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Title:
INJECTABLE/IN SITU FORMING TISSUE POLYURETHANE COMPOSITES AND METHODS THEREOF
Document Type and Number:
WIPO Patent Application WO/2011/075183
Kind Code:
A1
Abstract:
Present inventions present composites of tissue components and/or components and polyurethane(s), as well as methods of making such composite and uses thereof. A porous composite comprises a plurality of tissue particle components; and polyurethanes with which the tissue particle and/or components are combined. To prepare a porous composite, a composition comprises a plurality of tissue components and/or components, polyurethane precursors including polyisocyanate prepolymers and polyols, water and catalyst. A composition is either naturally moldable and/or injectable, or it can be made moldable and/or injectable and is three-dimensionally conforming to the recipient site. After implantation or injection, a composition may be set to form a porous composite that provides mechanical strength and supports the in-growth of cells. Inventive composites have the advantage of being able to fill irregularly shape implantation site while at the same time being settable to provide appropriate functional biomechanical strength and moduli for most tissue applications.

Inventors:
GUELCHER SCOTT A (US)
HAFEMAN ANDREA E (US)
DAVIDSON JEFFREY M (US)
Application Number:
PCT/US2010/032327
Publication Date:
June 23, 2011
Filing Date:
April 24, 2010
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
UNIV VANDERBILT (US)
GUELCHER SCOTT A (US)
HAFEMAN ANDREA E (US)
DAVIDSON JEFFREY M (US)
International Classes:
A61F2/00
Foreign References:
US20090093565A12009-04-09
US20050238683A12005-10-27
US20050043808A12005-02-24
US20050027033A12005-02-03
US20090130173A12009-05-21
US20040014830A12004-01-22
Other References:
DATABASE PMC BING ET AL.: "The Effect of the Local Delivery of Platelet-derived Growth Factor from Reactive Two-Component Polyurethane Scaffolds on the Healing in Rat Skin Excisional Wounds", Database accession no. PMC2683181
Attorney, Agent or Firm:
MYERS, Richard, S., Jr. (PLLC401 Commerce Street,Suite 80, Nashville TN, US)
Download PDF:
Claims:
WHAT IS CLAIMED IS:

1. A composite comprising:

at least one tissue component and a polyurethane with which at least one tissue has been combined.

2. The composite of claim 1 , wherein the porosity ranges from at least about 10% to at least about 90%.

3. The composite of claim 2, wherein the porosity ranges from about 30% to about

45%.

4. The composite of claim 1 , wherein the composite, after implantation, has pores or channels that can support the in-growth of cells and tissue.

5. The composite of claim 1 , wherein the tissue components comprise mammalian cells, extracellular matrix and components, intracellular matrix components, or synthetic analogs or combinations thereof.

6. The composite of claim 1 , wherein the tissue particle and/or components comprise autogenous tissue, allogenic tissue, xenogenic tissue, or combinations thereof.

7. The composite of claim 1 , wherein the tissue components comprise mammalian tissue, human tissue, or both.

8. The composite of claim 1 , wherein the tissue components and/or components comprise bovine, porcine, rabbit tissue-derived or synthetically produced, or combinations thereof.

9. The composite of claim 1, wherein the tissue component is a surface-treated particulate tissue component.

10. The composite of claim 9, wherein a mean average particle size of the tissue component ranges from about 10 to about 1000 microns.

1 1. The composite of claim 1 , comprising wherein the tissue component ranges from at least about 10 vol% to about 60 vol% of the composite.

12. The composite of claim 1 , wherein the tissue component is a particulate tissue component and wherein the length of at least 90% of the tissue component is between approximately 100 microns and approximately 1000 microns in its greatest dimension.

13. The composite of claim 12, wherein at least 90% of the tissue component is between approximately 200 microns and approximately 800 microns in their greatest dimension.

14. The composite of claim 1 , further comprising an inorganic material.

15. The composite of claim 1 , further comprising one or more of serum albumin, collagen, an extracellular matrix component, a synthetic polymer, and a naturally-derived polymer.

16. The composite of claim 1 , wherein the polyurethanes comprise a polymer selected from the group consisting of poly(caprolactones), poly(lactide), poly(glycolide), polyglyconate, poly(arylates), poly(anhydrides), poly(hydroxy acids), polyesters, poly(ortho esters), poly(alkylene oxides), polycarbonates, poly(propylene fumarates), poly(propylene glycol-co fumaric acid), polyamides, polyesters, polyethers, polyureas, polyamines, polyamino acids, polyacetals, poly(orthoesters), poly(pyrolic acid), poly(glaxanone), poly(phosphazenes), poly(organophosphazene), polylactides, polyglycolides,

poly(dioxanones), polyhydroxybutyrate, polyhydroxyvaiyrate, polyhydroxybutyrate/valerate copolymers, poly(vinyl pyrrolidone), polycyanoacrylates, polyurethanes, polysaccharides, RYPTON1TE, and combinations thereof.

17. The composite of claim 1 , wherein the polyurethanes comprise

poly(caprolactone), poly(lactide), poly(glycolide), and/or combinations thereof.

18. The composite of claim 24, wherein the polyurethanes comprise

poly(caprolactone-co-lactide-co-glycolide),

wherein a percentage of capro lactone in the polyol ranges from approximately 10% to

60%;

wherein a percentage of lactide in the polyol ranges from approximately 10% to approximately 80%; and

wherein a percentage of glycolide in the polyol ranges from approximately 10% to approximately 60%.

19. The composite of claim 24, wherein the polylactide is poly(D,L-lactide) or poly(L-lactide).

20. The composite of claim 1 , wherein the polyurethanes comprise poly(ethylene glycol) (PEG).

21. The composite of claim 27, wherein the PEG has an average molecule weight in a range of approximately 100 to 500 g/mol.

22. The composite of claim 1 , wherein the polyurethanes further comprise a chain extender.

23. The composite of claim 1 , further comprising a catalyst.

24. The composite of claim 23, wherein the catalyst comprises a blowing catalyst, a gelling catalyst, or combinations thereof.

25. The composite of claim 23, wherein the catalyst comprises a tertiary amine.

26. The composite of claim 23, wherein the catalyst is selected from the group consisting of bis(2-demethylaminoethyl)ether (DMAEE), triethylene diamine (TEDA), Tegoamin33, stannous octoate, dibutyltin dilaurate, and a bismuth-based organometallic catalyst.

27. The composite of claim 1, further comprising an additive selected from the group consisting of a plasticizer, a porogen, a stabilizer, a nonionic or an anionic surfactant, a solvent/diluents, a bioactive agent, a filler, and mixtures thereof.

28. The composite of claim 1 , further comprising a porogen.

29. The composite of claim 1 , further comprising a pore opener.

30. The composite of claim 27, wherein

the stabilizer is selected from the group consisting of polyethersiloxane, sulfonated caster oil, and sodium ricinoleicsulfonate;

wherein the bioactive agent is selected from the group consisting of antiviral agent, antimicrobial agent, antibiotic agent, amino acid, peptide, protein, glycoprotein, lipoprotein, antibody, steroidal compound, antibiotic, antimycotic, cytokine, vitamin, carbohydrate, lipid, extracellular matrix, extracellular matrix component, chemotherapeutic agent, cytotoxic agent, growth factor, anti-rejection agent, analgesic, anti-inflammatory agent, viral vector, protein synthesis co-factor, hormone, endocrine tissue, synthesizer, enzyme, polymer-cell scaffolding agent with parenchymal cells, angiogenic drug, collagen lattice, antigenic agent, cytoskeletal agent, mesenchymal stem cells, antitumor agent, cellular attractant, fibronectin, growth hormone cellular attachment agent, immunosuppressant, nucleic acid, surface active agent, and penetraction enhancer; and

wherein the filler is selected from the group consisting of hyaluronic acid (HA), carboxymethylcellulose (CMC), or combinations thereof

31. The composite of claim 27, wherein the bioactive agent is a growth factor.

32. The composite of claim 1, wherein the growth factor is selected from PDGF, VEGF, CartilageGrowth factor and BMP-2.

33. The composite of claim 1 , being configured for the repair of a simple, complex, tissue void or tissue augmentation or tissue obliteration, for reconstruction, or repair or therapeutic delivery to the integument, subdermal tissue, breast tissue, vascular tissue, cardiac tissue.urogential-renal tissue, pulmonary tissue, hepatic tissue, gastrointestinal tissue, muscle tissue, ligament tissue, tendon tissue, facial tissue, gynecologic and female reproductive genital tissue, non-articular surface fibrocartilage tissue and cartilage tissue and special sensory tissues and neural tissue.

34. A method of preparing a porous composite comprising steps of:

providing a composition that comprises a polyol, a catalyst and water;

adding at least 30 wt% of a tissue component to form a compostion; and

contacting the composition with a polyisocyanate prepolymer under reaction conditions sufficient to form a porous composite.

35. The method of claim 34, wherein the porous composite formed has a porosity of at least 30%.

36. The method of claim 34, wherein the step of adding is before or after the step of contacting.

37. The method of claim 34, wherein the polyisocyanate prepolymer comprises at least one of lysine diisocyanate, an alkyl ester of lysine diisocyanate, lysine triisocyanate, hexamethylene diisocyanate, isophorone diisocyanate (IPDI), 4,4'-dicyclohexylmethane diisocyanate, cyclohexyl diisocyanate (Hn DI), 2,2,4-(2,2,4)-trimethylhexamethylene diisocyanate (TMDI), hexamethylene diisocyanate (HDI) trimer polyisocyanate, dimers prepared form aliphatic polyisocyanates or trimers prepared from aliphatic polyisocyanates.

38. The method of claim 37, wherein the polyisocyanate prepolymer comprises at least one of hexamethylene diisocyanate dimer, hexamethylene diisocyanate trimer, isophorone diisocyanate dimer, or isophorone diisocyanate trimer.

39. The method of claim 34, wherein the polyisocyanate prepolymer comprise lysine triisocyanate (LTI).

40. The method of claim 34, wherein the polyisocyanate prepolymer further comprise a biocompatible polymer.

41. The method of claim 40, wherein the biocompatible polymer is PEG.

42. The method of claim 34, wherein the polyol comprises hydroxy 1-terminated compounds having hydrolysable ester linkages.

43. The method of claim 42, wherein the polyol comprises a polyalkylene glycol ester or a polyester prepared from at least one cyclic ester.

44. The method of claim 34, wherein the polyol comprises poly(ethylene adipate), poly(ethylene glutarate), poly(ethylene azelate), poly(trimethylene glutarate),

poly(pentamethylene glutarate), poly(diethylene glutarate), poly(diethylene adipate), poly(triethylene adipate), poly(l ,2-propylene adipate), a mixture thereof, or a copolymer of at least two thereof.

45. The method of claim 34, wherein the polyol comprise poly(caprolactone), poly(lactide), poly(glycolide), and/or combinations thereof.

46. The method of claim 45, wherein the polyol comprise poly(caprolactone-co- lactide-co-glycolide).

47. The method of claim 34, wherein the polyol has a molecular weight in a range of 200 Da to 2500 Da.

48. The method of claim 34, wherein the water is present in from about at least 0.1 parts to about at least 10 parts per hundred parts polyol (pphp).

49. The method of claim 34, wherein the catalyst comprises a blowing catalyst, a gelling catalyst, or combinations thereof.

50. The method of claim 34, wherein the catalyst comprises a tertiary amine.

51. The method of claim 34, wherein the catalyst is present in from about at least 1 parts to about at least 3 parts per hundred parts polyol (pphp).

52. The method of claim 34, further comprising the addition of a stabilizer to the composition, wherein the stabilizer is present in from about at least 1 part to at least 2 parts per hundred parts polyol (pphp).

53. The method of claim 34, further comprising the addition of a porogen to the composition, wherein the porogen is present in from about at least 3 parts to about at least 5 parts per hundred parts polyol (pphp).

54. The method of claim 53, further comprising the addition of a filler selected from the group consisting of hyaluronic acid (HA) and carboxymethylcellulose (CMC).

55. An injectable or moldable composition comprising

at least one tissue component;

a polyisocyanate prepolymer;

a polyol; and

water.

56. A method comprising the step of:

administering a composite of claim 1 to a subject having a tissue defect.

57. The method of claims 56, wherein the subject suffers from a genetic disease, a congenital abnormality, a soft tissue deheisence or fracture, an iatrogenic defect, a tissue cancer, a tissue metastasis, an inflammatory disease, an autoimmune disease, a metabolic disease, or a degenerative tissue disease.

58. The method of claims 58, wherein the subject has a soft tissue non osseous defect.

59. The method of claim 59, wherein the tissue defect site comprises at least one tissue void, and wherein the step of administering comprises filling at least part of the tissue void with the composite.

60. A method comprising the step of:

administering a composite of claim 55 to a subject having a tissue defect.

61. The method of claims 60, wherein the subject suffers from a genetic disease, a congenital abnormality, a soft tissue deheisence or fracture, an iatrogenic defect, a tissue cancer, a tissue metastasis, an inflammatory disease, an autoimmune disease, a metabolic disease, or a degenerative tissue disease.

62. The method of claims 61 , wherein the subject has a soft tissue non osseous defect.

63. The method of claim 62, wherein the tissue defect site comprises at least one tissue void, and wherein the step of administering comprises filling at least part of the tissue void with the composite.

64. A kit for making of a porous composite comprising

at least one tissue component;

a polyisocyanate prepolymer;

a polyol; and

a solution comprising a catalyst and water.

Description:
INJECT ABLE/IN SITU FORMING TISSUE POLYURETHANE COMPOSITES AND METHODS THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. § 1 19(e) to U.S.

provisional patent application, U.S. S.N. 61/286,718, filed December 15, 2009, which is incorporated herein by reference.

GOVERNMENT SUPPORT

|0002] This invention was made with support from the Orthopaedic Extremity Trauma Research Program through the Department of Defense (W81 XWH-07- 1-021 1).

BACKGROUND

[0003] Tissue (and in particular Soft tissues) is a composite structure composed of cells and a supporting matrix of proteins, biopolymers, and vascular channels. Tissue and tissue components can be processed or collected via cell culture into an implantable biomaterial, such as an adipose graft or mesenchymal stem cell graft or a decellularized graft/implant, for example, leaving behind the extracellular matrix. The processed tissue biomaterial can have a variety of properties, depending upon the specific processes and treatments applied to it, and may incorporate characteristics of other biomaterials with which it is combined. For example, tissue -derived biomaterials may be processed into structural void grafts that support and integrate with the patient's own tissue or may alternatively be processed into soft, moldable, or flowable tissue biomaterials that have the ability to induce a cellular or tissue healing response.

[0004] The use of tissue grafts and tissue substitute materials in medicine is well known. While some types of tissue wounds can regenerate without the formation of scar tissue, many injuries take a long time to heal. The recipient wound or anatomic site tissue is unable to support contructive physiologic healing. Alloplastic implants and meshes are frequently needed to replace the deficient structure and function of injured tissue. However, alloplastic or tissue transplants are at times have significantly different moduli and subsequent healing than native tissue. Use of alloplastic or tissue transplants implants may result in decreased tissue density around the implant site due to healing reaction. Furthermore, most alloplastic and tissue implants are permanent and/or unable to participate in physiological remodeling. (0005] Soft tissue's cellular healing processes, through healing tissue formation by cells coordinated with tissue and graft/implant remodeling by mononuclear (macrophage lineage) cells, permit tissue grafts and certain soft tissue substitute materials to remodel into endogenous tissue that is almost indistinguishable from the original. However, the use of tissue grafts and alloplastic implants is limited by the available shape and size and the desire to optimize both biomechanical strength and degradation rate. Variations in tissue quality and availability among patients (and donors) also make tissue grafts a less optimal substitute material. tissue(and soft tissue) substitute materials and may be quickly remodeled but cannot immediately provide long term regenerative potential

[0006] Thus, it is desirable to have a biomaterial for tissue implants and grafts that may be produced in larger quantities than grafts derived solely from autologous tissue and that may be fabricated or molded into shapes without being limited by the shape of the originating tissue. It is also desirable to have injectable tissue implant and graft materials that may be implanted using minimally invasive techniques.

SUMMARY

(0007] The invention relates to injectable, in situ forming, three dimensionally conformal, and/or moldable composites/compositions including at least a tissue component or processed sub-components or an excipient bioactive agent, or a synthetically derived analog, or a combination thereof and a biodegradable polyurethane, methods of making such composites, methods of using such composites in tissue repair applications and various related compositions. The present invention provides porous composites which, when implanted or injected, promote cellular infiltration from adjacent tissues, thus accelerating the remodeling process. Inventive composites comprise tissue components and polymers, such as a biocompatible polyurethane, and may further comprise additional components. The present invention also provides compositions, methods and processes that can be used for the preparation of such composites. The invention also provides methods and kits for making and/or using such inventive porous materials.

