WO2007056316A2 - Injectable physiological temperature setting cement composites for spinal fusion and related method thereof - Google Patents

Abstract

Biodegradable composites and methods for the replacement of bone are described. The composites are made up of a polyphosphazene and an osteoconductive material. The composites are free flowing upon mixing and then later set under physiological tempertures. Methods for using the composites for fusing vertebrae during spinal fusion procedures are also described.

Description

Injectable Physiological Temperature Setting Cement Composites for Spinal Fusion and

Related Method Thereof Statement of Priority

[0001 ] This application claims priority to U.S. Provisional Patent Application Serial No.

60/733,967, filed November 4, 2005, and U.S. Provisional Patent Application Serial No. 60/844,863, filed September 15, 2006, whose disclosures are hereby incorporated by reference herein. Government Interest

[0002] The subject matter of this application was made with support from the United

States Government under Grant No. AR46560 from the National Institutes of Health. The United States Government may retain certain rights. Field of the Invention

[0003] The present invention relates to biodegradable composites for bone replacement and methods for using them. The biodegradable composites allow for support of the site to which they are administered while natural bone forms in place of the composite. Background of the Invention

[0004] There are over 200,000 spinal fusion surgeries performed using bone grafts every year in the United States [I]. Spinal fusion is used to stabilize and align the axial skeleton in many disease processes including, but not limited to, spinal trauma, spine instability, degenerative disk disease, degenerative spondylolisthesis, congenital deformities, and scoliosis [2]. Posterolateral lumbar intertransverse process arthrodesis is the most common spinal fusion procedure performed [I]. Even though it is a commonly performed procedure and multiple advances have occurred over time, there are still many well known complications. [0005] The most clinically relevant complication is nonunion which has been reported to occur between 5-45% of the time even with the use of iliac crest autografts [3]. Nonunions frequently lead to continued pain and instability and often require further surgical intervention depending on the patient's health state. Nonunion has been attributed to many factors including infection, excessive motion at the graft site, trauma, smoking, metabolic abnormalities, insufficient graft material, and poor surgical technique [3].

[0006] Currently the gold standard for spinal fusion grafting is iliac crest autograft.

However there are many complications associated with obtaining the bone graft including a secondary surgery, donor site pain, gait disturbances, nerve injury, infection, fracture, increased operative time, and increased costs. There is also a limited supply as it is difficult to obtain the amount of autograft needed in revision cases or multilevel primary fusions [4]. Allograft bone obtained from a human cadaver eliminates donor site pain and is available in large quantity. However allograft could lead to disease transmission [5], increased immune response [6], and when used alone shows higher rates of nonunion and delayed time to fusion [4]. In spinal fusion cases, allograft is primarily used as an osteoconductive scaffold in combination with autograft, bone marrow aspirates, or osteoinductive proteins such as bone morphogenic proteins (BMP) [2, I]. A final problem with both autograft and allograft is that these materials do not form fit into sites of complex tissue shapes.

[0007] An ideal bone graft substrate should be (1 ) both osteoconductive and osteoinductive, (2) biomechanically strong, (3) minimally antigenic, (4) injectable to conform to complex tissue shapes, and (5) synthetic thus eliminating donor site morbidity and quantity issues. Many different biomaterials are being evaluated to determine a synthetic material that meets all of the above requirements [8,9], However, there is no perfect bone-graft material that is known in the art.

[0008] Polymers have been used in a variety of medical applications such as controlled drug delivery [29 - 32], medical sutures, vascular grafts [33 - 35], and as tri-leaflet heart valve scaffolds [36 - 38]. In the field of orthopaedic surgery, there is a growing clinical interest in the ability of polymers to act as scaffolding to support bone growth, and repair bone defects. Typically, bone defect repair has relied on two options, autogenous and allogenic bone grafts [39-42], Autogenous bone graft is bone obtained from one site of the body and relocated to another area in the same individual [42]. As a whole, autogenous bone grafting has the advantage of optimal biological behavior, histocompatibility, and no risk of disease transmission [40, 42]. Unfortunately, limited availability of autogenous bone combined with donor site morbidity such as nerve and artery damage, chronic pain, and infection which can be associated with the autograft harvesting procedure suggests that autogenous bone graft is less than optimal [41]. [0009] Allogenic bone graft, or allograft, is tissue transferred between two genetically different individuals of the same species [39, 42]. Bone allografts are usually recovered from cadavers and have the advantage over autografts of nearly unlimited availability and the lack of donor site morbidity associated with the harvesting procedure. However, allografts in general have the disadvantage of having an associated risk of disease transmission, immunogenicity, decreased mechanical properties [43], and donor-to-donor variation in quality [44]. The limitations with autografts and allografts have fueled the interest in developing synthetic alternatives to current available bone graft materials.

[0010] Advances in materials science and polymer design have allowed scientists to maximize host cells' potential to generate functional replacement tissue [44], while utilizing polymeric scaffolds as an alternative to traditional methods of bone repair. To that end, many synthetic materials produced in the laboratory have been used to exploit the skeleton's capacity to regenerate and repair [45]. One class of polymers currently on the market are the polyesters, such as poly(lactic-co-glycolic) acid (PLAGA), that has been FDA approved for use as suture and pin fixation devices for fractures [44]. .

[0011] Another novel polymer that has received great attention as a biomaterial is the inorganic polyphosphazenes [46], Previously it was demonstrated that polyphosphazenes are a suitable biodegradable polymer to support the repair of bone in vitro. These polymers can be fashioned into three-dimensional matrices that attempt to simulate the physico-chemical and mechanical properties of cancellous bone [47]. Polyphosphazenes are high molecular weight polymers with an inorganic backbone consisting of nitrogen and phosphorous atoms linked by alternating single and double bonds [48]. Starting with the macromolecular intermediate poly(dichlorophosphazene), a number of different molecules can be formed by nucleophilic substitution of the chlorine atoms with various organic side groups. This process allows polymers to be synthesized which express different physical, chemical, and mechanical properties depending on which side groups are attached to the phosphorus atoms [13, 49], Upon degradation, molecules such as phosphate, ammonia, and amino acids are released [50-54]. These molecules have been observed to be non-toxic to animal tissue and may even cause cell adhesion and growth, thus promoting their potential use as a biocompatible scaffolding to support the development of new bone [55-57].

[0012] Hydroxyapatite (HA) is a major component of bone made up of calcium and phosphate salts [19]. Hydroxyapatite alone is brittle with low tensile strength, but combining hydroxyapatite with a polymer has shown improved mechanical properties [20], While acrylic cements are commonly used in many orthopaedic applications, they have the drawback of generating high temperatures upon polymerization that can lead to tissue disruption [21]. Further, these cements also require the use of toxic reagents which can lead to systemic toxicity if the monomer is absorbed [22].

