CA2264647C - Fragmented polymeric hydrogels for adhesion prevention and their preparation - Google Patents

Abstract

Molecular cross-linked gels comprise a variety of biologic and non-biologic polymers, such as proteins, polysaccharides, and synthetic polymers. Such molecular gels may be applied to target sites in a patient's body by extruding the gel through an orifice at the target site.
Alternatively, the gels may be mechanically disrupted and used in implantable articles, such as breast implants. When used in vivo, the compositions are useful for inhibiting post-surgical spinal and other tissue adhesions, for filling tissue divots, tissue tracts, body cavities, surgical defects, and the like.

Description

10152025303540CA 02264647 1999-02-26W0 98I08550 PCT/US97/15262RF§fI.¥g¥IqEN1'ED POLYMERIC HYDROGELS FOR ADHESION PREVENTION AND THEIR PREPA-BACKGROUND OF THE INVENTION1. Field of the InventionThe present invention relates generally to cross-linked polymeric compositions and to the use of suchcompositions for inhibiting tissue adhesion and otherpurposes.Tissue adhesions occur frequently following surgeryand may contribute to or cause compromised surgical resultsand post—surgical complications. Tissue adhesions may resultfrom unwanted or excessive scar tissue and occur in variousbody regions including pelvic, abdominal, spinal, tendon,ophthalmic, urinary, thoracic and cardiovascular tissues andare formed when normal tissue bonds to the surfaces ofinternal organs which have been traumatized or damaged duringsurgery. Such adhesions may join organs or other body tissuesthat normally are separate. Treating adhesions maynecessitate additional surgery with additional costs, dangerand/or discomfort to the patient.Of particular pertinence to the present application,tissue adhesions often occur after spinal surgery as theresult of scar tissue formation between the spinal cordnerves, and adjacent underlying tissues. Such scar tissueformation can compress the nerve roots, producing neuralcomplications such as persistent low back pain and sciatica.At present, peridural scar tissue must be treated withadditional surgery.101520253035CA 02264647 1999-02-26wo 93/03550 PCT/US97/152622Numerous procedures and materials have been proposedSuchprocedures include introducing barrier materials such asto minimize or eliminate post—surgical adhesions.metals, polymers, and natural materials over the target site.A woven material of regenerated cellulose is currentlymarketed for this purpose by Johnson & Johnson under thetrademark Interceed®. This product, however, does not conformwell to the underlying tissue. Other polymeric materials thathave been tried for this purpose include nylon, cellophane,PTFE, polyethylene, siloxane, elastomers and polylactic acidcopolymer films. Many of these materials are notbiodegradable and therefore, remain in the body withunpredictable and potentially undesirable consequences.The reduction and elimination of post—surgicalspinal adhesions has been particularly problematic. A varietyof permanently implanted devices have been proposed, such asthose described in U.S. Patent Nos. 5,437,672 and 4,013,078.The use of permanent implants, however, is undesirable. Theuse of resorbable barriers and films has also been proposed.Placement of such barriers and films, however, has also beenproblematic. The regions between adjacent vertebrae aredifficult to access, and it is very difficult to properlyplace and immobilize barriers and films. The use of non—solidanti—adhesive materials is also problematic since suchmaterials must be sufficiently fluid to enter and conform tothe regions being treated, while being sufficiently viscousand persistent so that they remain in the space until thetissue is healed. These objectives must further be balancedwith the requirements of biocompatibility and resorbability ofthe anti—adhesive compositions.For these reasons, it would be desirable to provideimproved compositions, methods, and articles for inhibitingthe formation of tissue adhesions following surgery and othertrauma. In particular, it would be desirable to providecompositions and methods for introducing such compositions invivo for the prevention and inhibition of peridural adhesionsfollowing laminectomies or other surgical procedures on thespinal column. It would be further desirable if such101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/152623compositions were useful for the prevention or inhibition ofadhesions elsewhere in the body and for other in vivopurposes, such as a filler for tissue voids such as divotsresulting from tissue biopsies or other blunt tissue trauma,the filling of implants, such as breast implants, the sealingand/or hemostasis of percutaneous penetrations, and thefilling and supplementation of other constrained regionswithin a patient's body. Moreover, the compositions andmethods of the present invention should be adaptable fordelivering drugs and other biologically active substances totissue surfaces adjacent to regions where the compositionshave been implanted. At least some of these objectives willbe met by the embodiments of the invention of the presentapplication described hereinafter.2. Description of the Background ArtBarrier films and materials used for preventing orinhibiting spinal and other adhesions are described in U.S.Patent Nos. 5,350,173, 5,140,016; 5,135,751; 5,134,229;5,126,141;, 5,080,893; 5,017,229; 5,007,916; PCT publicationsWO 95/21354; WO 92/15747; WO 86/00912; and Boyers et al.(1988) Fert. Ster. 49: 1066-1070. U.S. Patent Nos. 5,437,672and 4,013,078 each describe intervertebral protective deviceswhich remain as permanent implants along the patient's spinalcord.Collagen and other polymeric plugs intended forsealing percutaneous penetrations, such as tissue tractscreated by accessing the femoral artery, are described in anumber of patents, including U.S. Patent Nos. 5,540,715,5,531,759, 5,478,352; 5,275,616; 5,192,300; 5,108,421; and5,061,274.Collagen-containing compositions which have beenmechanically disrupted to alter their physical properties aredescribed in U.S. Patent Nos. 5,428,024; 5,352,715; and5,204,382.insoluble collagens.These patents generally relate to fibrillar andAn injectable collagen composition isdescribed in U.S. Patent No. 4,803,075. An injectablebone/cartilage composition is described in U.S. Patent No.101520253035CA 02264647 1999-02-26wo 98/08550 PCT/US97/1526245,516,532.particles in the size range from 5 pm to 850 pm which may beA collagen—based delivery matrix comprising drysuspended in water and which has a particular surface chargedensity is described in WO 96/39159. A collagen preparationhaving a particle size from 1 pm to 50 pm useful as an aerosolspray to form a wound dressing is described in U.S. PatentNo. 5,196,185.A polymeric, non—erodible hydrogel that may becross—linked and injected via a syringe is described in WO96/06883. A polyoxyalkylene polymer for inhibiting adhesionis described in U.S. Patent No. 5,126,141.The following pending applications, assigned to theassignee of the present application, contain related subjectUSSN 60/050,437, filed on June 18, 1997; USSN08/704,852, filed on August 27, 1996; USSN 08/673,710, filedJune 19, 1996; USSN 60/011,898, filed February 20, 1996; USSN60/006,321, filed on November 7, 1996; USSN 60/006,322, filedon November 7, 1996; USSN 60/006,324, filed onNovember 7, 1996; and USSN 08/481,712, filed on June 7, 1995.The full disclosures of each of these applications ismatter:incorporated herein by reference.SUMMARY OF THE INVENTIONThe present invention provides improvedbiocompatible polymeric compositions and methods for applyingsuch compositions at target sites in a patient's body. Themethods and compositions will be particularly useful forpreventing or inhibiting the formation of tissue adhesions,such as spinal tissue adhesions, following surgery andtraumatic injury. In addition, the compositions and methodsmay also find use in stopping or inhibiting bleeding(hemostasis), particularly when combined with a suitablehemostatic agent, such as thrombin, fibrinogen, clottingfactors, and the like. The compositions will be furtheruseful for supplementing tissues, particularly for fillingsoft and hard tissue regions, including divots, tracts, bodycavities, etc., present in muscle, skin, epithelial tissue,connective or supporting tissue, nerve tissue, ophthalmic and101520253035CA 02264647 1999-02-26wo 98/08550 PCT/US97/152625other sense organ tissue, vascular and cardiac tissue,gastrointestinal organs and tissue, pleura and other pulmonarytissue, kidney, endocrine glands, male and female reproductiveorgans, adipose tissue, liver, pancreas, lymph, cartilage,bone, oral tissue, and mucosal tissue. The compositions ofthe present invention will be still further useful for fillingsoft implantable devices, such as breast implants, where thematerial will be protected from degradation by a cellular/enzyme—impermeable barrier or cover. The compositions willadditionally be useful in other procedures where it isdesirable to fill a confined space with a biocompatible andresorbable polymeric material. Additionally, the compositionsmay be combined with drugs and other biologically activeagents, where the drugs may be released at the target siteover time.The compositions of the present invention comprise amolecular, cross-linked hydrogel which is resorbable andcomprises small subunits having a size and other physicalproperties which enhance the flowability of the gel (e.g. theability to be extruded through a syringe) and the ability ofthe gel to flow onto and conform to sites on or in tissue,including tissue surfaces and defined cavities, e.g.intravertebral spaces, tissue divots, holes, pockets, and thelike.flow when the compositions are subjected to stresses above aIn particular, the subunits are sized to permit them tothreshold level, for example when extruded through an orificeor cannula or when packed into a delivery site using aspatula, or the like. The threshold stresses are typically inthe range from 3 x 104 Pa to 5 x 105 Pa. The compositions,however, will remain generally immobile when subjected tostresses below the threshold level.The compositions may be dry, partially hydrated orfully hydrated and will display a degree of swelling from 0%The fullyhydrated material will absorb from about 400% to about l300%to 100%, depending on the extent of hydration.water or aqueous buffer by weight, corresponding to a nominalincrease in diameter or width of an individual particle ofsubunit in the range from approximately 50% to approximately101520253035CA 02264647 1999-02-26wo 93/03550 PCT/US97/152626500%, usually from approximately 50% to approximately 250%.Thus, the size of particles in the dry powder startingmaterial (prior to hydration) will be determine the partiallyor fully hydrated size of the subunit (depending on thefactors described below). Exemplary and preferred size rangesfor the dry particles