(0008] In some aspects, the present invention provides compositions and composites including a plurality of components of an inorganic material, a tissue substitute material, a tissue-derived material, or any combination thereof, and a polymer with which the components are combined. The tissue-substitute material may be a purified or synthetically produced analog of tissue subcomponents in various forms and physical phases. The tissue graft -derived material may be in a solid or particulate form such as a lyophilized powder. More specifically, in one aspect, the invention features a composite including a tissue graft and biodegradable polyurethane (PUR). In some embodiments, a provided composite has a porosity of at least 30%. A "tissue graft" may denote, but is not limited to, a powdered form of a matrix (such as bladder or small intestinal submucosa) or a cell culture generated matrix.. A "tissue substitute" includes, but is not limited to, polysaccharides such as carboxymethylcellulose, hyaluronic acid, chitosan, or alginate or other synthetic analogs of naturally occurring substances.

[0009] A composition of tissue or tissue components and polymer is naturally moldable and/or injectable. Compositions may range from a thick, flowable liquid to a moldable, dough-like substance. In some embodiments, a composition has a low enough viscosity to be suitable for injection and in situ formation of a conformal volume. In some embodiments, a composition is workable so that it can be molded into an implantation site. Once cured, a composition may result in a porous composite including tissue components and polyurethane. In some embodiments, a composition may include tissue components and a reactive liquid. Such a reactive liquid can be a two-component composition for polyurethane include polyisocyanates, polyols, water and catalyst, and optionally additional components such as a stabilizer, a porogen, a plasticizer, a chain extender, a wetting agent, etc. In some embodiments, a composition may include bioactive agents to deliver such as antibiotics, growth factors, etc.

(0010] In some embodiments, provided porous composites have a porosity of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more than 90%. Porous composites of the present inventions may comprise pores or channels which, after implantation or injection, can support the in-growth of cell and/or the formation or remodeling of the recipient tissue site.

[0011] In some embodiments, provided porous composites have, when it is present in the composite, a tissue component weight percentage of between about 5 wt% and about 40 wt%. For example, a weight percentage of tissue components may be about 5 wt%, 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, or between any weight percentages of above.

[0012] Tissue components in a composite used in the present invention may have a variety of shapes and forms including spheroidal, plate, fiber, cuboidal, sheet, rod, ellipsoidal, string, elongated, polyhedral, etc., and may be incorporated as, for example, a liquid, a lyophilized solid, or a solution form of cells or biopolymers and mixtures thereof.

Components in the composite have a mean size of about 1 to about 5000 microns in diameter, for example, a mean size of about 20 to about 800 microns in diameter. Smaller or larger irregularly shaped components may also be found in composites. In certain embodiments, at least about 90% of the components have a mean size of about 100 microns to about 1000 microns in their greatest dimension.

[0013] Polyurethane (PUR) components used in preparing inventive composites may be selected from monomers, pre-polymers, oligomers, polymers, cross-linked polymers, partially polymerized polymers, partially cross-linked polymers, and any combinations thereof. For example, a composition may include polyurethane precursors. In some embodiments, polyurethane precursors include polyisocyanates prepolymers and polyols. In certain embodiments, polyisocyanates prepolymers may be prepared by reacting isocyanates with polyols. In certain embodiments, a polyol may include polyethylene glcol, PEG.

[0014] Polyisocyanates or multi-isocyanate compounds for use in the present invention include aliphatic polyisocyanates. Exemplary aliphatic polyisocyanates include, but are not limited to, lysine diisocyanate, an alkyl ester of lysine diisocyanate (for example, a methyl ester or an ethyl ester), lysine triisocyanate, hexamethylene diisocyanate, isophorone diisocyanate (1PDJ), 4,4'-dicyclohexylmethane diisocyanate (HuMDI), cyclohexyl diisocyanate, 2,2,4-(2,2,4)-trimethylhexamethylene diisocyanate (TMDI), dimers prepared form aliphatic polyisocyanates, trimers prepared from aliphatic polyisocyanates and/or mixtures thereof. In some embodiments, hexamethylene diisocyanate (HDI) trimer sold as Desmodur N3300A may be a polyisocyanate utilized in the present invention.

[0015] In some embodiments, polyols are polyester polyols. In some embodiments, polyester polyols may include poly(ethylene adipate), poly(ethylene glutarate), poly(ethylene azelate), poly(trimethylene glutarate), poly(pentamethylene glutarate), poly(diethylene glutarate), poly(diethylene adipate), poly(triethylene adipate), poly(l ,2-propylene adipate), mixtures thereof, and/or copolymers thereof. In some embodiments, polyester polyols can include, polyesters prepared from caprolactone, glycolide, D, L-lactide, mixtures thereof, and/or copolymers thereof. In some embodiments, polyester polyols can, for example, include polyesters prepared from castor-oil.

[0016] In some aspects, the present invention features methods including contacting tissue components with precursors of polyurethane to form porous composites. Water used in a composition may act as a blowing agent to generate a porous composite. [0017] In some aspects, the invention provides methods of administering an inventive composite and/or composition to a subject in need thereof. Among other things the invention provides composites, for example, comprising tissue components and polyurethanes, for use in medicine. Inventive composites are useful in clinical medicine. A composite may be used to repair a defect in a subject's soft tissues or organs. A composite may be used as tissue void fillers. A method includes providing a flowable or moldable composition of a polyurethane, a plurality of a tissue component and any additional components; administering the composition or composite to a subject in need thereof; and resulting in a porous three dimensionally conformal composite to set in situ. Before administration, the composite may be made flowable or moldable, for example, by heating the composite or adding a solvent to the composite. A composite may be administered into an implantation site (e.g., a breast of other soft tissue defect) followed by setting the composite. A composite may be allowed to remain at a target site providing the strength desired while at the same time promoting healing of the tissue and/or tissue growth. Polymer components of a composite may degraded or be resorbed as new tissue is formed at the implantation site. In some embodiments, a composite may be resorbed over approximately 1 month to approximately 6 years. In some embodiments, a porous composite may start to be remodeled in as little as a week as the composite is infiltrated with cells or new tissue in-growth. The remodeling process may continue for weeks, months, or years.

[0018] In some embodiments there is a specific "initial porosity" (such as created by gas foaming) and a specifically engineered "latent porosity" (that resulting from dissolution or degradation of the tissue component). In certain embodiments of the invention a low initial porosity from 0% to 5% by volume% and high tissue component providing latent porosity of 60-80 volume%. In this case the tissue component would create pores by diffusing (or cell- mediated degradation) from the injected material. A plurality of initial porosity ranging from 0 - 95% volume in design with a tissue component ranging from 5 - 80 wt% will useful in tissue repair and regenration.

[0019] In some embodiments, the present invention provides kits for the treatment of tissue. A kit includes a composition including a plurality of tissue component, which includes tissue particles as discussed herein, and polyurethane with which the tissue components are combined. In some embodiments, a kit may include a composition being contained within a delivery system for delivering the composite by injection (e.g., a syringe). A kit may also include a high pressure injection device for implanting composition of higher viscosity. A kit may also include components of the composite packaged separately for mixing just prior to implantation or injection. In some embodiments, components of a composition used in accordance with the present invention are sterilely packaged separately. A kit may also include a heating apparatus for warming the composite to a temperature where it is moldable. A kit may also include a solvent, a diluent, or pharmaceutically acceptable excipient for combining with the composite. A kit may further include instructions for using the composite.

[0020] Embodiments may include one or more of the following features or advantages. Composites can allow and encourage direct recipient tissue in-growth and remodeling, which can improve patient outcome. Composites can be formed into a variety of shapes and sizes. Composite can be porous as-prepared and/or the porosity of the composite can change (e.g., increase) over time to support in-growth of healing recipient tissues.

[00211 Other aspects, features and advantages will be apparent from the description of the following embodiments and from the claims.

DEFINITIONS

[0022 J The term ''bioactive agent" is used herein to refer to compounds or entities that alter, promote, speed, prolong, inhibit, activate, or otherwise affect biological or chemical events in a subject (e.g., a human or mammalian). For example, bioactive agents may include, but are not limited to adipogenic, adipoinductive, and adipoconductive agents, vasculogenic, vasculoinductive, and vasculoconductive agents, chondrogenic,

chondroinductive, and chondroconductive agents anti-HIV substances, anti-cancer substances, antibiotics, immunosuppressants, anti-viral agents, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants, anti-Parkinson agents, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite agents, anti -protozoal agents, and/or anti-fungal agents, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA, or protein synthesis, anti-hypertensives, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, angiogenic factors, anti-secretory factors, anticoagulants and/or antithrombotic agents, local anesthetics, reactive oxygen species inhibitors, chelating agents, ophthalmics, prostaglandins, anti-depressants, anti-psychotics, targeting agents, chemotactic factors, receptors, neurotransmitters, proteins, cell response modifiers, cells, peptides, polynucleotides, viruses, and vaccines. In certain embodiments, the bioactive agent is a drug. In certain embodiments, the bioactive agent is a small molecule.

[0023] A more complete listing of bioactive agents and specific drugs suitable for use in the present invention may be found in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel leemann and Jurgen Engel, Thieme Medical Publishing, 1999; the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", Edited by Susan Budavari et al., CRC Press, 1996, the United States Pharmacopeia-25/National Formulary- 20, published by the United States Pharmcopeial Convention, Inc., Rockville MD, 2001 , and the "Pharmazeutische Wirkstoffe", edited by Von Keemann et al. , Stuttgart/New York, 1987, all of which are incorporated herein by reference. Drugs for human use listed by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. §§ 330.5, 331 through 361 , and 440 through 460, and drugs for veterinary use listed by the FDA under 21 C.F.R. §§ 500 through 589, all of which are incorporated herein by reference, are also considered acceptable for use in accordance with the present invention.

[0024] The terms, "biodegradable", "bioerodable", or "resorbable" materials, as used herein, are intended to describe materials that degrade under physiological conditions to form a product that can be metabolized or excreted without damage to the subject. In certain embodiments, the product is metabolized or excreted without permanent damage to the subject. Biodegradable materials may be hydrolytically degradable, may require cellular and/or enzymatic action to fully degrade, or both. Biodegradable materials also include materials that are broken down within cells. Degradation may occur by hydrolysis, oxidation, enzymatic processes, phagocytosis, or other processes.

[0025] The term "biocompatible" as used herein, is intended to describe materials that, upon administration in vivo, do not induce undesirable side effects. In some embodiments, the material does not induce irreversible, undesirable side effects. In certain embodiments, a material is biocompatible if it does not induce long term undesirable side effects. In certain embodiments, the risks and benefits of administering a material are weighed in order to determine whether a material is sufficiently biocompatible to be administered to a subject.

[0026] The term "biomolecules" as used herein, refers to classes of molecules (e.g., proteins, amino acids, peptides, polynucleotides, nucleotides, carbohydrates, sugars, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, natural products, etc.) that are commonly found or produced in cells, whether the molecules themselves are naturally- occurring or artificially created {e.g. , by synthetic or recombinant methods). For example, biomolecules include, but are not limited to, enzymes, receptors, glycosaminoglycans, proteoglycans, neurotransmitters, hormones, cytokines, cell response modifiers such as growth factors and chemotactic factors, antibodies, vaccines, haptens, toxins, interferons, ribozymes, anti-sense agents, plasmids, DNA, and R A. Exemplary growth factors include but are not limited to tissue growth factors and their active fragments or subunits, such as platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), stromal- derived factor (SDF), vascular endothelial growth factor (VEGF),and Cartilage Growth Factor (BMP-4). In some embodiments, the biomolecule is a growth factor, chemotactic factor, cytokine, extracellular matrix molecule, or a fragment or derivative thereof, for example, a cell attachment sequence such as a peptide containing the sequence, RGD.

[0027] The term "carbohydrate" as used herein, refers to a sugar or polymer of sugars. The terms "saccharide", "polysaccharide", "carbohydrate", and "oligosaccharide", may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula C n H2 n O n . A carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates may contain modified saccharide units such as 2'-deoxyribose wherein a hydroxyl group is removed, 2'-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2 ' - fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

[0028] The term "composite" as used herein, is used to refer to a unified combination of two or more distinct materials. The composite may be homogeneous or heterogeneous. For example, a composite may be a combination of tissue component (which includes a tissue subcomponent or particle) and a polymer; or a combination of tissue component, polymers and antibiotics; or the polymer and an excipient molecule or other structure. In certain embodiments, the composite has a particular orientation. 10029] The term "decellularized" is used herein to refer to tissue (e.g., tissue

subcomponent such as cells or matrix components) that have been subjected to a process that causes a decrease in living cell content.

[0030| The term deorganified" as herein applied to matrices, components, etc., refers to tissue or cartilage matrices, components, etc., that were subjected to a process that removes at least part of their original organic content. In some embodiments, at least 1 %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the organic content of the starting material is removed. Deorganified tissue from which substantially all the organic components have been removed is termed "anorganic."

[0031] The term "flowable polymer materiaF' as used herein, refers to a flowable composition including one or more of monomers, pre-polymers, oligomers, low molecular weight polymers, uncross-linked polymers, partially cross-linked polymers, partially polymen ' zei/po/ymers, polymers, or combinations thereof that have been rendered formable. One skilled in the art will recognize that a flowable polymer material need not be a polymer but may be polymerizable. In some embodiments, flowable polymer materials include polymers that have been heated past their glass transition or melting point. Alternatively or in addition, a flowable polymer material may include partially polymerized polymer, telechelic polymer, or prepolymer. A pre-polymer is a low molecular weight oligomer typically produced through step growth polymerization. The pre-polymer is formed with an excess of one of the components to produce molecules that are all terminated with the same group. For example, a diol and an excess of a diisocyanate may be polymerized to produce isocyanate terminated prepolymer that may be combined with a diol to form a polyurethane. Alternatively or in addition, a flowable polymer material may be a polymer material/solvent mixture that sets when the solvent is removed.

(0032] The term "mineralized" as used herein, refers to tissue that has been subjected to a process that caused a decrease in their original organic content (e.g., de-fatting, de-greasing). Such a process can result in an increase in the relative inorganic mineral content of the tissue. Mineralization may also refer to the mineralization of a matrix such as extracellular matrix or demineralized tissue matrix. The mineralization process may take place either in vivo or in vitro.

[0033] The term "non-demineralized" as herein applied to tissue or tissue Component, refers to tissue or tissue-derived material that have not been subjected to a demineralization process (i.e., a procedure that totally or partially removes the original inorganic content of tissue). [0034] The term "nontoxic" is used herein to refer to substances which, upon ingestion, inhalation, or absorption through the skin by a human or animal, do not cause, either acutely or chronically, damage to living tissue, impairment of the central nervous system, severe illness or death.

f 00351 The term "tissue conductive" as used herein, refers to the ability of a substance or material to provide surfaces which are receptive to the growth of new tissue.

[0036] The term "tissue-genic" as used herein, refers to the ability of a substance or material that can induce or accelerate new or remodeled tissue formation.

[0037] The term "tissue inductive" as used herein, refers to the quality of being able to recruit cells (e.g., fibroblasts, endothelial, mesenchymal stem cells) from the host that have the potential to stimulate new tissue formation. In general, tissue-inductive materials are capable of inducing heterotopic tissue formation in dissimilar terminally differentiated soft tissues (e.g., muscle).

[0038] The term "STimplant" or "soft tissue-implant"is used herein in its broadest sense and is not intended to be limited to any particular shapes, sizes, configurations, compositions, or applications. STimplant refers to any device or material for implantation that aids or augments tissue formation or healing. STimplants are often applied at a tissue defect site, e.g., one resulting from injury, defect brought about during the course of surgery, infection, malignancy, inflammation, or developmental malformation. STimplants can be used in a variety of surgical procedures such as the repair of simple and complex tissue defects from tumor removal as in mastectomy or sarcoma excions or traumatic such as liver laceration or facial soft tissue defects or chronic disease states, etc.

[0039] The terms "polynucleotide" ', "nucleic acid", or "oligonucleotide" as used herein, refer to a polymer of nucleotides. The terms "polynucleotide", "nucleic acid", and

"oligonucleotide", may be used interchangeably. Typically, a polynucleotide comprises at least three nucleotides. DNAs and RNAs are exemplary polynucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thithymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyriboses, arabinose, and hexose), or modified phosphate groups (e.g. , phosphorothioates and 5 '-N-phosphoramidite linkages). The polymer may also be a short strand of nucleic acids such as R Ai, siRNA, or shRNA.

[0040] The terms "polypeptide", "peptide", or "protein" as used herein, include a string of at least three amino acids linked together by peptide bonds. The terms "polypeptide", "peptide", and "protein", may be used interchangeably. In some embodiments, peptides may contain only natural amino acids, although non-natural amino acids (i.e. , compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. In one embodiment, the modifications of the peptide lead to a more stable peptide (e.g. , greater half- life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.