[0013] Previously, the inventors synthesized a polyphosphazene calcium deficient hydroxyapatite composite which was found to support the adherence, proliferation, and maturation of MC3T3 osteoblast like cells [12]. The inventors have also shown the ability of these composites to support bone regeneration and in-growth in a rabbit tibial defect model [12]. Further, the inventors have shown that the combination of polyphosphazene and hydroxyapatite allows for ionic interactions between the polyphosphazene and the calcium ions in hydroxyapatite, which increases the overall strength of the composite [10,11]. [0014] While the composites described above have provided significant advances in the art, they have only been used with varying levels of success. One drawback of many of the previous composites is that they must be allowed to set outside of the patient's body. After these composites are set in a container, they may then be manipulated and shaped to try and form a structure that fits into the cavity to which they are to be applied. However, it is difficult if not impossible for the surgeon to know the exact shape of the area to which the composite will be applied. As such, the composites are not always a good fit for the area and may not be structurally sound or able to cause the formation of structurally sound bone. [0015] Accordingly, there is a need in the art for low temperature setting composites that set at physiological temperatures without any additives. Such composites could be administered while still in a free flowing form, which would allow them to conform to the exact shape of the area to which they are to be applied. Summary of the Invention

[0016] It is an object of the present invention to provide biodegradable composites for the replacement of bone. The composites include a polyphosphazene and an osteoconductive material. The composites are free flowing upon mixing, allowing them to form to the shape of the cavity to which they are applied. Further, the composites are able to set at a physiological temperature, allowing them to be applied in a free flowing state to a cavity in vivo. [0017] It is a further obj ect of the present invention to provide methods for the replacement of bone using the composites of the present invention. The composites may be applied to fill a cavity in a bone or a space between bones. After application, the composites will set to form a structure with a density and mechanical strength similar to that of natural bone. The composites of the present invention can support a bone structure while the body produces replacement natural bone. As the replacement natural bone is formed, the composites slowly degrade to form non-toxic products which can be easily cleared from the body. [0018] It is a still further object of the present invention to provide a method for fusing vertebrae during a spinal fusion procedure using the composites of the present invention. The composites may be applied in an amount sufficient to cause fusion of vertebrae in the space between the vertebrae to be fused. After the composite sets, natural bone formation and biodegradation of the composite begin as described. Eventually, substantially all of the composite between the vertebrae will degrade, leaving the vertebrae fused together with natural bone.

Brief Description of the Figures [0019] Figure 1. shows immediate post-operative radiographs showing the presence of the grafts: [A] PNEA₅₀PhPh₅₀-CDHA, [B] PNEA₅₀PhPh₅₀-CDSHA, [C] Allograft (human cancellous bone), [D] Autograft, and [E] Negative control (sham);

[0020] Figure 2. shows adiographs 1 week post operation: [A] PNEA₅₀PhPh₅₀-CDHA,

[B] PNEA₅₀PhPh₅₀-CDSHA, [C] Allograft (human cancellous bone), [D] Autograft, and [E]

Negative control (sham);

[0021] Figure 3. shows adiographs 2 weeks post, operation [A] PNEA₅₀PhPh₅₀-CDHA,

[B] PNEA₅₀PhPh₅₀-CDSHA, [C] Allograft (human cancellous bone), [D] Autograft, and [E]

Negative control (sham);

[0022] Figure 4. shows radiographs 3 weeks post operation: [A] PNEA₅₀PhPh₅O-

CDHA, [B] PNEA₅₀PhPh₅₀-CDSHA, [C] Allograft (human cancellous bone), [D] Autograft, and

[E] Negative control (sham);

[0023] Figure 5. shows radiographs 4 weeks post operation: [A] PNEA₅oPhPh_5O-

CDHA, [B] PNEA₅₀PhPh₅₀-CDSHA, [C] Allograft (human cancellous bone), [D] Autograft, and

[E] Negative control (sham);

[0024] Figure 6. shows radiographs 5 weeks post operation: [A]

PNEA₅₀PhPh₅₀-CDHA, [B] PNEA₅₀PhPh₅₀-CDSHA, [C] Allograft (human cancellous bone), [D] Autograft, and [E] Negative control (sham);

[0025] Figure 7. shows radiographs 6 weeks post operation: [A] PNEA₅₀PhPh₅₀-

CDHA, [B] PNEA₅₀PhPh₅₀-CDSHA, [C] Allograft (human cancellous bone), [D] Autograft, and

[E] Negative control (sham);

[0026] Figure 8. shows a radiograph of rabbit femurs and location of metaphyseal bone defect; [0027] Figure 9. shows the low-power histomorphological progression of the metaphyseal femur rabbit defect study over a twelve week period: representative samples from time intervals of one, two, four, and twelve weeks are shown for comparison, control represents a defect with no implant which is compared to PLAGA, PPHOS-50, and PPHOS-100 matrix implants;

[0028] Figure 10. shows representative micrographs of metaphyseal rabbit femur defect without polymeric matrices (control) over twelve weeks: one week micrographs (A) demonstrate granulation tissue in the region of the defect, the two week interval (B) shows new bone developing at the defect margins, at four weeks (C), defects contain prominent woven bone, at twelve weeks (D), micrographs demonstrate the accumulation of bone at the defect site (Original

Magnification=20X);

[0029] Figure 11. shows representative micrographs of metaphyseal bone defect study in rabbits with poly(lactideco-glycolide) (PLAGA) polymeric matrix over twelve weeks: the one week interval (A) demonstrates minimal bone formation at the defect site, PLAGA matrix at two weeks (B) reveals few trabeculae of new bone in contact with the implant surface, at four weeks

(C), adjacent tissue shows fibrosis, lymphocytes, and ill-defined granulomas around the defect site, vacuolated cells associated with giant cells, lymphocytes, and plasma cells resemble organizing fat necrosis, at twelve weeks (D) a thin fibrous membrane with only rare giant cells and adequate bone formation is seen at the implant site (Original magnification=20X, FN=F at

Necrosis);

[0030] Figure 12. shows micrographs of metaphyseal bone defect study in rabbits with poly[bis(ethyl glycinato) phosphazene (PPHOS-100) at one week (A), PPHOS-100 implanted samples demonstrate woven bone and fibrous tissue formation, the two week time point (B) shows accumulation of bone matrix as well as mild inflammation, at four weeks (C), macrophages and giant cells are associated with irregular vacuoles along with woven and lamellar bone formation and moderate inflammation, at twelve weeks (D), PPHOS-100 matrix implants demonstrate mature bone formation at the implant site along with a mild fibrous response; and

[0031] Figure 13. shows micrographs of a metaphyseal bone defect study in rabbits with poly[(50% pmethylphenoxy)-(50%ethyl glycinato) phosphazene] (PPHOS-50) matrices over a twelve week period, at one week (A), there is an area of woven bone immediately adjacent to the implant, at two weeks (B), inflammation was mild and a network of interconnecting woven bone was formed, at four weeks (C), an increase in bone formation extends into the implant site, at the twelve week time interval (D), sufficient mature bone formation is observed at the defect site, with mild inflammation (Original magnification=20X),

Detailed Description of the Invention

[0032] The present invention provides a novel biodegradable polymer composite that chemically and mechanically mimics bone. The polymer composites of the present invention are free flowing and may be injected or otherwise applied to the site to be treated. Further, the polymer composites of the present invention are capable of setting at physiological temperatures.

In a preferred embodiment, this composite contains at least one polyphosphazene and an osteoconductive material.

[0033] In a preferred embodiment, the composites of the invention contain at least one polyphosphazene. Polyphosphazenes are high molecular weight biodegradable polymers with an inorganic backbone consisting of alternate nitrogen and phosphorus atoms with each phosphorus atom is attached to two organic side groups, having a general structure as shown in Formula I,

[0034] Polyphosphazenes are biocompatible, biodegradable, and the rate of their degradation can be modulated by changing the side groups attached to the phosphorus atom [13,14]. Polyphosphazenes degrade in the body into products that are non-toxic and easily disposed of by the body. Such degradation products include phosphates, ammonia, alcohol, and the corresponding side chains [15]. Because of their degredation properties, polyphosphazenes have been used as drug delivery vehicles, for example with the drugs colchicine [16], calcitonin [17], and naproxen [18].

