.

13. The method of claim 9, wherein the first aqueous salt solution or the second aqueous salt solution has an osmolarity value of between 400 and 1,200 mOsm.

14.-23. (canceled)

24. The method of claim 9, wherein the peptide is a cell adhesion peptide selected from: TABLE-US-00008 (SEQ ID NO. 1, ″P-15″) Gly-Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg- Gly-Val-Val, (SEQ ID NO: 2) Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, (SEQ ID NO: 3) Gln-Gly-Ile-Ala-Gly-Gln, (SEQ ID NO: 4) Gln-Gly-Ile-Ala-Gly-Gln-Arg, (SEQ ID NO: 5) Phe-Gly-Ile-Ala-Gly-Phe, (SEQ IID NO: 6) Gly-Ile-Ala-Gly-Gln, (SEQ ID NO: 7) Gln-Gly-Ala-Ile-Ala-Gln, (SEQ ID NO: 8) Phe-Gly-Ile-Ala-Gly-Phe, (SEQ ID NO: 9) Cys-Gly-Ile-Ala-Gly-Cys, (SEQ ID NO: 10) Glu-Gly-Ile-Ala-Gly-Lys, (SEQ ID NO: 11) N-Acetyl Ile-Ala-Ala, (SEQ ID NO: 12) Ile-Ala-.beta.Ala, (SEQ ID NO: 13) N-Acetyl Ile-Ala NMe, (SEQ ID NO: 14) Asp-Gly-Glu-Ala, (SEQ ID NO: 15) Asp-Gly-Glu-Ala-Gly-Cys, (SEQ ID NO: 16) Gly-Phe-Pro*-Gly-Glu-Arg, (SEQ ID NO: 17) Gly-Leu-Pro*-Gly-Glu-Arg, (SEQ ID NO: 18) Gly-Met-Pro*-Gly-Glu-Arg, (SEQ ID NO: 19) Gly-Ala-Ser-Gly-Glu-Arg, (SEQ ID NO: 20) Gly-Leu-Ser-Gly-Glu-Arg, and (SEQ ID NO: 21) Gly-Ala-Pro*-Gly-Glu-Arg, wherein Pro* is hydroxyproline.

25.-38. (canceled)

39. A method for coating calcium phosphate particles with a peptide, said method comprising: (i) producing pretreated calcium phosphate particles by (a) combining the calcium phosphate particles with a first aqueous salt solution having an osmolarity value greater than 290 mOsm and a pH of between 6.5, (b) freezing said first aqueous salt solution, and (c) drying the calcium phosphate particles; (ii) combining the pretreated calcium phosphate particles with a second aqueous salt solution containing the peptide and having an osmolarity value greater than 290 mOsm and a pH of between 6.5 and 8.5 to produce coated calcium phosphate particles; and (iii) separating the coated calcium phosphate particles from the second aqueous salt solution.

40. A method for coating calcium phosphate particles with a peptide, said method comprising: (i) providing pretreated calcium phosphate particles prepared by (a) combining the calcium phosphate particles with a first aqueous salt solution having an osmolarity value greater than 290 mOsm and a pH of between 6.5 and 8.5, (b) freezing said first aqueous salt solution, and (c) drying the calcium phosphate particles; (ii) combining the pretreated calcium phosphate particles with a second aqueous salt solution containing the peptide and having an osmolarity value greater than 290 mOsm and a pH of between 6.5 and 8.5 to produce coated calcium phosphate particles; and (iii) separating the coated calcium phosphate particles from the second aqueous salt solution.

41. The method of claim 39, wherein the first aqueous salt solution or the second aqueous salt solution comprise HEPES buffer, TAPS buffer, Bicine buffer, Tris buffer, TAPSO buffer, TES buffer, MOPS buffer, PIPES buffer, or phosphate buffer.

42. The method of claim 39, wherein the first aqueous salt solution or the second aqueous salt solution comprises from 300 to 600 mM NaCl.