and fully hydrated subunits are asfollows:Particle/Subunit SizeExemplary Range Preferred RangeDry Particle 0.01 mm — 1.5 mm 0.05 mm - 1 mmFully Hydrated 0.05 mm - 3 mm 0.25 mm — 1.5 mmIiydrogelsubunfiCompositions of the present invention will usuallybe in the form of a dry powder, a partially hydrated gel, or afully hydrated gel. The dry powder (having a moisture contentbelow 20% by weight) will be useful as a starting material forpreparation of the hydrogels, as described below. Thepartially hydrated gels, typically having from 50% to 80%hydration, are useful for applications where it is desiredthat the material further swell upon application to a moisttarget site, e.g. a tissue divot. The fully hydrated formswill be useful for applications where in situ swelling is notdesired, such as in the spinal column and other areas wherenerves and other sensitive structures are present.The dimensions of the subunits may be achieved in avariety of ways. For example, a cross—linked hydrogel havingdimensions larger than the target range (as defined below) maybe mechanically disrupted at a variety of points during theproduction process. In particular, the composition may bedisrupted (1) before or after cross—linking of a polymerstarting material and (2) before or after hydration of thecross—linked or non—cross—linked polymer starting material,e.g. as a fully or partially hydrated material or as a dryparticulate powder. The term "dry" will mean that the101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/152627moisture content is sufficiently low, typically below 20% byweight water, so that the powder will be free—flowing and thatthe individual particles will not aggregate. The term"hydrated" will mean that the moisture content is sufficientlyhigh, typically above 50% of the equilibrium hydration level,usually in the range from 80% to 95% of the equilibriumhydration level, so that the material will act as a hydrogel.Mechanical disruption of the polymer material in thedry state is preferred in cases where it is desired to controlIt iseasier to control comminution of the dry particles than thethe particle size and/or particle size distribution.hydrated hydrogel materials, and the size of the resultingreduced particles is thus easier to adjust. Conversely,mechanical disruption of the hydrated, cross-linked hydrogelsis generally simpler and involves fewer steps than doesThus, thedisruption of hydrated gels may be preferred when the ultimatecomminution of a dry polymer starting material.gel subunit size and/or size distribution is not critical.In a first exemplary production process, a dry, non-cross—linked polymer starting material, e.g. dry gelatinpowder, is mechanically disrupted by a conventional unitoperation, such as homogenization, grinding, coacervation,milling, jet milling, and the like. The powder will bedisrupted sufficiently to achieve dry particle sizes whichproduce hydrogel subunit sizes in the desired ranges when theproduct is partially or fully hydrated. The relationshipbetween the dry particle size and the fully hydrated subunitsize will depend on the swellability of the polymericmaterial, as defined further below.Alternatively, a particulate polymeric startingmaterial may be formed by spray drying. Spray dryingprocesses rely on flowing a solution through a small orifice,such as a nozzle, to form droplets which are released into acounter—current or co—current gas stream, typically a heatedgas stream. The gas evaporates solvent from the liquidstarting material, which may be a solution, dispersion, or thelike.material is an alternative to mechanical disruption of theUse of spray drying to form a dry powder starting101520253035CA 02264647 1999-02-26WO 98108550 PCT/US97/152628starting material. The spray drying operation will usuallyproduce a non—cross—1inked dry powder product with a highlyuniform particle size. The particles may then be cross-linked, as described below.In many instances, the mechanical disruptionoperation can be controlled sufficiently to obtain both theparticle size and particle size distribution within a desiredrange. In other cases, however, where more precise particlesize distributions are required, the disrupted material can befurther treated or selected to provide the desired particlesize distribution, e.g. by sieving, aggregation, or the like.The mechanically disrupted polymeric starting material is thencross—linked as described in more detail below, and dried.The dried material may be the desired final product, where itmay be rehydrated and swollen immediately prior to use.Alternatively, the mechanically disrupted, cross—linkedmaterial may be rehydrated, and the rehydrated materialpackaged for storage and subsequent use. Particular methodsfor packaging and using these materials are described below.Where the subunit size of the fragmented hydrogel isless important, the dried polymeric starting material may behydrated, dissolved, or suspended in a suitable buffer andcross—linked prior to mechanical disruption. Mechanicaldisruption of the pre—formed hydrogel will typically beachieved by passing the hydrogel through an orifice, where thesize of the orifice and force of extrusion together determineWhile thismethod is often operationally simpler than the mechanicalthe particle size and particle size distribution.disruption of dry polymeric particles prior to hydration andcross—linking, the ability to control the gel particle size ismuch less precise.In a particular aspect of the mechanical disruptionof pre—formed gels, the gels may be packed in a syringe orother applicator prior to mechanical disruption. Thematerials will then be mechanically disrupted as they areapplied through the syringe to the tissue target site, asdiscussed in more detail below. Alternatively, a non-disrupted, cross—linked polymeric material may be stored in a101520253035CA 02264647 1999-02-26wo 98/08550 PCT/US97/152629dry form prior to use. The dry material may then be loadedinto a syringe or other suitable applicator, hydrated withinthe applicator, and mechanically disrupted as the material isdelivered to the target site, again typically being through anorifice or small tubular lumen.The polymer will be capable of being cross-linkedand of being hydrated to form a hydrogel, as described in moredetail below.from gelatin, collagen (e.g. soluble collagen), albumin,Exemplary polymers include proteins selectedhemoglobin, fibrinogen, fibrin, fibronectin, elastin, keratin,laminin, casein and derivatives and combinations thereof.Alternatively, the polymer may comprise a polysaccharide, suchas a glycosaminoglycan, a starch derivative, a cellulosederivative, a hemicellulose derivative, xylan, agarose,alginate, chitosan, and combinations thereof. As a furtheralternative, the polymer may comprise a non—biologic hydrogel-forming polymer, such as polyacrylates, polymethacrylates,polyacrylamides, polyvinyl polymers, polylactide—glycolides,polycaprolactones, polyoxyethylenes, and derivatives andcombinations thereof.Cross—linking of the polymer may be achieved in anyconventional manner. For example, in the case of proteins,cross-linking may be achieved using a suitable cross-linkingagent, such as an aldehyde, sodium periodate, epoxy compounds,and the like.exposure to radiation, such as 7—radiation or electron beamAlternatively, cross-linking may be induced byradiation. Polysaccharides and non—biologic polymers may alsobe cross-linked using suitable cross-linking agents andradiation. Additionally, non—biologic polymers may besynthesized as cross-linked polymers and copolymers. Forexample, reactions between mono— and poly—unsaturated monomerscan result in synthetic polymers having controlled degrees ofcross-linking. Typically, the polymer molecules will eachhave a molecular weight in the range from 20kD to 200 kD, andwill have at least one link to another polymer molecule in thenetwork, often having from 1 to 5 links, where the actuallevel of cross-linking is selected in part to provide a101520253035CA 02264647 1999-02-26wo 93/03550 PCT/US97ll526210desired rate of biodegradability in the ranges set forthbelow.The extent of cross—linking of the polymer has aneffect on several functional properties of the hydrogelincluding extrudability, absorptiveness of surroundingbiological fluids, cohesiveness, ability to fill space,swelling ability and ability to adhere to the tissue site.The extent of cross~linking of the polymeric gel compositionmay be controlled by adjusting the concentration of cross-linking agent, controlling exposure to cross—linkingradiation, changing the relative amounts of mono- and poly-unsaturated monomers, varying reaction conditions, and thelike.adjusting the concentration of cross—linking agent.Typically, the degree of cross—linking is controlled byExposure to radiation, such as 7—radiation, may alsobe carried out in order to sterilize the compositions beforeor after packaging. When the compositions are composed ofradiation—sensitive materials, it will be necessary to protectthe compositions from the sterilizing radiation. For example,in some cases, it will be desirable to add ascorbic acid inorder to inhibit further cross—linking of the materials fromfree radical mechanisms.The hydrogel compositions of the present inventionwill typically have a solids content in the range from 1% byweight to 70% by weight, preferably from 5% by weight to 20%by weight, preferably from 5% by weight to 16% by weight. Forgels having a higher solid content, typically above 16% byweight, it is preferred to include a plasticizer in thecomposition, typically from 0.1% by weight to 30% by weight,preferably from 1% by weight to 5% by weight. Suitableplasticizers include polyethylene glycols, sorbitol, glycerol,and the like.The equilibrium swell of the cross—linked polymersof the present invention will generally range from 400% tol300%, preferably being from 500% to 1lOO%, depending on itsintended use. Such equilibrium swell may be controlled byvarying the degree of cross—linking, which in turn is achievedby varying the cross—linking conditions, such as the type of101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/15262llcross¥linking method, duration of exposure of a cross—linkingagent, concentration of a cross—linking agent, cross—linkingtemperature, and the like.Materials having equilibrium swell values from about400% to l300% were prepared and are described in theExperimental section hereinafter. It was found that materialshaving differing equilibrium swell values perform differentlyin different applications. For example, the ability toinhibit bleeding in a liver divot model was most readilyachieved with cross-linked gelatin materials having a swell inthe range from 700% to 950%. For a femoral artery plug, lowerequilibrium swell values in the range from 500% to 600% weremore successful. Thus, the ability to control cross—linkingand equilibrium swell allows the compositions of the presentinvention to be optimized for a variety of uses.In addition to equilibrium swell, it is alsoimportant to control the hydration of the material immediatelyprior to delivery to a target site. Hydration and equilibriumswell are, of course, intimately connected. A material withA material with 100%hydration will be at its equilibrium water content.0% hydration will be non-swollen.Hydrations between 0% and 100% will correspond to swellingbetween the minimum and maximum amounts. As a practicalmatter, many dry, non—swollen materials according to thepresent invention will have some residual moisture content,usually below 20% by weight, more usually from 8% to 15% byweight. When the term "dry" is used herein, it will specifymaterials having a low moisture content where the individualparticles are free flowing and generally non—swollen.Hydration can be adjusted very simply by controllingthe amount of aqueous buffer added to a dry or partiallyhydrated cross—linked material prior to use. Usually, at aminimum, it will be desirable to introduce sufficient aqueousbuffer to permit extrusion through a syringe or other deliverydevice. In other cases, however, it may be desirable toutilize a spatula or other applicator for delivering lessfluid materials. The intended use will also help determinethe desired degree of hydration. In cases where it is desired, _,___,,_,,_,_,“,____,______,,_,M,_,,_. ,. . ._ .....