[0041] The terms "polysaccharide" or "oligosaccharide" as used herein, refer to any polymer or oligomer of carbohydrate residues. Polymers or oligomers may consist of anywhere from two to hundreds to thousands of sugar units or more. "Oligosaccharide" generally refers to a relatively low molecular weight polymer, while "polysaccharide" typically refers to a higher molecular weight polymer. Polysaccharides may be purified from natural sources such as human, animal (e.g., hyaluronic acid), or other species (e.g., chitosan) and plants (e.g., alginate) or may be synthesized de novo in the laboratory. Polysaccharides isolated from natural sources may be modified chemically to change their chemical or physical properties (e.g. , reduced, oxidized, phosphorylated, cross-linked). Carbohydrate polymers or oligomers may include natural sugars (e.g. , glucose, fructose, galactose, mannose, arabinose, ribose, xylose, etc.) and/or modified sugars (e.g. , 2'-fluororibose, 2 '- deoxyribose, etc.). Polysaccharides may also be either straight or branched. They may contain both natural and/or unnatural carbohydrate residues. The linkage between the residues may be the typical ether linkage found in nature or may be a linkage only available to synthetic chemists. Examples of polysaccharides include cellulose, maltin, maltose, starch, modified starch, dextran, poly(dextrose), and fructose. In some embodiments,

glycosaminoglycans are considered polysaccharides. Sugar alcohol, as used herein, refers to any polyol such as sorbitol, mannitol, xylitol, galactitol, erythritol, inositol, ribitol, dulcitol, adonitol, arabitol, dithioerythritol, dithiothreitol, glycerol, isomalt, and hydrogenated starch hydrolysates.

[0042] The term "porogen" as used herein, refers to a chemical compound that may be part of the inventive composite and upon implantation/injection or prior to

implantation/injection diffuses, dissolves, and/or degrades to leave a pore in the osteoimplant composite. A porogen may be introduced into the composite during manufacture, during preparation of the composite (e.g. , in the operating room), or after implantation/injection. A porogen essentially reserves space in the composite while the composite is being molded but once the composite is implanted the porogen diffuses, dissolves, or degrades, thereby inducing porosity into the composite. In this way porogens provide latent pores. In certain embodiments, the porogen may be leached out of the composite before

implantation/injection. This resulting porosity of the implant generated during manufacture or after implantation/injection (i.e. , "latent porosity") is thought to allow infiltration by cells, tissue formation, tissue remodeling, osteoinduction, osteoconduction, and/or faster degradation of the osteoimplant. A porogen may be a gas (e.g. , carbon dioxide, nitrogen, or other inert gas), liquid (e.g. , water, biological fluid), or solid. Porogens are typically water soluble such as salts, sugars (e.g. , sugar alcohols), polysaccharides (e.g., dextran

(poly(dextrose)), water soluble small molecules, etc. Porogens can also be natural or synthetic polymers, oligomers, or monomers that are water soluble or degrade quickly under physiological conditions. Exemplary polymers include polyethylene glycol,

poly(vinylpyrollidone), pullulan, poly(glycolide), poly(lactide), poly(lactide-co-glycolide), other polyesters, and starches. In certain embodiments, tissue and/or sub components or a synthetic analog excipient utilized in provided composites or compositions may act as porogens.

|0043] In some embodiments, porogens may refer to a blowing agent (i.e. , an agent that participates in a chemical reaction to generate a gas). Water may act as such a blowing agent or porogen.

[0044] The term "porosity" as used herein, refers to the average amount of non-solid space contained in a material (e.g. , a composite of the present invention). Such space is considered void of volume even if it contains a substance that is liquid at ambient or physiological temperature, e.g., 0.5 °C to 50 °C. Porosity or void volume of a composite can be defined as the ratio of the total volume of the pores (i.e. , void volume) in the material to the overall volume of composites. In some embodiments, porosity (ε), defined as the volume fraction pores, can be calculated from composite foam density, which can be measured gravimetrically. Porosity may in . certain embodiments refer to "latent porosity" wherein pores are only formed upon diffusion, dissolution, or degradation of a material occupying the pores. In such an instance, pores may be formed after implantation/injection. It will be appreciated by these of ordinary skill in the art that the porosity of a provided composite or composition may change over time, in some embodiments, after implantation/injection (e.g., after leaching of a porogen, when the porogen degrades either by dissolution, hydrolytic, or cell-mediated degradation via tissue remodeling mononuclear/multi-nucleated cell resorbing a graft tissue, etc.). For the purpose of the present disclosure, implantation/injection may be considered to be "time zero" (To). In some embodiments, the present invention provides composites and/or compositions having a porosity of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more than 90%, at time zero. In certain embodiments, pre-molded composites and/or compositions may have a porosity of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more than 90%, at time zero. In certain embodiments, injectable composites and/or compositions may have a porosity of as low as 3% at time zero. In certain embodiments, injectable composites and/or compositions may cure in situ and have a porosity of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more than 90% after curing.

[00451 The term "remodeling" as used herein, describes the process by which native tissue, processed tissue allograft, whole tissue sections employed as grafts, and/or other tissues are replaced with new cell-containing host tissue by the action of local mononuclear and multinuclear cells. Remodeling also describes the process by which non-osseous native tissue and tissue grafts are removed and replaced with new, cell-containing tissue in vivo. Remodeling also describes how inorganic materials (e.g., calcium-phosphate materials, such as β-tricalcium phosphate) is replaced with living tissue. ;

[0046] The term "setting time" as used herein, is approximated by the tack-free time (TFT), which is defined as the time at which the material could be touched with a spatula with no adhesion of the spatula to the foam. At the TFT, the wound could be closed without altering the properties of the material.

|0047| The term "shaped" as used herein, is intended to characterize a material (e.g., composite) or a soft tissue-implant refers to a material or soft tissue-implant of a determined or regular form, 3-D conformation or configuration in contrast to an indeterminate or vague form or configuration (as in the case of a lump or other solid matrix of special form).

Materials may be shaped into any shape, configuration, or size. For example, materials can be shaped as sheets, blocks, plates, disks, cones, pins, screws, tubes, teeth, tissues, portions of tissues, wedges, cylinders, threaded cylinders, and the like, as well as more complex geometric configurations.

[0048| The term "small molecule" as used herein, is used to refer to molecules, whether naturally-occurring or artificially created {e.g., via chemical synthesis), that have a relatively low molecular weight. In some embodiments, small molecules have a molecular weight of less than about 2,500 g/mol, for example, less than 1000 g/mol. In certain embodiments, small molecules are biologically active in that they produce a local or systemic effect in animals, such as mammals, e.g., humans. In certain embodiments, a small molecule is a drug. In certain embodiments, though not necessarily, a drug is one that has already been deemed safe and effective for use by an appropriate governmental agency or body (e.g., the U.S. Food and Drug Administration).

|0049] The term "transformation" as used herein, describes a process by which a material is removed from an implant site and replaced by host tissue after implantation.

Transformation may be accomplished by a combination of processes, including but not limited to remodeling, degradation, resorption, and tissue growth and/or formation. Removal of the material may be cell-mediated or accomplished through chemical processes, such as dissolution and hydrolysis.

[0050] The term "wet compressive strength " as used herein, refers to the compressive strength of a soft tissue implant (STimplant) after being immersed in physiological saline (e.g., phosphate-buffered saline (PBS), water containing 0.9 g NaCl/100 ml water, etc.) for a minimum of 12 hours (e.g., 24 hours). Compressive strength and modulus are well-known measurements of mechanical properties and is measured using the procedure described herein.

[0051] The term "working time" as used herein, is defined in the IS09917 standard as "the period of time, measured from the start of mixing, during which it is possible to manipulate a dental material without an adverse effect on its properties" (Clarkin et al. , J Mater Sci: Mater Med 2009;20: 1563 - 1570). In some embodiments, the working time for a two-component polyurethane is determined by the gel point, the time at which the crosslink density of the polymer network is sufficiently high that the material gels and no longer flows. According to the present invention, the working time is measured by loading the syringe with the reactive composite and injecting <0.25ml every 30s. The working time is noted as the time at which the material was more difficult to inject, indicating a significant change in viscosity.

DESCRIPTION OF THE FIGURES

[0052] Figure 1 illustrates an example of the injectability of PUR scaffolds: time-lapse photographs showing injection of the reactive liquid system.

[0053J Figure 2 illustrates SEM images of HA (left) and CMC (right) components.

|0054] Figure 3 displays the temperature profile for PUR foams during synthesis, where the initial time (t = 0) is immediately after missing is complete.

|0055] Figure 4 illustrates SEM images of injectable LT1-PEG PUR scaffolds with no additive (top left) and 35 wt-% HA (top right). The HA (or CMC) granules rise with the foam and become bridged in the pore walls, as indicated by the arrows, and magnified at bottom left. Some of the HA dissolved (arrows) when the PUR is foamed in a high-moisture environment, as would occur in vivo (bottom right).

[0056] Figure 5 illustrates representative compressive stress-strain profiles until 50% strain of LT1-PEG scaffolds with 0, 15, and 30 wt-% CMC.

[0057] Figure 6 illustrates representative stress-strain profiles of LTl-PEG scaffolds in DMA tension mode until failure.

|0058| Figure 7 displays the degradation of injectable LTl-PEG scaffolds in PBS at 37 °C. The wt-% filler affects overall extent of degradation due to CMC dissolution, but at parallel rates (n = 3).

[0059] Figure 6 illustrates trichrome-stained histological sections show the progression of dermal wound healing with the injectable PUR scaffold (35 wt-% HA). Cells migrate into scaffold edges by day 4 (top), with PUR degradation, granulation tissue and angiogenesis by day 14 (middle). Extensive collagen deposition and epidermal closure appear by days 21 and 28 (bottom).

[0060] Figure 7 illustrates trichrome histological sections of the large dermal wound healing show the PUR scaffold (25 wt-% CMC) in position at 7 days, but significantly degraded by 17 days. The 10X image at 17 days shows angiogenesis within the new matrix. The scaffold has almost completely degraded by day 35 with complete neoepidermal coverage. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0061 ] As used herein and in the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0062] Tissue/polyurethane composites (or soft tissue/polyurethane composites described herein include a tissue component (e.g. , tissue component and/or subcomponents as defined), polyurethane, and in some embodiments, one or more additional components (e.g. , a porogen and/or a bioactive agent). As described below, tissue and biodegradable polyurethanes are combined to form a porous composite (e.g., a tissue-implant or STimplant). In some embodiments, porous composites retain strength and/or release bioactive agents when present in a body. In some embodiments, composites degrade and are replaced by new tissue.

[0063] Inventive composites can be used in a large variety of clinical applications, for example, as soft tissue (i.e., non osseous tissue) void fillers, to repair or help healing of tissue or organ deficiencies resulting from trauma, tumors, surgery, iatrogenic, congenital, genetic, metabolic and degenerative or abnormal development, and inflammatory infection. In some embodiments, inventive composites promote cellular infiltration from adjacent tissues, thus accelerating the remodeling process. The composites may be used for the repair of a simple, complex, tissue void or tissue augmentation or tissue obliteration, for reconstruction, or repair or therapeutic delivery to the integument, subdermal tissue, breast tissue, vascular tissue, cardiac tissue,urogential-renal tissue, pulmonary tissue, hepatic tissue, gastrointestinal tissue, muscle tissue, ligament tissue, tendon tissue, facial tissue, gynecologic and female reproductive genital tissue, non-articular surface fibrocartilage tissue and cartilage tissue and special sensory tissues and neural tissue.

[0064] The invention also provides methods of preparing and using inventive composites as well as kits for preparing and/or administering inventive composites. Inventive porous composites may be prepared using any of a variety of methods. In some embodiments, inventive composites are prepared using a method that includes water as a blowing agent. In one embodiment, tissue or tissue components or other tissue substitute materials are combined with polyurethanes and injected, extruded, molded, or similarly delivered to a tissue site (e.g., soft tissue defect such as a breast lumpectomy) of a subject. Inventive composites are engineered to set in situ to form a solid composite that may have a desired or predetermined mechanical strength and are conformal to the tissue site. In certain embodiments, polyurethane present in a composition or composite may include monomers or pre-polymers. In some embodiments, polyurethane is a polymer that has been rendered formable through combination of two liquid components (i.e., a polyisocyanate prepolymer and a polyol).

Tissue Component

[0065) Tissue components used in accordance with the present invention may include a tissue-derived material, an inorganic material, a synthetic analog or animal or plant species tissue component, a tissue substitute material, a composite material, or any combinations thereof. As discussed below a tissue component may refer to autologous, allogenic, xenogenic tissue or a tissue subcomponent such as, but not limited to, a purified cell population; or extra-cellular matrix(ECM) component; or an intra-cellular matrix (ICM) component that may or may not be purified or a synthetically produced analog. Additionally, refined, purified, or synthetic analogs of polysaccharides, proteoglycans, cellulose species or other bio-mimetic molecules or derived from animal or plant sources should be considered as part of a tissue component. As discussed, the tissue component may be in particulate form. It may also act as a porogen when removed from the polyurethane matrix.

[0066] Any kind of tissue and/or tissue-derived components may be used in the present invention. In some embodiments, tissue components employed in the preparation of tissue component containing composites are obtained from tissue. A tissue component may be obtained from any vertebrate, or non-vertebrate animal or plant species. Tissue components may be of autogenous, allogenic, and/or xenogeneic origin. In certain embodiments, tissue components are autogenous, that is, tissue components are from the subject being treated. In other embodiments, tissue components are allogenic (e.g., from donors). In certain embodiments, the source of tissue may be matched to the eventual recipient of inventive composites (i.e., the donor and recipient are of the same species). For example, human tissue components are typically used in a human subject. In certain embodiments, tissue components are obtained from tissue of allogenic origin. In certain embodiments, tissue components are obtained from tissue of xenogeneic origin. Porcine and bovine tissue are types of xenogeneic tissue tissue that can be used individually or in combination as sources for tissue components and may offer advantageous properties. Xenogenic tissue tissue may be combined with allogenic or autogenous tissue.

[0067] In preferred embodiments of the invention the Tissue Component is extracellular matrix sub-component or sub-components (e.g., collagen or other matrix proteins, hyaluronic acid or other polysaccharides), or synthetic analog components (e.g., carboxymethylcellulose). In such embodiments the Tissue Component absorbs moisture from the wound bed, thus limiting over-expansion of the foam due to diffusion of water from the host tissue into the injected material. The Tissue Component also precludes both the formation of non-functional excessively large voids, as well as an undesirable pore morphology due to the excessively large pores that result from the diffusion of water or interstitial fluids from the wound bed into the reacting PUR portion of the composite. The tissue component is specifically engineered to absorb moisture from the wound bed, resulting in controlled expansion and pore morphology formation. Either during or after cure of the PUR component, the Tissue Component is removed from the injected material either through the process of dissolution or by cell-mediated degradation, thereby creating additional pores. Therefore in preferred embodiments the Tissue Component also functions as a porogen. The Tissue Component also allows for adhesive type of binding to host tissue.

[0068] In some embodiments, the tissue component may be a carbohydrate, which may also serve as a porogen. A carbohydrate may be a monosaccharide, disaccharide, or polysaccharide. The carbohydrate may be a natural or synthetic carbohydrate. In some embodiments, the carbohydrate is a biocompatible, biodegradable carbohydrate. In certain embodiments, the carbohydrate is a polysaccharide. Exemplary polysaccharides include cellulose, starch, amylose, dextran, poly(dextrose), glycogen, etc. In certain embodiments, a polysaccharide is dextran. Very high molecular weight dextran has been found particularly useful as a porogen. For example, the molecular weight of the dextran may range from about 500,000 g/mol to about 10,000,000 g/mol, preferably from about 1 ,000,000 g/mol to about 3,000,000 g mol. In certain embodiments, the dextran has a molecular weight of approximately 2,000,000 g/mol. Dextrans with a molecular weight higher than 10,000,000 g/mol may also be used as porogens. Dextran may be used in any form (e.g., particles, granules, fibers, elongated fibers) as a porogen. In certain embodiments, fibers or elongated fibers of dextran are used as a porogen in inventive composites. Fibers of dextran may be formed using any known method including extrusion and precipitation. Fibers may be prepared by precipitation by adding an aqueous solution of dextran {e.g. , 5-25% dextran) to a less polar solvent such as a 90-100% alcohol {e.g., ethanol) solution. The dextran precipitates out in fibers that are particularly useful as porogens in the inventive composite. Once the composite with dextran as a tissue component porogen is used, the dextran dissolves away very quickly. Within approximately 24 hours, substantially all of dextran is out of composites leaving behind pores in the composite. An advantage of using dextran in a composite is that dextran exhibits a hemostatic property in extravascular space. Therefore, dextran in a composite can decrease bleeding at or near the site of use.

