Methods of treating delayed union and nonunion fractures using platelet-derived growth factor compositions

Abstract

The present disclosure provides methods for treating delayed union or nonunion fractures in a bone More specifically, the present disclosure provides methods of inducing bone formation in a delayed union or nonunion fracture in a bone comprising: applying to a site of delayed union or nonunion fracture a composition comprising a platelet-derived growth factor (PDGF) solution disposed in a biocompatible matrix.

Description

[0001] E3383-03646

[0002] METHODS OF TREATING DELAYED UNION AND NONUNION FRACTURES USING PLATELET-DERIVED GROWTH FACTOR COMPOSITIONS

[0003] BACKGROUND

[0004] Bone fractures typically can heal completely. Nevertheless, in some cases, a fracture does not heal, resulting in a nonunion. In other cases, the fracture takes far longer to heal than usual, which is called a delayed union. For a fracture to heal properly the bone needs to be stabilized, usually with a cast or, in some cases, surgery is required to use plates, screws or nails to stabilize the bone. The bone also needs a steady blood supply to bring oxygen, healing cells and growth factors to the fracture site.

[0005] Factors including the use of tobacco or nicotine can impede bone healing and increase the risk of nonunion. Older individuals and those with diabetes, severe anemia or an infection also face an increased risk of nonunion. Medications such as anti-inflammatory drugs, including aspirin, ibuprofen, and prednisone, also may increase the risk of nonunion, since they affect blood flow. Nutrition can also affect the risk of nonunion, since bones need protein, calcium, vitamin C and D, and other minerals to ensure proper healing.

[0006] Currently there is a lack of standard and effective treatment for nonunion and delayed union fractures. Treatments range from wait and see to ultrasound and electrical stimulation. Other treatments may include resetting hardware, autograft, or micro-fracturing. Accordingly, there is a need for minimally invasive and reliable methods for treating such fractures. The present invention addresses those needs.

[0007] SUMMARY

[0008] One aspect of the present disclosure provides method of inducing bone formation at a site of delayed union or nonunion fracture in a bone comprising:

[0009] applying to the site of delayed union or nonunion fracture a composition comprising a platelet-derived growth factor (PDGF) solution disposed in a biocompatible matrix. In some embodiments, the biocompatible matrix comprises a bone scaffolding material and aE3383-03646

[0010] biocompatible binder.

[0011] In some embodiments, the bone scaffolding material comprises calcium phosphate, such as β-tricalcium phosphate (β-TCP). In some embodiments, the biocompatible binder comprises collagen, such as type I collagen, and more particularly, Type I bovine collagen.

[0012] In some embodiments, inducing bone formation comprises inducing bone bridging at the site of delayed union or nonunion fracture. In other embodiments, the method induces periosteal coverage at the site of delayed union or nonunion fracture. In other embodiments, the method induces osteon formation at the site of delayed union or nonunion fracture, the method induces osteon formation at the site of delayed union or nonunion fracture. In other embodiments, the method promotes woven bone formation at the site of delayed union or nonunion fracture.

[0013] In certain embodiments, the composition comprises particles of β-TCP and collagen, wherein the particles of β-TCP have a porosity of at least 90% and a size ranging from about 100 to about 300 microns, wherein the collagen is present in an amount of about 20% by weight of the biocompatible matrix, and wherein the PDGF solution has a concentration of about 0.3 mg / rnL.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Colored elements of the Figures below are presented in crosshatch and grayscale pursuant to 37 CFR 1.84.

[0016] Figure 1 shows X-ray radiographs of (a) lateral and (b) rostral caudal of chronic treatment group (Group D) taken prior to injection with Al to confirm the presence of nonunion (dashed box).

[0017] Figures 2A-D show representative DEXA radiographs of groups (a) A, (b) B, (c) C, and (d) D. Red arrows (arrows with vertical crosshatch design) point to absence of bone bridging, yellow arrows (arrows with crisscross crosshatch design) indicate presence of fibrotic connective tissue and the green arrow (arrow with diagonal crosshatch design) depicts complete bone bridging across the defect site.

[0018] Figure 3A-D show representative volumetric reconstructions of groups (a) A, (b) B, (c) C, and (d) D, with bone and calcified fibrotic tissue and fixation plate / screws in purple (darkE3383-03646

[0019] crosshatched sections). Red arrows (arrows with vertical crosshatch design) point to absence of bone bridging, the white arrow indicates presence of fibrotic connective tissue, and the green arrow (arrow with diagonal crosshatch design) depicts complete bone bridging across the defect site.

[0020] Figure 4A-D show representative low magnification histomicrographs of groups (a) A, (b) B, (c) C, and (d) D. Dashed black line depicts the induced 2 mm defect (gap). Blue arrows (wide arrows with horizontal crosshatch design) point towards surgical hardware (fixation plate), red arrows (arrows with vertical crosshatch design) highlight the inorganic β-TCP, yellow arrows (arrows with crisscross crosshatch design) indicate bone-related tissue.

[0021] Figures 5A-D show representative high magnification histomicrographs of groups (a) A, (b) B, (c) C, and (d) D in the region of interest. Dashed black line depicts the width of the induced defect (2 mm). Red arrows (arrows with vertical crosshatch design) indicate presence of β-TCP particles, blue arrows (wide arrows with horizontal crosshatch design) denote bone remodeling sites, cyan arrows (wide arrows with dashed vertical crosshatch design) show woven bone formation (in group C), green arrows (arrows with diagonal crosshatch design) point towards primary oseteon formation, and yellow (arrows with crisscross crosshatch design) arrow's show' the presence of periosteal coverage in group D.

[0022] Figure 6 is a graph showing the comparison of the presence of bone within the defect site (bone %) among the different treatment groups. p<0.05 is statistically significant. Data presented as median and respective interquartile ranges (IQR).

[0023] Figure 7 is a schematic depicting an exemplary method of preparing a composition for use in the present methods.

[0024] DETAILED DESCRIPTION

[0025] Definitions

[0026] As used herein, “septic nonunion” refers to delay of the union or nonunion or delayed union of the fracture due to bacterial infection to the bone. Fixation elements may play a key pathogenetic role in the infection.E3383-03646

[0027] As used herein, “pseudarthrosis nonunion” refers to a fracture with nonunion or delayed union that results in a joint like motion is called as Pseudarthrosis or a “false joint”.

[0028] As used herein, “hypertrophic nonunion” refers to nonunion or delayed union caused due to poor and improper fixation fracture joints, even though callus is formed.

[0029] As used herein, “atrophic nonunion” refers to poor or no callus formation due to lack of blood supply or low metabolic rate or impaired metabolic rate due to diabetes, hyperthyroidism, and smoking, etc.,

[0030] As used herein, “oligotrophic nonunion”: refers to nonunion or delayed union having poor callous formation. They are vascular and may have severely dislodged fracture or fixation without accurate positioning of bone fragments.