-.........,......,.., .101520253035CA 02264647 1999-02-26WO 98/08550 PCT/U S97/ 1526212to fill or seal a moist cavity, it is generally desirable toemploy a partially hydrated gel which can swell and fill thecavity by absorbing moisture from the target site.Conversely, fully or substantially fully hydrated gels arepreferred for application in the brain, near the spine, and totarget sites near nerves and other sensitive body structuresIt wouldalso be possible to prepare the gel compositions of thewhich could be damaged by post—placement swelling.present invention with excess buffer, resulting in a two—phasecomposition having a fully hydrated gel and a free bufferphase.A preferred hydrogel material according to thepresent invention is a gelatin which has been cross-linked toachieve from 700% to 950% swell at equilibrium hydration. Thematerial will be disrupted to have a gel particle size in therange from 0.01 mm to 1.5 mm, preferably 0.05 mm to 0.5 mm,and will preferably be hydrated at a level sufficient toachieve 70% to 100% of the equilibrium swell prior toapplication to the site.In some cases, the hydrogel compositions of thepresent invention may contain a combination of two or moredifferent materials, e.g combinations of proteins andpolysaccharides and/or non—biologic polymers, as well ascombinations of two or more individual materials from each ofthe polymer types, e.g. two or more proteins, polysaccharides,etc.The polymeric compositions of the present inventionmay also comprise combinations of the disrupted, cross-linkedpolymer hydrogels described above and non-cross—linkedpolymeric materials. The disrupted, cross—linked polymerichydrogels consist of a plurality of subunits having a sizedetermined by preparation method. The size is selected to beuseful for packing a confined volume, having both theflowability and the rate of biodegradability described below.The discrete nature of the cross—linked subunits, however,will leave void areas which may be filled by combination witha non—cross—linked polymeric material. The non—cross—linkedpolymeric or other filler material may comprise any of the101520253035CA 02264647 1999-02-26WO 98108550 PCT/US97/15262l3polymeric materials listed above, and may optionally but notnecessarily be the same polymeric material which has beencross—linked to form the cross—linked mechanically disruptedgel. The relative amounts of cross—linked polymer and non-cross—linked polymer will be selected to provide a relativelycontinuous (free of voids) composition after optionalmechanical disruption and delivery to a target site, typicallyhaving a weight ratio in the range from 20:1 to 1:1 (cross-linked polymer:non—cross—linked polymer), usually in the rangefrom 10:1 to 2:1, preferably from 5:1 to 2:1.The hydrogels of the present application may beapplied using a syringe, a spatula, a brush, a spray, manuallyby pressure, or by any other conventional technique. Usually,the gels will be applied using a syringe or similar applicatorcapable of extruding the gel through an orifice, aperture,needle, tube, or other passage to form a bead, layer, orsimilar portion of material. Mechanical disruption of thegels can occur as the gel is extruded through an orifice inthe syringe or other applicator, typically having a size inthe range from 0.01 mm to 5.0 mm, preferably 0.5 mm to 2.5 mm.Preferably, however, the polymeric hydrogel will be initiallyprepared from a powder having a desired particle size (whichupon hydration yields hydrogel subunits of the requisite size)or will be partially or entirely mechanically disrupted to therequisite size prior to a final extrusion or other applicationstep.The compositions may be applied at varying degreesof hydration, usually but not necessarily being at leastpartially hydrated. If applied in a non—hydrated form, thecompositions will swell to their full equilibrium swell value,Whenapplied at their equilibrium hydration level, the compositionsi.e. from about 400% to about 1300% as set forth above.will display substantially equilibrium hydration and little orno swelling when applied to tissue. Swelling of the non-hydrated and partially hydrated compositions results fromabsorption of moisture from the tissue and surroundings towhich the composition is being applied.l01520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97I1526214The present invention still further provides kitscomprising any of the hydrated or non—hydrated gel materialsdescribed above in combination with written instructions foruse (IFU) which set forth any of the methods described abovefor applying the gel onto a target site on tissue. Thecomposition and written instructions will be included togetherin a conventional container, such as a box, jar, pouch, tray,or the like.separate sheet of paper or other material and packaged on orThe written instructions may be printed on awithin the container or may be printed on the containeritself.separate, sterile bottle, jar, vial, or the like.Usually, the composition(s) will be provided in aWhen thegel material is non—hydrated, the kit may optionally include aseparate container with a suitable aqueous buffer forhydration. Other system components such as the applicator,e.g. syringe, may also be provided.BRIEF DESCRIPTION OF THE DRAWINGSFig. 1 illustrates the application of the molecularcross—linked polymeric gel of the present invention to asurgically created defect in the vertebral body for preventingpost—surgical spinal adhesions.Figs. 2A and 2B illustrate application of themolecular cross—linked polymeric gel compositions of thepresent invention to a defect in soft tissue, where thetreated region is optionally covered with a protective patchafter the defect is filled with the polymeric composition.Figs. 3A and 3B illustrate use of the molecularcross—linked polymeric compositions of the present inventionfor filling a percutaneous tissue penetration to a bloodvessel, such as a tissue tract formed as part of anintravascular catheterization procedure.Fig. 4 illustrates a kit comprising a sterilepackage for an applicator containing the molecular cross-linked polymeric composition of the present invention.Fig. 5 illustrates the correlation between percentswell and the percent solids of the polymeric gel.101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526215DESCRIPTION OF THE SPECIFIC EMBODIMENTSCompositions according to the present inventioncomprise resorbable biocompatible molecular cross—linkedhydrogels. By "biocompatible" is meant that the materialswill meet the criteria in standard # ISO 10993-1 promulgatedby the International Organization for Standardization (NAMSA,Northwood, Ohio).compositions will degrade or solubilize, when placed directlyBy "resorbable," it is meant that theinto a target site in a patient's body (and not protectedwithin an implant device such as a breast implant), over atime period of one year or less, usually from 1 day to 1 year,more usually from 1 day to 120 days. A particular protocolfor measuring resorption and degradation is set forth in theExperimental section hereinafter. By "molecular cross-linked", it is meant that the materials comprise polymermolecules (i.e. individual chains) which are attached bybridges composed of either an element, a group, or a compound,where the backbone atoms of the polymer molecules are joinedby primary chemical bonds. Cross—linking may be effected in avariety of ways, as will be described in greater detail below.By "hydrogel," it is meant that the compositioncomprises a single phase aqueous colloid in which a biologicor non—biologic polymer, as defined in more detail below,absorbs water or an aqueous buffer. The hydrogel comprisesmultiple sub—networks, where each sub—network is a molecularcross—linked hydrogel having dimensions which depend on thedegree of hydration and are within the ranges set forth above.Preferably, the hydrogels will have little or no free water,i.e. water cannot be removed from the hydrogel by simplefiltration.By "percent swell," it is meant that the dry weightis subtracted from the wet weight, divided by the dry weightand multiplied by 100, where wet weight is measured after thewetting agent has been removed as completely as possible fromthe exterior of the material, e.g. by filtration, and wheredry weight is measured after exposure to an elevatedtemperature for a time sufficient evaporate the wetting agent,e.g., 2 hours at 120°C.101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526216"Equilibrium swell," is defined as the percent swellat equilibrium after the polymeric material has been immersedin a wetting agent for a time period sufficient for watercontent to become constant, typically 18 to 24 hours."Target site" is the location to which the gelmaterial is to be delivered. Usually, the target site will bethe tissue location of interest, but in some cases the gel maybe administered or dispensed to a location near the locationof interest, e.g. when the material swells in situ to coverthe location of interest.The hydrogels of the present invention may be formedfrom biologic and non—biologic polymers. Suitable biologicpolymers include proteins, such as gelatin, soluble collagen,albumin, hemoglobin, casein, fibrinogen, fibrin, fibronectin,elastin, keratin, laminin, and derivatives and combinationsthereof. Particularly preferred is the use of gelatin orsoluble non—fibrillar collagen, more preferably gelatin, andOthersuitable biologic polymers include polysaccharides, such asexemplary gelatin formulations are set forth below.glycosaminoglycans, starch derivatives, xylan, cellulosederivatives, hemicellulose