[0069] Tissue components can be formed by any process known to break down tissue into small pieces or subcomponents. Exemplary processes for forming such components include tissue graft harvesting, milling, cell purification, or ECM or 1CM purification or synthesis. Exemplary particulate shapes include spheroidal, plates, shards, fibers, cuboidal, sheets, rods, oval, strings, elongated components, wedges, discs, rectangular, polyhedral, etc.

[0070] As for irregularly shaped tissue components, recited dimension ranges may represent the length of the greatest or smallest dimension of the particle. As examples, tissue components can be pin shaped, with tapered ends having an average diameter of from about 100 microns to about 500 microns. As will be appreciated by one of skill in the art, for injectable composites, the maximum particle size will depend in part on the size of the cannula or needle through which the material will be delivered.

[0071) In some embodiments, size distribution of tissue components utilized in accordance with the present inventions with respect to a mean value or a median value may be plus or minus, e.g., about 10% or less of the mean value, about 20% or less of the mean value, about 30% or less of the mean value, about 40% or less of the mean value, about 50% or less of the mean value, about 60% or less of the mean value, about 70% or less of the mean value, about 80% or less of the mean value, or about 90% or less of the mean value.

|0072| In some embodiments, particulate tissue components may have a median or mean diameter or a median or mean length of about 1200 microns, 1 100 microns, 1000 microns, 900 microns, 800 microns, 700 microns, 600 microns, 500 microns, 400 microns, 300 microns, 200 microns, 100 microns, but are to include a plurity from 5000 microns down to 1 micron etc. In some embodiments, diameters of tissue components are within a range between any of such sizes. For example, median or mean diameters or lengths of tissue components have a range from approximately 1 micron to approximately 5000 microns. In some embodiments, about 70, about 80 or about 90 percent of tissue components possess a median or mean diameter or a median or mean length within a range of any of such sizes.

[0073] For tissue components that are fibers or other elongated components, in some embodiments, at least about 90 percent of the components possess a median or mean length in their greatest dimension in a range from approximately 100 microns to approximately 1000 microns. Components may possess a median or mean length to median or mean thickness ratio from at least about 5: 1 up to about 500: 1 , for example, from at least about 50: 1 up to about 500: 1 , or from about 50: 1 up to about 100: 1 ; and a median or mean length to median or mean width ratio of from about 10: 1 to about 200: 1 and, for example, from about 50: 1 to about 100: 1. In certain embodiments, tissue components may short fibers having a cross- section of about 300 microns to about 100 microns and a length of about 0.1 mm to about 1 mm.

[0074| Processing of tissue components to provide sub-components may be adjusted to optimize for the desired size and/or distribution of tissue components or components. The properties of resulting inventive composites (e.g. , mechanical properties or degradation profile) may also be engineered by adjusting weight percent, shapes, sizes, distribution, etc. of tissue components or components or other components. For example, an inventive composite may be made more viscous and load bearing by including a higher percentage of components.

J0075] The surfaces of particulate tissue components utilized in accordance with the present invention may be optionally treated to enhance their interaction with polyurethanes and/or to confer some properties to particle surface. While some particulate tissue components will interact readily with monomers and be covalently linked to polyurethane matrices, it may be desirable to modify the surface of tissue components to facilitate their incorporation into polymers that do not bond well to tissue, such as poly(lactides). Surface modification may provide a chemical substance that is strongly bonded to the surface of tissue, e.g., covalently bonded to the surface. Particualte issue components may, alternatively or additionally, be coated with a material to facilitate interaction with polymers of inventive composites.

[0076J Alternatively or additionally, biologically active compounds such as a biomolecule, a small molecule, or a bioactive agent may be attached to tissue components through a linker. For example, mercaptosilanes will react with sulfur atoms in proteins to attach them to tissue components. Aminated, hydroxylated, and carboxylated silanes will react with a wide variety functional groups. Of course, the linker may be optimized for the compound being attached to tissue components.

[0077J Biologically active molecules can modify non-mechanical properties of inventive composites as they degrade. For example, immobilization of a drug on tissue components allows it to be gradually released at an implant site as the composite degrades. Antiinflammatory agents embedded within inventive composites will control inflammatory response long after an initial response to injection of the composites. For example, if a piece of the composite fractures several weeks after injection, immobilized compounds will reduce the intensity of any inflammatory response, and the composite will continue to degrade through hydrolytic or physiological processes. In some embodiments, compounds may also be immobilized on the tissue components that are designed to elicit a particular metabolic response or to attract cells to injection sites.

[0078] Some biomolecules, small molecules, and bioactive agents may also be incorporated into polyurethane matrices used in inventive composites. For example, many amino acids have reactive side chains. The phenol group on tyrosine has been exploited to form polycarbonates, polyarylates, and polyiminocarbonates (see Pulapura, et al. ,

Biopolymers, 1992, 32: 41 1 -417; and Hooper, et al, J. Bioactive and Compatible Polymers, 1995, 10:327-340, the entire contents of both of which are incorporated herein by reference). Amino acids such as lysine, arginine, hydroxylysine, proline, and hydroxyproline also have reactive groups and are essentially tri-functional. Amino acids such as valine, which has an isopropyl side chain, are still difunctional. Such amino acids may be attached to the silane and still leave one or two active groups available for incorporation into a polymer.

[0079] Non-biologically active materials may also be attached to tissue components. For example, radiopaque {e.g. , barium sulfate), luminescent {e.g., quantum dots), or magnetically active components {e.g. , iron oxide) may be attached to tissue components using the techniques described above. Mineralized tissue components are an inherently radiopaque component of some embodiments of present inventions, whereas demineralized tissue components, another optional component of inventive composites, are not radiopaque. To enhance radiopacity of inventive composites, mineralized tissue components can be used. Another way to render radiopaque the polymers utilized in accordance with the present invention is to chemically modify them such that a halogen {e.g. , iodine) is chemically incorporated into the polyurethane matrices, as in U.S. Patent Publication No. 2006-0034769, whose content is incorporated herein by reference.

(0080] If a material, for example, an alloplastic or tissue transplant atom or cluster, cannot be produced as a silane or other group that reacts with tissue components, then a chelating agent may be immobilized on tissue particle surface and allowed to form a chelate with the atom or cluster. As tissue components and polymers used in the present invention are resorbed, these non-biodegradable materials may be removed from tissue sites by natural metabolic processes, allowing degradation of the polymers and resorption of the tissue components to be tracked using standard medical diagnostic techniques.

|0081 ] Collagen fibers exposed by demineralization are typically relatively inert but have some exposed amino acid residues that can participate in reactions. Collagen may be rendered more reactive by fraying triple helical structures of the collagen to increase exposed surface area and number of exposed amino acid residues. This not only increases surface area of tissue components available for chemical reactions but also for their mechanical interactions with polymers as well. Rinsing partially demineralized tissue components in an alkaline solution will fray collagen fibrils. For example, tissue components may be suspended in water at a pH of about 10 for-about 8 hours, after which the solution is neutralized. One skilled in the art will recognize that this time period may be increased or decreased to adjust the extent of fraying. Agitation, for example, in an ultrasonic bath, may reduce the processing time. Alternatively or additionally, tissue components may be sonicated with water, surfactant, alcohol, or some combination of these.

[0082] In some embodiments, collagen fibers at tissue component particle surface may be cross-linked. A variety of cross-linking techniques suitable for medical applications are well known in the art (see, for example, U.S. Patent 6, 123,781 , the contents of which are incorporated herein by reference). For example, compounds like l -ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride, either alone or in combination with N- hydroxysuccinimide (NHS) will crosslink collagen at physiologic or slightly acidic pH (e.g., in pH 5.4 MES buffer). Acyl azides and genipin, a naturally occurring bicyclic compound including both carboxylate and hydroxyl groups, may also be used to cross-link collagen chains (see Simmons, et al, Biotechnol. Appl. Biochem. , 1993, 17:23-29; PCT Publication W098/1971 8, the contents of both of which are incorporated herein by reference).

Alternatively or additionally, hydroxymethyl phosphine groups on collagen may be reacted with the primary and secondary amines on neighboring chains (see U.S. Patent No.

5,948,386, the entire contents of which are incorporated herein by reference). Standard cross-linking agents such as mono- and dialdehydes, polyepoxy compounds, tanning agents including polyvalent metallic oxides, organic tannins, and other plant derived phenolic oxides, chemicals for esterification or carboxyl groups followed by reaction with hydrazide to form activated acyl azide groups, dicyclohexyl carbodiimide and its derivatives and other heterobifunctional crosslinking agents, hexamethylene diisocyanate, and sugars may also be used to cross-link collagens. Tissue components are then washed to remove all teachable traces of materials. In other embodiments, enzymatic cross-linking agents may be used. Additional cross-linking methods include chemical reaction, irradiation, application of heat, dehydrothermal treatment, enzymatic treatment, etc. One skilled in the art will easily be able to determine the optimal concentrations of cross-linking agents and incubation times for the desired degree of cross-linking. [0083] Both frayed and unfrayed collagen fibers may be derivatized with monomer, pre- polymer, oligomer, polymer, initiator, and/or biologically active or inactive compounds, including but not limited to biomolecules, bioactive agents, small molecules, inorganic materials, minerals, through reactive amino acids on the collagen fiber such as lysine, arginine, hydroxylysine, proline, and hydroxyproline. Monomers that link via step polymerization may react with these amino acids via the same reactions through which they polymerize. Vinyl monomers and other monomers that polymerize by chain polymerization may react with these amino acids via their reactive pendant groups, leaving the vinyl group free to polymerize. Alternatively, or in addition, tissue components may be treated to induce calcium phosphate deposition and crystal formation on exposed collagen fibers. Calcium ions may be chelated by chemical moieties of the collagen fibers, and/or calcium ions may bind to the surface of the collagen fibers. James et al , Biomaterials 20:2203-23 13, 1999; incorporated herein by reference. The calcium ions bound to the collagen provides a biocompatible surface, which allows for the attachment of cells as well as crystal growth. The polymer will interact with these fibers, increasing interfacial area and improving the wet strength of the composite.

[0084] In some embodiments, the surface treatments described above or treatments such as etching may be used to increase the surface area or surface roughness of particulate tissue components. Such treatments increase the interfacial strength of the particle/polymer interface by increasing the surface area of the interface and/or the mechanical interlocking of tissue components and polyurethane. Such surface treatments may also be employed to round the shape or smooth the edges of tissue components to facilitate delivery of the inventive composite. Such treatment is particularly useful for injectable composites.

|0085] In some embodiments, surface treatments of tissue components are optimized to < enhance covalent attractions between tissue components and polyurethanes. In some embodiments, the surface treatment may be designed to enhance non-covalent interactions between tissue particle and polyurethane matrix. Exemplary non-covalent interactions include electrostatic interactions, hydrogen bonding, pi-bond interactions, hydrophobic interactions, van der Waals interactions, and mechanical interlocking. For example, if a protein or a polysaccharide is immobilized on tissue particle, the chains of polymer matrix will become physically entangled with long chains of the biological macromolecules when they are combined. Charged phosphate sites on the surface of tissue components, produced by washing the tissue components in basic solution, will. interact with the amino groups present in many biocompatible polymers, especially those based on amino acids. The pi- orbitals on aromatic groups immobilized on a tissue particle will interact with double bonds and aromatic groups of the polymer.

[0086] In some embodiments, a tissue component may be employed in combination with other materials. For example, inorganic materials such as those described, for example, in U.S. patent applications ser. nos. 10/735,135; 10/681 ,651 ; and 10/639,912; (incorporated herein by reference) may be combined with proteins such as bovine serum albumin (BSA), collagen, or other extracellular matrix ECM or 1CM components to form a composite. In some embodiments, the inventive compositions and/or composites may also include and or be combined with a solid filler (e.g., carboxymethylcellulose (CMC) and hyaluronic acid (HA)). For example, when composites used in wound healing, solid fillers can help absorb excess moisture in the wounds from blood and serum and allow for proper foaming.

Polymer Component

[0087J Synthetic polymers can be designed with properties targeted for a given clinical application. According to the present invention, polyurethanes (PUR) are a useful class of biomaterials due to the fact that they can be injectable or moldable as a reactive liquid that subsequently cures to form a porous composite. These materials also have tunable degradation rates, which are shown to be highly dependent on the choice of polyol and isocyanate components (Hafeman et al., Pharmaceutical Research 2008;25(10):2387-99; Storey et al., J Poly Sci Pt A: Poly Chem 1994;32:2345-63; Skarja et al., J App Poly Sci 2000;75: 1522-34). Polyurethanes have tunable mechanical properties, which can also be enhanced with the addition of tissue components or subcomponents and/or other components (Adhikari et al., Biomaterials 2008;29:3762-70; Gorna et al, J Biomed Mater Res Pt A 2003;67A(3):813-27) and exhibit elastomeric rather than brittle mechanical properties.

[0088] Polyurethanes can be made by reacting together the components of a two- component composition, one of which includes a polyisocyanate while the other includes a component having two or more hydroxyl groups (i.e., polyols) to react with the

polyisocyanate. For example, U.S. Pat. No. 6,306,177, discloses a method for repairing a tissue site using polyurethanes, the content of which is incorporated by reference.

[0089] It is to be understood that by "a two-component composition" it means a composition comprising two essential types of polymer components. In some embodiments, such a composition may additionally comprise one or more other optional components. |0090| In some embodiments, polyurethane is a polymer that has been rendered formable through combination of two liquid components (i.e. , a polyisocyanate prepolymer and a polyol). In some embodiments, a polyisocyanate prepolymer or a polyol may be a molecule with two or three isocyanate or hydroxyl groups respectively. In some embodiments, a polyisocyanate prepolymer or a polyol may have at least four isocyanate or hydroxyl groups respectively.

(00911 Synthesis of porous polyurethane results from a balance of two simultaneous reactions. Reactions, in some embodiments, are illustrated below in Scheme 1. One is a gelling reaction, where an isocyanates and a polyester polyol react to form urethane bonds. The one is a blowing reaction. An isocyanate can react with water to form carbon dioxide gas, which acts as a lowing agent to form pores of polyurethane foam. The relative rates of these reactions determine the scaffold morphology, working time, and setting time.

[0092] Exemplary gelling and blowing reactions in forming of polyurethane are shown in Scheme 1 below, where Rj, R2 and R3, for example, can be oligomers of caprolactone, lactide and glycolide respectively.

Blowing reaction

[0093] Biodegradable polyurethane scaffolds synthesized from aliphatic polyisocyanates been shown to degrade into non-toxic compounds and support cell attachment and proliferation in vitro. A variety of polyurethane polymers suitable for use in the present invention are known in the art, many of which are listed in commonly owned applications: U.S. Ser. No. 10/759,904 filed on January 16, 2004, entitled "Biodegradable polyurethanes and use thereof and published under No. 2005-0013793; U.S. Ser. No. 1 1/667,090 filed on November 5, 2005, entitled "Degradable polyurethane foams" and published under No. 2007- 0299151 ; U.S. Ser. No. 12/298,158 filed on April 24, 2006, entitled "Biodegradable polyurethanes" and published under No. 2009-0221784; all of which are incorporated herein by reference. Polyurethanes described in U.S. Ser. No. 1 1/336, 127 filed on January 19, 2006 and published under No. 2006-0216323, which is entitled "Polyurethanes for Osteoimplants" and incorporated herein by reference, may be used in some embodiments of the present invention.

|0094| Polyurethanes foams may be prepared by contacting an isocyanate-terminated prepolymer (component 1 , e.g, polyisocyanate prepolymer) with a hardener (component 2) that includes at least a polyol (e.g., a polyester polyol) and water, a catalyst and optionally, a stabilizer, a porogen, pore opener, PEG, etc. In some embodiments, multiple polyurethanes (e.g., different structures, difference molecular weights) may be used in a

composite/composition of the present invention. In some embodiments, other biocompatible and/or biodegradable polymers may be used with polyurethanes in accordance with the present invention. In some embodiments, biocompatible co-polymers and/or polymer blends of any combination thereof may be exploited.