[0031] As used herein, “inducing bone formation” refers to formation of bone bridging at the site of delayed union or nonunion fracture, formation of periosteal coverage at the site of delayed union or nonunion fracture, osteon formation at the site of delayed union or nonunion fracture, promotion of woven bone formation at the site of delayed union or nonunion fracture, or any combination thereof. Furthermore, desirable effects of method of treatment include but are not limited to, for example, one or more of: increase in degree of bone density and / or acceleration of bone formation (e.g. acceleration of bone density ) at the site of the delayed union or nonunion fracture, increase in degree of bony union or bone bridging and / or acceleration of bony union or bony bridging at the site of the delayed union or nonunion fracture, improvement in composition and / or structure of bone at the site delayed union or nonunion fracture (for example, closer resemblance to natural bone at the site of delayed union or nonunion fracture).

[0032] As used herein, the term “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An effective amount can be provided in one or more administration of the compositions disclosed herein.

[0033] Reference to “about” a value or parameter herein also includes (and describes) embodiments that are directed to that value or parameter per se.

[0034] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise. For example, referenceE3383-03646

[0035] to a “PDGF homodimer’’ is a reference to one or multiple PDGF homodimers, and includes equivalents thereof known to those skilled in the art, and so forth.

[0036] It is understood that all aspects and embodiments of the invention described herein may include “comprising,’’ “consisting,’’ and “consisting essentially of’ aspects and embodiments. It is to be understood that methods or compositions “consisting essentially of’ the recited elements include only the specified steps or materials and those that do not materially affect the basic and novel characteristics of those methods and compositions.

[0037] “As used herein, the term "fibrillar collagen" means a collagen of a type which can normally form collagen fibrils. The fibrillar collagens are collagen types I-III, V, and XI. The collagen monomers that make up the fibrillar collagens contain "telopeptide" regions at the amino (N) and carboxy (C) terminal ends of the monomers which are non-helical in the collagen trimer. These collagens self-assemble into fibrils with the C-terminal end of the helical domain and the C-propeptide of one collagen triple helix overlapping with the N telopeptide and the N-terminal end of the triple helical domain of an adjacent collagen molecule. The monomers that make up the fibrillar collagens are made as preproproteins, including an N-terminal secretion signal sequence and N and C-terminal propeptide domains. The signal sequence is normally cleaved by signal peptidase, as with most secreted proteins, and the propeptides are removed by specific proteolytic processing enzymes after association, folding and secretion of trimeric procollagen. The term fibrillar collagen encompasses both native ( i.e., naturally occurring) and variant fibrillar collagens ( i.e., fibrillar collagens with one or more alterations in the sequence of one or more of the fibrillar collagen monomers). Unless the context clearly indicates otherwise (e.g., the term is modified by the word "monomer") "fibrillar collagen" refers to triple helical fibrillar collagen.” “Fibrillar collagen” also may be referred to herein as “insoluble collagen or polymeric collagen.”

[0038] " Soluble collagen" refers to individual tropocollagen molecules that are soluble in acidic aqueous environments. Tropocollagen may be considered the monomeric unit of fibrillary collagen fibers.

[0039] “Volume to mass ratio” as used herein refers to ratio of the liquid volume of PDGF solution in milliliters (mL) to the mass of biocompatible matrix in grams (g). For example,E3383-03646

[0040] the volume to mass ratio can range from about 1: 1 to about 5:1, about 1: 1 to about 4:1, about 1:1, about 3:1, about 2:1 to about 3:1, or about 3:1.

[0041] Methods of inducing bone formation at a site of delayed union or nonunion fracture

[0042] The present invention provides methods of inducing bone formation at a site of delayed union or nonunion fracture. In some embodiments, a method of inducing bone formation at a site of delayed union or nonunion fracture procedure comprises providing a composition comprising a PDGF solution incorporated in a biocompatible matrix and applying the composition to a site of delayed union or nonunion.

[0043] One aspect of the present disclosure provides method of inducing bone formation at a site of delayed union or nonunion fracture in a bone comprising:

[0044] applying to the site of delayed union or nonunion fracture a composition comprising a platelet-derived growth factor (PDGF) solution disposed in a biocompatible matrix. In some embodiments, the biocompatible matrix comprises a bone scaffolding material and a biocompatible binder.

[0045] In some embodiments, the bone scaffolding material comprises calcium phosphate, calcium sulfate, demineralized freeze-dried bone allograft, demineralized bone matrix, or any combination thereof. Preferably, the bone scaffolding material comprises calcium phosphate. More preferably, the calcium phosphate is selected from the group consisting of β-tricalcium phosphate (β-TCP), hydroxyapatite, poorly crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate, octacalcium phosphate, and any combination thereof, and most preferably, the calcium phosphate is P-TCP.

[0046] In some embodiments, the biocompatible binder comprises collagen, a protein, a polysaccharide, a nucleic acid, a carbohydrate, a synthetic polymer, or any combination thereof. Preferably, the biocompatible binder comprises collagen.

[0047] In some embodiments, the collagen is type I, type II, ty pe III, or any combination thereof. Preferably, the collagen is type I collagen, and more preferably, the collagen is bovine collagen, most preferably bovine Type I collagen. The collagen may be fibrillar or soluble. Preferably, the collagen is soluble.E3383-03646

[0048] In some embodiments, the biocompatible matrix comprises about 5% to about 40%, about 10% to about 30%, about 15% to about 25%, or about 20% by weight of the biocompatible binder.

[0049] In some embodiments, the PDGF solution and the biocompatible matrix are present in the composition in a liquid volume to mass ratio of about 1:1 to about 5: 1, 1:1 to about 4:1, about 1:1 to about 3:1, about 2:1 to about 3:1, or about 3:l. Prefereably, the liquid volume to mass ratio is about 3:1.

[0050] In some embodiments, the PDGF solution has PDGF concentration of about 0.1 to about 0.01 to about 10.0 mg / mL, about 0.05 to about 5 mg / mL, about 0.1 to about 1 mg / mL, about 0.1 to about 0.5 mg / mL, or about 0.3 mg / mL. Preferably, the PDGF concentration is about 0.1 to about 1 mg / mL, more preferably about 0.1 to about 0.5 mg / mL, and most preferably about 0.3 mg / mL.

[0051] In some embodiments, the PDGF solution is aqueous. In more particular embodiments, the aqueous solution comprises a buffer. Preferably, the buffer comprises sodium acetate.

[0052] In some embodiments, the PDGF comprises PDGF-BB. In preferred embodiments, the PDGF comprises recombinant human PDGF-BB (rhPDGF-BB). In some embodiments, the rhPDGF-BB comprises at least 65% intact rhPDGF-BB.

[0053] In some the bone scaffolding material comprises particles having an average diameter of about 100 microns to about 300 microns.