[0035] Various polyphosphazene compounds are contemplated for use in the composites of the present invention. In a most preferred embodiment, the polyphosphazene contained in the composites of the invention is poly[(50% ethyl alanato)(50% phenylphenoxy) phosphazene] as shown in Formula II.

[0036] Other preferred polyphosphazene compounds for use in the composites of the present invention include:

[0037] Poly[bis(ethyl alanato) phosphazene] as shown in Formula III;

(III) [0038] Poly[(50% ethyl alanato) (50% ethyl glycinato) phosphazene] as shown in

Formula IV; and

NHGH(CH₃)COOC₂H₅ NHCH_fCGOCj)H₆ /jyx

[0039] Poly[(50% ethyl alanato) (50% methyl phenoxy) phosphazene] as shown in

Formula V,

[0040] It is also contemplated that other polyphosphazene compounds can be used in compositions of the present invention, including the polyphosphazenes described in U.S. Patent No. 6,235,061, which is hereby incorporated by reference herein.

[0041 ] The polyphosphazenes of the present invention may be synthesized by various methods known in the art. Preferred methods of synthesis include those described by Chasin et al. [29] and the macromolecular substitution synthesis described by Singh et al. [65], which are hereby incorporated by reference herein.

[0042] Various osteoconductive materials are contemplated for use in the composites of the invention. In general, osteoconductive materials are substances which are conducive to the regeneration, growth and support of bone.

[0043] In a preferred embodiment the present invention, the osteoconductive material is hydroxyapatite. In a more preferred embodiment of the invention, the hydroxy apatite compounds used are calcium deficient hydroxyapatite compounds with Ca / P ratios ranging from about 1.0 to about 1.6. In the most preferred embodiment of the invention, the hydroxyapatite compounds used in the present invention are calcium deficient hydroxyapatite (CDHA - Ca/P = 1.5) or calcium deficient stochiometric hydroyapatite (CDSHA - Ca/P = 1.6). These types of hydroxyapatite compounds are well known in the art and are commercially available. Other types of hydroxyapatite compounds are also contemplated by the invention, both calcium deficient and non-calcium deficient. Further, hydroxyapatite compounds with various Ca/P ratios are contemplated, including ratios lower and higher than the preferred embodiments. Various hydroxyapatite compounds are well known in the art and many of them are commercially available. The hydroxyapatite compounds of the invention may be sintered or non-sintered. Other non- limiting examples of hydroxyapatite compounds that can be used in the composites of the present invention include those described by R.Z. Legeros in "Biological and Synthetic Apatites" and by D. K. Smith in "Calcium Phosphate Apatites In Nature," both of which are published in Hydroxyapatites and Related Materials [67] and are hereby incorporated by reference herein.

[0044] Other osteoconductive materials are also contemplated by the present invention, including other apatite compounds, calcium phosphates, bioactive glasses and other bioactive ceramics. Non-limiting examples of osteoconductive compounds contemplated by the invention include fluorapatite, oxyapatite, Wollastonite, anorthite, calcium sulfate, calcium fluoride, calcium oxide, silicon dioxide, sodium oxide, phosphorous pentoxide, agrellite, devitrite, canasite, phlogopite, monotite, brushite, octocalcium phosphate, Whitlockite, tetracalcium phosphate, cordierite, Berlinite and the like. Calcium phosphates contemplated by the present invention include mono-, di-, octa-, α-tri-, β-tri, tetra- calcium phosphate and the like. Commercially available calcium phosphates contemplated include Ca_I0(PO₄)_O(OH)₂ (CERAP ATITE®, SYNATITE®) tricalcium phosphate Ca₄(PO₄)₂ (BIOSORB®, CALCIRESORB®, CHRONOS®), and biphasic calcium phosphate for mixtures with hydroxyapatite (BIOSEL®, CERAFORM®, EUROCER®, MBCP®, HATRIC®, TRIBONE 80®, TRIOSITE®, TRICOS®). Bioactive glass and ceramic compounds contemplated include BIOGLASS® and glass-ceramic A-w, and bioactive glass compositions such as 45S5, 58S, S53P4 and S70C30. Further, non-limiting examples of osteoconductive materials that are contemplated for use in the composites of the present invention can be found in An Introduction to Bioceramics, [66] which is hereby incorporated by reference herein. [0045] It is further contemplated that pieces of natural bone may be added as osteocondcutive inaterial to the composites of the invention. The source of the bone to be added may be the patient to which the composites will be administered (autograft) or may be another subject, including subjects of other species (allograft). Natural bone may be added to the composites of the present invention is various size pieces that do not interfere with the free flowing nature of the composite. As a non-limiting example, the pieces of natural bone may be added as shards or fragments or may be added as a ground bone powder. [0046] It should also be apparent to one of skill in the art that the present invention further contemplates that one or more osteoconductive material may be used in the composites, in combination.

[0047] It is contemplated that the ratio by weight of polyphosphazene to osteoconductive material in the composite can vary from about 100% polyphosphazene to about 0% polyphosphazene in the composite. In a preferred embodiment, the polyphosphazene and osteoconductive materials are present in a 50:50 ratio by weight, but the composites of the invention may also vary widely in this ratio without detracting from the function of the composite. [0048] The composites of the present invention are preferably free flowing after mixing for a consistent period of time before setting, in contrast to previously described composites which are allowed to set outside of the body and are then cut or shaped to be fit in the desired area. The composites are contemplated to be free flowing enough to conform to the shape of the container or cavity in which they are placed. However, it is also contemplated that the composites may be viscous enough or may set at a fast enough rate so that they do not conform to the shape of a container or cavity before they set.

[0049] It is especially preferred the composites of the present invention be injectable, i.e. that they may be injected, such as from a syringe, into the area to be treated, where they will then set. The composites may be injected from syringes with or without needles, or they may be injected from other devices with a similar function. Further, it is also contemplated that the composites may be applied by other means, such as with a spatula or brush. [0050] To make the composites of the present invention free flowing, it may be necessary to add a solvent in an amount necessary to form a paste or other mixture. Solvents used in the composites of the present invention may be acids, bases, buffers, salt solutions, organic solvents and the like. If a solvent is used, a preferred solvent is phosphoric acid. Preferably, the phosphoric acid is of a concentration of between about 0.01 to 10 %, more preferably from 0.1 to 5%. When a solvent is added to the composites of the invention, it may be added in an amount sufficient to form a substance with a paste-like consistency. It may also be added to greater or lesser amounts to form a substance that is more or less viscous than a paste. [0051] Because the composites of the present invention are free flowing, they will form to fill the shape of the area to which they are applied. After mixing and application, the composites of the present invention will then set to form structures that have similar strength, structure, and composition of trabecular bone. The compounds of the present invention may set within varying times after application, such as within minutes to hours after their application, or as fast as seconds after their application.

[0052] The rate at which the composites of the present invention set may be varied by varying the components of the composite. Preferably, variations the rate of setting are obtained by varying the polyphosphazene polymer that is present in the compound, as described by Greish et al. [10, 11, 68].