[0095] Polyurethanes used in accordance with the present invention can be adjusted to produce polymers having various physiochemical properties and morphologies including, for example, flexible foams, rigid foams, elastomers, coatings, adhesives, and sealants. The properties of polyurethanes are controlled by choice of the raw materials and their relative concentrations. For example, thermoplastic elastomers are characterized by a low degree of cross-linking and are typically segmented polymers, consisting of alternating hard

(diisocyanates and chain extenders) and soft (polyols) segments. Thermoplastic elastomers are formed from the reaction of diisocyanates with long-chain diols and short-chain diol or diamine chain extenders. In some embodiments, pores in tissue/polyurethanes composites in the present invention are interconnected and have a diameter ranging from approximately 50 to approximately 1000 microns.

|0096] Prepolymer. Polyurethane prepolymers can be prepared by contacting a polyol with an excess (typically a large excess) of a polyisocyanate. The resulting prepolymer intermediate includes an adduct of polyisocyanates and polyols solubilized in an excess of polyisocyanates. Prepolymer can, in some embodiments, be formed by using an approximately stoichiometric amount of polyisocyanates in forming a prepolymer and subsequently adding additional polyisocyanates. The prepolymer therefore exhibits both low viscosity, which facilitates processing, and improved miscibility as a result of the polyisocyanate-polyol adduct. Polyurethane networks can, for example, then be prepared by reactive liquid molding, wherein the prepolymer is contacted with a polyester polyol to form a reactive liquid mixture (i.e., a two-component composition) which is then cast into a mold and cured. [0097| Polyisocyanates or multi-isocyanate compounds for use in the present invention include aliphatic polyisocyanates. Exemplary aliphatic polyisocyanates include, but are not limited to, lysine diisocyanate, an alkyl ester of lysine diisocyanate (for example, the methyl ester or the ethyl ester), lysine triisocyanate, hexamethylene diisocyanate, isophorone diisocyanate (IPD1), 4,4'-dicyclohexylmethane diisocyanate (H12MDI), cyclohexyl diisocyanate, 2,2,4-(2,2,4)-trimethylhexamethylene diisocyanate (TMDI), dimers prepared form aliphatic polyisocyanates, trimers prepared from aliphatic polyisocyanates and/or mixtures thereof. In some embodiments, hexamethylene diisocyanate (HDI) trimer sold as Desmodur N3300A may be a polyisocyanate utilized in the present invention. In some embodiments, polyisocyanates used in the present invention includes approximately 10 to 55% NCO by weight (wt % NCO= 100*(42/Mw)). In some embodiments, polyisocyanates include approximately 15 to 50% NCO.

(0098] Polyisocyanate prepolymers provide an additional degree of control over the structure of biodegradable polyurethanes. Prepared by reacting polyols with isocyanates, NCO-terminated prepolymers are oligomeric intermediates with isocyanate functionality as shown in Scheme 1 . To increase reaction rates, urethane catalysts (e.g., tertiary amines) and/or elevated temperatures (60-90 °C) may be used (see, Guelcher, Tissue Engineering: Part B, 14 (1 ) 2008, pp 3-17).

[0099] Polyols used to react with polyisocyanates in preparation of NCO-terminated prepolymers refer to molecules having at least two functional groups to react with isocyanate groups. In some embodiments, polyols have a molecular weight of no more than 1000 g/mol. In some embodiments, polyols have a range of molecular weight between about 100 g/mol to about 500 g/mol. In some embodiments, polyols have a range of molecular weight between about 200 g/mol to about 400 g/mol. In certain embodiments, polyols (e.g., PEG) have a molecular weight of about 200 g/mol. Exemplary polyols include, but are not limited to, PEG, glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, 1 ,2,4-butanetriol, trimethylolpropane, 1 ,2,3-trihydroxyhexane, myo-inositol, ascorbic acid, a saccharide, or sugar alcohols (e.g., mannitol, xylitol, sorbitol etc.). In some embodiments, polyols may comprise multiple chemical entities having reactive hydrogen functional groups (e.g. , hydroxy groups, primary amine groups and/or secondary amine groups) to react with the isocyanate functionality of polyisocyanates.

[00100] In some embodiments, polyisocyanate prepolymers are resorbable. Zhang and coworkers synthesized biodegradable lysine diisocyanate ethyl ester (LDI)/glucose polyurethane foams proposed for tissue engineering applications. In those studies, NCO- terminated prepolymers were prepared from LDI and glucose. The prepolymers were chain- extended with water to yield biocompatible foams which supported the growth of rabbit tissue marrow stromal cells in vitro and were non-immunogenic in vivo, (see Zhang, et al. ,■ Biomaterials l Y. 1247-1258 (2000), and Zhang, et al , Tiss. Eng., 8(5): 771 -785 (2002), both of which are incorporated herein by reference).

[00101] In some embodiments, prepared polyisocyanate prepolymer can be a fiowable liquid at processing conditions. In general, the processing temperature is no greater than 60 °C. In some embodiments, the processing temperature is ambient temperature (25 °C).

[00102] Polyols. Polyols, which are biocompatible, utilized in accordance with the present invention can be amine- and/or hydroxyl-terminated compounds and include, but are not limited to, polyether polyols (such as polyethylene glycol (PEG) or polyethylene oxide (PEO), polytetramethylene etherglycol (PTMEG), polypropylene oxide glycol (PPO));

amine-terminated polyethers; polyester polyols (such as polybutylene adipate, caprolactone polyesters, castor oil); and polycarbonates (such as poly(l ,6-hexanediol) carbonate). In some embodiments, polyols may be ( 1 ) molecules having multiple hydroxyl or amine functionality, such as glucose, polysaccharides, and castor oil; and (2) molecules (such as fatty acids, triglycerides, and phospholipids) that have been hydroxylated by known chemical synthesis techniques to yield polyols.

[00103] Polyols used in the present invention may be polyester polyols. In some embodiments, polyester polyols may include polyalkylene glycol esters or polyesters prepared from cyclic esters. In some embodiments, polyester polyols may include poly(ethylene adipate), poly(ethylene glutarate), poly(ethylene azelate), poly(trimethylene glutarate), poly(pentamethylene glutarate), poly(diethylene glutarate), poly(diethylene adipate), poly(triethylene adipate), poly( l ,2-propylene adipate), mixtures thereof, and/or copolymers thereof. In some embodiments, polyester polyols can include, polyesters prepared from caprolactone, glycolide, D, L-lactide, mixtures thereof, and/or copolymers thereof. In some embodiments, polyester polyols can, for example, include polyesters prepared from castor-oil. When polyurethanes degrade, their degradation products can be the polyols from which they were prepared from.

|00104] In some embodiments, polyester polyols can be miscible with prepared prepolymers used in reactive liquid mixtures {i.e., two-component composition) of the present invention. In some embodiments, surfactants or other additives may be included in the reactive liquid mixtures to help homogenous mixing. [00105] The glass transition temperature (Tg) of polyester polyols used in the reactive liquids to form polyurethanes can be less than 60 °C, less than 37 °C (approximately human body temperature) or even less than 25 °C. In addition to affecting flowability at processing conditions, Tg can also affect degradation. In general, a Tg of greater than approximately 37 °C will result in slower degradation within the body, while a Tg below approximately 37 °C will result in faster degradation.

[00106] Molecular weight of polyester polyols used in the reactive liquids to form polyurethanes can, for example, be adjusted to control the mechanical properties of polyurethanes utilized in accordance with the present invention. In that regard, using polyester polyols of higher molecular weight results in greater compliance or elasticity. In some embodiments, polyester polyols used in the reactive liquids may have a molecular weight less than approximately 3000 Da. In certain embodiments, the molecular weight may be in the range of approximately 200 to 2500 Da or 300 to 2000 Da. In some embodiments, the molecular weight may be approximately in the range of approximately 450 to 1800 Da or 450 to 1200 Da. In some embodiments, a polyester polyol comprise poly(caprolactone-co- lactide-co-glycolide), which has a molecular weight in a range of 200 Da to 2500 Da, or 300 Da to 2000 Da.

[00107] In some embodiments, polyols may include multiply types of polyols with different structures, molecular weight, properties, etc.

[00108] Additional Components. In accordance with the present invention, two- component compositions (i.e., polyprepolymers and polyols) to fonn porous composites may be used with other agents and/or catalysts. Zhang et al. have found that water may be an adequate blowing agent for a lysine diisocyanate PEG/glycerol polyurethane (see Zhang, et al., Tissue Eng. 2003 (6): 1 143-57) and may also be used to form porous structures in polyurethanes. Other blowing agents include dry ice or other agents that release carbon dioxide or other gases into the composite. Alternatively, or in addition, porogens (see detail discussion below) such as salts may be mixed in with reagents and then dissolved after polymerization to leave behind small voids.

[00109] Two-component compositions and/or the prepared composites used in the present invention may include one or more additional components. In some embodiments, inventive compositions and/or composites may includes, water, a catalyst (e.g., gelling catalyst, blowing catalyst, etc.), a stabilizer, a plasticizer, a porogen, a chain extender (for making of polyurethanes), a pore opener (such as calcium stearate, to control pore morphology), a wetting or lubricating agent, etc. (See, U.S. Ser. No. 10/759,904 published under No. 2005- 0013793, and U.S. Ser. No. 1 1/625,1 19 published under No. 2007-0191963; both of which are incorporated herein by reference).

[00110] Water. Water may be a blowing agent to generate porous polyurethane-based composites. Porosity of tissue/polymer composites increased with increasing water content, and biodegradation rate accelerated with decreasing polyester half-life, thereby yielding a family of materials with tunable properties that are useful in the present invention. See, Guelcher et al.. Tissue Engineering, 13(9), 2007, pp232 1 -2333, which is incorporated by reference. In some embodiments, an amount of water is about 0, 0.5, 1 , 1 .5, 2, 3, 4 5, 6, 7, 8, 9, 10 parts per hundred parts (pphp) polyol. In some embodiments, water has an approximate range of any of such amounts.

(00111] Catalyst. In some embodiments, at least one catalyst is added to form reactive liquid mixture {i.e., two-component compositions). A catalyst, for example, can be non-toxic (in a concentration that may remain in the polymer). A catalyst can, for example, be present in two-component compositions in a concentration in the range of approximately 0.5 to 5 parts per hundred parts polyol (pphp) and, for example, in the range of approximately 0.5 to 2, or 2 to 3 pphp. A catalyst can, for example, be an amine compound. In some

embodiments, catalyst may be an organometallic compound or a tertiary amine compound. In some embodiments the catalyst may be stannous octoate (an organobismuth compound), triethylene diamine, bis(dimethylaminoethyl)ether, dimethylethanolamine, dibutyltin dilaurate, and Coscat organometallic catalysts manufactured by Vertullus (a bismuth based catalyst), or any combination thereof.

[00112] Stabilizer. In some embodiments, a stabilizer is nontoxic (in a concentration remaining in the polyurethane foam) and can include a non-ionic surfactant, an anionic surfactant or combinations thereof. For example, a stabilizer can be a polyethersiloxane, a salt of a fatty sulfonic acid or a salt of a fatty acid. In certain embodiments, a stabilizer is a polyethersiloxane, and the concentration of polyethersiloxane in a reactive liquid mixture can, for example, be in the range of approximately 0.25 to 4 parts per hundred polyol. In some embodiments, polyethersiloxane stabilizer are hydrolyzable.

(00113] In some embodiments, the stabilizer can be a salt of a fatty sulfonic acid.

Concentration of a salt of the fatty sulfonic acid in a reactive liquid mixture can be in the range of approximately 0.5 to 5 parts per hundred polyol. Examples of suitable stabilizers include a sulfated castor oil or sodium ricinoleicsulfonate.

[00114] Stabilizers can be added to a reactive liquid mixture of the present invention to, for example, disperse prepolymers, polyols and other additional components, stabilize the rising carbon dioxide bubbles, and/or control pore sizes of inventive composites. Although there has been a great deal of study of stabilizers, the operation of stabilizers during foaming is not completely understood. Without limitation to any mechanism of operation, it is believed that stabilizers preserve the thermodynamically unstable state of a polyurethane foam during the time of rising by surface forces until the foam is hardened. In that regard, foam stabilizers lower the surface tension of the mixture of starting materials and operate as emulsifiers for the system. Stabilizers, catalysts and other polyurethane reaction components are discussed, for example, in Oertel, G nter, ed., Polyurethane Handbook, Hanser Gardner Publications, Inc. Cincinnati, Ohio, 99- 108 ( 1994). A specific effect of stabilizers is believed to be the formation of surfactant monolayers at the interface of higher viscosity of bulk phase, thereby increasing the elasticity of surface and stabilizing expanding foam bubbles.

[00115| Chain extender. To prepare high-molecular-weight polymers, prepolymers are chain extended by adding a short-chain (e.g., <500 g/mol) poly amine or polyol. In certain embodiments, water may act as a chain extender. In some embodiments, addition of chain extenders with a functionality of two {e.g., diols and diamines) yields linear alternating block copolymers.

[00116] Plasticizer. In some embodiments, inventive compositions and/or composites include one or more plasticizers. Plasticizers are typically compounds added to polymers or plastics to soften them or make them more pliable. According to the present invention, plasticizers soften, make workable, or otherwise improve the handling properties of polymers or composites. Plasticizers also allow inventive composites to be moldable at a lower temperature, thereby avoiding heat induced tissue necrosis during implantation. Plasticizer may evaporate or otherwise diffuse out of the composite over time, thereby allowing composites to harden or set. Without being bound to any theory, plasticizer are thought to work by embedding themselves between the chains of polymers. This forces polymer chains apart and thus lowers the glass transition temperature of polymers. In general, the more plasticizer added, the more flexible the resulting polymers or composites will be.

[00117) In some embodiments, plasticizers are based on an ester of a polycarboxylic acid with linear or branched aliphatic alcohols of moderate chain length. For example, some plasticizers are adipate-based. Examples of adipate-based plasticizers include bis(2- ethylhexyl)adipate (DOA), dimethyl adipate (DMAD), monomethyl adipate (MMAD), and dioctyl adipate (DOA). Other plasticizers are based on maleates, sebacates, or citrates such as bibutyl maleate (DBM), diisobutylmaleate (DIBM), dibutyl sebacate (DBS), triethyl citrate (TEC), acetyl triethyl citrate (ATEC), tributyl citrate (TBC), acetyl tributyl citrate (ATBC), trioctyl citrate (TOC), acetyl trioctyl citrate (ATOC), trihexyl citrate (THC), acetyl trihexyl citrate (ATHC), butyryl trihexyl citrate (BTHC), and trimethylcitrate (TMC). Other plasticizers are phthalate based. Examples of phthalate-based plasticizers are N-methyl phthalate, bis(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), bis(n- buty phthalate (DBP), butyl benzyl phthalate (BBzP), diisodecyl phthalate (DOP), diethyl phthalate (DEP), diisobutyl phthalate (DIBP), and di-n-hexyl phthalate. Other suitable plasticizers include liquid polyhydroxy compounds such as glycerol, polyethylene glycol (PEG), triethylene glycol, sorbitol, monacetin, diacetin, and mixtures thereof. Other plasticizers include trimellitates (e.g. , trimethyl trimellitate (TMTM), tri-(2-ethylhexyl) trimellitate (TEHTM-MG), tri-(n-octyl,n-decyl) trimellitate (ATM), tri-(heptyl,nonyl) trimellitate (LTM), n-octyl trimellitate (OTM)), benzoates, epoxidized vegetable oils, sulfonamides (e.g., N-ethyl toluene sulfonamide (ETSA), N-(2-hydroxypropyl) benzene sulfonamide (HP BSA), N-(n-butyl) butyl sulfonamide (BBSA-NBBS)), organophosphates (e.g. , tricresyl phosphate (TCP), tributyl phosphate (TBP)), glycols/polyethers (e.g. , triethylene glycol dihexanoate, tetraethylene glycol diheptanoate), and polymeric plasticizers. Other plasticizers are described in Handbook of Plasticizers (G. Wypych, Ed., ChemTec Publishing, 2004), which is incorporated herein by reference. In certain embodiments, other polymers are added to the composite as plasticizers. In certain particular embodiments, polymers with the same chemical structure as those used in the composite are used but with lower molecular weights to soften the overall composite. In other embodiments, different polymers with lower melting points and/or lower viscosities than those of the polymer component of the composite are used.

[00118] In some embodiments, a polymers used as plasticizer are poly(ethylene glycol) (PEG). PEG, which also may be used as a plasticizer, is typically a low molecular weight PEG such as those having an average molecular weight of 1000 to 10000 g/mol, for example, from 4000 to 8000 g/mol. In certain embodiments, as discussed here and above, PEG 4000, PEG 5000, PEG 6000, PEG 7000, PEG 8000 or combinations thereof may be used in inventive composites. For example, plasticizer (PEG) is useful in making more moldable composites that include poly(lactide), poly(D,L-lactide), poly(lactide-co-glycolide), poly(D,L-lactide-co-glycolide), or poly(caprolactone). Plasticizer may comprise 1 -40% of inventive composites by weight. In some embodiments, the plasticizer is 10-30% by weight. In some embodiments, the plasticizer is approximately 10%, 15%, 20%, 25%, 30% or 40% by weight. In other embodiments, a plasticizer is not used in the composite. For example, in some polycaprolactone-containing composites, a plasticizer is not used. [00119] 1° some embodiments, inert plasticizers may be used. In some embodiments, a plasticizer may not be used in the present invention.

