WO1999011196A1 - Injectable tissue reconstruction material - Google Patents

Abstract

An injectable and flowable material for tissue repair, reconstruction and bulking is provided, comprising microspheres having an average diameter in the range of about 10 to 100 microns. The close packing of the microspheres at the site of application and the biodegradation of the materials allow for residency of the particles at the target site to permit tissue repair and in-growth into the implant. Active components, such as drugs or growth factors may be incorporated into the material to elicit a specific tissue response or to provide local delivery of a therapeutic agent.

Description

INJECTABLE TISSUE RECONSTRUCTION MATERIAL

This is a continuation-in-part of Serial No. 08/923,623, filed September 4, 1997, which is incorporated by reference in its entirety.

The invention is directed to compositions which are biodegradable and in ectable for implantation into tissues to facilitate repair, reconstruction and bulking of damaged or deficient tissue areas in the body.

Background of the Invention There are several situations which require the efficient repair, reconstruction and augmentation of biological tissues. For example, during surgery, various materials such as sutures, staples, tissue adhesives, natural and synthetic polymers and autologous tissues have been used to provide reconstructive scaffolds for the repair of damaged tissues or to bulk tissues into the appropriate anatomically correct configuration. In these applications the use of degradable materials allows the reconstruction to be performed without a follow-up surgical procedure to remove non-degraded materials which may provoke long term tissue irritation or infection. However, when utilizing the techniques in the field of minimally invasive surgery, methods to augment and repair tissues through small bore cannulae are required in order to permit surgical repair with lower operational time and cost.

There are certain diseases and conditions which may be treated or remedied by tissue reconstruction. In one example, tissue segments of the myocardium (heart muscle) are damaged and possibly rendered completely non-viable as a result of myocardial infarction. Utilizing a degradable reconstruction material tailored to provide a scaffold for tissue repair as well a reservoir for substances to stimulate various healing responses, these damaged areas may be re-vitalized to provide some utility xn maxntaxnxng coronary output. There are no materials other than difficult to obtain tissue grafts to repair non-viable cardiac tissue.

Another example pertains to urinary incontinence due to intrinsic sphincter deficiency (ISD) where the urethral sphincter is partially or totally incompetent, causing leakage of urine from the bladder. There are several modalities used to treat ISD, including external devices, such as pads and diapers, and more complex internal devices, including surgical intervention to correct the anatomic defects. Sphincter augmentation involves the injection of a substance into the tissues surrounding the urethra in order to increase the periurethral tissue mass, bringing the tissues into apposition and therefore increasing resistance to urine flow. Such substances which have been tested for this procedure include autologous fat, silicon elastomer particles, Teflon particles and dispersions of bovine derived collagen. The biodegradable materials which have been used, such as collagen, requxre re-xnj ectxon perxodically because of their shorter residence time as compared to non-biodegradable materials, such as silicon and Teflon.

Other utilizations for tissue reconstruction materials include the repair of depressed scars or wrinkles on the skin, repair of bone fractures or defects, repair of damaged cartilage tissues and the embolization of vascular deformities.

Summary of the Invention An xnjectable and flowable material for tissue reconstruction is provided comprising microparticles, preferably microspheres formed of a biomaterial which sets at the site of application into a cohesive mass containing an interconnected porous network. The biomaterial is biodegradable, can be delivered at a high solids content and is cross-linked to a sufficient extent such that the in vivo residency of the particles at the site of application is sufficient to stimulate tissue repair and in-growth. The extent of cross-linking may be adjusted to tailor the material for specific applications. The microparticles may optionally contain active compounds or drugs to stimulate tissue repair such as the incorporation of growth or angiogenic factors, or the incorporation of anti-infective or anti-inflammatory agents to limit adverse tissue response. A method of inducing tissue repair in a tissue defect or wound site is provided comprising the step of applying an effective amount of the described injectable compositions to a site of desired tissue reconstruction . In particular, a method is provided to induce tissue repair and in-growth to treat damaged myocardial tissue, structurally defective sphincters and vocal cords, depressed scars or skin wrinkles, damaged bone or cartilage and vascular deformities.