[0053] It is preferred that the composites of the present invention are formulated so that they set at physiological temperature. Preferred setting temperatures are from about 20° to about 50° C. More preferred setting temperatures are from about 32° to about 42° C. [0054] Without wishing to be bound by theory, it is postualted that the novel polyphosphazene calcium deficient hydroxyapatite composites will allow for ingrowth, proliferation, and differentiation of osteoprogenitor cells that will be present in the local environment following the formation of a unicortical defect in the transverse process. This will also allow for localized increases in concentration of osteoinductive proteins such as BMPs. It is further hypothesized that the polyphosphazene calcium deficient hydroxyapatite composites of the present invention will show superior mechanical strength and decreased rate of non-union in comparison to allograft and show a similar strength and union rate to autograft, but without the donor-site morbidity and complications of iliac crest harvest.

[0055] As natural bone forms in area to which the composites have been applied, the composites will slowly degrade to natural products, as described. This rate of degradation may be varied by changing the properties of the polyphophazene polymers used in the composites. [65] Preferably, the composites of the present invention will degrade over a time period of months. However, shorter or longer time periods are also contemplated by the present invention, including time periods of days or years.

[0056] Although the composites of the present invention will allow for ingrowth of osteoprogenitor cells as described, it may be desired to add other components to the composites of the present invention before their application. Such components may include cells, proteins and peptides, polysaccharides, or therapeutic agents and may be used alone or in combination. Non-limiting examples of cells that may be added to the composites include osteoprogenitor cells, osteoblasts and stem cells. Proteins and peptides of various types may be added, especially those that are osteoinductive, such as BMPs. Other types of proteins including various hormones may be added, for example, insulin and growth factors. Polysaccharides, such as heparin, may also be administered. When therapeutic agents are added to the composites, the therapeutic agents may be drug compounds. Potential drug compounds that may be added to the composites include anesthetics, antibiotics, antivirals, chemotherapeutic agents, anti-angiogenic agents, drugs that effect vascular flow and antiinflammatories. Therapuetic agents added to the composites may also include antibodies and nucleic acids, such as antisense nucleic acids, vectors bearing genes, and ribozymes. In all cases, additives may be directly to the composites during their formation or may be encapsulated in microparticles as is well known in the art. [0057] It is also contemplated that diagnostic agents may be added to the composites of the present invention to allow for monitoring of bone repair following implantation. Suitable imaging agents include commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI). Non-limiting examples of suitable materials for use as contrast agents in MRI include the gadolinium chelates currently available, such as diethylene triamine pentaacetic acid (DTPA) and gadopentotate dimeglumine, as well as iron, magnesium, manganese, copper, and chromium. Non-limiting examples of materials useful for CAT and x-rays include iodine based materials, such as ionic monomers such as diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexol, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte. [0058] Further additives are also contemplated by the invention, including those described in U.S. Patent 6,077,916, which is hereby incorporated by reference herein.

[0059] Methods of use of the composites

\ [0060] The composites of the present invention may be used in various methods for replacing bone. The composites replace bone temporarily and provide mechanical support while allowing for natural bone to form in their place.

[0061] In a preferred embodiment, the composites of the present invention are used in spinal fusion surgery. Most preferably, the composites of the present invention are used in a procedure similar to those requiring an iliac crest autograft, however, the composites of the invention completely or partially replace the need for an autograft.

[0062] In general, spinal fusion surgery is a procedure for fusing one or more vertebrae of the spine. This technique is typically performed whereby the vertebrae to be fused are exposed using standard surgical techniques, followed by inclusion of material between the vertebrae to promote bone growth and fusion of the vertebrae. As discussed, the material included between the vertebrae is typically autograft or allograft bone.

[0063] Methods and composites of the present invention are compatible with standard spinal fusion surgery procedures. Preferably, the spinal fusion surgery procedures used in the methods of the present invention are those described by Inneke et al. [69], Pitzen et al. [70] and Jenis et al. [71], which are hereby incorporated by reference herein. [0064] A preferred spinal fusion method of the present invention can be described generally as:

1. Exposure of the vertebrae to be fused through standard surgical techniques;

^■2. Injection of a composite of the present invention between the desired vertebrae in an amount sufficient to cause fusion of the treated vertebrae; and

3. Closure of the wound.

[0065] After their injection, the composites will set to a hardness and density consistent with natural bone. Although patient's spine will need to be stabilized because of the healing of the surgical wound, it is also contemplated that special stabilization, such as through a brace, may be desired immediately after the surgery while the compositions are setting. [0066] As the patient recovers, it may be desirable to monitor the progress of healing and bone formation in the spine. This can be done using common medical techniques such as MRI, PET, x-ray, fluoroscopy and CAT. As mentioned above, diagnostic additives may be added to the composites to help facilitate this type of monitoring.

[0067] The amount sufficient to cause fusion of the vertebrae may vary depending on the positioning and condition of the vertebrae themselves. It is contemplated that the space between the vertebrae may be completely or partially filled. Further, it is contemplated that methods of spinal fusion of the present invention may include use of composites containing additives, such as natural bone, as described above. The composites and methods of the present invention may be combined with other traditional spinal surgery techniques and devices, such as the use of metal rods to stabilize the vertebrae and spine. [0068] As described above, natural bone will form in the area supported by the composite, causing a fusion of the vertebrae. As the composites of the present invention are biodegradable, no further surgical intervention is required.

[0069] It is also contemplated that the composites of the present invention may be used for bone replacement in other procedures. As a non-limiting example, the composites may be used for filling cavities in bone, or for filing fractures. Further, the composites may be used for supporting weak bone sections.

Examples

I. Spinal Fusion in Rabbits

[0070] Preparation for Surgery

[0071] The experimental protocol was reviewed and approved by the Animal Care and

Use Committee at University of Virginia. Fifty male New Zealand White rabbits weighing 3.5-

4.5 kg were obtained from Robinson Services Inc. (Mocksville, North Carolina). All animals were maintained in a temperature controlled facility accredited by the Association for

Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The guidelines as described in The Animal Welfare Act and the Policy on Humane Care and Use of

Laboratory Animals were followed.

[0072] The rabbits were randomly divided into five groups as shown in table 1. All animal underwent bilateral intertransverse process spinal fusion surgery as previously described

[1, 23-27].

Table 1. Shows the different groups that were used in the study

[0073] B. Intertransverse Process Spinal Fusion Surgical Procedure:

[0074] Each rabbit was pre-medicated with the antibiotic enrofloxacin (Baytril® (Bayer,

Germany)) at a dose of 5 mg/kg intramuscularly for prophylaxis twenty-four hours prior to surgery. The rabbit also received a second dose of enrofloxacin (5 mg/kg) immediately prior to the operation. On the day of surgery, the rabbit was sedated with an intramuscular injection of ketamine (Butler Animal Health Supply, Norcross, Georgia) at a dose of 60 mg/kg, xylazine (Butler Animal Health Supply) at a dose of 6 mg/kg, bupronex (Butler Animal Health Supply) at a dose of 0.5 mg/kg, and acepromazine (Butler Animal Health Supply) at a dose of 1 mg/kg. The rabbit's back was shaved and prepared for surgery with a povidone iodine cleansing solution (Allegiance Healthcare Corporation, McGaw Park, Illinois) followed by an alcohol wash. The L6 vertebral body was localized by fluoroscopy and a sterile 23 gauge needle was placed for incision planning. The rabbit was then placed in a prone position on the operating table and sterile drapes were placed in a standard fashion. Anesthesia was maintained using an anesthesia mask ventilator system with 1.5 - 2.5 % isoflurane gas (Butler Animal Health Supply) given to effect.