43. The method of claim 39, wherein the first aqueous salt solution or the second aqueous salt solution has an osmolarity value of between 400 and 1,200 mOsm.

44.-53. (canceled)

54. The method of claim 39, wherein the peptide is a cell adhesion peptide selected from: TABLE-US-00009 (SEQ ID NO. 1, ″P-15″) Gly-Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg- Gly-Val-Val, (SEQ ID NO: 2) Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, (SEQ ID NO: 3) Gln-Gly-Ile-Ala-Gly-Gln, (SEQ ID NO: 4) Gln-Gly-Ile-Ala-Gly-Gln-Arg, (SEQ ID NO: 5) Phe-Gly-Ile-Ala-Gly-Phe, (SEQ IID NO: 6) Gly-Ile-Ala-Gly-Gln, (SEQ ID NO: 7) Gln-Gly-Ala-Ile-Ala-Gln, (SEQ ID NO: 8) Phe-Gly-Ile-Ala-Gly-Phe, (SEQ ID NO: 9) Cys-Gly-Ile-Ala-Gly-Cys, (SEQ ID NO: 10) Glu-Gly-Ile-Ala-Gly-Lys, (SEQ ID NO: 11) N-Acetyl Ile-Ala-Ala, (SEQ ID NO: 12) Ile-Ala-.beta.Ala, (SEQ ID NO: 13) N-Acetyl Ile-Ala NMe, (SEQ ID NO: 14) Asp-Gly-Glu-Ala, (SEQ ID NO: 15) Asp-Gly-Glu-Ala-Gly-Cys, (SEQ ID NO: 16) Gly-Phe-Pro*-Gly-Glu-Arg, (SEQ ID NO: 17) Gly-Leu-Pro*-Gly-Glu-Arg, (SEQ ID NO: 18) Gly-Met-Pro*-Gly-Glu-Arg, (SEQ ID NO: 19) Gly-Ala-Ser-Gly-Glu-Arg, (SEQ ID NO: 20) Gly-Leu-Ser-Gly-Glu-Arg, and (SEQ ID NO: 21) Gly-Ala-Pro*-Gly-Glu-Arg, wherein Pro* is hydroxyproline.

55.-61. (canceled)

Description

DETAILED DESCRIPTION

[0037] The invention features methods and compositions for (i) controlling the amount of peptide bound to calcium phosphate particles; and (ii) for tightly binding peptide to the surface of calcium phosphate particles. The methods and compositions can be useful for preparing implants useful for promoting bone deposition at the site of implantation, and for repairing a variety of orthopedic conditions. The invention features methods for pretreating calcium phosphate particles to increase the number of sites on the surface of the particles to which a peptide can tightly bind.

[0038] Calcium Phosphate Particles

[0039] The formulations of the invention include a particulate calcium phosphate. The calcium phosphate may be any biocompatible, calcium phosphate material known in the art. The calcium phosphate material may be produced by any one of a variety of methods and using any suitable starting components. For example, the calcium phosphate material may include amorphous, apatitic calcium phosphate. Calcium phosphate material may be produced by solid-state acid-base reaction of crystalline calcium phosphate reactants to form crystalline hydroxyapatite solids. Other methods of making calcium phosphate materials are known in the art, some of which are described below. Alternatively, the calcium phosphate material can be crystalline hydroxyapatite (HA). Crystalline HA is described, for example, in U.S. Pat. Nos. Re. 33,221 and Re. 33,161. These patents teach preparation of calcium phosphate remineralization compositions and of a finely crystalline, non-ceramic, gradually resorbable hydroxyapatite carrier material based on the same calcium phosphate composition. A similar calcium phosphate system, which consists of tetracalcium phosphate (TTCP) and monocalcium phosphate (MCP) or its monohydrate form (MCPM), is described in U.S. Pat. Nos. 5,053,212 and 5,129,905. This calcium phosphate material is produced by solid-state acid-base reaction of crystalline calcium phosphate reactants to form crystalline hydroxyapatite solids. Carbonate substituted crystalline HA materials (commonly referred to as dahllite) may be prepared (see U.S. Pat. No. 5,962,028). These HA materials (commonly referred to as carbonated hydroxyapatite) can be formed by combining the reactants with an aqueous liquid to provide a substantially uniform mixture, shaping the mixture as appropriate, and allowing the mixture to harden in the presence of water. During hardening, the mixture crystallizes into a solid and essentially monolithic apatitic structure. The reactants will generally include a phosphate source, e.g., phosphoric acid or phosphate salts, an alkali earth metal, particularly calcium, optionally crystalline nuclei, particularly hydroxyapatite or calcium phosphate crystals, calcium carbonate, and a physiologically acceptable lubricant. The dry ingredients may be pre-prepared as a mixture and subsequently combined with aqueous liquid ingredients under conditions where substantially uniform mixing occurs.