Detailed Description of the Invention

The invention provides an injectable composition and methods of its use for repairing damaged or incompetent tissues by administering via injection an effective amount of biomaterial at the site of the defect or structural deformity.

The terms "damaged" and "incompetent" are used to describe tissues damaged due to vascular ischemia; muscles, tendons, or cartilage that are inadequately functioning or non-functioning due to deterioration; or a structural deformity caused by an anatomic disruption of a muscle or mechanism.

The term "effective amount" means the quantity of biomaterial needed to repair tissue or to achieve improved continence, or the quantity of wound healing agents needed to achieve improved healing. The effective amount of biomaterial administered may vary depending upon the patient ' s ability to absorb or breakdown the biomaterial, the consistency and concentration of the material, and the site and condition being treated. The biomaterial may be administered over a number of treatment sessions to achieve and maintain the desired results .

The biomaterial used in the present invention is in a form which is injectable, biocompatible, non- immunogenic and in a physical and chemical state that allows it to persist at the site of placement for at least 7 days. These include, but are not limited to, biodegradable materials such as collagen, gelatin, elastin, fibrin, fibrinogen, glycosoaminoglycans, polyhydroxybutyrate, polylactic acid, polyglycolic acid, polyesters and combinations of these materials.

Highly purified, high molecular weight gelatin or collagen is particularly useful. Commercially available laboratory grades may be used, or gelatin derived from collagen obtained by known purification processes of collagenous materials available from sources such as bovine or porcine corium, bone or tendon. It is preferred that the gelatin material be of high molecular weight of at least 200 to 300 bloom and substantially free of extraneous proteins, proteoglycans, lipids or other processing residuals which may adversely effect biological response.

The biomaterial will be in the form of solid or hollow microparticles which can be delivered to the target site as a high solids content slurry without the inherent high viscosity found in dispersions and emulsions of other particulates . Typically, the microparticles will be in the form of microspheres. The microspheres are stabilized by cross-linking, so that they are insoluble in an aqueous media. Typically, the diameters of the microspheres will be in the range of about 10 to 100 microns, and can be conveniently injected through a cannula having an inner diameter of lOx the average microsphere diameter.

The microspheres may be produced by spray drying, spray coagulation or emulsion methods. Microspheres produced by spray drying are usually hollow whereas those produced by spray coagulation are usually solid. While the usual particle diameter will be in the range of about 10 to 100 microns, particles in the range of 10 to 55 microns are also useful, particularly for treating tissues through small bore needles.

For a gelatin raw material, the biomaterial will typically be solubilized in an aqueous solution, either water or a buffered aqueous solution, ranging from about pH 2 to pH 10 depending on the source of the gelatin and isoelectric point. The gelatin concentration in the solution is typically between about 2 and 20 percent weight/volume.

The microspheres are cross-linked to increase their stability and resistance to in-vivo degradation. Various cross-linking procedures may be utilized, either singly or in combination, including but not limited to, cross- linking using carbodiimides, aldehydes, dehydrothermal

(DHT) or other methods known to those skilled in the art. The extent of cross-linking can be adjusted to achieve the desired in-vivo degradation rate of the material, with higher cross-linked materials lasting for a longer time period than lower cross-linked materials. A preferred method for cross-linkxng to achxeve good txssue compatxbxlxty and medxum term resorptxon rates xs to cross-lxnk wxth carbodxxmxdes . A co-solvent system usxng 2-propanol (IPA) and dxlute hydrochlorxc acxd (HCL) , xn a concentratxon rangxng from 1 to 100 rtiM, xn a solutxon ratxo rangxng from 99:1% to 70:30% (IPA/HCL) xs used. The cross-lxnkxng agent xs then added, preferably 1- (3-dxmethylamxnopropyl) -3-ethylcarbodxxmxde hydrochlorxde (EDC) , xn a concentratxon of typxcally 0.5 to 5 mg/ml. The cross-lxnkxng reactxon may be run for approxxmately 4 to 96 hours. Subsequent to cross-lxnkxng the mxcrospheres are washed wxth dxlute HCL, xn a concentratxon range from 10 - 100 mM, and IPA. The mxcrospheres are then collected and drxed. Another method for cross-lxnkxng to achxeve hxgh enzymatxc resxstance and low fluxd uptake (swell) xnvolves the use of two dxfferent cross-lxnkxng chemxstrxes xn successxon. The fxrst cross-lxnkxng xs performed xn a co-solvent system usxng IPA and dexonxzed water (DI) xn a solutxon ratxo rangxng from 99:1% to 70:30% (IPA/DI) . The dexonxzed water xs acxdxfxed, typxcally wxth HCL, to an unbuffered pH xn a range of about 4.0 to 6.5. The cross-lxnkxng agent xs then added, preferably EDC, xn a concentratxon of typxcally 0.5 to 5 mg/ml. Cross-lxnkxng may be run for approxxmately 4 to 48 hours.