[0054] In some embodiments, the biocompatible binder has a porosity greater than about 50%, greater than about 60%, greater than about 70%, about than about 80%, or greater than about 90%.

[0055] In some embodiments, the method induces bone bridging at the site of delayed union or nonunion fracture. In other embodiments, the method induces periosteal coverage at the site of delayed union or nonunion fracture. In other embodiments, the method induces osteon formation at the site of delayed union or nonunion fracture, the method induces osteon formation at the site of delayed union or nonunion fracture. In other embodiments, the method promotes woven bone formation at the site of delayed union or nonunion fracture.

[0056] In some embodiments, the bone is an appendicular bone. In more particular embodiments, the bone is selected from a humerus, a radius, an ulna, a carpals, a metacarpals,E3383-03646

[0057] a phalanges, a femur, a tibia, a fibula, and a tarsals. In more particular embodiments, the bone is selected from the tibia, femur, humerus, fibula, radius, and ulna.

[0058] In some embodiments, the delayed union or nonunion fracture is selected from a septic nonunion fracture, a pseudoarthrosis nonunion fracture, a hypertrophic nonunion fracture, an atrophic nonunion fracture and an oligotrophic nonunion fracture.

[0059] In some embodiments, applying the composition to the site of delayed union or nonunion comprises injecting the composition into the site.

[0060] In some embodiments, the method comprises:

[0061] a) creating a stab incision at the site of delayed union or nonunion fracture; b) accessing the site of delayed union or nonunion percutaneously with a delivery needle and a trocar, optionally using fluoroscopic guidance;

[0062] c) optionally disrupting fibrous tissue with the trocar, thereby creating an injury response in the fracture site;

[0063] d) removing and replacing the trocar with a periosteal elevator, thereby creating a space subperiosteally or within the soft tissues surrounding the fracture site.

[0064] e) providing a syringe containing the composition

[0065] f) removing the periosteal elevator and docking the syringe to the delivery needle g) injecting the graft in and / or around the fracture site;

[0066] h) withdrawing the needle from the site of delayed union or nonunion; and i) closing the incision.

[0067] In some embodiments, the needle is withdrawn from the site after a period of time (such as about 3 minutes to about 5 minutes).

[0068] In some embodiments, the method further comprises combining the PDGF solution with the biocompatible matrix to form the composition prior to applying the composition to the site of nonunion or delayed union.

[0069] In some embodiments, combining the PDGF solution comprises mixing the PDGF solution and the biocompatible to form a paste, particularly a flowable paste.

[0070] In some embodiments, the composition comprises particles of β-TCP and collagen, wherein the particles of β-TCP have a porosity of at least 90% and a size ranging from aboutE3383-03646

[0071] 100 to about 300 microns, wherein the collagen is present in an amount of about 20% by weight of the biocompatible matrix, and wherein the PDGF solution has a concentration of about 0.3 mg / mL.

[0072] Compositions

[0073] PDGF Solutions

[0074] In some embodiments, the aqueous liquid comprises a solution comprising PDGF, and the solution is disposed or incorporated into the biocompatible matrix, for example by manual or mechanical mixing in order to form the desired consistency.

[0075] In some embodiments, the PDGF is present in the solution in a concentration ranging from about 0.01 mg / ml to about 10 mg / ml, from about 0.05 mg / ml to about 5 mg / ml, or from about 0.1 mg / ml to about 1.0 mg / ml. PDGF may be present in the solution at any concentration within these stated ranges, including the upper limit and lower limit of each range. In other embodiments, PDGF is present in the solution at any one of the following concentrations: about 0.05 mg / ml; about 0.1 mg / ml; about 0.15 mg / ml; about 0.2 mg / ml; about 0.25 mg / ml; about 0.3 mg / ml; about 0.35 mg / ml; about 0.4 mg / ml; about 0.45 mg / ml; about 0.5 mg / ml; about 0.55 mg / ml; about 0.6 mg / ml; about 0.65 mg / ml; about 0.7 mg / ml; about 0.75 mg / ml; about 0.8 mg / ml; about 0.85 mg / ml; about 0.9 mg / ml; about 0.95 mg / ml; or about 1.0 mg / ml. It is to be understood that these concentrations are simply examples of particular embodiments, and that the concentration of PDGF may be within any of the concentration ranges stated above, including the upper limit and lower limit of each range.

[0076] The concentration of PDGF or other growth factors in some embodiments of the present disclosure can be determined by using an enzyme-linked immunoassay as described in U. S. Pat. Nos. 6,221,625, 5,747,273, and 5,290,708, incorporated herein by reference, or any other assay known in the art for determining PDGF concentration. When provided herein, the molar concentration of PDGF is determined based on the molecular weight (MW) of PDGF dimer (e g., PDGF-BB; MW about 25 kDa).

[0077] PDGF may comprise PDGF homodimers and / or heterodimers, including PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD, and mixtures and derivatives thereof. In some embodiments, PDGF comprises PDGF-BB. In another embodiment PDGF comprises a recombinant human (rh) PDGF, such as rhPDGF-BB.E3383-03646

[0078] PDGF, in some embodiments, can be obtained from natural sources. In other embodiments, PDGF can be produced by recombinant DNA techniques. In other embodiments, PDGF or fragments thereof may be produced using peptide synthesis techniques known to one of ordinary skill in the art, such as solid phase peptide synthesis. When obtained from natural sources, PDGF can be derived from biological fluids. Biological fluids, according to some embodiments, can comprise any treated or untreated fluid associated with living organisms including blood.

[0079] Biological fluids, in another embodiment, can also comprise blood components including platelet concentrate (PC), apheresed platelets, platelet-rich plasma (PRP), plasma, serum, fresh frozen plasma (FFP), and buffy coat (BC). Biological fluids, in a further embodiment, can comprise platelets separated from plasma and resuspended in a physiological fluid.

[0080] When PDGF is produced by recombinant DNA techniques, a DNA sequence encoding a single monomer (e.g., PDGF B-chain or A-chain), in some embodiments, can be inserted into cultured prokaryotic or eukaryotic cells for expression to subsequently produce the homodimer (e.g. PDGF-BB or PDGF-AA). In other embodiments, a PDGF heterodimer can be generated by inserting DNA sequences encoding for both monomeric units of the heterodimer into cultured prokaryotic or eukaryotic cells and allowing the translated monomeric units to be processed by the cells to produce the heterodimer (e.g. PDGF-AB). Research grade rhPDGF-BB can be obtained from multiple sources including R& D Systems, Inc. (Minneapolis, Minn.), BD Biosciences (San Jose, Calif.), Sigma Aldrich (Darmstadt, Germany), Invitrogen (Gibco – unknown location), and Chemicon, International (Temecula, Calif).