[0040] Cell Adhesion Peptides

[0041] The calcium phosphate particles in the formulations of the invention can be coated with one or more cell adhesion peptides. Cell adhesion peptides can include any of the proteins of the extracellular matrix which are known to play a role in cell adhesion, including fibronectin, vitronectin, laminin, elastin, fibrinogen, and collagens, such as types I, II, and V, as well as their bioactive fragments. Additionally, the cell adhesion peptides may be any peptide derived from any of the aforementioned proteins, including derivatives or fragments containing the binding domains of the above-described molecules. Exemplary peptides include those having integrin-binding motifs, such as the RGD (arginine-glycine-aspartate) motif, the YIGSR (tyrosine-isoleucine-glycine-serine-arginine) motif, and related peptides that are functional equivalents. For example, peptides containing RGD sequences (e.g., GRGDS) and WQPPRARI sequences are known to direct spreading and migrational properties of endothelial cells (see V. Gauvreau et al., Bioconjug Chem. 16:1088 (2005)). REDV tetrapeptide has been shown to support endothelial cell adhesion but not that of smooth muscle cells, fibroblasts, or platelets, and YIGSR pentapeptide has been shown to promote epithelial cell attachment, but not platelet adhesion (see Boateng et al., Am. J. Physiol. Cell Physiol. 288:30 (2005). Other examples of cell-adhesive sequences are the NGR tripeptide, which binds to CD13 of endothelial cells (see L. Holle et al., Oncol. Rep. 11:613 (2004)) and DGEA that binds Type I collagen (see Hennessy et.al. Biomaterials, 30:1898 (2009)).

[0042] Cell adhesion peptides that can be used in the methods and compositions of the invention include, without limitation, those mentioned above, and the peptides disclosed in U.S. Pat. No. 6,156,572; U.S. patent publication No. 2003/0087111; and U.S. patent publication No. 2006/0067909, each of which is incorporated herein by reference.

[0043] Alternatively, the cellular adhesion peptides can be obtained by screening peptide libraries for adhesion and selectivity to specific cell types (e.g. endothelial cells) or developed empirically via Phage display technologies.