The second cross-lxnkxng step xs preferably accomplxshed xn a co-solvent system usxng (IPA) and dxlute phosphate solutxon (PHOS) at a concentratxon rangxng from 0.05 to 0.5 M, at a pH rangxng from 5.0 to 8.0, xn a solutxon ratxo rangxng from 99:1% to 5:95% (IPA/PHOS) . Glutaraldehyde (GTA) xs added as the cross- lxnkxng agent xn the concentratxon rangxng from about 0.001 to 1.0 percent. Thxs reactxon xs run for a perxod of txme from about 4 to 48 hours. Subsequent to cross-linking the microspheres are washed with de-ionized water and isopropyl alcohol, then collected and dried.

Residuals of cross-linking agents and break-down products from such agents must be sufficiently minimal so as not to adversely affect tissue response. Sterilization may be accomplished using standard practice such as electron beam irradiation, gamma irradiation or ethylene oxide gas exposure. The cross-linked microspheres are typically formulated into a flowable slurry with a biologically acceptable vehicle, such as saline or phosphate buffer. The concentration (solids-content) range may be determined so that the flow rate is suitable for the application, which is typically in the range of about 2 to 70% weight/volume of solids. Typically, the solids content will be at least 10%, which may be delivered through cannula as small as about 30 gauge.

Additives may be incorporated into the fluid vehicle to promote flow properties of the slurry, such as, but not limited to, uncrosslinked gelatin, collagen, hyaluronic acid, polyethylene glycol, surfactants or other flow promoting agents. The fluid vehicle may also be formulated with physiologically acceptable ionic compounds to adjust for toxicologically acceptable ionic strength and pH.

While not intending to be bound by any theory, upon injection of the microsphere slurry into tissues, the microspheres are believed to aggregate into a mass having an interconnected porosity created by the close-packing of the essentially spherical particles, providing areas for cellular in-growth and the resulting tissue integration via cellular proliferation between the particles. Alternatively, some of the particles may be cross-linked to a lesser extent than the others and therefore be susceptible to a faster degradation to allow for the creation of a network of passages within the implant to further encourage new tissue in-growth and tissue formation. Other variations of preparation of the microspheres may be accomplished. For example, the cross-linked microspheres may be resuspended in gelatin and resprayed or recoagulated to create a larger set of particles containing a cross-linked core and an uncross-linked or lightly cross-linked outer shell. This multi-layered approach may be used for products such as tissue adhesives, where a soft and sticky outer shell is provided which allows cohesion of the particles to each other and adhesion to the tissues to which they have been applied. The multi-layered particle may also incorporate either as the core or the shell, other materials, such as polyhydroxybutyrate, polylactic acid, polyglycolic acid, polyesters, elastins, fibrin, fibrinogen or collagen. The particles are prepared by dxssolving the biomaterial raw material into solution, suitably buffered, if required, depending upon the nature of the biomaterial. In the case of gelatin, typically the solution will be 100% water or a solution buffered within the range of about pH 2 to 10. A useful concentration for forming microspheres by spraying methods is a solution having a concentration of the biopolymer in the range of about 2 - 20% weight/volume. Thxs solution may be mixed with active ingredients such as growth factors or angiogenesis factors. Fine sprayed droplets of the solution are thereafter formed to produce solid microspheres havxng average diameters in the range of about 10 to 100 microns. Suitable techniques for forming droplets include spray drying, spray coagulation, emulsification, extrusion, electrostatic droplet formation and other known droplet forming methodology.