[0081] In some embodiments of the present invention, PDGF comprises PDGF fragments. In some embodiments rhPDGF-B comprises the following fragments: amino acid sequences 1-31, 1-32, 33-108, 33-109, 1-108 and / or 1-109 of the entire B chain. The complete amino acid sequence (1-109) of the B chain of PDGF is provided in FIG. 15 of U. S. Pat. No. 5,516,896, the disclosure of which is hereby incorporated by reference in its entirety. It is to be understood that the rhPDGF-BB compositions of the present invention may comprise a combination of intact rhPDGF-B (1-109) and fragments thereof. Other fragments of PDGFE3383-03646

[0082] may be employed such as those disclosed in U. S. Pat. No. 5,516,896. In accordance with one embodiment, the rhPDGF-BB comprises at least 65% of intact rhPDGF-B (1-109). In another embodiment, the rhPDGF-BB comprises at least 75%, 80%, 85%, 90%, 95%, or 99% of intact rhPDGF-B (1-109). Methods of producing PDGF are described in US Publication No.

[0083] 20140308332, the contents of which are hereby incorporated by reference in their entirety.

[0084] In some embodiments, PDGF can be purified. Purified PDGF, as used herein, comprises compositions having greater than about 95% by weight PDGF prior to incorporation in solutions of the present invention. The solution may be any pharmaceutically acceptable solution. In other embodiments, the PDGF can be substantially purified.

[0085] Substantially purified PDGF, as used herein, comprises compositions having about 5% to about 95% by weight PDGF prior to incorporation into solutions of the present invention. In some embodiments, substantially purified PDGF comprises compositions having about 65% to about 95% by weight PDGF prior to incorporation into solutions of the present invention. In other embodiments, substantially purified PDGF comprises compositions having about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%. or about 90% to about 95%, by weight PDGF. prior to incorporation into solutions of the present invention.

[0086] In a further embodiment. PDGF can be partially purified. Partially purified PDGF, as used herein, comprises compositions having PDGF in the context of platelet rich plasma (PRP). fresh frozen plasma (FFP), or any other blood product that requires collection and separation to produce PDGF. Embodiments of the present invention contemplate that any of the PDGF isoforms provided herein, including homodimers and heterodimers, can be purified or partially purified. Compositions of the present invention containing PDGF mixtures may contain PDGF isoforms or PDGF fragments in partially purified proportions. Partially purified and purified PDGF, in some embodiments, can be prepared as described in U. S. patent application Ser. No. 11 / 159,533 (Publication No: 20060084602).

[0087] In some embodiments, solutions comprising PDGF are formed by solubilizing PDGF in aqueous media or in one or more buffers. Buffers suitable for use in PDGF solutions of the present invention can comprise, but are not limited to, carbonates, phosphates (e.g. phosphate buffered saline), histidine, acetates (e g. sodium acetate), acidic buffers such as acetic acidE3383-03646

[0088] and HCl, and organic buffers such as lysine, Tris buffers (e.g. tris(hydroxymethyl)aminoethane), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and 3-(N-morpholino) propanesulfonic acid (MOPS). Buffers can be selected based on biocompatibility with PDGF and the buffer's ability to impede undesirable protein modification. Buffers can additionally be selected based on compatibility with host tissues. In some embodiments, sodium acetate buffer is used. The buffers can be employed at different molarities, for example, about 0.1 mM to about 100 mM, about 1 mM to about 50 mM, about 5 mM to about 40 mM, about 10 mM to about 30 mM, or about 15 mM to about 25 mM. or any molarity within these ranges. In some embodiments, an acetate buffer is employed at a molarity of about 20 mM.

[0089] In another embodiment, solutions comprising PDGF are formed by solubilizing lyophilized PDGF in water, wherein prior to solubilization the PDGF is lyophilized from an appropriate buffer.

[0090] The pH of solutions comprising PDGF, in some embodiments, can be controlled by the buffers and pH modifiers recited herein. Various proteins demonstrate different pH ranges in which they are stable. Protein stabilities are primarily reflected by isoelectric points and charges on the proteins. The pH range can affect the conformational structure of a protein and the susceptibility of a protein to proteolytic degradation, hydrolysis, oxidation, and other processes that can result in modification to the structure and / or biological activity of the protein.

[0091] In some embodiments, solutions comprising PDGF can further comprise additional components, such as other biologically active agents. In other embodiments, solutions comprising PDGF can further comprise cell culture media, other stabilizing proteins such as albumin, antibacterial agents, protease inhibitors [e.g., ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA), aprotinin,.epsilon.-aminocaproic acid (EACA), etc.] and / or other growth factors such as fibroblast growth factors (FGFs), epidermal growth factors (EGFs), transforming growth factors (TGFs), keratinocyte growth factors (KGFs), insulin-like growth factors (IGFs), bone morphogenetic proteins (BMPs), or other PDGFs including compositions of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and / or PDGF-DD.E3383-03646

[0092] Biocompatible Matrix

[0093] The implant material comprises a biocompatible matrix. In some embodiments, the biocompatible matrix comprises a porous calcium phosphate and fibrillar collagen.

[0094] The calcium phosphate provides a framework or scaffold for new bone and tissue growth to occur. A calcium phosphate can be used to permanently or temporarily replace bone. Following implantation, the calcium phosphate can be retained by the body or it can be resorbed by the body and replaced with bone. Exemplary calcium phosphates include, e.g., a calcium phosphate (e.g., tricalcium phosphate, such as β-tricalcium phosphate (β-TCP), hydroxyapatite, poorly crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate, and octacalcium phosphate), calcium sulfate, and allograft (e.g. mineralized bone, mineralized deproteinized xenograft, or demineralized bone (e.g., demineralized freeze-dried cortical or cancellous bone), and any combination thereof.

[0095] In some embodiments, the calcium phosphate comprises P-TCP. In other embodiments, the composition comprises a plurality of calcium phosphates. Calcium phosphates suitable for use have a calcium to phosphorus atomic ratio ranging from 0.5 to 2.0.

[0096] In some embodiments, biocompatible matrices may include calcium phosphate particles with or without or bone allograft such as demineralized freeze dried bone allograft (DFDBA) or particulate demineralized bone matrix (DBM). In another embodiment, biocompatible matrices may include bone allograft such as DFDBA or DBM. In an embodiment, the biocompatible matrix is bioresorbable. In some embodiment, a biocompatible matrix comprises an allograft such as DFDBA or particulate DBM.

[0097] Non-limiting examples of suitable calcium phosphates suitable comprise amorphous calcium phosphate, monocalcium phosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA), dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA). octacalcium phosphate (OCP). a-tricalcium phosphate, P-TCP, hydroxyapatite (OHAp), poorly crystalline hydroxyapatite, tetracalcium phosphate (TTCP), heptacalciumE3383-03646

[0098] decaphosphate, calcium metaphosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate, carbonated calcium phosphate, or any mixture thereof.