[0044] In certain embodiments, the cell adhesion peptide is a collagen mimetic peptide. The integrin αa2β1 consists of two non-identical subunits, α2 and β1, members of the integrin family each with a single trans-membrane domain, and α2β1 is known to bind to collagen via a specialized region of the α2-subunit. There are several known α2β1 recognition sites within collagens. This knowledge arises from the use of collagen fragments derived from purified αchains cleaved into specific and reproducible peptides. Collagen mimetic peptides that can be used in the implantable compositions of the invention include, without limitation, those described in PCT Publication Nos. WO/1999/050281; WO/2007/017671; and WO/2007/052067, each of which is incorporated herein by reference. Collagen mimetic peptides include, without limitation, peptides including the peptide sequences of any of SEQ ID NOS. 1-21: Gly-Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val (SEQ ID NO. 1, also known as “P-15”), Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln -Arg (SEQ ID NO: 2), Gln-Gly-Ile-Ala-Gly-Gln (SEQ ID NO: 3), Gln-Gly-Ile-Ala-Gly-Gln-Arg (SEQ ID NO: 4), Phe-Gly-Ile-Ala-Gly-Phe (SEQ ID NO: 5), Gly-Ile-Ala-Gly-Gln (SEQ IID NO: 6), Gln-Gly-Ala-Ile-Ala-Gln (SEQ ID NO: 7), Phe-Gly-Ile-Ala-Gly-Phe (SEQ ID NO:8), Cys-Gly-Ile-Ala-Gly-Cys (SEQ ID NO:9), Glu-Gly-Ile-Ala-Gly-Lys (SEQ ID NO:10), N-Acetyl Ile-Ala-Ala (SEQ ID NO:11), Ile-Ala-.beta.Ala (SEQ ID NO:12), N-Acetyl Ile-Ala NMe (SEQ ID NO:13), Asp-Gly-Glu-Ala (SEQ ID NO:14), Asp-Gly-Glu-Ala-Gly-Cys (SEQ ID NO:15), Gly-Phe-Pro*-Gly-Glu-Arg (SEQ ID NO:16, where Pro*=hydroxyproline), Gly-Leu-Pro*-Gly-Glu-Arg (SEQ ID NO:17, where Pro* =hydroxyproline), Gly-Met-Pro*-Gly-Glu-Arg (SEQ ID NO:18, where Pro*=hydroxyproline), Gly-Ala-Ser-Gly-Glu-Arg (SEQ ID NO:19), Gly-Leu-Ser-Gly-Glu-Arg (SEQ ID NO:20), Gly-Ala-Pro*-Gly-Glu-Arg (SEQ ID NO:21, where Pro*=hydroxyproline), and any other collagen mimetic peptides described in U.S. Pat. No. 7,199,103, incorporated herein by reference.

[0045] For example, the cell adhesion peptide can be coated onto ABM particles have a mean particle diameter of 300 microns, and nearly all will fall within a range between 200 microns to 425 microns. However, a particle size range between 50 microns to 2000 microns may also be used.

[0046] As an alternative to anorganic bone mineral (ABM), a synthetic alloplast matrix or some other type of xenograft or allograft mineralized matrix that might not fit the definition of “anorganic” may be used in the methods and compositions of the invention as a substitute for calcium phosphate particles. The alloplast could be a calcium phosphate material or it could be one of several other inorganic materials that have been used previously in bone graft substitute formulations, e.g., calcium carbonates, calcium sulphates, calcium silicates, used in a mixture that includes calcium phosphate and that could function as biocompatible, osteoconductive matrices. The anorganic bone mineral, synthetic alloplast matrix, and xenograft or allograft mineralized matrix can be the particulate bone graft substitute and can be used to bind a cell adhesion peptide to their surface.

[0047] Formulations

[0048] The invention features peptide-coated calcium phosphate particles which are useful for the treatment of bone defects. The coated particles can be incorporated into putties and lyophilized implants. Such implants can include, e.g., the coated particles suspended in a hydrogel carrier. Polysaccharides that may be utilized as a carrier include, for example, any suitable polysaccharide within the following classes of polysaccharides: celluloses/starch, chitin and chitosan, hyaluronic acid, alginates, carrageenans, agar, and agarose. Certain specific polysaccharides that can be used include agar methylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, oxidized cellulose, chitin, chitosan, alginic acid, sodium alginate, and xanthan gum.

[0049] The hydrogels will typically include a solvent to control the viscosity of the material. The solvent may be an alcohol or alcohol ester, including for example, glycerol, triacetin, isopropyl alcohol, ethanol, and ethylene glycol, or mixtures of these. The paste or gel can include others components, such as surfactants, stabilizers, pH buffers, and other additives (e.g., growth factors, antibiotics, analgesics, etc.). For example, a suitable gel or paste can be prepared using water, glycerin and sodium carboxymethylcellulose.