Typically the dispersion droplets may be sprayed through a micro-droplet forming apparatus into a non- solvent of the biomaterial that is immiscible with water, such as isopropanol, hexene or chloroform, at a temperature above the freezing point of the non-solvent in order to physically stabilize the droplets. Alternatively, the dispersion droplets may be sprayed into liquefied gas, such as liquid nitrogen, to stabilize the droplets.

Once the microspheres have been formed, cross- linking is effected to increase stability and resistance to degradation when implanted into a body. The microspheres must be of high purity materials, free of potentially toxic additives which may impair tissue growth or preclude complete resorption upon implantation. Additives such as protease inhibitors may be formulated into the microspheres either prior to or after microsphere formation to extend the residence time in- vivo .

After cross-linking the microspheres are washed to remove unbound cross-linking agent, collected and dried. Washing is conducted typically in mild acidic solutions, deionized water and/or isopropanol.

Finally, the microspheres may be sterilized using standard practices such as electron beam irradiation, gamma irradiation or ethylene oxide gas exposure. Alternatively, the raw material components may have been sterilized and the microspheres fabricated aseptically with sterile equipment.

The cross-linked microspheres are formulated into a fully flowable slurry with a biologically acceptable vehicle such as saline or phosphate buffer. The injectable formulations may contain macromolecular materials for promoting tissue repair, ingrowth or angiogenesis . Materials may be chosen from, but not limited to, hyaluronic acid, FGF (fibroblast growth factor) , TGF-beta or PDGF (platelet derived growth factor), angiogenic growth factors, vascular endothelial growth factor. Drug components loaded into the microspheres provide for a sustained delivery and biological effect, enhancing drug performance and tissue reconstruction. The formulations may also contain therapeutic agents for local delivery around the area of application. Drug components such as growth factors, protease inhibitors, anti-infective agents, anti- inflammatory agents, anti-proliferative agents, anti- tumor agents and the like may be incorporated in the materials used to fabricate the microspheres or preferably incorporated into the microspheres after fabrication and stabilization by crosslinking . The drug components may be placed in a solvent system to allow diffusion into the microspheres, utilizing ionic strength and pH conditions to control drug loading. Alternatively, the drug components may be incorporated into the fluid vehicle and allowed to diffuse into the microspheres prior to use. The materials according to the invention are particularly applicable for repairing damaged cardiac tissue, augmenting defective urinary sphincters or vocal cords; for filling scars, skin wrinkles or puncture wounds, delivering therapeutic compounds to tumors, delivering compounds to vascular tissues to prevent restenosis and repairing damaged cartilage or ossiferous tissue. In one example, the material may be formulated with a suitable growth factor chosen for angiogenic potential and then injected into a region of non- functional myocardial tissue to promote repair of the site after an ischemic episode. In another example, the material without any additives may be injected into the periurethral tissues under endoscopic visualization to repair incompetent urethral sphincter functionality. Multiple injections may be performed in order to bring the urethral tissues into apposition. For the repair of depressed scars or wrinkles, the microsphere formulation is injected utilizing known methods for treating cutaneous depressions as with such materials as a collagen-based commercial product. The material will provide similar tissue bulking properties.

Example 1 A solution of high purity gelatxn of 300 bloom was made up at 10% weight/volume solids. The dry gelatin was added to 100% de-ionxzed water and placed xn an oven at 60° C. for 1 hour, mixing occasxonally . A spraying apparatus consisting of two co-axial stainless steel tubes was set-up. The inner tube was connected to a temperature controlled reservoxr containing the gelatin solution and means to dispense the solution at a constant rate. The outer tube was connected to a pressurized gas tank, preferably nitrogen, to supply the carrier gas for sprayxng. The carrxer gas was dispensed through a heating element attached to a temperature controller to provide constant temperature gas. The spray head was positioned above a tank containing 100% isopropyl alcohol (IPA) .