derivatives, agarose, alginate,Suitablenon—biologic polymers will be selected to be degradable bychitosan, and derivatives and combinations thereof.either of two mechanisms, i.e. (1) break down of the polymericbackbone or (2) degradation of side chains which result inaqueous solubility. Exemplary nonbiologic hydrogel—formingpolymers include synthetics, such as polyacrylates,polymethacrylates, polyacrylamides, polyvinyl resins,polylactide—glycolides, polycaprolactones, polyoxyethylenes,and derivatives and combinations thereof.The polymer molecules may be cross—linked in anymanner suitable to form an aqueous hydrogel according to thepresent invention. For example, polymeric molecules may becross—linked using bi- or poly—functional cross—linking agentswhich covalently attach to two or more polymer moleculeschains. Exemplary bifunctional cross—linking agents includealdehydes, epoxies, succinimides, carbodiimides, maleimides,azides, carbonates, isocyanates, divinyl sulfone, alcohols,101520253035CA 02264647 1999-02-26WO 98/08550 PCT/U S97/ 1526217amines, imidates, anhydrides, halides, silanes, diazoacetate,aziridines, and the like. Alternatively, cross-linking may beachieved by using oxidizing and other agents, such asperiodates, which activate side—chains or moieties on thepolymer so that they may react with other side—chains orAn additionalmethod of cross-linking comprises exposing the polymers tomoieties to form the cross-linking bonds.radiation, such as gamma radiation, to activate the polymer topermit cross—linking reactions. Dehydrothermal cross—linkingmethods would also be suitable. Dehydrothermal cross-linkingof gelatin can be achieved by holding it at an elevatedtemperature, typically 120°C, for a period of at least 8hours. Increasing the extent of cross—linking, as manifestedin a decline in percent swell at equilibrium, can be achievedby elevating the holding temperature, extending the durationof the holding time, or a combination of both. Operatingunder reduced pressure can accelerate the cross-linkingreaction. Preferred methods for cross-linking gelatinmolecules are described below.Optionally, the molecular cross—linked hydrogel mayinclude a plasticizer to increase the malleability,flexibility, and rate of degradation of the gel. Theplasticizer may be an alcohol, such as polyethylene glycol,sorbitol, or glycerol, preferably being polyethylene glycolhaving a molecular weight ranging from about 200 to 1000 D,preferably being about 400 D. The plasticizers will bepresent in the compositions at from about 0.1% by weight toabout 30% by weight, preferably from 1% by weight to 5% byweight of the composition. The plasticizers are particularlybeneficial for use with gels having a high solids content,typically above 10% by weight of the composition (withoutplasticizer).Exemplary methods for producing molecular cross-linked gelatins are as follows. Gelatin is obtained andplaced in an aqueous buffer to form a non—cross-linked gel,typically having a solids content from 1% to 70% by weight,usually from 3% to 10% by weight. The gelatin is cross-linked, typically by exposure to either glutaraldehyde (e.g.101520253035CA 02264647 1999-02-26wo 93/03550 PCTIUS97/15262180.01% to 0.05% w/w, overnight at 0°—8°C in aqueous buffer),sodium periodate (e.g. 0.05 M, held at 0° C to 8° C for 48hours) or 1—ethyl—3— (3—dimethylaminopropyl) carbodiimide(IIEDCII)temperature), or by exposure to about 0.3 to 3 megarads of(e.g., 0.5% to 1.5% w/w, overnight at roomgamma or electron beam radiation. Alternatively, gelatinparticles can be suspended in an alcohol, preferably methylalcohol or ethyl alcohol, at a solids content of 1% to 70% byweight, usually 3% to 10% by weight, and cross-linked byexposure to a cross-linking agent, typically glutaraldehyde(e.g., 0.01% to 0.1% w/w, overnight at room temperature).When cross-linking with glutaraldehyde, the cross-links areformed via Schiff bases which may be stabilized by subsequenttreatment with sodium borohydride. In the case of aldehydes,the pH should be held from about 6 to 11, preferably from 7 to10. After cross-linking, the resulting granules may be washedin distilled water and optionally rinsed in an alcohol, driedand resuspended to a desired degree of hydration in an aqueousmedium having a desired buffer and pH. The resultinghydrogels may then be loaded into the applicators of thepresent invention, as described in more detail hereinafter.Alternatively, the hydrogels may be mechanically disruptedprior to or after cross-linking, also as described in moredetail hereinafter.Exemplary methods for producing molecular cross-linked gelatin compositions having equilibrium percent swellsin the range from about 400% to about l300%, preferably 600%to 950%, are as follows. Gelatin is obtained and placed in anaqueous buffer (typically at a pH of 6 to 11, preferably at apH between 7 and 10) containing a cross-linking agent insolution (typically glutaraldehyde, preferably at aconcentration of 0.01% to 0.1% w/w) to form a gel, typicallyhaving a solids content from 1% to 70% by weight, usually from3% to 10% by weight. The gel is well mixed and held overnightat 0°—8°C as cross-linking takes place. It is then rinsedthree times with deionized water, twice with an alcohol(preferably methyl alcohol, ethyl alcohol, or isopropylalcohol) and allowed to dry at room temperature. Optionally,101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526219the gel may be treated with sodium borohydride to furtherstabilize the cross—linking.The compositions of the present invention may befurther combined with other materials and components, such asbioactive component(s) to be delivered to the patient,viscosity modifiers, such as carbohydrates and alcohols, andother materials intended for other purposes, such as tocontrol the rate of resorption. Exemplary bioactivecomponents include, but are not limited to, proteins,carbohydrates, nucleic acids, and inorganic and organicbiologically active molecules such as enzymes, antibiotics,antineoplastic agents, bacteriostatic agents, bacteriocidalagents, antiviral agents, hemostatic agents, localanesthetics, anti~inflammatory agents, hormones,antiangiogenic agents, antibodies, neurotransmitters,psychoactive drugs, drugs affecting reproductive organs andSuchbioactive components will typically be present at relativelyoligonucleotides, such as antisense oligonucleotides.low concentrations, typically below 10% by weight of thecompositions, usually below 5% by weight, and often below 1%by weight.Exemplary hemostatic agents include thrombin,fibrinogen and clotting factors. Hemostatic agents likethrombin may be added in concentrations ranging from 50 to10,000 Units thrombin per ml gel, preferably from about 100Units thrombin per ml gel to about 1000 Units thrombin per mlgel.The molecular cross—linked hydrogels of the presentinvention can be mechanically disrupted at the time they aredelivered to a target site by extrusion through an orifice orother flow restriction, or they can be mechanically disruptedin a batch process prior to delivery to a target site. Theprimary purpose of this mechanical disruption step is tocreate multiple subunits of hydrogel having a size whichenhances the ability to fill and pack the space to which it isbeing delivered. Another purpose of the mechanicaldisruption is to facilitate passage of the gel down smalldiameter tubes, cannulas, and/or other applicators to the101520253035CA 02264647 1999-02-26wo 93/03550 PCT/US97/1526220target site. Without mechanical disruption, the molecularcross—linked hydrogels will have difficulty conforming to andfilling the irregularly target spaces which are being treated,e.g. intravertebral spaces in the spinal column, tissuedivots, percutaneous tissue tracks, and the like. By breakingthe gel down to smaller sized sub-units, such spaces can befilled much more efficiently while retaining the mechanicalintegrity and persistence of the cross—linked gel which areessential for it to act as an anti—adhesive agent, tissuefiller, or the like.single manual extrusion of the composition, typically using aSurprisingly, it has been found that asyringe having an orifice in size in the range from 0.01 mm to5.0 mm, preferably from 0.1 mm to 2.5 mm, provides the properamount of mechanical disruption to enhance the gel propertiesas described above.Alternatively, the gel compositions of the presentinvention may be mechanically disrupted prior to their finaluse or delivery. Molecular cross-linking of the polymerchains of the gel can be performed before or after itsmechanical disruption. The gels may be mechanically disruptedin batch operations, such as mixing, so long as the gelcomposition is broken down into sub—units having a size in theWhen the gelcomposition is disrupted prior to use, the gel can be applied0.01 mm to 5.0 mm range set forth above.or administered by techniques other than extrusion e.g. usinga spatula, spoon, or the like. Other batch mechanicaldisruption processes include pumping through a homogenizer ormixer or through a pump which compresses, stretches, or shearsthe gel to a level which exceeds a fractural yield stress ofthe hydrogel. In some cases, extrusion of the polymericcomposition causes the gel to be converted from asubstantially continuous network, i.e. a network which spansthe dimensions of the original gel mass, to a collection ofsub—networks or sub—units having dimensions in the ranges setforth above. In other cases it may be desirable to partiallydisrupt the gel compositions prior to packaging in the syringeor other applicator. In such cases, the gel material willachieve the desired sub—unit size prior to final extrusion.101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526221In a presently preferred embodiment, the polymer maybe initially prepared (e.g. by spray drying) and/or bemechanically disrupted prior to being cross—linked, oftenusually prior to hydration to form a gel. The polymer may beprovided as a finely divided or powdered dry solid which maybe disrupted by further comminution to provide particleshaving a desired size, usually being narrowly confined withina small range. Further size selection and modification steps,such as sieving, cyclone classification, etc., may also beperformed. For the exemplary gelatin materials describedhereinafter, the dry particle size is preferably in the rangefrom 0.01 mm to 1.5 mm, more preferably from 0.05 mm to 1.0mm. An exemplary particle size distribution will be such thatgreater than 95% by weight of the particles are in the rangefrom 0.05 mm to 0.7 mm. Methods for comminuting the polymericstarting material include homogenization, grinding,coacervation, milling, jet milling, and the like. Powderedpolymeric starting materials may also be formed by spraydrying. The particle size distribution may be furthercontrolled and refined by conventional techniques such assieving, aggregation, further grinding, and the like.The dry