[0050] Therapy

[0051] The compositions of the invention can be used in bone graft substitutes which are implanted into a subject. The compositions of the invention can include a cell adhesion peptide to promote rapid ossification of the implant.

[0052] The compositions of the invention can be useful for repairing a variety of orthopedic conditions. For example, the compositions may be injected into the vertebral body for prevention or treatment of spinal fractures, injected into long bone or flat bone fractures to augment the fracture repair or to stabilize the fractured fragments, or injected into intact osteoporotic bones to improve bone strength. The compositions can be useful in the augmentation of a bone-screw or bone-implant interface. Additionally, the compositions can be useful as bone filler in areas of the skeleton where bone may be deficient. Examples of situations where such deficiencies may exist include post-trauma with segmental bone loss, post-bone tumor surgery where bone has been excised, and after total joint arthroplasty (e.g., impaction grafting and so on). The compositions may be formulated as a paste prior to implantation to hold and fix artificial joint components in patients undergoing joint arthroplasty, as a strut to stabilize the anterior column of the spine after excision surgery, as a structural support for segmented bone (e.g., to assemble bone segments and support screws, external plates, and related internal fixation hardware), and as a bone graft substitute in spinal fusions.

[0053] The compositions of the invention can be used to coat prosthetic bone implants. For example, where the prosthetic bone implant has a porous surface, the composition may be applied to the surface to promote bone growth therein (i.e., bone ingrowth). The composition may also be applied to a prosthetic bone implant to enhance fixation within the bone.

[0054] The compositions of the invention can be used as a remodeling implant or prosthetic bone replacement, for example in orthopedic surgery, including hip revisions, replacement of bone loss, e.g. in traumatology, remodeling in maxillofacial surgery or filling periodontal defects and tooth extraction sockets, including ridge augmentation and sinus elevation. The compositions of the invention may thus be used for correcting any number of bone deficiencies at a bone repair site.

[0055] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0056] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention

EXAMPLE 1

Preparation of Peptide-Coated Calcium Phosphate Particles

[0057] The increased binding is achieved by pretreating, coating and washing the ABM in a buffer solution to equilibrate and stabilize the surface of the ABM particle before, during and after coating to increase the concentration of peptide tightly bound to the particles. The steps for coating the particles is described below.

[0058] Step 1: PRETREATMENT—ABM is pretreated in a Hypertonic (˜460 mM NaCl) HEPES buffer, pH 7.2, at a 10:1 ratio (ml solution to g ABM) for 24 hours. This solution is decanted off after 24 hours. This wash is repeated with fresh solution for 1 hour or until there is no pH change upon exposure to fresh solution. The final wash solution is decanted and the ABM is vacuum dried. The pretreatment step is performed with agitation of some kind (roller, rotation, etc.).

[0059] Step 2: COATING—A P-15 coating buffer (concentration depends on final desired P-15 coated on ABM) is made using hypertonic HEPES, pH 7.2. Pretreated ABM is coated using a 2:1 ratio (mL solution to g ABM) for 24 hours. This step is performed statically, with no agitation.

[0060] Step 3: WASHING—After the 24 hour coating step, the solution is removed by a rapid vacuum filtration process. The coated ABM is washed 6-12 times in a 10:1 ratio of solution to ABM using hypertonic HEPES buffer, pH 7.2. Vacuum filtration is performed between each wash to remove the solution. After the final wash, the washed ABM is then vacuum dried overnight.

[0061] The pretreatment step equilibrates the surface of the ABM particles to a desired pH and osmolarity (ionic strength). The coating process is also performed under conditions similar to those used to pretreat the ABM to provide an improved coating environment for the peptide. Finally, the wash step can be performed using the same buffer to remove any extraneous peptide not bound to the ABM surface.