[00120] Additional Porogens. Porosity of inventive composites may be accomplished using any means known in the art. Exemplary methods of creating porosity in a composite include, but are not limited to, particular leaching processes, gas foaming processing, supercritical carbon dioxide processing, sintering, phase transformation, freeze-drying, cross- linking, molding, porogen melting, polymerization, melt-blowing, and salt fusion (Murphy et al, Tissue Engineering 8(l):43-52, 2002; incorporated herein by reference). For a review, see Karageorgiou et al, Biomaterials 26:5474-5491 , 2005; incorporated herein by reference. Porosity may be a feature of inventive composites during manufacture or before implantation, or porosity may only be available after implantation. For example, a implanted composite may include latent pores. These latent pores may arise from including porogens in the composite. While the tissue component may act as a porogen, some embodiments of the invention will incorporate other porogens to control, or to further control, porosity.

[00121] Porogens may be any chemical compound that will reserve a space within the composite while the composite is being molded and will diffuse, dissolve, and/or degrade prior to or after implantation or injection leaving a pore in the composite. Porogens may have the property of not being appreciably changed in shape and/or size during the procedure to make the composite moldable. For example, a porogen should retain its shape during the heating of the composite to make it moldable. Therefore, a porogen does not melt upon heating of the composite to make it moldable. In certain embodiments, a porogen has a melting point greater than about 60 °C, greater than about 70 °C, greater than about 80 °C, greater than about 85 °C, or greater than about 90 U C.

[00122] Porogens may be of any shape or size. A porogen may be spheroidal, cuboidal, rectangular, elonganted, tubular, fibrous, disc-shaped, platelet-shaped, polygonal, etc. In certain embodiments, the porogen is granular with a diameter ranging from approximately 100 microns to approximately 800 microns. In certain embodiments, a porogen is elongated, tubular, or fibrous. Such porogens provide increased connectivity of pores of inventive composite and/or also allow for a lesser percentage of the porogen in the composite.

[00123] Amount of porogens may vary in inventive composite from 1% to 80% by weight. In certain embodiments, the plasticizer makes up from about 5% to about 80% by weight of the composite. In certain embodiments, a plasticizer makes up from about 10% to about 50% by weight of the composite. Pores in inventive composites are thought to improve the cell and tissue inductivity or conductivity of the composite by providing holes for cells such as mononuclear and macrophage, fibroblasts, cells of the mesechymal lineage, stem cells, etc. Pores provide inventive composites with biological in growth capacity. Pores may also provide for easier degradation of inventive composites as tissue is formed and/or remodeled. In some embodiments, a porogen is biocompatible.

[00124] A porogen may be a gas, liquid, or solid. Exemplary gases that may act as porogens include carbon dioxide, nitrogen, argon, or air. Exemplary liquids include water, organic solvents, or biological fluids (e.g., blood, lymph, plasma). Gaseous or liquid porogen may diffuse out of the implant before or after implantation thereby providing pores for biological in-growth. Solid porogens may be crystalline or amorphous. Examples of possible solid porogens include water soluble compounds. Exemplary porogens include carbohydrates (e.g. , sorbitol, dextran (poly(dextrose)), starch), salts, sugar alcohols, natural polymers, synthetic polymers, and small molecules.

[00125] Small molecules including pharmaceutical agents may also be used as porogens in the inventive composites. Examples of polymers that may be used as plasticizers include polyvinyl pyrollidone), pullulan, poly(glycolide), poly(lactide), and poly(lactide-co- glycolide). Typically low molecular weight polymers are used as porogens. In certain embodiments, a porogen is poly(vinyl pyrrolidone) or a derivative thereof. Plasticizers that are removed faster than the surrounding composite can also be considered porogens.

[00126] In some embodiments, a pore opener can be used to faciliate an interconnected, or open, pore structure. Such pore openers are preferably nontoxic. Exemplary pore openers are described, for example, in US Published application 2009-0130174-A1 , which is incorporated herein by references.

[00127J For example, powdered divalent salts of stearic acid can be used, as they cause a local disruption of the pore structure during the foaming process and thereby gaps in the pore walls for an open pore structure.

[00128] Components to Deliver: Alternatively or additionally, composites of the present invention may have one or more components to deliver when implanted, including biomolecules, small molecules, bioactive agents, etc., to promote tissue growth and regeneration, and/or to accelerate healing. Examples of materials that can be incorporated include chemotactic factors, angiogenic factors, tissue cell inducers and stimulators, including the general class of cytokines such as the TGF-β superfamily of tissue growth factors, the family of tissue morphogenic proteins, osteoinductors, and/or tissue marrow or tissue forming precursor cells, isolated using standard techniques. Sources and amounts of such materials that can be included are known to those skilled in the art.

[00129] Biologically active materials, comprising biomolecules, small molecules, and bioactive agents may also be included in inventive composites to, for example, stimulate particular metabolic functions, recruit cells, or reduce inflammation. For example, nucleic acid vectors, including plasmids and viral vectors, that will be introduced into the patient's cells and cause the production of growth factors such as tissue morphogenetic proteins may be included in a composite. Biologically active agents include, but are not limited to, antiviral agent, antimicrobial agent, antibiotic agent, amino acid, peptide, protein, glycoprotein, lipoprotein, antibody, steroidal compound, antibiotic, antimycotic, cytokine, vitamin, carbohydrate, lipid, extracellular matrix, extracellular matrix component, chemotherapeutic agent, cytotoxic agent, growth factor, anti-rejection agent, analgesic, antiinflammatory agent, viral vector, protein synthesis co-factor, hormone, endocrine tissue, synthesizer, enzyme, polymer-cell scaffolding agent with parenchymal cells, angiogenic drug, collagen lattice, antigenic agent, cytoskeletal agent, mesenchymal stem cells, tissue digester, antitumor agent, cellular attractant, fibronectin, growth hormone cellular attachment agent, immunosuppressant, nucleic acid, surface active agent, hydroxyapatite, and penetraction enhancer. Additional exemplary substances include chemotactic factors, angiogenic factors, analgesics, antibiotics, anti-inflammatory agents, tissue morphogenic proteins, and other growth factors that promote cell-directed degradation or remodeling of the polymer phase of the composite and/or development of new tissue (e.g. , tissue). RNAi or other technologies may also be used to reduce the production of various factors.

[00130] In some embodiments, inventive composites include antibiotics. Antibiotics may be bacteriocidial or bacteriostatic. An anti-microbial agent may be included in composites. For example, anti-viral agents, anti-protazoal agents, anti-parasitic agents, etc. may be include in composites. Other suitable biostatic/biocidal agents include antibiotics, povidone, sugars, and mixtures thereof. Exemplary antibiotics include, but not limit to, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin, Geldanamycin, Herbimycin, Loravabef, etc. (See, The Merck Manual of Medical Information - Home Edition, 1999).

[00131] Inventive composites may also be seeded with cells. In some embodiments, a patient's own cells are obtained and used in inventive composites. Certain types of cells (e.g. , osteoblasts, fibroblasts, stem cells, cells of the osteoblast lineage, etc.) may be selected for use in the composite. Cells may be harvested from marrow, blood, fat, bone, muscle, connective tissue, skin, or other tissues or organs. In some embodiments, a patient's own cells may be harvested, optionally selected, expanded, and used in the inventive composite. In other embodiments, a patient's cells may be harvested, selected without expansion, and used in the inventive composite. Alternatively, exogenous cells may be employed.

Exemplary cells for use with the invention include mesenchymal stem cells and connective tissue cells, including osteoblasts, osteoclasts, fibroblasts, preosteoblasts, and partially differentiated cells of the osteoblast lineage. Cells may be genetically engineered. For example, cells may be engineered to produce a tissue morphogenic protein.

[00132] In ' some embodiments, inventive composites may include a composite material comprising a component to deliver. For example, a composite material can be a biomolecule (e.g., a protein) encapsulated in a polymeric microsphere or nanocomponents. In certain embodiments, BMP-2 encapsulated in PLGA microspheres may be embedded in a tissue/polyurethane composite used in accordance with the present invention. Sustained release of BMP-2 can be achieved due to the diffusional barriers presented by both the PLGA and Polyurethane of the inventive composite. Thus, release kinetics of growth factors (e.g., BMP-2) can be tuned by varying size of PLGA microspheres and porosity of polyurethane composite.

[00133] In some embodiments, inventive composites may include a composite material comprising a component to deliver locally for oncologic or chronic disease management. For example, a composite materials can be a biomolecule (e.g., a protein) encapsulated in a polymeric microsphere or nanocomponents. In certain embodiments, anti-Her2 and anti- VGEF(Avastin® (bevacizumab) Herceptin® (Trastuzumab)) (Genentech, South San Francisco, CA) or similar bio therapeutic agents may be encapsulated in PLGA microspheres or nano-particlate spheres and embedded in the injectable polyurethane composite used in accordance with the present invention. In a patient with local or metastatic disease with positive receptor profile the tumor may be infiltrated or removed and via a minimally invasive approach fill the tumor site/tissue void with the composite of the invention. Tunable sustained release of can be achieved due to the diffusional barriers presented by both the PLGA microsphere or other nano particulate microspheres and polyurethane of the inventive composite.

|00134] To enhance biodegradation in vivo, composites of the present invention can also include different enzymes. Examples of suitable enzymes or similar reagents are proteases or hydrolases with ester-hydrolyzing capabilities. Such enzymes include, but are not limited to, proteinase , bromelaine, pronase E, cellulase, dextranase, elastase, plasmin streptokinase, trypsin, chymotrypsin, papain, chymopapain, collagenase, subtilisin, chlostridopeptidase A, ficin, carboxypeptidase A, pectinase, pectinesterase, an oxireductase, an oxidase, or the like. The inclusion of an appropriate amount of such a degradation enhancing agent can be used to regulate implant duration.

f00135] Components to deliver may not be covalently bonded to a component of the composite. In some embodiments, components may be selectively distributed on or near the surface of inventive composites using the layering techniques described above. While surface of inventive composite will be mixed somewhat as the composite is manipulated in implant site, thickness of the surface layer will ensure that at least a portion of the surface layer of the composite remains at surface of the implant. Alternatively or in addition, biologically active components may be covalently linked to the tissue components or components before combination with the polymer. As discussed above, for example, silane coupling agents having amine, carboxyl, hydroxyl, or mercapto groups may be attached to the tissue components through the silane and then to reactive groups on a biomolecule, small molecule, or bioactive agent.

Preparation of Composite

[00136] In general, inventive composites are prepared by combining components, polymers and optionally any additional components. To form inventive composites, components as discussed herein may be combined with a reactive liquid (i.e., a two- component composition) thereby forming a naturally injectable or moldable composite or a composite that can be made injectable or moldable. Alternatively, components may be combined with polyisocyanate prepolymers or polyols first and then combined with other components.

[00137J In some embodiments, components may be combined first with a hardener that includes polyols, water, catalysts and optionally a solvent, a diluent, a stabilizer, a porogen, a pore opener, a plasticizer, etc., and then combined with a polyisocyanate prepolymer. In some embodiments, a hardener (e.g., a polyol, water and a catalyst) may be mixed with a prepolymer, followed by addition of components. In some embodiments, in order to enhance storage stability of two-component compositions, the two (liquid) component process may be modified to an alternative three (liquid)-component process wherein a catalyst and water may be dissolved in a solution separating from reactive polyols. For example, polyester polyols may be first mixed with a solution of a catalyst and water, followed by addition of tissue components or components, and finally addition of NCO-terminated prepolymers. [00138] In some embodiments, additional components or components to be delivered may be combined with a reactive liquid prior to injection. In some embodiments, they may be combined with one of polymer precursors (i.e., prepolymers and polyols) prior to mixing the precursors in forming of a reactive liquid/paste.

f00139] Porous composites can be prepared by incorporating a small amount (e.g., <5 wt%) of water which reacts with prepolymers to form carbon dioxide, a biocompativle blowing agent. Resulting reactive liquid/paste may be injectable through a 12-ga syringe needle into molds or targeted site to set in situ. In some embodiments, gel time is great than 3 min, 4 min, 5 min, 6 min, 7 min, or 8 min. In some embodiments, cure time is less than 20 min, 18 min, 16 min, 14 min, 12 min, or 10 min.

[00140] In some embodiments, catalysts can be used to assist forming porous composites. In general, the more blowing catalyst used, the high porosity of inventive composites may be achieved.

[00141] Polymers and components may be combined by any method known to those skilled in the art. For example, a homogenous mixture of polymers and/or polymer precursors (e.g., prepolymers, polyols, etc.) and components may be pressed together at ambient or elevated temperatures. At elevated temperatures, a process may also be accomplished without pressure. In some embodiments, polymers or precursors are not held at a temperature of greater than approximately 60°C for a significant time during mixing to prevent thermal damage to any biological component (e.g., growth factors or cells) of a composite. In some embodiments, temperature is not a concern because components and polymer precursors used in the present invention have a low reaction exotherm.

[00142] Alternatively or in addition, components may be mixed or folded into a polymer softened by heat or a solvent. Alternatively, a moldable polymer may be formed into a sheet that is then covered with a layer of components. Components may then be forced into the polymer sheet using pressure. In another embodiment, components are individually coated with polymers or polymer precursors, for example, using a tumbler, spray coater, or a fluidized bed, before being mixed with a larger quantity of polymer. This facilitates even coating of the components and improves integration of the components and polymer component of the composite.

[00143] After combination with components, polymers may be further modified by further cross-linking or polymerization to form a composite in which the polymer is covalently linked to the components. In some embodiments, composition hardens in a solvent-free condition. In some embodiments, compositions are a polymer/solvent mixture that hardens when a solvent is removed (e.g. , when a solvent is allowed to evaporate or diffuse away). Exemplary solvents include but are not limited to alcohols (e.g. , methanol, ethanol, propanol, butanol, hexanol, etc.), water, saline, DMF, DMSO, glycerol, and PEG. In certain embodiments, a solvent is a biological fluid such as blood, plasma, serum, marrow, etc. In certain embodiments, an inventive composite is heated above the melting or glass transition temperature of one or more of its components and becomes set after implantation as it cools. In certain embodiments, an inventive composite is set by exposing a composite to a heat source, or irradiating it with microwaves, IR rays, or UV light. Components may also be mixed with a polymer that is sufficiently pliable to combine with the components but that may require further treatment, for example, combination with a solvent or heating, to become a injectable or moldable composition. For example, a composition may be combined and injection molded, injected, extruded, laminated, sheet formed, foamed, or processed using other techniques known to those skilled in the art. In some embodiments, reaction injection molding methods, in which polymer precursors (e.g. , polyisocyanate prepolymer, a polyol) are separately charged into a mold under precisely defined conditions, may be employed. For example, tissue components or components may be added to a precursor, or it may be separately charged into a mold and precursor materials added afterwards. Careful control of relative amounts of various components and reaction conditions may be desired to limit the amount of unreacted material in a composite. Post-cure processes known to those skilled in the art may also be employed. A partially polymerized polyurethane precursor may be more completely polymerized or cross-linked after combination with hydroxylated or aminated materials or included materials (e.g., a particulate, any components to deliver, etc.).

[00144] In some embodiments, an inventive composite is produced with a injectable composition and then set in situ. For example, cross-link density of a low molecular weight polymer may be increased by exposing it to electromagnetic radiation (e.g., UV light) or an alternative energy source. Alternatively or additionally, a photoactive cross-linking agent, chemical cross-linking agent, additional monomer, or combinations thereof may be mixed into inventive composites. Exposure to UV light after a composition is injected into an implant site will increase one or both of molecular weight and cross-link density, stiffening polymers (i.e. , polyurethanes) and thereby a composite. Polymer components of inventive composites used in the present invention may be softened by a solvent, e.g. , ethanol. If a biocompatible solvent is used, polyurethanes may be hardened in situ. In some embodiments, as a composite sets, solvent leaving the composite is released into surrounding tissue without causing undesirable side effects such as irritation or an inflammatory response. In some embodiments, compositions utilized in the present invention become moldable at an elevated temperature into a pre-determined shape. Composites may become set when composites are implanted and allowed to cool to body temperature (approximately 37 °C).

[00145] The invention also provides methods of preparing inventive composites by combining tissue components and components and polyurethane precursors and resulting in naturally flowable compositions. Alternatively or additionally, the invention provides methods to make a porous composite include adding a solvent or pharmaceutically acceptable excipient to render a flowable or moldable composition. Such a composition may then be injected or placed into the site of implantation. As solvent or excipient diffuses out of the composite, it may become set in place.