The gas supply was actxvated and set to deliver a constant flow rate at a constant temperature and pressure. The pumping means was actxvated to deliver the temperature controlled solution into the spray head. The spray head produced small droplets of the gelatin solution, which coalesced into spherxcal droplets during thexr fall to the IPA bath. Upon contact with the IPA, the water component of the droplets was removed vxa solvent exchange and the droplets became stabxle mxcrospheres. The mxcrospheres were collected by passxng through precxsxon sxeves to xsolate the sxze fractxon(s) desxred for the product. The collected mxcrospheres were then placed xn a low humxdxty dryxng chamber untxl all of the IPA was evaporated.

A cross-lxnkxng solutxon was prepared usxng IPA and 1 mM HCL xn a ratxo of 92/8% (IPA/HCL) . EDC was added at a concentratxon of 50 mg per ml of cross-lxnk solutxon.

The solutxon was mxxed untxl the EDC was fully dxssolved. The solutxon was added to a contaxner wxth the dry mxcrospheres xn a concentratxon of 50 ml solutxon per gram of mxcrospheres. The contaxner was mxxed by rotatxng for a perxod of 48 hours.

Subsequent to the cross-lxnkxng step, the solutxon contaxnxng cross-lxnked mxcrospheres was fxltered through an 8 mxcron fxlter to collect the mxcrospheres. The mxcrospheres were then washed to remove resxdual cross- lxnkxng agent. The mxcrospheres were placed xnto a solutxon of 50 mM HCL and mxxed for a perxod of 24 hours. The mxcrospheres were fxlter collected and then placed xnto a solutxon of 100% IPA for a perxod of 4 hours. These washxng steps were repeated one more txme and then the collected mxcrospheres were drxed xn a low humxdxty chamber.

The mxcrospheres were packaged by wexght xnto glass or piastre vxals and sterxlxzed usxng 2.5 MRad equxvalent electron beam irradiation. Example 2 Mxcrospheres were fabrxcated usxng a spray system xnto an IPA bath as detaxled xn Example 1 above. The mxcrospheres were cross-lxnked usxng a combxnatxon of two cross-lxnk chemxstrxes xn successxon. The fxrst cross- lxnkxng step was accomplxshed accordxng to Example 1 above. After the mxcrospheres were collected, they were drxed xn a low humxdxty chamber xn preparatxon for the second cross-lxnkxng step. A solutxon of IPA and 50 mM phosphate solutxon (sodxum phosphate monobasxc plus sodxum phosphate dxbasxc) at a pH of 7.0, xn a solutxon ratxo of 92/8% (IPA/Phos) was made up. Glutaraldehyde was added at a concentratxon of 0.1%. The mxcrospheres were placed xnto a contaxner and the cross-lxnk solutxon was added xn a concentratxon of 100 ml solutxon per gram of mxcrospheres. The contaxner was mxxed by rotatxng for a perxod of 48 hours. Subsequent to the second cross- lxnkxng step, the mxcrospneres were washed and sterxlxzed accordxng to Example 1 above.

Example 3 A fxbrxnogen based mxcrosphere txssue adhesxve was fabrxcated as follows. A solutxon of fxbrxnogen (Fractxonal type 1-S from bovxne plasma, Sxgma Chemxcals) was made up xn 100% DI at a concentratxon of 10% wexght/volume . The fxbrxnogen was solubxlxzed at a temperature of 37° C. for 25-35 mxnutes. The resultxng solutxon was then spray coagulated as detaxled xn Example 1 above. The collected fxbrxnogen mxcrospheres were then drxed xn a low humxdxty cnamber untxl all the IPA was evaporated.

A solutxon was made up of bovxne thrombxn (Sxgma Chemxcals) and 40 mM calcxum chlorxde (CaCl,) at pH 7.0. 1,000 unxts of thrombxn were added to 2 ml of the CaCl solutxon. The thrombxn solutxon was added to a sample of the fxbrxnogen mxcrospheres. The fxbrxnogen/thrombxn reactxon created a fxrm solxd mass from the slurry wxthxn 1 mxnute .Mxcroscopxc examxnatxon xndxcated a fully fused "clot" mass resultxng from the mxxture.