EXAMPLE 2

In Vivo Performance of P-15 Coated ABM Particles

[0062] The performance of three test articles (a-c) were evaluated in a pre-clinical critical-defect, ovine drill hole model.

[0063] Materials:

[0064] a) Control Article #1: uncoated ABM.

[0065] b) Test Article #1: P-15 coated ABM produced without pretreating the particles (<20 ng-P-15/g-ABM).

[0066] c) Test Article #2: ABM coated with P-15 using the pretreatment processing (ca. 250 ng-P-15/g-ABM).

[0067] Animal Model:

[0068] The Control and Test samples were implanted into a condyle site on the sheep femur. The drill hole was 8 mm by 15 mm is size. The samples were implanted for 4-weeks and 8-weeks. An empty defect was also included in the study. At the indicted end point the biopsies were harvested and prepared for histology. The amount of new bone formation generated by the samples was determined by histomophometry. In addition, a histological evaluation was performed to determine if any of the samples elicited an inflammatory response.

[0069] The uncoated ABM was delivered in a collagen carrier as a putty. The ABM with low P-15 concentration was delivered in a hydrogel carrier as a putty. The ABM with high P-15 was delivered in a collagen carrier as a putty.

[0070] The measurement of the inflammatory response was determined by the following scoring system:

[0071] 0=none present

[0072] 1=rare: 1-5 cells per high power field

[0073] 2=moderate: 6-10 cell per high power field

[0074] 3=heavy infiltration

[0075] 4=significant infiltration

[0076] The histomorphometric measurements quantitated the proportion of new bone formation within the original defect size as follows:

[0077] Total defect areas (TA)

[0078] New bone formation area (BA)

[0079] New bone formation: BA/TA

[0080] Results:

[0081] Tables 1 and 2 display the histopathologist's review of the Test and Control samples at 4-weeks and 8-weeks with regards to new bone formation and infiltration of immune cells.

TABLE-US-00001 TABLE 1 Histopathology & Histomorphometry Analysis - 4-weeks ABM ABM Low P-15 coating High P-15 coating Measurement Empty Defect ABM (<20 ng-P-15/g-ABM) (ca. 250 ng-P-15/g-ABM) PMN 1.00 ± 0.0 0.0 0.0 0.0 Lymphocytes 1.00 ± 0.0 0.0 0.0 0.0 Macrophages 2.00 ± 0.0  1.0 ± 0.0 1.25 ± 0.50 1.00 ± 0.0  New Bone 26.8 ± 3.7 31.7 ± 7.3 47.3 ± 17.4 68.4 ± 13.4 Formation

TABLE-US-00002 TABLE 2 Histopathology & Histomorphometry Analysis - 8-weeks ABM ABM Low P-15 coating High P-15 coating Measurement Empty Defect ABM (<20 ng-P-15/g-ABM) (ca. 250 ng-P-15/g-ABM) PMN  0.50 ± 0.71 0.0 0.0 0.0 Lymphocytes 0.0 0.0 0.0 0.0 Macrophages 1.00 ± 0.0 1.00 ± 0.0 1.00 ± 0.0  1.00 ± 0.0 New Bone 48.4 ± 7.1 50.5 ± 9.3 63.1 ± 13.0 92.6 ± 4.4 Formation

[0082] Conclusions:

[0083] The increased in concentration of P-15 bound to the ABM did not elicit any significant inflammatory response. Furthermore, the higher levels (>20 ng-P-15/g-ABM) of P-15 tightly bound to the ABM yielded a significant increase in new bone formation as compared to the ABM coated with a low (<20 ng-P-15/g-ABM) P-15 concentration.