[0075] A 7 cm dorsal midline skin incision was made over vertebrate corresponding to

L4-L7. Bilateral paramedian incisions were made in the lumbodorsal fascia. The intermuscular plane between the multifϊdus and longissimus muscles was split and the transverse processes of L5 and L6 were exposed. A small self-retaining retractor was used to maintain exposure of the two transverse processes. Using a nitrogen gas powered burr, a 3mm uni cortical defect was made in each transverse process. Care was taken to avoid damage to the L5 ventral nerve root. The graft was then placed between the defects in the L5 and L6 transverse processes over the intertransverse ligament. Graft materials consisted of Group 1 - Polyphosphazene-CDHA composite matrix, Group 2 - Polyphosphazene-CDSHA composite matrix, Group 3 - human cancellous bone allograft, Group 4 - autograft obtained from iliac crest, and Group 5 - negative control (sham surgery with no graft). Exposure and type of graft were completed in a bilateral symmetric fashion for all groups.

[0076] The precursor powders were prepared as explained previously [I]. One gram of the composite precursor powder was finely ground in a mortar and pestle and ImI of 0.5% phosphoric acid was added to make a paste. The paste was loaded onto a 3ml sterile syringe and was injected between the L5-L6 transverse processes. The cement paste was localized to the desired area of fusion.

[0077] C. Harvest of Iliac Crest for Autograft [0078] Using the same skin incision a separate lunibodorsal fascial incision was made over the right iliac crest. The iliac wing was exposed by subperiosteal dissection using a small elevator to raise the muscles on the inner and outer tables of the crest. A bone cutter was then used to harvest the complete right iliac wing cephalad to the sacroiliac joint. Approximately 1.2 gm of cortico-cancellous bone was broken into small pieces with a rongeur prior to implantation. Next, 0.6 gm of the autograft was implanted on each side. The pieces of bone were placed between the defects in the L5 and L6 transverse processes over the intertransverse ligament.

[0079] D. Wound Closure

[0080] The bilateral paramedian fascial incisions were closed with a running 3-0 Vicryl®

(Ethicon, Somerville, New Jersey) suture. The skin incision was then closed with a running 0

Vicryl® (Ethicon) subcutaneous suture. One milliliter of bupivacaine (Butler Animal Health Supply) was injected around the wound for post-operative pain control. A post-operative posterior-anterior lumbar spine radiograph was obtained. The animals were placed in a recovery chamber until the anesthesia had completely worn off. A Duragesic® patch (Janssen

Pharmaceutica, Titusville, New Jersey) at a dose of 25 meg was placed on the ear of each rabbit for post-operative pain control. The animals were then transferred back to their individual cages and normal care was resumed. Ten rabbits were completed for each group, No animal showed clinical signs of weakness or paralysis after recovery from surgery.

[0081] E. Analysis

[0082] Spinal fusion will be assessed by weekly radiographs (Figures 1-7). The animals will be followed for 12 weeks and then euthanized. The animals will be euthanized with Euthasol® (Butler Animal Health Supply) at a toxic dose of 175 mg/kg. Endpoint data will

include physical examination of spine stability, micro CT of the spine, histology of the fused vertebrae, and mechanical strength testing of the fusion [1, 23-25, 28].

II. Filling of Bone Cavities in Rabbits

[0083] Materials and Methods

[0084] Materials

[0085] Poly[(50% p-methylphenoxy)-(50% ethyl glycinato) phosphazene] (PPHOS-50) and poly[bis(ethyl glycinato) phosphazene] (PPHOS-100) were synthesized according to a procedure reported previously [47]. American Cyamid provided the 50:50 poly(lactide-co- glycolide) (PLAGA).

[0086] Preparation of cylindrical matrices

[0087] The PPHOS matrices were prepared as follows: 4 grams of polymer were dissolved in 40 mL of tetrahydrofuran. The polymer solution was poured into water (40OmL) to precipitate out the polymer and form a putty-like material. The putty was extruded through a glass cylinder (5 mm in diameter) and then air dried. For PPHOS-50 and PPHOS-100, the matrices were placed in a stainless steel cylindrical mold (5 mm in diameter) and heated for three hours at 70⁰C. The matrices were then cut into 5 mm long plugs and lyophilized for 48 hours prior to use. The PLAGA matrices were prepared by heating the polymer above its glass transition temperature and placing it in a 5 mm stainless steel cylindrical mold.

[0088] In vivo study [0089] The cylindrical matrices were exposed to UV light for one hour on all sides prior to implantation in an effort to minimize bacterial contamination. Sixteen adult New Zealand White rabbits weighing four kilograms each were used for each type of polymer. A 2.5 cm lateral incision near the distal femur of the right leg was made. The soft tissue and muscle was then dissected and the lateral distal femur was exposed to locate the metaphysis. A 5 mm defect was created using a drill bit and the polymeric matrix was inserted. A similar defect without the implant was made in the left leg as a control (Figure 8). The soft tissue and muscle was then closed to stabilize the implant site. At designated time points of one, two, four, and twelve weeks, four animals from each polymer group were sacrificed by CO2 asphyxiation. The femurs were isolated and processed for histological examination. [0090] Histological Preparation and Evaluation

[0091 ] At predetermined time points of one, two, four, and twelve weeks, animals were sacrificed following the American Association of Laboratory Animal Care guidelines and the distal femurs were harvested. The superficial tissue and skin were surgically dissected and the samples were fixed in paraformaldehyde. Specimens were then cut on a band saw, each 1-2 mm in thickness in the region of the distal femoral defects. Samples were processed for embedding as each segment was decalcified, dehydrated in a graded series of alcohols, embedded in paraffin, and stained with haematoxylin and eosin. Sectioned samples 4-5 μm thick were then visualized under a light microscope to evaluate the inflammatory response. The response was described as either minimal, mild, or moderate based on a pathological description previously developed in our laboratory [58-60]. Minimal response was described as the presence of neutrophils, erythrocytes, and lymphocytes. [0092] Mild inflammation consists of macrophages, fibroblasts or giant cells. Moderate inflammatory response was described by the abundance of macrophages, giant cells and tissue exudates [58].

[0093] General Observations

[0094] Observation of the implant sites at one, two, four and twelve weeks after surgical

[0095] implantation demonstrated no gross inflammation or obvious signs of physical impairment due to the use of the novel polyphosphazene matrices. No signs of systemic or neurological toxicity were observed in the rabbits.

[0096] A summary describing the inflammatory response of the polymeric materials in vivo at the bone polymer defect site is characterized in Table II. The inflammatory response is described as either minimal, mild, or moderate as read by a certified pathologist [58]. The PLAGA polymer was found to have a mild inflammatory response over the predetermined time interval. The polyphosphazene matrices were found to show similar histological responses to PLAGA and the control. The PPHOS-100 demonstrated a moderate inflammatory response at the early time point of four weeks which resolved by the twelve week interval.

TABLE II. Inflammatory Response of Implanted Polymeric Matrices in Rabbit Metaphyseal Defect Study

Time Interval (Weeks)

Polymer Group 1 2 4 12

PLAGA Min Mild Mild Mild PPHOS-50 Min Mild Mild Mild PPHOS-100 Min Mild Mod Mild Min=Minimal Response, MiId=MiId Response; Mod=Moderate Response.