[00146] Polymer processing techniques may also be used to combine components with a polyurethane or precursors (e.g., polyisocyanates and polyols). In some embodiments, a composition of polyurethane may be rendered formable (e.g., by heating or with a solvent) and combined with components by injection molding or extrusion forming. Alternatively, polyurethanes and tissue components and components may be mixed in a solvent and cast with or without pressure. For example, a solvent may be dichloromethane. In some embodiments, a composition of particle and polymer utilized in the present invention is naturally injectable or moldable in a solvent-free condition.

[00147] In some embodiments, components may be mixed with a polymer precursor according to standard composite processing techniques. For example, regularly shaped components may simply be suspended in a precursor. A polymer precursor may be mechanically stirred to distribute the components or bubbled with a gas, preferably one that is oxygen-, and moisture-free. Once components of a composition are mixed, it may be desirable to store it in a container that imparts a static pressure to prevent separation of the components and the polymer precursor, which may have different densities. In some embodiments, distribution and particle/polymer ratio may be optimized to produce at least one continuous path through a composite along components.

[00148] Interaction of polymer components with tissue components and components may also be enhanced by coating individual components with a polymer precursor before combining them with bulk precursors. The coating enhances the association of the polymer component of the composite with the components. For example, individual components may be spray coated with a monomer or prepolymer. Alternatively, the individual components may be coated using a tumbler— components and a solid polymer material are tumbled together to coat the components. A fluidized bed coater may also be used to coat the components. In addition, the components may simply be dipped into liquid or powdered polymer precursor. All of these techniques will be familiar to those skilled in the art.

[00149] In some embodiments, it may be desirable to infiltrate a polymer or polymer precursor into vascular and/or interstitial structure of tissue components or into tissue-derived tissues. Vascular structure of tissue includes such structures for example the hepatic or renal vessels. Many of monomers and precursors (e.g., polyisocyanate prepolymers, polyols) suggested for use with the invention are sufficiently flowable to penetrate through the channels and pores . Thus, it may be necessary to incubate tissue components and components in polyurethane precursors for a period of time to accomplish infiltration. In certain embodiments, polyurethane itself is sufficiently flowable that it can penetrate channels and pores of tissue. Other ceramic materials and/or other tissue-substitute materials employed as a particulate phase may also themselves include pores that can be infiltrated as described herein.

|00150] Inventive composites utilized in the present invention may include practically any ratio of polyurethane and tissue components, for example, between about 0 wt% and about 95 wt% tissue components. In some embodiments, composites may include about 10 wt% to about 15 wt% tissue components, about 15 wt% to about 20 wt% tissue components, about 20 wt% to about 25 wt% tissue components or about 25 wt% to about 30 wt% tissue components. Jn some embodiments, composites may include about 30 wt% to about 35 wt% tissue components. Jn some embodiments, composites may include at least approximately 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, or 35 wt%,40 wt%, or 45 wt%, 50 wt%, or 55 wt%, 60 wt%, or 65 wt% ,70 wt%, or 75 wt%, 80 wt%, or 85 wt% of tissue components. In certain embodiments, such weight percentages refer to weight of tissue components and may include other various components such as discussed above.

|00151 ] Desired proportion may depend on factors such as injection sites, shape and size of the components, how evenly polymer is distributed among components, desired flowability of composites, desired handling of composites, desired moldability of composites, and mechanical and degradation properties of composites. The proportions of polymers and components can influence various characteristics of the composite, for example, its mechanical properties, including fatigue strength, the degradation rate, and the rate of biological incorporation. In addition, the cellular response to the composite will vary with the proportion of polymer and components. In some embodiments, the desired proportion of components may be determined not only by the desired biological properties of the injected material but by the desired mechanical properties of the injected material. That is, an W

increased proportion of components will increase the viscosity of the composite, making it more difficult to inject or mold. A larger proportion of components having a wide size distribution may give similar properties to a mixture having a smaller proportion of more evenly sized components.

I 0152J Inventive composites of the present invention can exhibit high degrees of porosity over a wide range of effective pore sizes. Thus, composites may have, at once,

macroporosity, mesoporosity and microporosity. Where the tissue component is the only porogen present, however, the initial porosity may be 0%. Macroporosity is characterized by pore diameters greater than about 100 microns. Mesoporosity is characterized by pore diameters between about 100 microns about 10 microns; and microporosity occurs when pores have diameters below about 10 microns. In some embodiments, the composite has a an initial porosity of at least about 30%. For example, in certain embodiments, the composite has a porosity of more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90%. In some embodiments, inventive composites have a porosity in a range of 70% - 80%, 80% - 85%, or 85% - 90%. Advantages of a porous scaffold over non-porous scaffold include, but are not limited to, more extensive cellular and tissue in-growth into the composite, more continuous supply of nutrients, more thorough infiltration of therapeutics, and enhanced revascularization, allowing tissue growth and repair to take place more efficiently. Furthermore, in certain embodiments, the porosity of the composite may be used to load the composite with biologically active agents such as drugs, small molecules, cells, peptides, polynucleotides, growth factors, etc, for delivery at the implant site. Porosity may also render certain composites of the present invention compressible.

[00153] In some embodiments, pores of inventive composite may be over 100 microns wide for the invasion of cells and tissue in-growth (Klaitwatter et al., J. Biomed. Mater. Res. Symp. 2: 161 , 1971 ; incorporated herein by reference). In certain embodiments, the pore size may be in a range of approximately 50 microns to approximately 1000 microns, for example, of approximately 100 microns to approximately 500 microns.

[00154] In some embodiments, compressive strength of dry scaffolds may be in an approximate range of 17-97 kPa, while compressive modulus may be in an approximate range of 25 - 216 kPa.

[00155J After implantation, inventive composites are allowed to remain at the site providing the strength and modulus desired while at the same time promoting healing of the tissue and/or tissue growth. Polyurethane of composites may be degraded or be resorbed as new tissue is formed at the implantation site. Polymer may be resorbed over approximately 2 weeks to approximately 2 years. Composites may start to be remocleled in as little as a week as the composite is infiltrated with cells or new tissue in-growth. A remodeling process may continue for weeks, months, or years. For example, polyurethanes used in accordance with the present invention may be resorbed within about 4-8 weeks, 2-6 months, 6- 12 months, 12- ] 8months, or 18-24 months. A degradation rate is defined as the mass loss as a function of time, and it can be measured by immersing the sample in phosphate buffered saline or medium and measuring the sample mass as a function of time.

[00156] One skilled in the art will recognize that standard experimental techniques may be used to test these properties for a range of compositions to optimize a composite for a desired application. For example, standard mechanical testing instruments may be used to test the compressive strength and stiffness of composites. Cells may be cultured on or transplanted as part of composites for an appropriate period of time, and metabolic products and amount of proliferation (e.g. , the number of cells in comparison to the number of cells seeded) may be analyzed. Weight change of composites may be measured after incubation in saline or other fluids. Repeated analysis will demonstrate whether degradation of a composite is linear or not, and mechanical testing of incubated materials will show changes in mechanical properties as a composite degrades. Such testing may also be used to compare enzymatic and non-enzymatic degradation of a composite and to determine levels of enzymatic degradation. A composite that is degraded is transformed into living tissue upon implantation or transplantation from cell/tissue culture or bio-reactor.

Use and Application of Composite

[00157] As discussed above, polymers or polymer precursors, and components and tissue components may be supplied separately, e.g. , in a kit, and mixed immediately prior to implantation, injection or molding. A kit may contain a preset supply of tissue components and components having, e.g. , certain sizes, shapes,and physical form. Surface of tissue components and components may have been optionally modified using one or more of techniques described herein. Alternatively, a kit may provide several different types of components of varying sizes, shapes, and levels of demineralization and that may have been chemically modified in different ways. A surgeon or other health care professional may also combine components in a kit with autologous tissue and components derived during surgery or biopsy. For example, a surgeon may want to include autogenous tissue or cells, (e.g., marrow or tissue grafts) generated while preparing an implant site, into a composite). [00158] Composites of the present invention may be used in a wide variety of clinical applications. A method of preparing and using polyurethanes for orthopedic applications utilized in the present invention may include the steps of providing a curable

tissue/polyurethane composition, mixing parts of a composition, and curing a composition in a tissue site wherein a composition is sufficiently flowable to permit injection by minimally invasive techniques. In some embodiments, a flowable composition to inject may be pressed by hand or machine. In some embodiments, a moldable composition may be pre-molded and implanted into a target site. Injectable or moldable compositions utilized in the present invention may be processed (e.g., mixed, pressed, molded, etc.) by hand or machine.

[00159] Inventive composites and/or compositions may be used as injectable materials with or without exhibiting high- mechanical strength [i.e., load-bearing or non-load bearing, respectively). In some embodiments, inventive composites and/or compositions may be used as moldable materials. For example, compositions (e.g., prepolymer, monomers, reactive liquids/pastes, polymers, tissue components and components, as well as additional components, etc.) in the present invention can be pre-molded into pre-determined shapes. Upon implantation, the pre-molded composite may further cure in situ and provide tissue specific functional mechanical strength (i.e., load-bearing). A few examples of potential applications are discussed in more detail below.

[00160] In some embodiments, compositions and/or composites of the present invention may be used as a tissue void filler. Tissue defects, which result from trauma, injury, infection, malignancy or developmental malformation can be difficult to heal in certain circumstances. If a defect or gap is larger than a certain critical size, natural tissue is unable to bridge or fill the defect or gap. These are several deficiencies that may be associated with the presence of a void in a tissue. A tissue void may compromise mechanical integrity of the tissue, making the tissue potentially susceptible to dehiscence or chronic infection or inflammation until the void becomes ingrown with native tissue. Accordingly, it is of interest to fill such voids with a substance which helps voids to eventually fill with naturally or endogenously generated tissue. Open defects in practically any tissue may be filled with composites according to various embodiments. Even where a composite is not required to support full function; physiological forces will tend to encourage remodeling of a composite to a shape reminiscent of original tissues.

[00161] Many Soft tissue defects are created in surgery for trauma, oncology, and aesthetic procedures. One example is in Breast surgery for cancer whereby a "lumpectomy" is performed. The size of the defect can be quite significant and impact body symmetry— the use of the invention to fill the defect and have the tissue component encourage regeneration of adipose type tissue of equivalent differentiated and mechanical functional tissue is desired. In oncology there may be metastatic cancer deposits in bone or liver, and these lesions can be therapueticlly addressed by treatment with the composite of the invention containing a therapeutic agent that is slowly released locally. During aging there is thought to be a loss of subdermal tissue volume and the invention can be used for augmentation restoration of the facial area, (see Coleman-Fat transplantion)

[00162] Many orthopedic, periodontal, neurosurgical, oral and maxillofacial surgical procedures require drilling or cutting into tissue in order to harvest autologous implants used in procedures or to create openings for the insertion of implants. In either case voids are created in tissues. In addition to all the deficiencies associated with tissue void mentioned above, surgically created tissue voids may provide an opportunity for incubation and proliferation of any infective agents that are introduced during a surgical procedure. Another common side effect of any surgery is ecchymosis in surrounding tissues which results from bleeding of the traumatized tissues. Finally, surgical trauma to tissue and surrounding tissues is known to be a significant source of post-operative pain and inflammation. Surgical tissue voids are sometimes filled by the surgeon with autologous tissue chips that are generated during trimming of bony ends of a graft to accommodate graft placement, thus accelerating healing. However, the volume of these chips is typically not sufficient to completely fill the void. Composites and/or compositions of the present invention, for example composites comprising anti-infective and/or anti-inflammatory agents, may be used to fill surgically created tissue voids.

[00163] Inventive composites may be administered to a subject in need thereof using any technique known in the art. A subject is typically a patient with a disorder or disease related to tissue. In certain embodiments, a subject has a tissue defect such as a fracture. In some embodiment, a subject is typically a mammal although any animal with tissues may benefit from treatment with the inventive composite. In certain embodiments, a subject is a vertebrate (e.g., mammals, reptiles, fish, birds, etc.). In certain embodiments, a subject is a human. In other embodiments, the subject is a domesticated animal such as a dog, cat, horse, etc. Any tissue disease or disorder may be treated using inventive composites/compositions including genetic diseases, congenital abnormalities, fractures, iatrogenic defects, tissue cancer, tissue metastases, inflammatory diseases (e.g., rheumatoid arthritis), autoimmune diseases, metabolic diseases, and degenerative tissue disease (e.g., osteoarthritis). In certain embodiments, inventive implant composites are formulated for repair or bio-therapy of a simple fracture, compound fracture, or non-union;; for joint reconstruction, arthrodesis, arthroplasty, or cup arthroplasty of hips; for femoral or humeral head replacement; for femoral head surface replacement or total joint replacement; for repair of vertebral column, spinal fusion or internal vertebral fixation; for tumor surgery; for deficit filling; for discectomy; for laminectomy; for excision of spinal tumors; for an anterior cervical or thoracic operation; for the repairs of a spinal injury; for scoliosis, for lordosis or kyphosis treatment; for intermaxillary fixation of a fracture; for mentoplasty; for temporomandibular joint replacement; for alveolar ridge augmentation and reconstruction; as an inlay osteoimplant; for implant placement and revision; for sinus lift; for a cosmetic procedure; and, for the repair or replacement of the ethmoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus, radius, ulna, carpal tissues, metacarpal tissues, phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal tissues, or metatarsal tissues, and for repair of tissue surrounding cysts and tumors.

[00164] Composites and/or compositions of the present invention can be used as tissue void fillers either alone or in combination with one or more other conventional devices, for example, to fill the space between a device and tissue. Examples of such devices include, but are not limited to, tissue fixation plates (e.g., cranofacial, maxillofacial, orthopedic, skeletal, and the like); screws, tacks, clips, staples, nails, pins or rods, anchors (e.g., for suture, tissue, and the like), scaffolds, scents, meshes (e.g., rigid, expandable, woven, knitted, weaved, etc), sponges, implants for cell encapsulation or tissue engineering, drug delivery (e.g., carriers, tissue ingrowth induction catalysts such as tissue morphogenic proteins, growth factors (e.g., PDGF, VEGF and BMP-2), peptides, antivirals, antibiotics, etc), monofilament or multifilament structures, sheets, coatings, membranes (e.g., porous, microporous, resorbable, etc), foams (e.g., open cell or close cell), screw augmentation, cranial, reconstruction, and/or combinations thereof.

[00165] These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims.

EXAMPLES

Example 1

[00166] Polyester acrotriol synthesis and characterization. e-Caprolactone, the blowing catalysts bis (2-dimethylaminoethyl) ether (DMAEE) and bis(2-dimethylaminoethyl)ether (BDAE), the gelling catalyst triethylene diamine (TEDA), dipropylene glycol (DPG), and poly(ethylene glycol) (PEG, MW 200-Da) were all obtained from Sigma-Aldrich (St. Louis, MO). GlycoHde and D,L-lactide were purchased from Polysciences, Inc. (Warrington, PA), and a tertiary amine gelling catalyst (TEGOAMIN33) from Goldschimidt (Hopewell, VA). Lysine Triisocyanate (LTI) was obtained from Kyowa Hakko USA. Sodium hyaluronate (HA) and carboxymethy!cellulose (CMC) were purchased from Acros Organics (Morris Plains, NJ). With the exception of e-caprolactone, PEG, DMAEE, and glycerol, all materials were used as received. Prior to use, PEG and glycerol were dried at 10 mm Hg for at least 4 hours at 80°C, and e-caprolactone was dried over anhydrous magnesium sulfate. DMAEE was blended with DPG at a 70:30 mass ratio.

[00167] Polyester triols of 900-Da molecular weight, T6C3G1L900, were prepared with a trifunctional glycerol starter and 60 wt% e-caprolactone, 30% glycolide, 10% D,L-lactide, and stannous octoate catalyst (0.1%), as previously described. An alternate polyester triol, T7C2G 1L900, was similarly prepared with the trifunctional glycerol starter and 70 wt% e- caprolactone, 20% glycolide, 10% D,L-lactide, and stannous octoate catalyst (0.1%). These components were mixed with mechanical stirring in a three-neck flask for 36 hours under argon at 140°C. The product was then dried under vacuum for at least 24 hours at 80°C, followed by preparing a concentrated solution in dichloromethane and washing 3x with hexane (Storey at el, Journal of Polymer Science, Part A: Polymer Chemistry

1994;32(12):2345-2363).

[00168] The OH number was measured by titration according to ASTM D 4274-99 Method C and the molecular weight was measured by GPC (Waters Breeze) using two MesoPore 300x7.5mm columns (Polymer Laboratories, Amherst, MA) in series and a dichloromethane (DCM) mobile phase. The polyol hardener was produced by mixing the appropriate amounts of T6C3G 1L900, deionized (Dl) water, DMAEE, and TEGOAMIN33 in a Hauschild SpeedMixer™ DAC 150 FVZ-K vortex mixer (FlackTek, Inc., Landrum, SC). The %NCO of the prepolymer was measured by titration using ASTM D 2572-97, and the hydroxyl number calculated from the mass balance and measured %NCO.