Example 4 Sterxle mxcrospheres accordxng to Example 1 above were dxspersed xn phosphate buffered salxne (PBS) at a solxds content of 15% wexght/volume . The dxspersxon was loaded xnto lcc sterxle syrxnges (Becton-Dxckxnson Corp. (BD) ) . 26 gage xntra-dermal needles (BD) were attached to the syrxnges. New Zealand Whxte rabbxts, 3-4 months old, wexghxng 2-4 kxlograms were prepared by anesthetxzxng wxth Ketamxne and Xylazxne, then shavxng thexr dorsal spxne from the plane of the shoulder blades dxstally for approxxmately 8 cm. The mxcrosphere dxspersxons were injected in 6 locations, 3 each on either side of the spine, approximately 1-2 cm laterally from the spine. Injection of control materials comprising two commercially available tissue bulking agents (Contigen, CR Bard Inc and Fibrel, Mentor Corp.) were made xn 2 locatxons totalxng 8 injection sites. Injections of 0.25 cc of material were made at each site. The materials were injected subcutaneously m each location. A total of 8 animals were studied for time points of 7, 14, 28 and 56 days. The animals are euthanized according to protocol and the implant sites excised. At necropsy all mxcrosphere xmplant sxtes showed remarkable coherence of xmplant materxal, wxth no xndxcatxons of mxgratxon of the partxcles. Implant sxtes were xntact at 7 and 14 days, wxth some sxtes showxng resorptxon begxnnxng between 14 and 28 days. By 56 days all of the sxtes showed some degree of degradatxon xncludxng complete resorptxon xn some cases. The sxtes were carefully excxsed and the explants placed xn buffered formalxn for fxxatxon.

After a mxnxmum of 14 days fxxatxon, the explants were sectxoned and staxned for hxstologxcal examxnatxon. Examxnatxon of the xmplant sxtes from 7 days xmplantatxon xndxcated the begxnnxngs of cellular xnfxltratxon xnto the mxcrosphere matrxx. Varyxng degrees of xnflammatory response as xndxcated by xnflammatory cell types could be seen xn each sxte. By the 14 day xmplantatxon, the sxtes showed remarkable cellular xngrowth xnto the mxcrosphere matrxx. Cellular xnfxltratxon between the mxcrospheres was seen throughout the margxns of the xmplants and appeared to be contxnuxng at a rapxd rate. By the 28 day xmplantatxon, the begxnnxngs of mxcrovasculature could be noted by the presence of voxds xn the matrxx filled with red blood cells. These indications continued to the 56 day implant .

Example 5 Sterile microspheres fabricated according to Example 2 above were dispersed into syringes according to Example 4 above. The dispersion concentration was made up at 20% weight/volume solids. New Zealand White rabbits, 3-4 months old, weighing 2-4 kilograms were prepared by anesthetizing with Ketamme and Xylazme, then shaving their abdominal region from the central abdomen to the genitalia. An incision approximately 2 cm long was made beginning about 0.5 cm from the genitalia and proceeding caudally. The incision was made through rhe cutaneous layers. The skin was retracted and the bladder was exposed and pulled from the abdominal cavity.

Injections of the microsphere dispersions were made into the wall of the bladder using the 26 gage mtra- dermal needles (BD) . Injections were maαe into 2 sites in the bladder wall in 4 animals for time Domts of 7 ana 14 days. Injections of 0.1 cc were made at each site. The surgical sites were closed with 2 layers continuous suture plus a cutaneous layer of interrupted sutures.

The animals were euthanized according to protocol at the proper time points. The bladders of each animal were explanted and fixed in buffered formalin for histological evaluation. The fixed bladders were sectioned and stained. Microscopic examination showed similar tissue response as m Example 4 above, however with the smaller implant quantity, indications were that full resorption of the implant would occur m fewer days than in Example 4.