EXAMPLE 3

Effect of Osmolarity on the Binding of P-15 Peptide to ABM Particles

[0084] ABM particles were incubated in a buffered solution that contained varying amounts of NaCl to modulate the final osmolarity. The ABM particles were incubated in the solutions for 18-24 hours, rinsed a second time in the same buffer, and dried. The pre-treated surface modified ABM particles were then incubated with P-15 dissolved in the same buffer as used during the pre-treatment step to coat the particles with P-15 peptide. Following the P-15 coating step, the ABM particles were washed repeatedly with the corresponding buffer solution. The amount of bound P-15 was determined using an ELISA test method. The results are provided in Table 3. The different runs represent different preparations of the same ABM source.

TABLE-US-00003 TABLE 3 Effect of Osmolarity on P-15 Coating of ABM Granules P-15 Concentration Bound to ABM Particles for Different ABM Sources (ng-P-15/g-ABM) Bovine Bovine Porcine Buffer osmolarity Cortical Cancellous Cancellous (Hepes buffer, pH 7.2) Run #1 Run #2 Run #1 Run #2 Run #1 Hypotonic Solution 105 ng 64 ng 68 ng 651 ng 231 ng (0% saline) Isotonic Solution 116 ng 66 ng 129 ng 611 ng 283 ng (0.9% saline) Hypertonic Solution 153 ng 105 ng 149 ng 1024 ng 376 ng (2.7%saline)

[0085] This data demonstrated that the treatment of ABM with a buffering solution that has a hypertonic saline concentration yields a higher level of bound P-15 peptide. This effect was applicable to all types of ABM (calcium phosphate) particles tested.

EXAMPLE 4

The Effect of Rate of Freezing (Rapid vs. Slow Freeze) on the Amount of P-15 Bound to the ABM Particles

[0086] Increased P-15 binding is achieved by freezing the pretreated ABM particles. The pretreated

[0087] ABM particles are produced as described in step 1 of Example 1, where step 1 further includes freezing and then drying the ABM particles subjected to hypertonic buffer.

[0088] Paired samples of pretreated ABM particles were freeze-dried (F/D) by two different methods, and a third sample was simply air dried (without any freezing). One sample was placed on a controlled ambient temperature F/D shelf and a vacuum was immediately pulled to initiate rapid evaporative freezing (FD-EC). The second sample was placed on an ambient F/D shelf and slowly frozen by ramped freezing (˜1-2° C. per minute). Once the sample was frozen the vacuum was initiated; this process is considered standard freeze-drying. The F/D process was used for both (a) the pretreatment step 1 and the washing (post-coating) step 3. The third samples was not frozen; the water was removed from the sample by air-drying at both drying steps. The following table indicates the amount of bound P15 on the final washed product. Table 4 indicates the amount of bound P-15 on the final washed ABM particles.

TABLE-US-00004 TABLE 4 The effect of rate of freezing on the amount of P-15 bound to the ABM particles. F/D Technique P-15 ng/g ABM Rapid freeze in freeze-dryer 1521 Slow freeze in freeze-dryer 366 Air drying 74

[0089] Conclusions:

[0090] A rapid rate of freezing, regardless of vacuum drying condition, resulted in higher amount of P-15 bound on the P-15 ABM particles. Simply drying the pretreated particles without freezing them (i.e., air drying) produced only modest improvements in the levels of P-15 binding compared to ABM not pretreated with hypertonic buffer.

Example 5

The Effect of Rapid Freeze in Freeze-Dry vs. Intermediate Freeze in Vacuum Oven on the Amount of P-15 Bound to the ABM Particles

[0091] Increased P-15 binding is achieved by rapidly freezing the pretreated ABM particles. The pretreated ABM particles are produced as described in step 1 of Example 1, where step 1 further includes freezing and then drying the ABM particles subjected to hypertonic buffer.

[0092] Paired samples of pretreated ABM particles were F/D by two different methods. One sample was placed on a controlled ambient temperature F/D shelf and a vacuum was immediately pulled to initiate rapid evaporative freezing. The second sample was placed on an uncooled shelf and a vacuum was immediately pulled to initiate evaporative cooling (vacuum oven drying). It should be noted that the rate of cooling is slower for this process due to the shelf temperature and the strength of vacuum. The F/D process was used for both (a) the pretreatment step 1 and the washing (post-coating) step 3. Table 5 indicates the amount of bound P-15 on the final washed ABM particles.