[0099] In another embodiment, the calcium phosphate has a porous composition. Porosity is a desirable characteristic as it facilitates cell migration and infiltration into the implant material so that the infiltrating cells can secrete extracellular bone matrix. Porosity also provides access for vascularization. Porosity also provides a high surface area for enhanced resorption and release of active substances, as well as increased cell-matrix interaction. In a preferred embodiment, the bone substituting agent is a calcium phosphate (e.g., (β-TCP). Porous calcium phosphates, according to some embodiments, can comprise pores having diameters ranging from about 1 μm to about 1 mm. In some embodiments, porous calcium phosphates comprises macropores having diameters ranging from about 100 μm to about 1 mm. In another embodiment, porous calcium phosphates comprises mesopores having diameters ranging from about 10 μm to about 100 μm. In a further embodiment, porous calcium phosphates comprises micropores having diameters less than about 10 μm.

[0100] Embodiments of the present invention contemplate Porous calcium phosphates comprising macropores, mesopores, and micropores or any combination thereof. In some embodiments, the bone scaffolding material comprises interconnected pores. In some embodiments, the porous calcium phosphates comprise non-interconnected pores. In some embodiments, the porous calcium phosphates comprise interconnected and non-interconnected pores.

[0101] The porous calcium phosphates, in some embodiments, has a porosity greater than about 25% or greater than about 40%. In another embodiment, porosity is greater than about 50%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 80%, or greater than about 85%. In a further embodiment, porosity greater than about 90%. In some embodiments, the porous calcium phosphate comprises a porosity that facilitates cell migration into the scaffolding material.

[0102] In some embodiments, the porous calcium phosphates comprise a plurality of particles. Particles of the porous calcium phosphates, in some embodiments, can individually demonstrate any of the pore diameters and porosities provided herein. In other embodiments, particles of the porous calcium phosphates can form an association to produce a matrix having any of the pore diameters or porosities provided herein.E3383-03646

[0103] Porous calcium phosphate particles may be mm, pm or submicron (nm) in size. Porous calcium phosphate particles, in some embodiments, have an average diameter ranging from about 1 μm to about 5 mm. In other embodiments, particles have an average diameter ranging from about 1 mm to about 2 mm, from about 1 mm to about 3 mm. or from about 250 μm to about 750 μm. In another embodiment, the particles have an average diameter ranging from about 100 μm to about 300 μm. In a further embodiment, the particles have an average diameter ranging from about 75 μm to about 300 μm. In additional embodiments, porous calcium phosphate particles have an average diameter less than about 25 μm, less than about 1 μm and, in some cases, less than about 1 mm. In some embodiments, porous calcium phosphate particles have an average diameter ranging from about 100 μm to about 5 mm or from about 100 μm to about 3 mm. In other embodiments, porous calcium phosphate particles have an average diameter ranging from about 250 μm to about 2 mm, from about 250 μm to about 1 mm, from about 200 μm to about 3 mm. Particles may also be in the range of about 1 nm to about 1000 nm, less than about 500 nm or less than about 250 nm.

[0104] Porous calcium phosphate particles, in some embodiments, have a diameter ranging from about 1 μm to about 5 mm. In other embodiments, particles have a diameter ranging from about I mrn to about 2 mm, from about 1 mm to about 3 mm, or from about 250 μm to about 750 μm. Porous calcium phosphate particles, in another embodiment, have a diameter ranging from about 100 μm to about 300 μm. In a further embodiment, the particles have a diameter ranging from about 75 μm to about 300 μm. In additional embodiments, porous calcium phosphate particles have a diameter less than about 25 μm, less than about 1 μm and, in some cases, less than about 1 mm. In some embodiments, porous calcium phosphate particles have a diameter ranging from about 100 μm to about 5 mm or from about 100 μm to about 3 mm. In other embodiments, porous calcium phosphate particles have a diameter ranging from about 250 μm to about 2 mm, from about 250 μm to about 1 mm, from about 200 μm to about 3 mm. Particles may also be in the range of about 1 nm to about 1000 nm, less than about 500 nm or less than about 250 nm.

[0105] In some embodiments, porous calcium phosphates are bioresorbable. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 75% or 90% resorbed within one year subsequent to in vivo implantation. In another embodiment, the material can be resorbed atE3383-03646

[0106] least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or 90% within 1, 3, 6, 9, 12, or 18 months of in vivo implantation. Bioresorbability will be dependent on: (1) the nature of the matrix material (i.e., its chemical make-up, physical structure and size); (2) the location within the body in which the matrix is placed; (3) the amount of matrix material that is used; (4) the metabolic state of the patient (diabetic / non-diabetic, osteoporotic, smoker, old age, steroid use. etc.); (5) the extent and / or type of injury treated; and (6) the use of other materials in addition to the matrix such as other bone anabolic, catabolic and anti-catabolic factors.

[0107] The biocompatible matrix includes a fibrillar collagen, for example about 10 to about 40 percent by weight of fibrillar collagen. The biocompatible further comprises non-fibrillar (soluble) collagen) in an amount up to about 10% by weight.

[0108] Biocompatible matrices may be shredded. In preferred embodiments, the matrix is shredded, thus advantageously providing a flowable paste when combined with the aqueous liquid.

[0109] β-Tricalcium Phosphate

[0110] In some embodiments, the porous calcium phosphate is β-TCP. β-TCP, according to some embodiments, can comprise a porous structure having multidirectional and interconnected pores of varying diameters. In some embodiments, β-TCP comprises a plurality of pockets and non-interconnected pores of various diameters in addition to the interconnected pores. The porous structure of β-TCP, in some embodiments, comprises macropores having diameters ranging from about 100 μm to about 1 mm, mesopores having diameters ranging from about 10 μm to about 100 μm, and micropores having diameters less than about 10 μm. Macropores and micropores of the β-TCP can facilitate osteoinduction and osteoconduction while macropores, mesopores and micropores can permit fluid communication and nutrient transport to support bone regrowth throughout the β-TCP biocompatible matrix.

[0111] In comprising a porous structure, β-TCP, in some embodiments, can have a porosity greater than 25% or greater than about 40%. In other embodiments, β-TCP can have a porosity greater than 50%, greater than about 60%, greater than about 65%, greater thanE3383-03646

[0112] about 70%, greater than about 75%, greater than about 80%, or greater than about 85%. In a further embodiment, β-TCP can have a porosity greater than about 90%. In some embodiments, (β-TCP can have a porosity that facilitates cell migration into the β-TCP.