Example 6 Microspheres fabricated according to Example 1 were cross-linked using different time end points to vary the extent of cross-linking. The extent of cross-linking was measured by the amount of fluid uptake (swell) experienced by the particles when hydrated with water. Samples of the fabricated microspheres were cross-linked with EDC for 24, 47 and 73 hours. The microspheres were washed and dried as m Example 1. The samples for each time point were then hydrated with an excess of water, the excess water was removed and the hydrated microspheres weighed. The samples were then dried in an oven at 120° C for 1 hour and then re-weighed to obtain the dry weights. The swell was calculated as a percentage. The microspheres cross-linked for 24 hours had a swell of 553%, those cross-lxnked for 47 hours had a swell of 462% and the 73 hour samples had a swell value of 441%. The extent of cross-lxnkxng of the mxcrospheres xs expected to relate to the m-vivo degradatxon rate of the materxal, wxth hxgher cross-lxnked materxals lastxng for a longer txme perxod than lower cross-lxnked materxals. Example 7 Dispersions of microspheres fabricated according to Example 1 above were made using PBS in concentrations of 15 and 20% weight/volume solids. The dispersions were placed into 1 cc syringes and 26 gage needles attached. Commercially available dispersions of collagen (Contigen) at 3.5% weight/volume solids were placed in similar syringe/needle combinations. The force required to expel the dispersions was estimated manually. It was found that the microsphere dispersions were able to be expelled with similar forces to the commercial product even with the solids content being significantly higher.

Example 8 Sterile microspheres according to Example 1 were dispersed in phosphate buffered saline with 0.1% bovine serum albumin with varying amounts of recombinant basic fibroblast growth factor (bFGF) . A slurry was prepared, composed of 17% by weight of microspheres with 0, 1, 25, or 50 micrograms of bFGF per cc. Injections of 0.2 cc of the microsphere slurry were injected through a 18 1/2 gauge needle into the subcutaneous tissue of rabbit dorsum. On one side of the spine, injections with 0, 1, 25, 50 micrograms per cc bFGF was injected, formulated in the microsphere slurry aseptically. On the other side of the spine, the corresponding doses of bFGF were injected, formulated in buffer alone. The rabbits were necropsied and the tissues around the injections examined after 7 and 14 days. The tissues exposed to bFGF in buffer alone were unremarkable and appeared normal. The microsphere samples had in all cases formed a single mass without trace particles. The samples all showed only traces of inflammation and appeared well accepted by surrounding tissue. At the 7 day examination, the microsphere samples were all approximately the same size. The samples with bFGF appeared more translucent and attached to surrounding tissues, especially at the two highest dosages. At the 14 day examination, the microsphere samples were progressively smaller with increasing bFGF dosage as compared to the sample without bFGF. The samples had integrated with surrounding tissue, especially when samples were found partially intramuscular .

Claims

What xs claxmed xs:

1. An xnjectable and flowable composxtxon for txssue reconstructxon comprxsxng mxcropartxcles formed of a bxomaterxal whxch xs bxodegradable and cross-lxnked to a densxty such that the in vivo retentxon perxod of saxd partxcles at the sxte of applxcatxon xs suffxcxent to stxmulate txssue repaxr and xn-growth.

2. A composxtxon accordxng to claxm 1 wherexn saxd the average dxameter of saxd mxcropartxcles xs xn the range of 1 to 100 mxcrons.

3. A composxtxon accordxng to claxm 1 wherexn saxd mxcropartxcles are xn the form of mxcrospheres.

4. A composxtxon accordxng to claxm 2 wherexn saxd dxameter xs relatxvely unxform such that close packxng rules apply to the packxng of saxd mxcropartxcles at the sxte of applxcatxon to form an xntegral mass wxth xnterconnectxng porosxty.

5. A composxtxon accordxng to claxm 1 further comprxsxng a bxologxcally acceptable medxum xn whxch saxd mxcropartxcles are dxspersed at a solxds content of at least 10% wexght per volume.

6. A composxtxon accordxng to claxm 1 wherexn saxd bxomaterxal xs selected from the group consxstxng of gelatxn, collagen, fxbrxnogen, fxbrxn, polylactxc acxd, polyglycolxc acxd, polyhydroxybutyrate, polyesters, elastxn, glycosomamxnoglycans and combxnatxons of two or more of saxd bxomaterxals .

7. A composxtxon accordxng to claxm 1 wherexn saxd bxomaterxal comprxses gelatxn.

8. A composxtxon accordxng to claxm 1 wherexn saxd bxomaterxal xs cross-lxnked wxth l-(3- dimethylammopropyl ) -3-ethylcarbod╬╣╬╣m╬╣de .