TABLE-US-00005 TABLE 5 The effect of rapid freeze in freeze-drier vs. intermediate freeze in vacuum oven on the amount of P-15 bound to the ABM particles. F/D Technique P-15 ng/g ABM Rapid freeze in freeze-dryer 4201 Intermediate freeze in vacuum oven 308

[0093] Conclusions:

[0094] A rapid rate of freezing, regardless of the use of evaporative cooling at the onset, resulted in higher amount of P-15 bound to the ABM particles.

EXAMPLE 6

The Effect of Freezing During the Pretreatment Step Versus the P-15 Coating Step

[0095] Increased P-15 binding is achieved by rapidly freezing the pretreated ABM particles relative to rapidly freezing only during the washing (post-coating) step 3.

[0096] Four paired samples of pretreated ABM particles were F/D by two methods, alternately performed at the two separate drying steps. Two samples were F/D by vacuum oven drying after the pretreatment step and then dried by either the vacuum oven drying process or by FD-EC (placed on a controlled ambient temperature F/D shelf and a vacuum was immediately pulled to initiate rapid evaporative freezing) method after the P-15 coating step. The other two samples were dried by FD-EC after the pretreatment step and then dried by either the vacuum oven drying process or by FD-EC method after the P-15 coating Table 6 table indicates the amount of bound P-15 on the final washed ABM particles.

TABLE-US-00006 TABLE 6 FD-ED rapid drying procedure. F/D Technique Pretreatment Step P-15 Coating Step P-15 ng/g ABM Vacuum oven Vacuum oven 72 Vacuum oven FD-EC 77 FD-EC Vacuum oven 261 FD-EC FD-EC 351

[0097] Conclusions:

[0098] The implementation of the FD-EC rapid freezing procedure only produced large changes in P-15 binding when applied to the ABM particles during the pretreatment step. While there was some decrease of the P-15 concentration when the slower freezing method was used following the coating step, effective and higher levels of P-15 coating were still achieved.

EXAMPLE 7

Blast-Freeze Followed by Freeze-Dryer vs. Intermediate Freeze in Vacuum Oven

[0099] Increased P-15 binding is achieved by rapidly freezing the pretreated ABM particles. The pretreated ABM particles are produced as described in step 1 of Example 1, where step 1 further includes freezing and then drying the ABM particles subjected to hypertonic buffer.

[0100] Paired samples of pretreated ABM particles were F/D by two different methods. One sample was rapidly frozen using a blast-freezer. The frozen sample was placed on a cold F/D shelf and a vacuum was immediately pulled. The second samples was placed on an uncooled shelf and a vacuum was immediately pulled to initiate evaporative cooling. The F/D process was used for both (a) the pretreatment step 1 and the washing (post-coating) step 3. Table 7 indicates the amount of bound P-15 on the final washed ABM particles.

TABLE-US-00007 TABLE 7 Blast-freeze followed by freeze-dryer vs. intermediate freeze in vacuum oven. F/D Technique P-15 ng/g ABM Blast-freeze followed by standard freeze-dryer 260 Intermediate freeze in vacuum oven 80

[0101] Conclusions:

[0102] A rapid rate of freezing yielded a high concentration of P-15 bound to ABM and the rapid freezing at the pretreatment step can be achieved by multiple methods. The use of a blast-freezer to rapidly freeze the ABM worked similarly to the FD-EC method.

Other Embodiments

[0103] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

[0104] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0105] This application claims the benefit of U.S. Provisional Ser. No. 62/366,370, filed Jul. 25, 2016, which is incorporated herein by reference.

[0106] Other embodiments are within the claims.