[0097] Histological Analysis

[0098] Control

[0099] Representative samples of the control femurs (defects without implants) are shown in Figure 9. The biopsy site was easily identified in sections obtained at one and two weeks, but was difficult to clearly visualize in the four and twelve week samples. Histologically, the one week samples contained hemorrhage, loose fibrous connective tissue, granulation tissue, and a few spicules of dead bone. Small nodules of proliferating cartilage were also present, especially near the periosteum.

[00100] Occasional trabeculae of woven bone were present around the edge of the defect

(Figure 10A). At two weeks the defect contained more prominent proliferating cartilage, as well as areas of woven bone (Figure 10B).

[00101] At four weeks the control defects began to fill in and were less well defined. New cortex formed beneath the periosteum. Edges of the original defect could be identified in some specimens as a refractile reversal line, but the defect itself contained variable amounts of woven bone, lamellar bone, and fat. No inflammation was observed at the defect site (Figure 10C). At twelve weeks the defect was difficult to identify and visualize in all specimens. In some cases a slight periosteal reaction or a linear orientation to the trabeculae was the only evidence of the defect. Most defects contained thin, lamellar trabeculae and fat. There was no granulation tissue or inflammation observed (Figure 10D).

[00102] Polylactic-co-glycolic Matrix (PLAGA) [00103] Representative photomicrographs of the defect sites are shown in Figure 9. The space occupied by the PLAGA material was obvious in the one and two week samples, while the four and twelve week samples show the defect to be filling in with new growth. Histological analysis of the one week samples showed the interface between bone and implant consisted of a thin layer of loose fibrous and granulation tissues (Figure 1 IA).

[00104] At two weeks the interface between implant and native bone was more irregular.

The pattern of bone formation suggested focal bone apposition around some of the implants.

Areas of woven bone and proliferating cartilage were observed at the defect site. Scattered lymphocytes and plasma cells were present around the implant (Figure HB). At four weeks, the tissue adjacent to the implant showed a combination of woven bone, granulation tissue and a mild inflammatory reaction.

[00105] Granulation tissue appeared to be associated with vacuoles, accompanied by scattered lymphocytes, giant cells, and plasma cells (Figure 11C). At twelve weeks it was difficult to visualize the implant or the defect site in several of the PLAGA specimens.

Periosteum appears to have formed new bone, containing the implant within the metaphysis. The

PLAGA implant was irregularly shaped, probably reflecting dissolution and/or fragmentation.

The implant was surrounded by only a thin fibrous membrane with rare giant cells. Adjacent tissue showed mild inflammation with rare lymphocytes. Bone adjacent to the defect site appeared histologically normal (Figure 1 ID).

[00106] Poly[bis(ethyl glycinato) phosphazene] (PPHOS-100)

[00107] Low power examination showed easy visualization of the implant site at one week that appears to fill in over time (Figure 9). The interface between implant and adjacent tissue contained primarily granulation tissue, new woven bone and loose fibrosis. Areas of apparent organizing fat formation were also present (Figure 12A). At two weeks the tissue adjacent to the implant contained loose fibrosis, scattered lymphocytes and plasma cells, and woven bone. In some regions, areas of apparent bone apposition to the implant could be identified. Granulation tissue was also present, with an appearance similar to organizing fat necrosis. A periosteal reaction was noted to be consistent with proliferating cartilage (Figures 12B). [00108] At four weeks, moderate inflammation and apparent degradation of the implant was evident. The interface between implant and bone contained numerous lymphocytes, plasma cells, and giant cells. Woven bone was not prominent. Strands of fibrous tissue appeared to extend into the area occupied by the implant, probably reflecting tissue in-growth. In other areas, relatively large vacuoles were present, adjacent to the implant site, which were often associated with foreign body giant cells and macrophages (Figure 12C). At twelve weeks the overall inflammatory reaction decreased, but lymphocytes and plasma cells persisted in tissue adjacent to the implant. The implant itself appeared more irregular. Bone apposition appears to have been minimal. The implant was primarily surrounded by a thin fibrous membrane with an inflammatory zone that extended for approximately 0.25- 0,50 millimeters. Bone peripheral to the inflammatory zone appeared histologically normal (Figure 12D).

[00109] Poly [(50% p-methylphenoxy)~(50%ethyl glycinato) phosphazene] (PPHOS-50)

[00110] Again, the implant site was obvious at one week, but appears to show tissue ingrowth during the time period of study (Figure 9). Histologically, the one week sample showed the interface between implant and adjacent bone was composed mostly of a thin layer of fibrous tissue with abundant woven bone formation. While granulation tissue was present, only a minimal inflammatory response was evident (Figure 13A). At two weeks, the implant interface contained mostly woven bone. While occasional lymphocytes and plasma cells were present there was no prominent inflammation. The implant appeared relatively intact, without histologic evidence of fragmentation (Figure 13B). At four weeks there was minimal change. Woven bone and more mature lamellar bone were also present around the implant with mild inflammation (Figure 13C). At twelve weeks, the interface showed areas of mature lamellar bone along with fat and bone marrow reconstitution at the implant site. Again mild inflammation was identified and the implant was contained within the defect site (Figure 13D).

[00111] Discussion

[00112] Without wishing to be bound by theory, the following discussion provides an analysis of the data presented in the examples.

[00113] The goal of this experiment was to examine the novel polyphosphazene polymers as candidates for tissue engineered matrices for musculoskeletal tissue regeneration in vivo. Previous studies by the inventors have demonstrated polyphosphazenes to be effective materials for musculoskeletal tissue growth in vitro [47]. They have also previously demonstrated suitable in vivo biodegradability and biocompatibility of amino acid ester substituted polyphosphazenes in a rat model [59]. To date no studies have examined these materials with various polymer compositions for bone regeneration in vivo. It was hypothesized that polyphosphazene based matrices would be suitable materials for bone regeneration based upon previous in vitro studies. At early time points, the steocompatability of polyphosphazenes was acceptable with minimal to mild inflammatory responses surrounding the implants. The most extensive inflammatory response observed histologically was associated with the PPHOS-100 implants. Lymphocytes, plasma cells, ill-defined granulomas, macrophages, and giant cells were present surrounding the implants at four weeks which decreased by twelve weeks. However, this cellmediated response is similar to that observed in the rat model [59] as well as with other tissue engineered matrices, such as polyester and poly( anhydride) based polymers [29,42]. The PPHOS-100 implants also appeared to undergo fragmentation resulting in vacuoles of polymeric material surrounded by macrophages and giant cells located adjacent to the implant insertion point. Although this chronic tissue response was prominent immediately adjacent to the PPHOS-100 implants, bone within 0.5 millimeters of the interface was histologically normal in appearance suggesting a very localized inflammatory response.

[00114] By twelve weeks, the PPHOS-100 and PPHOS-50 implants demonstrated some evidence of new lamellar bone formation along with a fibrous tissue response at the bone- polymer interface. Previously, the inventors have demonstrated long-term cellular growth on three dimensional PPHOS substituted ethyl glycinato materials [47]. Enhanced osteocompatibility of the matrix was noted by twenty-one days, as well as infiltration of cellular material throughout the matrix. This in vitro data supports the ability of polyphosphazenes to promote osteoinductivity and bone growth [47]. The in vivo data presented demonstrates a similar trend as observed by the presence of lamellar bone formation within the PPHOS-100 and PPHOS-50 matrices.