[0113] β-TCP particles, in some embodiments, can individually demonstrate any of the pore diameters and porosities provided herein for β-TCP. In other embodiments, β-TCP particles of a bone scaffolding material can form an association to produce a matrix having any of the pore diameters or porosities provided herein for the bone scaffolding material. Porosity may facilitate cell migration and infiltration into the matrix for subsequent bone formation. β-TCP particles, in some embodiments, have an average diameter ranging from about 1 μm to about 5 mm. In other embodiments, β-TCP particles have an average diameter ranging from about 1 mm to about 2 mm, from about 1 mm to about 3 mm, from about 250 μm to about 750 μm, from about 250 μm to about 1 mm, from about 250 μm to about 2 mm, or from about 200 μm to about 3 mm. In another embodiment, P-TCP particles have an average diameter ranging from about 100 μm to about 300 μm. In a further embodiment, P-TCP particles have an average diameter ranging from about. 75 pm to about 300 pm. In additional embodiments, P-TCP particles have an average diameter less than about 25 pm. average diameter less than about I pm, or less than about 1 mm

[0114] In some embodiments, β-TCP particles have an average diameter ranging from about 100 μm to about 5 mm or from about 100 μm to about 3 mm.

[0115] Flowable matrices may be applied through syringes, tubes, or spatulas or equivalent devices. Flowable β-TCP bone scaffolding materials, in some embodiments, can be applied to sites of delayed union or nonunion fracture using a needle and syringe or cannula. In some embodiments, a β-TCP bone scaffolding materials harden in vivo.

[0116] β-TCP, according to some embodiments, is bioresorbable. In some embodiments, a β-TCP bone scaffolding material can be at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% resorbed one year subsequent to in vivo implantation. In another embodiment, a β-TCP bone scaffolding material can be greater than about 90% resorbed one year subsequent to in vivo implantation.

[0117] CollagenE3383-03646

[0118] While not being bound by theory, it is believed that collagen can promote cohesion between combined substances. Collagen, for example, can promote adhesion between particles of calcium phosphate in the formation of a biocompatible matrix.

[0119] In some embodiments, the collagen comprises Type I collagen comprises any type of collagen, including Type I. Type II, and Type III collagens. In some embodiments, a collagen comprises a mixture of collagens, such as a mixture of Type I and Type II collagen. Other types of collagen present in bone or musculoskeletal tissues may be employed. Recombinant, synthetic and naturally occurring forms of collagen may be used in the present invention. In preferred embodiments, the collagen is a soluble Type I bovine collagen. The soluble bovine Type I collagen may be derived from the inner layer stratum corium of hides.

[0120] β-TCP particles suitable for use with a collagen can comprise any of the β-TCP particles described herein. In some embodiments, β-TCP particles suitable for combination with a collagen have an average diameter ranging from about 1 μm to about 5 mm. In another embodiment. 0-TCP particles suitable for combination with a collagen have an average diameter ranging from about. 1 pm to about 1 mm, from about I mm to about 2 mm, from about I mm to about 3 mm. from about 250 μm to about 750 μm. from about 250 pm to about 1 rnm, from about 250 pm to about 2 mm, from about. 200 pm to about I mm, or from about 200 pm to about 3 mm. 0-TCP particles, in other embodiments, have an average diameter ranging from about 100 μm to about 300 μm. In a further embodiment, β-TCP particles suitable for combination with a collagen have an average diameter ranging from about 75 μm to about 300 μm. In additional embodiments β-TCP particles suitable for combination with a collagen have an average diameter less than about 25 μm and, less than about 1 mm or less than about 1 μm. In some embodiments, β-TCP particles suitable for combination with a collagen have an average diameter ranging from about 100 μm to about 5 mm or from about 100 μm to about 3 mm. β-TCP particles, in some embodiments, can be adhered to one another by the collagen so as to produce a biocompatible matrix having a porous structure. In some embodiments, a biocompatible matrix comprising β-TCP particles and a collagen can comprise pores having diameters ranging from about 1 μm to about 1 mm. A biocompatible matrix comprising β-TCP particles and a collagen can comprise macropores having diametersE3383-03646

[0121] ranging from about 100 pm to about 1 mm, mesopores having diameters ranging from about 10 pm to 100 pm, and micropores having diameters less than about 10 μm.

[0122] A biocompatible matrix comprising β-TCP particles collagen can have a porosity greater than about 25% or greater than 40%. In another embodiment, the biocompatible matrix can have a porosity greater than about 50%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 80%, or greater than about 85%. In a further embodiment, the biocompatible matrix can have a porosity greater than about 90%. Porosity facilitates cell migration and infiltration into the matrix for subsequent bone formation.

[0123] A biocompatible matrix comprising β-TCP particles, in some embodiments, can comprise collagen in an amount ranging from about 10 weight percent to about 20 weight percent.

[0124] In a further embodiment, a collagen can be present in an amount of about 20 weight percent of the biocompatible matrix.

[0125] In a further embodiment, a collagen can be present in an amount of about 15 weight percent of the biocompatible matrix.

[0126] In a further embodiment, a collagen can be present in an amount of about 10 weight percent of the biocompatible matrix.

[0127] A biocompatible matrix comprising β-TCP particles and a collagen, according to some embodiments, can be flowable. In such embodiments, the biocompatible matrix can be delivered in a syringe using a needle, cannula or trocar.

[0128] A biocompatible matrix comprising β-TCP particles and a collagen can be resorbable. In some embodiments, a biocompatible matrix comprising β-TCP particles and a collagen can be at least 30%, 40%, 50%, 60%, 70%, 75%, or 90% resorbed one year subsequent to in vivo implantation. In another embodiment, this matrix can be resorbed at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% or 90% within 1, 3, 6, 9, 12, or 18 months subsequent to in vivo implantation.

[0129] The implant composition in some embodiments is flowable and may be applied to a desired site via a syringe with a needle. An exemplary composition is Augment ® Injectable, and is described in the AUGMENT® INJECTABLE PACKAGE INSERT dated May, 2018,E3383-03646

[0130] which is hereby incorporated by reference in its entirety Augment® Injectable (Al) consists of 2 components. Beta-tricalcium phosphate (β-TCP) / Bovine type I soluble collagen matrix and rhPDGF-BB (0.3 mg / ml solution of rhPDGF-BB in 20 mM sodium acetate solution, pH 6.0). Al is formulated for use by mixing the matrix and rhPDGF-BB solution in a volume to mass ratio of 3:1 (3 mL of rhPDGF-BB solution mixed with 1 gram of the matrix), as per the instructions for use (IFU) using the syringe provided as a part of the Al kit. The Al forms a paste-like formulation that is be applied to a site of delyed or nonunion through a cannula or blunt needle.

[0131] Incorporating PDGF Solution into a Biocompatible Matrix

[0132] PDGF may be disposed into a biocompatible matrix by providing a biocompatible matrix, and saturating the biocompatible matrix with a solution containing PDGF. PDGF solutions and biocompatible matrices suitable for combination are consistent with those described hereinabove.