powdered solid may then be suspended in anaqueous buffer, as described elsewhere herein, and cross-linked.aqueous buffer, cross—linked, and then dried.In other cases, the polymer may be suspended in anThe cross-linked, dried polymer may then be disrupted, and the disruptedIn allthe cases, the resulting material comprises a cross—linkedmaterial subsequently resuspend in an aqueous buffer.hydrogel having discrete sub-networks having the dimensionsset forth above.The compositions of the present invention, aftermechanical disruption, will be resorbable, i.e., they willbiodegrade in the patient's body, in a period of less than oneyear, usually from 1 to 120 days, preferably from 1 to 90days, and more preferably from 2 to 30 days following theirinitial application. This is particularly true when thematerials are used for preventing post-surgical and otheradhesions, where a barrier is necessary between the healing101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526222tissue surfaces only for so long as the tissue is healing.Techniques for measuring the length of time required forresorption are set forth in Example 11 in the Experimentalsection below. In other cases, such as when the compositionsare contained within an implantable device, such as a breastimplant, resorption of the material will be prevented by themembrane or other mechanical barrier surrounding thecompositions (unless the integrity of the barrier is broken).Referring now to Fig. 1, a method for preventingadhesions following a laminectomy procedure will be described.A syringe 10 containing the resorbable molecular cross—linkedgel of the present invention is used to apply the gel in sucha manner that all exposed dura is covered. Usually, the gelwill be resorbed over a time period in the range from 7 to 60days.Referring now to Figs. 2A and 2B, the molecularcross—linked hydrogels of the present invention may also beused to fill divots D in soft tissue T. A syringe 50comprising a barrel 52, plunger 54 and cannula 56 contains themolecular cross—linked hydrogel in the interior of the barrel52. The hydrogel G is extruded through the cannula 56 bydepressing the plunger 54 in a conventional manner.Sufficient gel is extruded to fill the divot, as shown in Fig.2B. Preferably, a partially hydrated hydrogel which willswell further upon exposure to the moist tissue environmentwill be used. It may be desirable to place a patch P over theexposed surface of the gel, as shown in Fig. 2B. The patchmay be an adhesive or other conventional self—securing patch.Preferably, however, the patch comprises a collagen, gelatin,or other film that may be immobilized by applying energy e.g.optical or radio frequency energy as described in publishedPCT applications WO 96/07355 and WO 92/14513.Referring now to Figs. 3A and 3B, compositions andmethods of the present invention may also be used to fillpercutaneous tissue tracts TT which were formed throughoverlying tissue to access blood vessels BV. A barrierelement 70 may be placed along the inner wall of the bloodvessel at the distal end of the tissue tract TT. Filament 72101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526223may be used to hold the barrier element 70 in place. Asyringe 74 comprising a barrel 76, plunger 78, and cannula 80is then used to extrude the molecular cross—linked hydrogelmaterial of the present invention into the tissue tract overThe hydrogel G will be used to fillthe entire interior volume of the tissue tract TT, as shown inthe barrier element 70.Fig. 3B, and will preferably be partially hydrated to permitpost-placement swelling as described above. Optionally, apatch or other cover may be placed over the exposed surface ofthe tissue tract (not shown). The barrier element 70 may thenbe removed.Referring now to Fig. 4, the present inventioncomprises kits including the hydrated, partially hydrated,and/or non—hydrated polymeric compositions described abovepackaged in a suitable container, usually with writteninstructions for use. For example, the composition may bepackaged in an applicator 90 which contains the pre—extrudedmolecular cross—linked hydrogel of the present invention. Theapplicator may take a wide variety of forms, includingsyringes as previously described. In Fig. 4, the applicator90 comprises a tube 92 having a neck 94 which defines anextrusion orifice. The gel is contained within the tube andmay be extruded through the neck 94 by squeezing the tube.The applicator 90 is preferably contained in a sterile package96. The sterile package may take a variety of forms, and isillustrated as an envelope comprising a backing sheet and aclear plastic cover. Such packages may be sterilized in aconventional manner. Optionally, the radiation used to cross-link the hydrogel may also be used to sterilize the entirepackage. The instructions for use may be printed on thepackaging or provided on a separate sheet placed in thepackage.The present invention may also be used to inhibitbleeding (cause hemostasis) on an abraded or damaged tissuesurface, e.g., any organ surface including the liver, spleen,heart, kidney, intestine, blood vessels, vascular organs, andthe like.cross-linked gel combined with a hemostasis agent is used toA syringe containing the resorbable molecular101520253035CA 02264647 1999-02-26wo 93/03550 PCT/US97/1526224apply the gel to the abraded or damaged tissue site. The gelis applied so that the actively bleeding abraded or damagedarea is completely covered with the resorbable molecularcross—linked gel. Suitable hemostatic agents includethrombin, fibrinogen, and other clotting factors, as describedfor example in U.S. Patent Nos. 5,411,885; 4,627,879;4,265,233; 4,298,598; 4,362,567; 4,377,572; and 4,442,655, thedisclosures of which are incorporated herein by reference.Conveniently, catalytic components of the hemostasis agent,e.g. thrombin, may be combined in the syringe immediatelyprior to use so that their combined activities are preserveduntil applied to the tissue.When used in regions surrounding nerves and othersensitive body structures, it is preferable to employ fullyhydrated hydrogels (i.e. with >95% of hydration at equilibriumswell) in order to avoid damage to the nerves from swelling inan enclosed environment.The following examples are offered by way ofillustration, not by way of limitation.EXPERIMENTALEXAMPLE 1: MATERIALS AND METHODS FOR PRODUCTION OF AFRAGMENTED POLYMERIC PRODUCTFragmented polymeric compositions are generallyprepared as follows:Using pyrogen-free glassware and distilled waterthroughout, food grade gelatin (300 Bloom, Woburn Co.,Woburn,MA.) at 10% solids was allowed to swell in 0.1 N aq. sodiumhydroxide and 0.05 sodium periodate and held at 0°C to 8°C for2-3 days. The swollen granules were washed in distilled wateruntil the pH reached 8. The neutralized swollen granules weredried in a laminar flow hood and re—suspended in 0.05 M sodiumphosphate, 0.15 M sodium chloride, pH 7.2 +/- 0.2, at 10%solids. The composition was then loaded into 3.0 cc syringesand irradiated at 3.0 megarad with electron beam to sterilize.10152025303540CA 02264647 1999-02-26WO 98/08550 PCT/U S97/ 1526225EXAMPLE 2: MATERIALS AND METHODS FOR PRODUCTION OF AFRAGMENTED POLYMERIC PRODUCTGelatin (Woburn) was allowed to swell in an aqueousbuffer (e.g. 0.05 M sodium phosphate, 0.15 M sodium chloride,pH 7.2 +/- 0.2) at 1—l0% solids and was cross-linked by eitherglutaraldehyde (0.01 - 0.05%, w/w, overnight, roomtemperature), by sodium periodate (0.05 M, 0°C to 8°C, 48hours) or by 0.3 - 3.0 megarads of gamma or electron beamirradiation. The gels were then extruded from a syringe usingnormal manual pressure.EXAMPLE 3: MATERIALS AND METHODS FOR PRODUCTION OF AFRAGMENTED POLYMERIC PRODUCTGelatin (Woburn) was allowed to swell in distilledwater at 1-10% solids (w/w) chilled to 5°C. The resultant gelwas fragmented by stirring with an impeller driven by a motor.Then, sodium periodate and sodium hydroxide were added andmixed to achieve 0.05 M sodium periodate and 0.10 M sodiumThe chilled mixture was held at 0°C to 8°C for 2-3The cross—linked gel fragments were then washed withhydroxide.days.5°C water to achieve pH 8. Finally the gel fragments werewashed with an aqueous buffer (e.g. 0.05 sodium phosphate and0.15 sodium chloride, pH 7.2 +/- 0.2) and left at 0°C to 8°Cto equilibrate with the buffer. Free buffer was decanted fromthe fragmented gel mass and the gel particles were loaded intosyringes and irradiated at 3.0 megarads by electron beam orgamma irradiation to sterilize. Such sterilized fragmentedwere then extruded directly from the syringe causing furtherfragmentation.EXAMPLE 4: MATERIALS AND METHODS FOR PREVENTION OF POSTSURGICAL SPINAL ADHESIONSThis study demonstrated the effectiveness of thefragmented polymeric composition to prevent or reducepostlaminectomy scar formation. New Zealand White rabbits (R&RRabbitry, Stanwood, WA) having weights of m 3.0 — 4.0 kg wereused for the study. Anesthesia was induced with anintramuscular injection of ketamine hydrochloride incombination with xylazine. Each rabbit received an injection101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526226The back ofthoraxof Baytril® intramuscularly at a dose of 5 mg/kg.each rabbit was shaved from the level of the midT-10) to the tail.(2The shaved area was extended far enoughThe rabbitsternalventrally to allow for adequate skin preparation.was placed on a circulating water heating pad inrecumbency. A small towel was placed ventral to the abdomento produce a slight lumbar flexion. The skin of thelumbosacral area was prepared with an iodophor scrub andrinsed with 70% alcohol.A midline skin incision was made from L—1 to L-5 andcarried down to the lumbosacral fascia. Hemostasis wasachieved with a combination of mechanical compression andelectrocautery. The fascia was incised to expose the tips ofthe spinous processes. The paraspinous muscles were dissectedfree from the spinous processes and laminae of L—4 with theuse of a periosteal elevator. The muscles were then held awayA total, dorsallaminectomy of L—4 was performed by removal of the spinouswith the use of a self—retaining retractor.process with rongeurs and careful excision of the lamina tothe base of the mammillary process bilaterally.Care was taken to avoid injury to the spinal cord orcauda equina. The laminectomy defect was irrigated withsterile saline and any remaining bone fragments were removed.The ligamentum flavum and epidural fat were removed, leavingclean dura exposed for the extent of the laminectomy.When the procedure at the L—4 site was completed, anidentical laminectomy was performed at the L—2 level. Duringthis time, the L-4 site was protected from desiccation withgauze sponges soaked in sterile saline. The L—3 space was notmanipulated to provide a soft tissue barrier between the twomanipulated sites. Once both sites were prepared, the siteswere treated with the test material or left as controlsaccording to the randomized key