[00115] PLAGA implants also demonstrated a mild chronic tissue inflammatory response with possible implant fragmentation, similar to, but less pronounced than PPHOS-100 implants by the four week time point. There was evidence of lymphocytes, plasma cells, granulomas, macrophages and giant cell reactions adjacent to the implant site. At twelve weeks, the PLAGA implants were surrounded by a thin fibrous membrane with rare giant cells, occasional lymphocytes and plasma cells. [00116] There was a mild inflammatory response associated with the PPHOS-50 implants that appeared less prominent than PPHOS-100 and similar to PLAGA. One week following PPHOS-50 implantation, a thin layer of fibrous tissue developed at the interface of the implant and adjacent bone with abundant woven bone formation observed. Granulation tissue was present but chronic inflammation was not prominent. At two weeks, the interface adjacent to the implants contained mostly woven bone, with mild inflammation. Although occasional lymphocytes were present, plasma cells were rare and no granulomas were observed. The sample at four weeks was similar to that at two weeks with woven bone as well as mature appearing lamellar bone being present around the implants. The interface at twelve weeks showed areas primarily of lamellar bone and histologically unremarkable fat and bone marrow cells adjacent to the implant. There was no histological evidence of implant fragmentation, fat necrosis or granulomas. The osteocompatibility of the PPHOS-50 matrix can possibly be explained by understanding the adhesion mechanism observed in vitro in which ethyl glycinato containing materials enhanced initial cellular adhesion in both a two and three dimensional polymeric model [47,49],

[00117] The difference in tissue responses between PPHOS-50 and PPHOS-100 may be attributed to their respective degradation rates. PPHOS-100 contains a higher loading of ethyl glycinato groups, conferring hydrolytic instability to the polymeric backbone thus allowing it to degrade faster than PPHOS-50. The rapid degradation of PPHOS-100 was associated with evidence of an increased local tissue response and possibly focal bone resorption within 0.5 mm of the bone-polymer interface. The slower degrading PPHOS-50 demonstrated a less prominent tissue response and greater bone apposition by twelve weeks. Previous in vitro studies demonstrated that PPHOS containing materials have specific degradation patterns that affect cellular adhesion, as well as the material structure [13, 60]. PLAGA degrades via bulk erosion which can lead to greater then 50% loss of mechanical strength in less than two months. On the other hand, polyanhydrides degrade primarily by surface erosion. Polyphosphazenes have been shown to degrade by both surface and bulk erosion [15, 61]. A combination of surface and bulk erosion allows for greater control of the degradation kinetics of polyphosphazenes by simple variance of the side-groups.

[00118] Studies examining drug release from polyanhydrides and polyphosphazenes have shown that both polymer classes show promise as localized drug carriers. Due to polyanhydrides surface eroding quality, a near zero-order drug release profile is seen. Polyanhydrides are extremely hydrolytic and in general degrade quickly with exposure to water. However, degradation can be controlled by variance of monomer type and ratio in the polymer backbone to provide degradation profiles in the range of days to years [62]. On the other hand, polyphosphazenes degradation occurs by both surface and bulk erosion and is regulated by varying the side groups. In vitro release of colchicine using polyphosphazene carriers suggested that drug release was proportional to the rate of polymer breakdown [16]. These studies suggest that biocompatible and biodegradable polymers that allow for regulation of their degradation rate are obvious candidates for short and long-term localized drug delivery [63]. [00119] Currently, one of the primary focuses of polymer research is on localized drug delivery. One quality of an ideal polymer is that it is biodegradable to non-toxic byproducts which undergo selfelimination. Polymers currently being studied include but are not limited to poly(ester), poly(anhydride), and poly(phosphazene). PLAGA implants are known to degrade into acidic components creating a localized acidic pH that may be toxic to surrounding tissues [60]. [00120] Polyanhydrides degrade into diacids which must be metabolized and eliminated.

However, polyanhydrides have also been shown to be non-cytotoxic and non-mutagenic. In vivo experiments implanting polyanhydride matrices into bone defects showed similar inflammatory . responses to PLAGA implants [64]. Polyphosphazenes degrade into the non-toxic natural products of phosphorus, ammonia, and the side-group ester or amino acid [15]. The data presented, along with previous in vitro [47] and in vivo [59] studies, suggests that polyphosphazenes show similar if not better biocompatibility profiles with minimal to mild inflammatory responses when compared to polyanhydrides and polyesters. [00121] Overall, this study demonstrates that biodegradable polyphosphazenes with ethyl glycinato side chains are biocompatible with bone and may prove to be a viable biomaterial for bone tissue engineering. The difference in degradation rates of PPHOS-50 and PPHOS-100 may explain the different tissue responses to these polymers. This suggests that PPHOS-50 may be a more suitable biomaterial for use in musculoskeletal regeneration or drug delivery applications. [00122] This study allows for a qualitative evaluation of the biocompatibility of the implants by examining the extent of localized tissue response at the bone-implant interface, as well as the bone remodeling potential in the presence of these implants.

[00123] While a preferred embodiment and various uses have been set forth above, those skilled in the art who have reviewed the present disclosure will readily appreciate that other embodiments can be realized within the scope of the invention. For example, disclosures of the type and ratios of compounds, as well as the optional additives to be used therein, are illustrative rather than limiting. Therefore, the present invention should be construed as limited only by the appended claims. References

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Claims

What is claimed is:

1. A biodegradable composite for the replacement of bone, comprising: a polyphosphazene; and one or more osteoconductive materials.

2. The composite of claim 1 , wherein the polyphosphazene is poly[(50% ethyl alanato)(50% phenylphenoxy) phosphazene] as shown in Formula II.

NKCH(CH₃)COOC_jjH_s

(II)

3. The composite of claim 1 , wherein the polyphosphazene is poly[bis(ethyl alanato) phosphazene] as shown in Formula III.

.JJJ.

4. The composite of claim 1, wherein the polyphosphazene is poly[(50% ethyl alanato) (50% ethyl glycinato) phosphazene] as shown in Formula IV.

NHCHtCH_j)COOC₂Ho NHCH₂COOCJH₆

5. The composite of claim 1 , wherein the polyphosphazene is poly[(50% ethyl alanato) (50% methyl phenoxy) phosphazene]

6. The composite of claim 1 , wherein the osteoconductive material is hydroxyapatite.

7. The composite of claim 6, wherein the hydroxyapatite is calcium deficient hydroxyapatite.

8. The composite of claim 7, wherein the calcium deficient hydroxyapatite has a Ca / P ratio of 1.5.

9. The composite of claim 7, wherein the calcium deficient hydroxyapatite has a Ca / P ratio of 1.6.

10. The composite of claim 1, wherein the osteoconductive agent is selected from the group consisting of: apatite compounds, calcium phosphates, bioactive glasses, bioactive ceramics and mixtures thereof.

11. The composite of claim 1 , wherein the osteoconductive agent is a bioactive glass.

12. The composite of claim 11 , wherein the bioactive glass is selected from the group consisting of:

45S5, 58S, S53P4, S70C30 and mixtures thereof.

13. The composite of claim 1 , wherein the osteoconductive agent is a calcium phosphate.

14. The composite of claim 13, wherein the calcium phosphate is selected from the group consisting of: mono-, di-, octa-, α-tri-, β-tri, and tetra- calcium phosphates and mixtures thereof.