9. A composition according to claim 1 wherein said biomaterial is cross-linked with glutaraldehyde .

10. A composition according to claim 1 wherein said biomaterial is cross-linked with a combination of l-(3- dimethylammopropyl) -3-ethylcarbod╬╣╬╣m╬╣de and glutaraldehyde .

11. A composition according to claim 1 further comprising growth factors, protease inhibitors, anti- mfective agents, anti-tumor agents, anti-proliferative agents, or anti-mflammatory agents.

12. A composition according to claim 11 comprising basic fibroblast growth factor or vascular endothelial growth factor.

13. A composition according to claim 1 in which the microparticles aggregate m-vivo to form a mass with interconnecting porosity capable of harboring tissue ingrowth .

14. A composition according to claim 1 wherein said microparticles comprise a plurality of layers.

15. A method of inducing tissue growth at a tissue defect or wound site comprising the step of applying an effective amount of an injectable composition comprising microparticles of biomaterial which is biodegradable and cross-linked to a density such that the in vivo retention period of said microparticles at the site of application is sufficient to stimulate tissue repair in-growth.

16. A method according to claim 15 wherein said site is cardiac tissue.

17. A method according to claim 15 wherein said site is cartilaginous tissue.

18. A method according to claim 15 wherein said site is ossiferous tissue.

19. A method according to claim 15 wherein said site is a structurally defective sphincter.

20. A method according to claim 15 wherein said site is a vocal cord.

21. A method according to claim 15 wherein said site is a scar.

22. A method according to claim 15 wherein said site is a skin wrinkle.

23. A method according to claim 15 wherein said site is a puncture wound.

24. A method according to claim 15 wherein said biomaterial is selected from the group consisting of gelatin, collagen, fibrinogen, fibrin, polylactic acid, polyglycolic acid, polyhydroxybutyrate, polyesters, elastin, glycosomaminoglycans and combinations of two or more of said biomaterials .

25. A method according to claim 15 wherein the average diameter of said microparticles are in the range of 1 to 100 microns.

26. A method according to claim 15 wherein said microparticles are in the form of microspheres.

27. A method according to claim 25 wherein said diameter is relatively uniform such that close packing rules apply to the packing of said microparticles at the site of application to form an integral mass with interconnecting porosity.

28. A method according to claim 15 wherein said composition further comprises a biologically acceptable medium in which said microparticles are dispersed at a solids content of at least 10% weight per volume.

29. A method according to claim 15 wherein said biomaterial comprises gelatin.

30. A method according to claim 15 wherein said biomaterial is cross-linked with l-(3- dimethylaminopropyl) -3-ethylcarbodiimide .

31. A method according to claim 15 wherein said biomaterial is cross-linked with glutaraldehyde.

32. A method according to claim 15 wherein said biomaterial is cross-linked with a combination of l-(3- dimethylaminopropyl) -3-ethylcarbodiimide and glutaraldehyde .

33. A method according to claim 15 wherein said composition further comprises growth factors, protease inhibitors, anti-infective agents or anti-inflammatory agents .

34. A method according to claim 15 wherein said microparticles comprise a plurality of layers.

35. A process for fabricating microspheres comprising the steps of dispersing a biomaterial into an aqueous solvent to form a dispersion, forming said dispersion into microdroplets, capturing said microdroplets in a non-solvent of said biomaterial to form said microspheres.

36. A process according to claim 35 wherein said aqueous solvent is miscible with said non-solvent.

37. A process according to claim 35 further comprising the step of cross-linking said microspheres.

38. A process according to claim 37 wherein said cross-linking is performed by contacting said microspheres with 1- (3-dimethylaminopropyl) -3- ethylcarbodiimide hydrochloride .

39. A process according to claim 37 wherein said cross-linking is performed by contacting said microspheres with glutaraldehyde.

40. A process according to claim 37 where said cross-linking is accomplish by dehydrothermal dehydration.

41. A process according to claim 37 wherein said cross-linking is performed by contacting said microspheres with a plurality of crosslinking agents.

42. A process according to claim 41 wherein said agents comprise 1- (3-dimethylaminopropyl) -3- ethylcarbodiimide hydrochloride and glutaraldehyde.