below.Rabbits were assigned to the experimental groups andafter laminectomies were performed, the followingmanipulations were carried out. The exposed dura at a lumbarsite was treated with 0.5-0.9 mls fragmented gelatincomposition of Example 1. The material was placed in such a1015202530CA 02264647 1999-02-26WO 98/08550 PCT/U S97/ 1526227manner that all exposed dura was covered with the testmaterial. The exposed dura at a different lumbar sitereceived no treatment.The wound was closed in layers without furtherirrigation. The lumbosacral fascia was closed with anabsorbable suture of an appropriate size (e.g. 4-0), in asimple interrupted pattern. The subcutaneous tissue wasclosed with an absorbable suture in a simple continuouspattern and the skin was closed with appropriate suturematerial or surgical clips.For the first five days post—operatively, theanimals received Baytril® at a dose of 5 mg/kg givenintramuscularly, twice daily.Animals were euthanized and a necropsy performed ateither day 7 or day 28 post operatively. The surgicalincision was dissected free and the area of laminectionexamined.Dural adhesions were graded and scored according toextent and severity and were scored as follows:Dural Adhesions: Connective tissue attachmentsbetween bone or deep scar and dura within the spinal canalThese were evaluated by inserting a probe between the bone anddura to separate the two structures.0 = absent, with anatomical plane evident 1 = thin adhesions2 = moderate adhesions 3 = thick & tenacious adhesionsIn no case in which the fragmented gelatincomposition was applied did post surgical adhesion develop.However, adhesions occurred in 71% of the control sites. Theresults from all animal sites tested are combined and aresummarized in Table 1 below.10152025CA 02264647 1999-02-26WO 98/08550 PCT/U S97/ 1526228TABLE1Site No. Vertebral Treatment AdhesionLocation1 L4 CONTROL/N ONE 02 L1 CON TROL/NONE 03 L3 CONTROL/N ONE 24 L4 CONTROL/N ONE 25 L4 CONTROL/NONE 26 L2 CONTROL/NONE 37 L2 CONTROL/NONE 28 L1 FRAGMENTED 0GELATINCOMPOSITION9 L2 FRAGMENTED 0GELATINCOMPOSITION10 L4 FRAGMENTED OGELATINCOMPOSITION11 L5 FRAGMENTED 0GELATINCOMPOSITION12 L5 FRAGMENTED 0GELATINCOMPOSITIONEXAMPLE 5: VESSEL PLUGThis study demonstrated the effectiveness of thefragmented polymeric composition to seal a vessel puncture.The femoral artery of a farm grade Hampshire/Yorkshire crosspig (Pork Power Farms, Turlock, California) was identified andcannulated using a needle (SmartNeedle”, CardiovascularDynamics, Irvine, California). After the guide wire wasplaced, a 9 French dilator was used to create a tunnel to thevessel and enlarge the femoral artery opening. The dilatorwas removed and a 7 French sheath was introduced into thePositioningfemoral artery. The guide wire was then removed.101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526229was checked by withdrawing blood into the sheath side arm.Pulsatile arterial bleeding was also observed at the point ofAs the sheath wasremoved, a 18 gauge Teflon catheter tip attached to ainsertion of sheath at the skin incision.hypodermic syringe was used to introduce the fragmentedgelatin composition of Example 1 into the tunnel. No bleedingwas observed at the point of exit demonstrating theeffectiveness of the fragmented gelatin composition in sealingthe vessel puncture site and surrounding tissue.EXAMPLE 6: FRAGMENTED POLYMERIC COMPOSITION AS A CARRIERThis study demonstrated the effectiveness of thefragmented polymeric composition of Example 1 as a carrier tofill and seal a tissue divot in the liver. Three wounds (2tissue divots and 1 tissue puncture) were induced in the liverof a farm grade Hampshire/Yorkshire cross pig (Pork PowerFarms, Turlock, CA).Liver tissue divot #1 was actively bleedingfollowing the surgical creation of a tissue divot. A syringe,containing approximately 1 ml of fragmented gelatincomposition containing approximately 500 U of thrombin (500 to1000 units/ml) was extruded from a syringe and applied toAfter 2-3 minutes, a bloodWhen theapplied composition was grasped with forceps, it appeared tocompletely fill the tissue defect.clot formed causing immediate cessation of bleeding.adhere quite well to the tissue and had good integrity. Thesealant was manually challenged and no additional bleeding wasobserved.Liver tissue divot #2 was actively bleedingfollowing the surgical creation of a tissue divot.Approximately 1 ml of fragmented gelatin compositioncontaining thrombin (approximately 500 units/ml) was extrudedfrom a syringe and applied to completely fill the tissuedefect. A Rapiseal” patch (Fusion Medical Technologies, Inc.,Mountain View, CA) was applied using an argon beam coagulator(Valleylab, Boulder, Colorado, or Birtcher Medical Systems,Irvine, California,). Immediate cessation of bleedingoccurred.101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/ 1526230Liver puncture #1, was actively bleeding followingthe surgical creation of a blunt puncture. Approximately 0.8ml of fragmented gelatin composition containing thrombin(approximately 500 units/ml) was extruded from a syringe andapplied to completely fill the tissue defect. Approximately 2minutes following the delivery of the fragmented gelatincomposition, all bleeding stopped.Spleen puncture #1 was actively bleeding followingthe surgical creation of a blunt puncture. Approximately 0.8ml of fragmented gelatin composition containing thrombin(approximately 500 units/ml) was extruded from a syringe andapplied to completely fill the tissue defect. Approximately 2minutes following the delivery of the fragmented gelatincomposition, all bleeding stopped.In the above four examples, the delivery system usedwas a 3 cc syringe (Becton Dickinson, Franklin Lakes, NewJersey). It contained the fragmented gelatin composition ofexample 1.A material according to the present invention forfilling tissue divots and other defects could be prepared asA thrombin solution (0.5 ml; 4,000 to 10,000 U/ml)is added to 4.5 ml of flowable gel to produce 5 ml of gelcontaining 400 to 1000 U/ml thrombin.follows.The gel can be used inany convenient amount, e.g. 0.5 ml to 5 ml.EXAMPLE 7: FRAGMENTED POLYMERIC COMPOSITION AS A TISSUEFILLER AND ANASTOMIC SEALANTThis study demonstrated the effectiveness of thefragmented gelatin composition as a wound closure system thatfills and seals tissue defects. Four tissue divots weresurgically induced, 1 in the lung, 2 in the liver and 1 in thespleen of a farm grade Hampshire/Yorkshire cross pig (PorkPower Farms, Turlock, CA).On the lung, following the surgical creation of thetissue divot, an air leak was observed. Approximately 1 ml ofthe fragmented gelatin composition of Example 1 was extrudedfrom a syringe and applied to completely fill the tissuedefect. A Rapiseal“ patch (Fusion Medical Technologies, Inc.,101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526231Mountain View, CA) was applied using an argon beam coagulator(Valleylab, Boulder, Colorado, or Birtcher Medical Systems,Irvine, California,). Immediate cessation of the air leakoccurred. When the applied patch was grasped with forceps, itappeared to adhere quite well to the tissue and had goodintegrity. The fragmented gelatin composition was challengedby ventilating the lung to a pressure of 28 cm water. No airleak was observed.On the liver, following the surgical creation of thetissue divot, excessive bleeding was observed. Approximately1 ml of fragmented gelatin composition was extruded from asyringe and applied to completely fill the tissue defect. Thefragmented composition swelled and adequately stopped thebleeding although some seepage bleeding was observed.On the liver, following the surgical creation of thetissue divot, excessive bleeding was observed. Approximately1 ml of fragmented gelatin composition was extruded from asyringe and applied to completely fill the tissue defect. ARapiseal” patch (Fusion Medical Technologies, Inc., MountainView, CA) was applied using an argon beam coagulator(Valleylab, Boulder, Colorado, or Birtcher Medical Systems,Irvine, California,). Immediate cessation of the bleedingoccurred. When the applied patch was grasped with forceps, itappeared to adhere quite well to the tissue and had goodintegrity.Spleen puncture #1 was actively bleeding followingthe surgical creation of a blunt puncture. Approximately 0.8ml of fragmented gelatin composition was extruded from asyringe and applied to completely fill the tissue defect.Approximately 2 minutes following the delivery of thefragmented gelatin composition, all bleeding stopped.A female juvenile farm grade goat (Clovertop Dairy,Madera, California) was used under appropriate anesthesia.The right cartoid artery was exposed. The vessel wascarefully dissected to remove any connective tissue. Thevessel was clamped using atraumatic vascular clamps, separatedby a distance of approximately 2-3 cm. The vessel wasdissected using a standard scalpel blade to expose 2 free101520253035CA 02264647 1999-02-26WO 98/08550 PCT/US97/1526232vessels ends. An end—to—end anastomosis was created using 6-Oprolene suture in an interrupted fashion. Followingcompletion of the anastomoses, the clamps were released.Bleeding was observed at the anastomotic site. Approximately2 cc of the fragmented gelatin composition containing thrombin(approximately 500 units/ml) was extruded from a syringearound the anastomoses. Gauze was placed against thecomposition. Approximately 3 minutes after the application ofthe fragmented gelatin composition, all bleeding was observedto have ceased. The incision was appropriately closed and theanimal was allowed to recover for subsequent follow—up.EXAMPLE 8: MATERIALS AND METHODS FOR PREVENTION OF POSTSURGICAL ABDOMINAL ADHESIONSThis study demonstrated the effectiveness of thefragmented gelatin composition in preventing/reducing theincidence of adhesions in the abdominal cavity when used aloneor in conjunction with the Rapisealm patch (Fusion MedicalTechnologies, Inc., Mountain View, CA)A standard animal model for evaluating surgicaladhesions has been developed using the Sprague Dawley rat(Harris, E.S. (1995) "Analysis of the kinetics of peritonealadhesion formation in the rat and evaluation of potentialanti—adhesive agents," Surgery 117:663—669). In this model, asingle, specific adhesion may be objectively measured.For this study, 15 Sprague Dawley rats were used.Anesthesia was induced with an intramuscular injection ofketamine hydrochloride in combination with xylazine.Following anesthesia and appropriate preparation for surgery,a midline was performed. A defect in the abdominal bodywallwas created approximately 1 cm lateral to the midlineincision. The defect was created by excising a 1 x 2 cmsegment of parietal peritoneum, including s superficial layerof muscle. A 1 x 2 cm defect was then created on the serosalsurface of the cecum. The cecum was abraded by scraping witha scalpel blade so that a homogeneous surface of petechialhemorrhage was created over the abraded