15. The composite of claim 13, wherein the calcium phosphate is selected from the group consisting of:

CERAP ATITE®, SYNATITE®, BIOSORB®, CALCIRESORB®, CHRONOS®, BIOSEL®, CERAFORM®, EUROCER®, MBCP®, HATRIC®, TRIBONE 80®, TRIOSITE®, TRICOS® and mixtures thereof.

16. The composite of claim 1 , wherein the osteoinductive agent is selected from the group consisting of: fluorapatite, oxyapatite, Wollastonite, anorthite, calcium sulfate, calcium fluoride, calcium oxide, silicon dioxide, sodium oxide, phosphorous pentoxide, agrellite, devitrite, canasite, phlogopite, monotite, brushite, octocalcium phosphate, Whitlockite, tetracalcium phosphate, cordierite, Berlinite, glass-ceramic A-w and mixtures thereof.

17. The composite of claim 1 , wherein the osteoinductive material is a bone fraction.

18. The composite of claim 17, wherein the bone fraction is an autograft bone fraction.

19. The composite of claim 17, wherein the bone fraction is an allograft bone fraction.

20. The composite of claim 1 , wherein the composite is free flowing upon mixing and sets after application.

21. The composite of claim 8, wherein the composite is injectable.

22. The composite of claim 1, wherein the composite sets at a physiological temperature.

23. The composite of claim 22, wherein the physiological temperature is from about 20 ⁰C to about 50⁰C.

24. The composite of claim 22, wherein the physiological temperature is from about 32 ⁰C to about 42°C.

25. The composite of claim 1, further comprising a solvent.

26. The composite of claim 25, wherein the solvent is selected from the group consisting of: acids, bases, buffers and salt solutions.

27. The composite of claim 25, wherein the solvent is a weak phosphoric acid.

28. The composite of claim 27, wherein the weak phosphoric acid has a concentration of about 0.01 to about 10 % phosphoric acid.

29. The composite of claim 27, wherein the weak phosphoric acid has a concentration of about 0.1 to about 5 % phosphoric acid.

' 30. The composite of claim 1 , further comprising an additive.

31. The composite of claim 30, wherein the additive is selective from the group consisting of: cells, proteins and peptides, polysaccharides, therapeutic agents and mixtures thereof.

32. A biodegradable composite for the replacement of bone, comprising: a polyphosphazene of formula II; and

a calcium deficient hydroxyapatite.

33. The composite of claim 32, wherein the calcium deficient hydroxyapatite has a Ca / P ratio of 1.5.

34. The composite of claim 32, wherein the calcium deficient hydroxyapatite has a Ca / P ratio of 1.6.

35. A method for causing the formation of bone in a cavity or space comprising: formulating a biodegradable composite comprising: a polyphosphazene; and one or more osteoconductive materials; and applying the composite to the space or cavity in an amount sufficient to cause the formation of bone.

36. A method for spinal fusion of vertebrae in a patient comprising: exposing the one or more vertebrae to be fused using a surgical technique; formulating a biodegradable composite comprising: a polyphosphazene; and one or more osteoconductive materials; and applying the composite between the one or more vertebrae to be fused in an amount sufficient to cause fusion of the vertebrae.

37. The method of claims 35 or 36, wherein the polyphosphazene is poly[(50% ethyl alanato)(50% phenylphenoxy) phosphazene] as shown in Formula II.

38. The method of claims 35 or 36, wherein the polyphosphazene is poly[bis(ethyl alanato) phosphazene] as shown in Formula III.

₍j^

39. The method of claims 35 or 36, wherein the polyphosphazene is poly[(50% ethyl alanato) (50% ethyl glycinato) phosphazene] as shown in Formula IV.

40. The method of claims 35 or 36, wherein the polyphosphazene is poly[(50% ethyl alanato) (50% methyl phenoxy) phosphazene]

41. The method of claims 35 or 36, wherein the osteoconductive material is hydroxyapatite.

42. The method of claim 41 , wherein the hydroxyapatite is calcium deficient hydroxyapatite.

43. The method of claim 42, wherein the calcium deficient hydroxyapatite has a Ca / P ratio of l .5.

44. The composite of claim 42, wherein the calcium deficient hydroxyapatite has a Ca / P ratio of 1.6.

45. The method of claims 35 or 36, wherein the osteoconductive agent is selected from the group consisting of: apatite compounds, calcium phosphates, bioactive glasses, bioactive ceramics and mixtures thereof.

46. The method of claims 35 or 36, wherein the osteoconductive agent is a bioactive glass.

47. The method of claim 46, wherein the bioactive glass is selected from the group consisting of:

45S5, 58S, S53P4, S70C30 and mixtures thereof.

48. The method of claims 35 or 36, wherein the osteoconductive agent is a calcium phosphate.

49. The method of claim 48, wherein the calcium phosphate is selected from the group consisting of: mono-, di-, octa-, α-tri-, β-tri, and tetra- calcium phosphates and mixtures thereof.

50. The method of claim 48, wherein the calcium phosphate is selected from the group consisting of:

CERAP ATITE®, SYNATITE®, BIOSORB®, CALCIRESORB®, CHRONOS®, BIOSEL®, CERAFORM®, EUROCER®, MBCP®, HATRIC®, TRIBONE 80®, TRIOSITE®, TRICOS® and mixtures thereof.

51. The method of claims 35 or 36, wherein the osteoconductive agent is selected from the group consisting of: fluorapatite, oxyapatite, Wollastonite, anorthite, calcium sulfate, calcium fluoride, calcium oxide, silicon dioxide, sodium oxide, phosphorous pentoxide, agrellite, devitrite, canasite, phlogopite, monotite, brushite, octocalciurn phosphate, Whitlockite, tetracalcium phosphate, cordierite, Berlinite, glass-ceramic A-w and mixtures thereof.

52. The method of claims 35 or 36, wherein the osteoconductive material is a bone fraction.

53. The method of claim 52, wherein the bone fraction is an autograft bone fraction.

54. The composite of claim 52, wherein the bone fraction is an allograft bone fraction.

55. The method of claims 35 or 36, wherein the composite is free flowing upon mixing and sets after application.

56. The method of claim 55, wherein the composite is injectable.

57. The method of claims 35 or 36, wherein the composite sets at a physiological temperature.

58. The method of claim 57, wherein the physiological temperature is from about 20 ⁰C to about 50⁰C.

59. The method of claim 57, wherein the physiological temperature is from about 32 ⁰C to about 42⁰C.

60. The method of claims 35 or 36, wherein the composite further comprises a solvent.

61. The method of claim 60, wherein the solvent is selected from the group consisting of: acids, bases, buffers and salt solutions.

62. The method of claim 60, wherein the solvent is a weak phosphoric acid.

63. The composite of claim 62, wherein the weak phosphoric acid has a concentration of about 0.01 to about 10 % phosphoric acid.

64. The composite of claim 62, wherein the weak phosphoric acid has a concentration of about 0.1 to about 5 % phosphoric acid.

65. The method of claims 35 or 36, wherein the composite further comprises an additive.

66. The method of claim 65, wherein the additive is selective from the group consisting of: cells, proteins and peptides, polysaccharides, therapeutic agents and mixtures thereof.

67. The method of claims 35 or 36, wherein after application of the composite, the composite sets to form a structure with a density and mechanical strength similar to that of natural bone.