[0133] In some embodiments, a PDGF solution can be incorporated into the biocompatible matrix by soaking the biocompatible matrix in the PDGF solution. A PDGF solution, in another embodiment. can be incorporated in a biocompatible matrix by injecting the biocompatible matrix with the PDGF solution In some embodiments, injecting a PDGF solution can comprise incorporating the PDGF solution in a syringe and expelling the PDGF solution into the biocompatible matrix to saturate the biocompatible matrix. The biocompatible matrix may be incubated for a period of time and then worked with a spatula or stirring device to homogenize the implant material. In some embodiments, a composition is prepared by providing a syringe containing a biocompatible matrix and a syringe containing a solution of PDGF. The syringes may be connected together with a Luer lock (e.g., a female to female Luer lock connector), and the PDGF solution transferred to the syringe containing the biocompatible matrix (See Figure 7). After the matrix is hydrated, the composition is further mixed by transferring the contents back and forth between the two syringes at least 20 times.

[0134] Compositions Comprising Additional Biologically Active AgentsE3383-03646

[0135] The compositions described herein for inducing bone formation at the site a delayed union or nonunion fracture, according to some embodiments, can further comprise one or more biologically active agents in addition to PDGF. Biologically active agents that can be incorporated into compositions of the present invention in addition to PDGF can comprise organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, small insert ribonucleic acids [si-RNAs], gene regulatory sequences, nuclear transcriptional factors, and antisense molecules), nucleoproteins, polysaccharides (e.g,, heparin), glycoproteins, and lipoproteins. Non-limiting examples of biologically active compounds that can be incorporated into compositions of the present invention, including, e.g., anti-cancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, hormones, muscle relaxants, prostaglandins, trophic factors, osteoinductive proteins, growth factors, and vaccines, are disclosed in U. S. patent application Ser. No. 11 / 159,533 (Publication No: 20060084602). In some embodiments, biologically active compounds that can be incorporated into compositions of the present invention include osteoinductive factors such as insulin-like growth factors, fibroblast growth factors, or other PDGFs. In accordance with other embodiments, biologically active compounds that can be incorporated into compositions of the present invention preferably include osteoinductive and osteostimulatory factors such as bone morphogenetic proteins (BMPs), BMP mimetics, calcitonin, calcitonin mimetics, statins, statin derivatives, or parathyroid hormone Preferred factors also include protease inhibitors, as well as osteoporotic treatments that decrease bone resorption including bisphosphonates, and antibodies to receptor activator of NF-kB ligand (RANK) ligand.

[0136] Standard protocols and regimens for delivery of additional biologically active agents are known in the art. Additional biologically active agents can be introduced into compositions of the present invention in amounts that allow delivery of an appropriate dosage of the agent to the implant site. In most cases, dosages are determined using guidelines known to practitioners and applicable to the particular agent in question. The amount of an additional biologically active agent to be included in a composition of the present invention can depend on such variables as the type and extent of the condition, the overall health status of the particular patient, the formulation of the biologically active agent, release kinetics, andE3383-03646

[0137] the bioresorbability of the biocompatible matrix. Standard clinical trials may be used to optimize the dose and dosing frequency for any particular additional biologically active agent.

[0138] EXAMPLES

[0139] Example 1: Non-union healing fracture model

[0140] In this pilot study, rhPDGF-BB was combined with a bovine type I collagen / β-TCP matrix (Augment® Injectable ) to evaluate its effectiveness in various models to replicate a delayed union or acute treatment for bone healing in a rabbit long-bone critical size defect model of non-union fracture healing.

[0141] Using an anteromedial approach, a sixty millimeter (60 mm) longitudinal incision will be made on the skin and underlying soft tissue. The surgeon will osteotomize the right or left distal tibia diaphysis, stripping the periosteum and drilling the marrow 15* mm proximal and distal to the osteotomy, after which, will fix a plate (2.5mm thickness + 4 locking screws) on the antero-medial aspect of the tibia, leaving a 2mm gap between the bone segments. After fixation and saline irrigation, Al will be delivered to the site (gap) for the Acute treatment groups.

[0142] Chronic treatment groups were treated with Al delivered to the site (gap) 4 weeks post-defect creation

[0143] Material

[0144] Grp. Arm Treatment Group Count Healing Time (Wks)

[0145] (Y(Yes) / N(No))

[0146] A Acute Negative Control N 6 8

[0147] B Acute Short Healing Y 6 4

[0148] C Acute Long Healing Y 6 8

[0149] D Chronic* Long healing N (time 0) 6 4 + 8 (w / material)

[0150]

[0151] *Note: 15 mm marrow drilling was used the initial cohort only (rabbit #2 and #4). After discussing concerns related to PDGF-BB contact with target cells, we reduced this to approx.

[0152] 5 mm marrow drilling in the 2nd cohort.E3383-03646

[0153] Results of the study are showing in Figures 1-6. Figure 1 shows X-ray radiographs of (a) lateral and (b) rostral caudal of chronic treatment group (Group D) taken prior to injection with Al to confirm the presence of non-union (dashed box).

[0154] Figures 2A-D show representative DEXA radiographs of groups (a) A, (b) B, (c) C, and (d) D. Red arrows (arrows with vertical crosshatch design) point to absence of bone bridging, yellow arrows (arrows with crisscross crosshatch design) indicate presence of fibrotic connective tissue and the green arrow (arrows with diagonal crosshatch design) depicts complete bone bridging across the defect site.

[0155] Figure 3A-D show representative volumetric reconstructions of groups (a) A, (b) B. (c) C, and (d) D, with bone and calcified fibrotic tissue and fixation plate / screws in purple (dark crosshatched sections). Red arrows (arrows with vertical crosshatch design) point to absence of bone bridging, the white arrow indicates presence of fibrotic connective tissue, and the green arrow (arrow with diagonal crosshatch design) depicts complete bone bridging across the defect site.

[0156] Figure 4A-D show representative low magnification histomicrographs of groups (a) A, (b) B, (c) C, and (d) D. Dashed black line depicts the induced 2 mm defect (gap). Blue arrows (wide arrows with horizontal crosshatch design) point towards surgical hardware (fixation plate), red arrows (arrows with vertical crosshatch design) highlight the inorganic β-TCP, yellow arrows (arrows with crisscross crosshatch design) indicate bone-related tissue.

[0157] Figures 5A-D show representative high magnification histomicrographs of groups (a) A, (b) B, (c) C, and (d) D in the region of interest. Dashed black line depicts the width of the induced defect (2 mm). Red arrows (arrows with vertical crosshatch design) indicate presence of β-TCP particles, blue arrows (wide arrows with horizontal crosshatch design) denote bone remodeling sites, cyan arrows (wide arrows with dashed vertical crosshatch design) show woven bone formation (in group C), green arrows (arrows with diagonal crosshatch design) point towards primary oseteon formation, and yellow (arrows with crisscross crosshatch design) arrows show the presence of periosteal coverage in group D.

[0158] In the present study, quantitative analysis of the presence of bone within the defect site (bone %) revealed no differences between negative control and the groups that receivedE3383-03646

[0159] Al treatment. Nevertheless, qualitatively and semi-quantitatively, chronic application of Al facilitated complete bridging of the induced non-union defect, while untreated defects presented evidence of non-healing at 8 weeks (see Figure 6).