surface. The cecumwas then elevated and positioned so that at closure, the cecumwould Contact the abdominal wall defect. The abdominal wall1015202530CA 02264647 1999-02-26wo 98/08550 PCT/US97/ 1526233defect was similarly abraded. Both abraded areas were exposedto air for 10 minutes.The following 3 experimental groups wereestablished. Each group had 5 animals.Group 1: Control/No treatment prior to closureGroup 2: Fragmented gelatin composition of Example 1Placed between abdominal wall and cecum defectsprior to closure.Group 3: Fragmented gelatin composition (of Example 1) +Rapisealm patchPlaced on cecum defect prior to closureThe midline incision was closed with 4-0 absorbablesuture and the skin was closed with 4-0 silk suture. Allanimals were recovered from surgery and observed for 7 days.At day 7 post—surgery, the rats were euthanized andthe abdomen opened to evaluate the surgically created defect.Adhesions between the abdominal wall defect and the cecumdefect, if present, were evaluated for tenacity and strengthby pulling the two tissues apart. A tensiometer was used tomeasure the force required to break the adhesions.Both the treatment with the fragmented gelatincomposition alone and with the Rapiseal patch resulted in areduction in the number of animals presenting with adhesionswhen compared to the control group. The percentage of animalsin each group that had adhesions are given in the Table 2below.'EABL£I2CHUDUP 'FREAJ%IENT' QEAUVHWAJASVVYFHADHESIONS1 Control 80%2 Fragmented gelatin composition 60%3 Fragmented gelatin composition + RapiSeal“‘ 40%patch101520253035CA 02264647 1999-02-26WO 98108550 PCT/US97/1526234EXAMPLE 9: MATERIALS AND METHODS OF ASCORBATE ADDITION TO GELPRIOR TO IRRADIATIONGelatin particles (300 Bloom, Woburn Co.,Woburn, MA) were suspended at 5%—l5% by weight in methylalcohol (Aldrich, Milwaukee, Wisconsin) containing 0.0l%—0.1%by weight glutaraldehyde (Sigma, St. Louis, MO) and stirredovernight at ambient temperature. Alternatively, gelatinparticles, obtained from an extract of calf hide(Spears Co., PA) were suspended at 5%—15% by weight in aqueousbuffer at pH 9 containing 0.01%—0.1% by weight glutaraldehyde(Sigma) to form a gel that was well—mixed and refrigeratedovernight. The cross—linked gelatin fragments were thenrinsed three times with alcohol and dried at ambienttemperature. Equilibrium swelling for the rinsed, cross-linked gelatin was then measured, and 0.5 g—l.O g portions of3.0 ml—4.5 mlof aqueous buffer containing ascorbic acid or a salt ofthis material were packed into 5 cc syringes.ascorbic acid, e.g. 0.02 M sodium phosphate (J.T. Baker,Phillipsburg, New Jersey), 0.15 M sodium chloride (VWR, WestChester, Pennsylvania), 0.005 M sodium ascorbate (Aldrich), pH7.0, was added to the syringes containing cross—linked gelatinusing a second syringe and a three-way stopcock, with caretaken not to introduce extraneous air into the syringes, toform a hydrogel within several syringes. Alternatively, anaqueous buffer that did not contain ascorbic acid or a salt ofascorbic acid but was otherwise of similar composition and pHwas added to other syringes containing cross—linked gelatin toform a hydrogel within them. The hydrogel-containing syringeswere then gamma—irradiated under refrigerated conditions at3.0 i 0.3 megarads. Equilibrium swell was measured for thehydrogel contained within the syringes after irradiation.Hydrogels that were formed using buffers that containedascorbic acid or a salt of ascorbic acid generally maintainedvalues for equilibrium swell upon irradiation within i 20%,and usually i 10%, of the value prior to irradiation, whilegels that were formed using buffers not containing ascorbic101520253035CA 02264647 1999-02-26wo 93/03550 PCT/US97/1526235acid or a salt of ascorbic acid experienced a decrease inequilibrium swell of 25-30% of its value prior to irradiation.EXAMPLE 10: MATERIALS AND METHODS OF CROSS—LINKING ANDMEASURING PERCENT SWELL.Gelatin particles were allowed to swell in anaqueous buffer (e.g., 0.2 M sodium phosphate, pH 9.2)containing a cross—linking agent (e.g., 0.005—O.5% by weightglutaraldehyde). The reaction mixture was held refrigeratedovernight and then rinsed three times with deionized water,twice with ethyl alcohol, and allowed to dry at ambienttemperature. The dried, cross—linked gelatin was resuspendedin an aqueous buffer at a low solids concentration (2—3%) atambient temperature for a fixed period of time. Buffer was inexcess of the concentration needed for equilibrium swelling,and two phases (a hydrogel phase and a buffer) were present.An aliquot of the suspension containing wet hydrogel was thenfiltered by applying vacuum on a 0.8 pm nominal cut-off filtermembrane (Millipore, Bedford, Massachusetts). After removalof extraneous buffer, the combined weight of the retained wethydrogel and wet filter membrane was recorded. The hydrogeland membrane were then dried at approximately 120°C for atleast two hours, and the combined weight of the dried hydrogelresidue and dried filter membrane was recorded. Severalmeasurements of samples of wet filter membrane withouthydrogel residue and dried filter membrane without hydrogelwere also performed and were used to deduce the net weight ofwet hydrogel and dry hydrogel. “Percent swell” was thencalculated as follows:percent swell = 100 X(wet weight of qel - dry weight of qel)dry weight of gelSwell measurements were conducted in triplicate and averagedfor a given sample of gelatin. The value of percent swell forsamples resuspended in buffer for 18 — 24 hr prior tomeasuring wet weight was defined as "equilibrium swell."The resulting cross—linked gelatin materialsdisplayed equilibrium swell values in the range from 400% to101520253035CA 02264647 1999-02-26WO 98/08550 PCT/U S97/ 1526236l300%.‘ The degree of equilibrium swell depended on the methodand extent of cross—linking.EXAMPLE ll: DEGRADATIONThirty rabbits (15 untreated control animals and 15animals treated with fragmented gelatin composition) underwentsurgery to mimic splenic injury and bleeding. A lesion on thespleen was created by making a controlled wound with a 6 mmbiopsy punch. In the "Treated" group, the experimentallycreated injury was immediately treated with the fragmentedgelatin composition to cause hemostasis of the wound."Control" group animals were not treated during the first 7.5minutes to demonstrate the amount of bleeding resulting fromthe lesion. At 7.5 minutes from the time the injury wascaused, the fragmented gelatin composition was then used tostop bleeding from the lesion to prevent spontaneousexsanguination and death of the animal. All animals wereallowed to recover. Ten animals each were euthanized on Days14 and 28 post-surgery. The final necropsy date for theremaining animals was determined after the Day 28 animals wereevaluated. In animals harvested at the Day 28 time point itwas difficult to determine via gross examination if the testmaterial was present or not, therefore half of the remaininganimals were harvested at Day 42 and the other half at Day 56.At the time of necropsy, the site of the splenic lesion andthe peritoneal cavity were evaluated macroscopically.Presence of fragmented gelatin composition in the peritonealcavity away from the site of placement was noted andevaluated, as well as its presence or absence at the spleniclesion. The presence or absence of postoperative adhesions atthe site of the splenic lesion was also evaluated and noted.The spleen was carefully dissected and processed forhistological evaluation of biocompatibility andbiodegradation.The application of the fragmented gelatincomposition to the surgically created wounds on the spleenresulted in good hemostatic tamponade. Following applicationof the fragmented gelatin composition at the time of surgery,101520253035CA 02264647 1999-02-26WO 98/08550 PCT/U S97/ 1526237rabbits were survived for 14, 28, 42, and 56 dayspostoperatively. One rabbit died of unrelated pneumonia atDay 5 postoperatively and the spleen was not harvested forhistopathological examination.At necropsy, the site of the splenic lesion as wellas the peritoneal cavity in general were evaluated grossly.Presence of the fragmented gelatin composition in theperitoneal cavity away from the site of placement wasevaluated, as well as the presence or absence of thefragmented gelatin composition at the splenic lesion. Thepresence or absence of adhesions at the site of the spleniclesion were evaluated and noted. The spleen was carefullydissected and processed for histological evaluation.Grossly, the site of the splenic lesion was visiblein all animals, at all time points. Macroscopically, thefragmented gelatin composition was absent in two of the tenDay 14 animals. At all other time points it was not possibleto identify the fragmented gelatin compositionmacroscopically. The macroscopic absence of the hydrogelmaterial as measured in this rabbit model defines thedegradation of the hydrogel as that term is used herein and inthe claims.In three of ten animals sacrificed at 14 dayspostoperatively, small amounts of the fragmented gelatincomposition were found free—floating in the abdominal cavity.This most likely represents the excess material that hadmigrated from its placement site at the splenic lesion. In nocase where this material was found away from the spleniclesion was there any evidence of tissue reaction from thevisceral surfaces or the omentum. No material was found awayfrom the site of the splenic lesion in animals that wereharvested at any other time point.No postoperative adhesions associated with thefragmented gelatin composition material were noted at the siteof the splenic lesion in any animal. In all animals, asexpected, there was omentum attached to the site of thesplenic lesion. Other adhesions involving the spleen were101520CA 02264647 1999-02-26W0 98l08550 PCT/US97/ 1526238rare, and when noted were incidental and usually associatedwith the incision of the body wall.The fragmented gelatin composition was absentmacroscopically and microscopically in two of the ten animalsfrom the 14 day time point. At 28 days post-implant, thefragmented gelatin composition was not visible on grossobservation and microscopically was completely absent in fiveout of ten rabbits examined and present in minimal amounts inthe remaining animals, showing that the fragmented gelatincomposition was composition was essentially biodegraded by 28days. The fragmented gelatin composition was completelyabsent in all five animals examined at 42 days post-implantand was found in minimal amounts in only one of four rabbitsexamined at 56 days post-implant. Healing of the splenicwound was proceeding in a normal fashion at Day 42 and more soat Day 56.Although the foregoing invention has been describedin some detail by way of illustration and example, forpurposes of clarity of understanding, it will be obvious thatcertain changes and modifications may be practiced within thescope of the appended claims.