[00169] The molecular weight and OH number of the polyester macrotriol are listed in Table 1. The number-average molecular weight was measured to be 1405 g/mol, compared to the theoretical value of 900 g/mol. However, GPC is a relative measure of molecular weight, and is therefore not as useful for formulating two-component polyurethanes, which requires the absolute molecular weight. The OH number is a more reliable value for formulating the PUR composition (Storey et al., Journal of Polymer Science, Part A: Polymer Chemistry 1994;32( 12):2345-2363). While the theoretical OH number was 187 mg KOH/g, the measured value was 153 mg KOH/g, and the calculated value from the prepolymer %NCO titration was 212 mg KOH/g. Considering that the theoretical value of the OH number was between the two measured values, the theoretical value was used to formulate the polyurethanes, as reported previously (Hafeman et al., Pharm Res

2008;25( 10):2387-99; Guelcher et al., Tissue Engineering 2007; 13(9):2321 -2333).

[00170] Table 1. Characterization of polyester macrotriol.

Example 2

|001711 Prepolymer synthesis and characterization. The LTI-PEG prepolymer was synthesized by adding poly(ethylene glycol) (200 g/mol, PEG200) dropwise over the course of I hour to LTI in a three-neck flask while stirring under argon. The mixture was then stirred for 24 hours at 45°C, and the subsequently dried under vacuum for at least 24 hours at 80°C. The NCO-.OH equivalent ratio of the prepolymer was 3.0: 1.0. The %NCO was measured by titration according to ASTM D 2572-97, the molecular weight distribution was measured by GPC as described previously, and the viscosity was determined using a Brookfield viscometer. The prepolymer was stored under argon at 4°C.

[00172] The %NCO of the prepolymer was measured to be 22.8%, which is in good agreement with the theoretical value of 23%. The viscosity was measured to be 21 ,000 cP using a Brookfield viscometer. As shown in Table 2, the molecular weight of the prepolymer is broadly distributed, ranging from monomeric LTI to the LTI-PEG-LT1-PEG-LTI-PEG- LTI-PEG-LT1 adduct comprising 4 molecules of LTI and 3 molecules of PEG. This observation is consistent with previously reported data for polyurethane prepolymers, which are typically characterized by a broad molecular weight distribution (Oertel G., Polyurethane Handbook. Berlin: Hanser Gardner Publications; 1994).

[00173] Table 2. Molecular weight distribution of LTI-PEG prepolymer. The "theoretical" value is calculated from the actual molecular weights of LTI and PEG200, and the

"calculated" value is calculated from the measured M„ of LTI and PEG and the structure of the component.

Example 3

|00174] Synthesis and characterization of the injectable PVR scaffold. To synthesize the PUR scaffolds, the soft segment hardener and LTI-PEG prepolymer were combined in a one- shot manner with 0 to 35-wt% CMC or HA. These were mixed either in syringes linked by a luer-lok connector, or with a spatula in a mixing cup, and injected into the particular mold or wound site. In the case of syringe-mixing, the polyol-based hardener and isocyanate-based resin were each loaded into separate syringes, which were then linked by a luer-lok connector. The contents were then passed back and forth between syringes to mix for 2 minutes. The experimental batch sizes could be mixed in 1 , 3, and 10-mL luer-lok syringes. Alternately, the resin and hardener could be hand-mixed with the HA or CMC using a spatula, especially for cases with high polysaccharide loading (> 20 wt-%). The resulting mixture could then either be spread directly onto the wound site, or transferred. into a syringe and injected into the wound site. Figure 1 illustrates the latter scenario, with the mixed reaction contents transferred to a 1-mL syringe and injected onto the benchtop.

[00175] The relative amounts of the prepolymer and hardener components were calculated assuming an index of 1 15 (the index is defined as 100 x (no. of NCO equivalents/no. of OH equivalents)) (Guelcher et al, Tissue Eng 2006; 12(5): 1247- 1259). The OH titration, NCO titration, and GPC measurement yielded different values of the OH number that bracketed the theoretical OH number; therefore, the theoretical OH number was used to formulate the composites. This approach has been reported to yield PUR scaffolds with minimal sol fraction when indexed at 1 15 (Guelcher et al, Tissue Eng 2006;12(5): 1247-1259).

100176] SEM images of the HA and CMC granules before incorporation into the PUR scaffolds are shown in Figure 2. These components vary heterogeneously in size and shape, with HA ranging from 200-800 mm in diameter, and CMC ranging from 30 to 200 mm in length or diameter.

[00177] Core densities were determined from mass and volume measurements of triplicate cylindrical foam cores, of 7 mm diameter x 10 mm height samples, at least 24 hours after foam synthesis to ensure full curing and drying. The core porosities (ec), defined as the volume fraction of pores, were calculated from the density values (pc), where pp = 1200 kg/m 3 is the polyurethane specific gravity and p A = 1.29 kg/m 3 is the specific gravity of air (Guelcher et al, Tissue Engineering 2006; 12(5): 1247- 1259), which was measured gravimetrically:

These core density and porosity values are given in Table 3. Scanning electron microscope (SEM) micrographs, used to determine pore size, were obtained using a Hitachi S-4200 (Finchampstead, UK).

[00178] Table 3 provides the PUR scaffold physical properties. The density and porosity of the injectable composites was adjusted primarily by varying the concentrations of the polysaccharide content.

6C3G 1 L (15) 161 ± 8 87 ± 0.6

7C2G1L (15) 139 ± 14 89 ± 1

6C3G 1L (30) 269 ± 8 78 ± 0.6

7C2G1L (30) 271 ± 20 79 ± 0.1

[00179] An important aspect of the two-component hardener process relates to the storage stability of the hardener component. When the hardener component comprising polyol, water, and catalyst was stored for >3 days at 37°C and subsequently used to prepare composite foams, the resulting materials exhibited dramatic (e.g., >10 - 2%) changes in porosity. In order to prepare an injectable polyurethane with acceptable storage stability, the two (liquid) component process was modified to an alternative three (liquid)-component process wherein the TEDA catalyst (0.8 pphp) and water were dissolved in a dipropylene glycol (DPG) solution. Another advantage of the three-component process is that the volume of DPG can be increased to yield a sufficiently large solution volume that can be reliably filled in a syringe (e.g., -200 ml for a clinically relevant batch size of 5g). Allograft/PUR composite foams were synthesized by first mixing the polyol and DPG+catalyst+water solution for 60s, followed by addition of allograft components, and finally addition of the LTI-PEG prepolymer. The resulting reactive paste was mixed for 30s, charged to a 3-ml syringe, and injected into a 3-ml polypropylene mold. There were no significant differences in the porosity of the composite foams between the two- and three-component processes.

[00180] The working and cure times were adjusted by varying the concentration of catalysts using the two-component process. At elapsed times shorter than the working time, the mixed components of the scaffold can be injected from the syringe and manipulated without disrupting the pore structure. The tack-free time is the amount of time required for the scaffold to sufficiently cure such that the surface can be touched with a probe that is , subsequently removed without adhering to the surface (analogous to the setting time of a calcium phosphate tissue cement). The tack-free times for the PUR scaffolds ranged from 10 minutes with catalyst concentrations of 1.125 pphp Tegoamin 33 and 0.75 pphp

BDAEE/DPG, to 30 minutes with catalyst concentrations of 0.625 pphp Tegoamin 33 and 0.375 pphp BDAEE/DPG. The working time varied correspondingly from 2 to 7 minutes. The tack-free time was influenced somewhat by the amount of moisture present in the wound bed. [00181 ) The internal temperature within the polyurethane mixture was recorded with a digital thermocouple at the center of the rising foams in triplicate during the exothermic foaming reaction. Batch sizes (3-g) were large enough to diminish the effects of heat loss from the exterior surfaces of the foam. Starting at room temperature (21.2 °C) when the reactants were first mixed, the maximum increase in temperature was 10.2 °C, as shown in Figure 3.

[00182] SEM images of the PUR scaffolds are shown in Figure 4 for composites with 0 and 35 wt-% HA. HA components are dispersed throughout the scaffold (as indicated by the arrows), and are generally separated from one another by a polymer film. The pores appear to be interconnected and 100-600 mm in diameter, and the pore structure seems to become slightly more irregular when HA is incorporated.

Example 4

[00183] Mechanical Testing. Dynamic mechanical properties were measured using a TA Instruments Q800 Dynamic Mechanical Analyzer (DMA) in compression and tension modes (New Castle, DE). Cylindrical 7 x 6 mm samples were compressed along the axis of foam rise. The temperature-dependent storage modulus and glass transition temperature (T g ) of each material was evaluated with a temperature sweep of -80 °C to 100 °C, at a compression frequency of 1 Hz, 20-μηι amplitude, 0.3-% strain, and 0.2-N static force. The T g values are given in Table 4 below.

[00184] Compressive stress-strain curves were generated by controlled-force compression of the cylindrical foam cores at 37 °C. With an initial force of 0.1 N, each sample was deformed at 0.1 N/min until it reached 50% strain (i.e., 50% of its initial height). The Young's (elastic) modulus was determined from the slope of the initial linear region of each stress-strain curve [ASTM-International. D695-02a. Standard test method for compressive properties of rigid plastics. 2007]. Due to their highly elastic properties, the scaffolds could not be compressed to failure. Therefore, as a measure of compressive strength, the compressive stress was evaluated as the stress at 50% strain in the stress-strain curves.

Calculated from the measured force and cross-sectional sample area, the compressive stress indicates material compliance such that more compliant materials require lower stress to induce a particular strain. The compressive moduli were determined as the relaxation modulus values after one minute at 50% strain was measured using the DMA stress relaxation mode at 37 °C. (00185] Table 4 provides the glass transition temperature, compressive Young's modulus, compressive stress, and compressive modulus values of triplicate PUR samples at 0, 15, and 30 wt-% HA (n = 3).

[00186] The Young's modulus values ranged from 69.8 kPa for scaffolds with no filler, to 140.9 kPa for those with 30 wt-% CMC. The differences were statistically significant (p < 0.05) only for the 30 wt-% scaffolds when compared to the 0 wt-%. The compressive stress, or stress measured at 50% strain, ranged from 16.9 to 95.3 kPa, with statistically significant differences between each of the 15 wt-% and 30 wt-% materials with respect to the 0 wt-% material. Representative stress-strain curves show how the fillers contribute greater strength and stiffness to the composite materials (Figure 5).

[00187] Tensile testing was performed on thin, rectangular scaffold samples (10 mm long x 5 mm wide x 1.7 mm thick). Stress-strain curves were generated by elongating the samples at 1 % strain per minute at 37 °C until failure, and show the long elongations at sample break. The strain at failure (%) is the strain value at which the sample fails, or tore apart. The Young's modulus was again calculated from the slope of the initial linear region of each stress-strain curve, and the tensile strength was determined as the stress (kPa) at failure. (00188| The representative tensile stress-strain curves are shown in Figure 6 for T6C3G 1 L scaffolds with 0, 15, and 30 wt-% CMC, and the extrapolated tensile mechanical properties are listed in Table 5. The Young's modulus values ranged from 60 to 280 kPa. The Young's modulus increased upon the incorporation of HA or CMC. The elongation (% strain) values at break were also less for these LTl-PEG materials, at 45 to 145 %, than for the LTI and HDIt scaffolds. The added fillers proportionately and significantly (p < 0.05) lowered the ultimate strain values, essentially acting as points of stress cracking in a nonhomogeneous composite material. The tensile strength, or tensile stress at sample failure, ranged from 95 to 200 kPa, and the wt-% filler did not seem to have a significant effect. |00189j Table 5. PUR scaffold mechanical properties as measured by DMA in tension mode (n=3).

Example 6

[00190] In Vitro Degradation. Samples (6mm diameter χ 5mm long) were individually placed in 2-mL vials, immersed in PBS, and stored at 37°C under mechanical agitation. At each time point samples were immersed in DI water for at least 1 hour for a total of 2 water changes at room temperature to remove any residual buffer or salt. The samples were then lyophilized at -50°C and 0.1 mbar for 24 hours, and weighed to determine mass lost. Data are presented as mean ± standard deviation of quadruplicate samples.

[00191] In vitro degradation data are presented in Figure 7. The degradation rates of the T6C3G 1 L/LTI-PEG and T7C2G 1 L/LTI-PEG materials in buffer at 37 °C, with 0, 15, and 35 wt-% CMC, were recorded for up to 24 weeks (Figure 7). After 24 weeks, the mass remaining ranged from 46.5% (30 wt-% CMC) to 92.4% (0 wt-% CMC). The primary mechanism of degradation is hydrolysis of the ester bonds within the polyester soft segment. The fillers caused the materials to initially lose more mass within the first few days, which presumably corresponds to the CMC dissolution, after which the rates of polymer degradation are parallel.

Example 7

[00192] In Vivo Study. The capacity of the scaffolds to facilitate dermal wound healing was evaluated in an excisional wound model (10-mm diameter) in adult male Sprague- Dawley rats. The materials were applied as a reactive liquid and expanded by gas foaming to fill the defects and cure in situ. The scaffolds were trimmed to be flush with the skin surface when they expanded beyond the wound outline. All materials were sterilized by gamma irradiation at 5 kGy prior to surgery. The wounds were harvested at 4, 14, 21 , and 28 days, and histological sections were stained with Gomori's trichrome or hematoxylin and eosin. The scaffolds were assessed for biocompatibility, biodegradation, cellular infiltration, and tissue regeneration. Other variables investigated included the performance of injectable scaffolds in comparison to the implants, and the possible benefit of HA over CMC.

[00193] The purpose of these experiments was to verify that the polyurethane reaction in situ does not elicit a substantial inflammatory response, and that the cellular infiltration and material biodegradation is similar to that of the corresponding implants. Trichrome histology indicated mononuclear cell infiltration and early granulation tissue by day 4 (Figure 8).

Collagen deposition and new tissue organization proceeded at 14 days and material remnants were transiently engulfed by macrophages with extensive angiogenesis. Mature granulation tissue and almost complete repithelialization were present by day 21 , followed by evidence of folliculogenesis in the neoepidermis by day 28. Inclusion of the solid hygroscopic fillers HA and CMC in the scaffolds improved adhesion between the material and wound bed by absorbing excess moisture, and its presence may have augmented the local healing response. Qualitatively, hyaluronic acid may have promoted more accelerated angiogenesis and material degradation than CMC.

[001941 Several healed wounds demonstrated evidence of appendage formation, specifically possible hair follicles and sebaceous glands, from the epidermis. Viewing dermal histological sections through a polarizing lens helps to distinguish mature collagen from newly deposited collagen. Specifically mature collagen fluoresces brightly, as in the unwounded tissue, while new collagen does not fluoresce. These possible appendages are positioned within areas of new collagen, suggesting that they have developed de novo instead of being a product of epidermal contraction from the wound boundaries.

100195] A second, larger dermal wound model was also evaluated, as it might be more realistic of a human dermal wound, with a larger surface available for tissue-material adherence. The injectable materials were applied as above to an unstented, 1 in 2 square wound on the dorsum of each rat. Each wound and scaffold was covered with a non-stick absorbent Release gauze (Johnson & Johnson) and Tegaderm. The wounds were harvested at four time points evenly spaced between 7 and 35 days (days 7, 17, 26, and 35). The wounds were processed and evaluated as described above.

[00196] The large excisional dermal wounds ( 1 x 1 in) were intended to be a more accurate model of a typical human wound. They demonstrated that the PUR scaffolds adhere sufficiently to the underlying wound bed, provided that the scaffolds are covered with Tegaderm dressing. Due to their size, these wounds healed at a slightly slower rate than the smaller 10-mm excisional wounds. Figure 9 shows representative trichrome-stained histological sections of PUR scaffolds with 25 wt-% CMC. At day 7, much of the scaffold remained, with cellular infiltration visible at the bottom and sides of the section. By 17 days, the material had degraded significantly and the wound bed was filled with granulation tissue and some collagen deposition. Extensive angiogenesis was apparent with small blood vessels permeating of the wound bed. An eschar covered most of the wound surface, as the epithelium began to migrate from the wound edges toward the center. At 26 days, PUR degradation had proceeded, with mature granulation tissue and increased collagen throughout the wound bed. The epithelium had nearly covered the wound, although it appeared to still be in its hypertrophic phase characteristic of initial healing. No scaffold remnants were visible at day 35, and the reepitheltali ' zation was complete. These results confirmed that the PUR scaffolds could be successful in various wound sizes, although the healing time corresponded to the wound size.

[00197] All references, such as patents, patent applications, and publications, referred to above are incorporated by reference in their entirety.