[0160] INCORPORATION BY REFERENCE

[0161] All of the U. S. patents and U. S. and PCT published patent applications cited herein are hereby incorporated by reference.

[0162] EQUIVALENTS

[0163] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall wi thin the scope of the appended claims. The advantages and obj ects of the invention are not necessarily encompassed by each embodiment of the invention.

Claims

E3383-03646CLAIMSWe claim:

1. A method of inducing bone formation in a delayed union or nonunion fracture in a bone comprising:applying to a site of delayed union or nonunion fracture a composition comprising a platelet-derived growth factor (PDGF) solution disposed in a biocompatible matrix.

2. The method of claim 1. wherein the biocompatible matrix comprises a bone scaffolding material and a biocompatible binder.

3. The method of claim 2, wherein the bone scaffolding material comprises calcium phosphate, calcium sulfate, demineralized freeze-dried bone allograft, demineralized bone matrix, or any combination thereof.

4. The method of claim 3, wherein the bone scaffolding material comprises calcium phosphate.

5. The method of claim 4. wherein the calcium phosphate is selected from the group consisting of β-tricalcium phosphate (β-TCP), hydroxyapatite, poorly crystalline hydroxyapatite, amorphous calcium phosphate, calcium metaphosphate, dicalcium phosphate dihydrate, heptacalcium phosphate, calcium pyrophosphate dihydrate, calcium pyrophosphate, octacalcium phosphate, and any combination thereof.

6. The method of claim 5, wherein the calcium phosphate is β-TCP.

7. The method of any one of claims 1 to 6, wherein the biocompatible binder comprises collagen, a protein, a polysaccharide, a nucleic acid, a carbohydrate, a synthetic polymer, or any combination thereof.

8. The method of claim 7, wherein the biocompatible binder comprises collagen.E3383-036469. The method of claim 8, wherein the collagen is ty pe I, type II, type III, or any combination thereof.

10. The method of claim 9, wherein the collagen is type I collagen.

11. The method of any one of claims 8-10, wherein the collagen is bovine collagen.

12. The method of any one of claims 1 to 11, wherein the biocompatible matrix comprises about 5% to about 40%, about 10% to about 30%, about 15% to about 25%, or about 20% by weight of the biocompatible binder.

13. The method of any one of claims 1 to 12, wherein the PDGF solution and the biocompatible matrix are present in the composition in a liquid volume to mass ratio of about 1: 1 to about 4:1, about 2: 1 to about 3:1, about 2: 1, or about 3:1.

14. The method of any one of claims 1 to 13, wherein the PDGF solution has PDGF concentration of about 0.1 to about 0.01 to about 10.0 mg / mL. about 0.05 to about 5 mg / mL, about 0.1 to about 1 mg / mL, about 0.1 to about 0.5 mg / mL, or about 0.3 mg / mL.

15. The method of any one of claims 1 to 14, wherein the PDGF solution is aqueous.

16. The method of claim 15, wherein the PDGF solution comprises a buffer.

17. The method of claim 1, wherein the buffer comprises sodium acetate.

18. The method of any one of claims 1 to 17, wherein the PDGF comprises PDGF-BB.

19. The method of claim 18, wherein the PDGF comprises recombinant human PDGF-BB (rhPDGF-BB).E3383-0364620. The method of claim 18, wherein the rhPDGF-BB comprises at least 65% intact rhPDGF-BB.

21. The method of any one of claims 2 to 20, wherein the bone scaffolding material comprises particles having an average diameter of about 100 microns to about 300 microns.

22. The method of any one of claims 2 to 21. wherein the biocompatible binder has a porosity greater than about 50%, greater than about 60%, greater than about 70%, about than about 80%. or greater than about 90%.

23. The method of any one of claims 1 to 22, wherein the method induces bone bridging at the site of delayed union or nonunion fracture.

24. The method of any one of claims 1 to 23, wherein the method induces periosteal coverage at the site of delayed union or nonunion fracture.

25. The method of any one of claims 1 to 24. wherein the method induces osteon formation at the site of delayed union or nonunion fracture.

26. The method of any one of claims 1 to 25, wherein the method induces osteon formation at the site of delayed union or nonunion fracture.

27. The method of any one of claims 1 to 26, wherein the method promotes woven bone formation at the site of delayed union or nonunion fracture.

28. The method of any one of claims 1 to 27, wherein the bone is an appendicular bone.

29. The method of claim 28, wherein the bone is selected from a humerus, a radius, an ulna, a carpals, a metacarpals, a phalanges, a femur, a tibia, a fibula, and a tarsals.E3383-0364630. The method of claim 29, wherein the bone is seslected from the tibia, femur, humerus, fibula, radius, and ulna.

31. The method of any one of claims 1 to 30, wherein the bone is in a mammal.

32. The method of claim 31, wherein the mammal is a human.

33. The method of any one of claims 1 to 32, wherein the delayed union or nonunion fracture is selected from a septic nonunion fracture, a pseudoarthrosis nonunion fracture, a hypertrophic nonunion fracture, an atrophic nonunion fracture and an oligotrophic nonunion fracture.

34. The method of any one of claims 1 to 33, wherein applying the composition to the site of delayed union or nonunion comprises injecting the composition into the site.

35. The method of any one of claims 1 to 34, wherein the method comprises:a) creating a stab incision at the site of delayed union or nonunion fracture; b) accessing the site of delayed union or nonunion percutaneously with a delivery needle and a trocar, optionally using fluoroscopic guidance;c) optionally disrupting fibrous tissue with the trocar, thereby creating an injury response in the fracture site;d) removing and replacing the trocar with a periosteal elevator, thereby creating a space subperiosteally or within the soft tissues surrounding the fracture site.e) providing a syringe containing the compositionf) removing the periosteal elevator and docking the syringe to the delivery needle g) injecting the graft in and / or around the fracture site;h) withdrawing the needle from the site of delayed union or nonunion; and i) closing the incision.E3383-0364636. The method of claim 34, wherein the needle is withdrawn from the site of delayed union or nonunion after a period of time of from about 3 minutes to about 5 minutes.

37. The method of any one of claims 1 to 36, wherein the method further comprises combining the PDGF solution with the biocompatible matrix to form the composition prior to applying the composition to the site of nonunion or delayed union.

38. The method of claim 37, wherein combining the PDGF solution comprises mixing the PDGF solution and the biocompatible to form a paste.

39. The method of any one of claims 1 to 38, wherein composition comprises particles of β-TCP and collagen, wherein the particles of β-TCP have a porosity of at least 90% and a size ranging from about 100 to about 300 microns, wherein the collagen is present in an amount of about 20% by weight of the biocompatible matrix, and wherein the PDGF solution has a concentration of about 0.3 mg / mL.

Images