Claims (46)

WHAT IS CLAIMED IS:

1. A fragmented polymeric composition comprising a biocompatible cross-linked hydrogel having:
a combination of two or more materials, wherein each material is selected from proteins, polysaccharides or non-biologic polymers;
a subunit size when fully hydrated in the range from 0.05 mm to 5 mm;
an equilibrium swell from 400% to 1300%; and an in vivo degradation time in a moist tissue environment in the range from 1 day to 1 year.

2. A fragmented polymeric composition comprising a biocompatible cross-linked hydrogel having:
one or more materials selected from the group consisting of proteins, polysaccharides and non-biologic polymers;
a subunit size when fully hydrated in the range from 0.05 mm to 5 mm;
an equilibrium swell from 400% to 1300%; and an in vivo degradation time in a moist tissue environment in the range from 1 day to 1 year.

3. A composition as in claim 1 or 2, wherein the hydrogel has a subunit size in the range from 0.05 mm to 1 mm and a moisture content below 20% by weight.

4. A composition as in claim 1 or 2, wherein the hydrogel has a degree of hydration in the range from 50% to 95% of the hydration at equilibrium swell.

5. A composition as in claim 1 or 2, wherein the hydrogel has a degree of hydration above 95%.

6. A composition as in any one of claims 1-5, further comprising a plasticizer selected from polyethylene glycol, sorbitol, or glycerol.

7. A composition as in claim 6, wherein the plasticizer is present at from 0.1% by weight to 30% by weight of the polymeric composition.

8. A composition as in any one of claims 1-5, further comprising an active agent selected from a protein, a carbohydrate, a nucleic acid, an enzyme, an antibiotic, an antineoplastic agent, a bacteriostatic agent, a bacteriocidal agent, an antiviral agent, a hemostatic agent, a local anesthetic, an anti-inflammatory agent, a hormone, an antiangiogenic agent, an antibody, a neurotransmitter, a psychoactive drug, an oligonucleotide, or an antisense oligonucleotide.

9. A composition as in claim 8, wherein the active agent is a hemostatic agent.

10. A composition as in claim 9, wherein the active agent is thrombin.

11. A composition as in any one of claims 1-5, wherein the cross-linked hydrogel comprises a cross-linked protein hydrogel.

12. A composition as in claim 11, wherein the protein is gelatin, soluble collagen, albumin, hemoglobin, fibrinogen, fibrin, casein, fibronectin, elastin, keratin, laminin, or derivatives thereof.

13. A composition as in any one of claims 1-5, wherein the cross-linked hydrogel comprises a cross-linked polysaccharide.

14. A composition as in claim 13, wherein the polysaccharide is selected from glycosaminoglycans, starch derivatives, cellulose derivatives, hemicellulose derivatives, xylan, agarose, alginate, chitosan, or derivatives thereof.

15. A composition as in any one of claims 1-5, wherein the cross-linked hydrogel comprises a cross-linked non-biologic polymer.

16. A composition as in claim 15, wherein the polymer is selected from polyacrylates, polymethacrylates, polyacrylamides, polyvinyl resins, polylactide-glycolides, polycaprolactones, polyoxyethlenes, or derivatives thereof.

17. A composition as in any one of claims 1-5, wherein the cross-linked hydrogel comprises at least two components selected from cross-linked proteins, cross-linked polysaccharides, or cross-linked non-biologic polymers.

18. A composition as in any one of claims 1-5, wherein the cross-linked hydrogel further comprises a cross-linking agent.

19. A method for making a polymeric composition, said method comprising:
providing a biocompatible resorbable polymer;
combining the polymer with an aqueous buffer to form a hydrogel;
cross-linking the hydrogel;
and disrupting the cross-linked hydrogel, wherein the disrupted cross-linked hydrogel has a subunit size between 0.01 and 5 mm.

20. A method for making a polymeric composition, said method comprising:
providing a biocompatible resorbable polymer;
disrupting the polymer;
cross-linking the disrupted polymer, and combining the cross-linked disrupted polymer with an aqueous buffer to form a hydrogel, wherein the disrupted polymer has a subunit size between 0.01 and 5 mm.

21. A method as in claim 20, wherein the cross-linking step comprises exposing the polymer to radiation.

22. A method as in claim 21, further comprising combining the cross-linked polymer with a stabilizer to inhibit modification of the polymer when exposed to the radiation.

23. A method as in claim 22, wherein the stabilizer is ascorbic acid, sodium ascorbate, other salts of ascorbic acid, or an antioxidant.

24. Use of the composition according to claim 1 or 2 for sealing a tissue tract.

25. Use of the composition according to claim 1 or 2 for inhibiting bleeding at a target site in a patient's body.

26. Use of the composition according to claim 1 or 2 for delivering a bioactive substance to a target site in a patient's body.

27. Use of the composition according to claim 1 or 2 for treating a patient, wherein the use is preventing surgical adhesions, drug delivery, stopping or inhibiting bleeding, filling soft and hard tissue regions, filling soft implantable devices or filling confined spaces said composition being hydrated at less than its equilibrium swell prior to application to the patient, wherein it swells to an equilibrium swell value subsequent to application to the patient.

28. A kit comprising:
the composition according to claim 1 or 2;
written instructions to apply the composition onto a target site on tissue; and a container holding the composition and the written instructions.

29. A kit as in claim 28, wherein the hydrogel is dehydrated.

30. A kit as in claim 28, wherein the hydrogel is hydrated.

31. Use of the composition according to claim 1 or 2 for treating a target site of a patient, wherein the hydrogel is suitable for extrusion through an orifice at the target site.

32. A use as in claim 31, wherein the hydrogel is suitable for extrusion through a syringe having an orifice size in the range from 0.01 mm to 5.0 mm.

33. A use as in claim 31, wherein the hydrogel has been disrupted prior to extruding the hydrogel.

34. A use as in claim 31, wherein the target site is in a tissue selected from muscle, skin, epithelial tissue, connective tissue, supporting tissue, nerve tissue, ophthalmic tissue, sense organ tissue, vascular tissue, cardiac tissue, gastrointestinal organ tissue, pleura, pulmonary tissue, kidney, endocrine glands, reproductive organs, adipose tissue, liver, pancreas, lymph, cartilage, bone, oral tissue, mucosal tissue, spleen, or abdominal organs.

35. A use as in claim 34, wherein the target site is a void region within the selected tissue.

36. A use as in claim 35, wherein the void region is selected from tissue divots, tissue tracts, intravertebral spaces, or body cavities.

37. A use as in claim 35, further comprising a barrier layer suitable for immobilizing over the void after the gel has been extruded.

38. A use as in claim 31, wherein extruding the hydrogel causes the hydrogel to fracture into subunits having a size in the range from 0.05 mm to 3.0 mm.

39. An applicator containing the composition according to claim 1 or 2, said applicator comprising:
an applicator body having an internal receptacle volume and an extrusion orifice; and a quantity of the composition in the internal receptacle.

40. An applicator as in claim 39, wherein the applicator body is a syringe having an orifice size in the range from 0.01 mm to 5.0 mm.

41. A sterile package comprising:

a container having a sealed interior; and the applicator as in either of claims 39 or 40, wherein the applicator is maintained in a sterile condition within the container.

42. A method as in claim 20, wherein the cross-linking step comprises reacting a monounsaturated and a polyunsaturated monomer.

43. The composition of claim 1 or 2, wherein the hydrogel has an in vivo degradation time in a moist tissue environment in the range from 1 day to 120 days.

44. The composition of claim 1 or 2, wherein the hydrogel has a subunit size in the range from 0.05 mm to 3 mm.

45. The composition of claim 1 or 2, wherein the hydrogel has a subunit size in the range from 0.25 mm to 1.5 mm.

46. The composition of claim 1 or 2, wherein the hydrogel has a subunit size in the range from 0.05 mm to 1 mm.