CA2223889A1 - Supplemented and unsupplemented tissue sealants, methods of their production and use - Google Patents

Abstract

This invention provides a fibrin sealant bandage or dressing, wherein said fibrin sealant may be supplemented with at least one composition selected from, for example, one or more regulatory compounds, antibody, antimicrobial compositions, analgesics, anticoagulants, antiproliferatives, antiinflammatory compounds, cytokines, cytotoxins, drugs, growth factors, interferons, hormones, lipids, demineralized bone or bone morphogenetic proteins, cartilage inducing factors, oligonucleotides, polymers, polysaccharides, polypeptides, protease inhibitors, vasoconstrictors or vasodilators, vitamins, minerals, stabilizers and the like. Also disclosed are methods of preparing and/or using the unsupplemented or supplemented fibrin sealant bandage or dressing.

Description

WO 96/40174 PCT~US96/10006 Title of the Il~v~ ;on Supplçmente-l and Unsupplemented Tissue Se~l~nt~, Methods of Their Production and Use l~ights of the United States Government in This Invention Under a Coop~,~live Research and Development Agltelll~llL belwt:el-The American National Red Cross and The U.S. Army T~ le of Dental Research, the U.S. Go~ l may have a non-exclusive, irrevocable, paid-up license in one or more embo~li"-~,lx of this invention.

0 Pield of Invention This invention is directed to unsupplemPntt~(l and supplemtont~(l Tissue Se~l~nt~ (TS), such as fibrin glue (FG), as well as to methods of their production and use. In one embo~im~ont, this invention is directed to TSs which do not inhibit full-thi~l~nPss skin wound healing. In another embo-liment this invention is directed to TSs which have been supplemPnt~l with a growth factor(s) and/or a drug(s), as well as to methods of their production and use. The particular growth factor(s) or drug(s) that is selected is a function of its use.

Background of the Invention A. Wound Healingand Growth~actors Wound healing, the repair of lesions, begins almost h~L~llLly after injury. It requires the ~,~cc~s~;ve coordil~Led function of a variety of cells and the close regulation of degradative and iege~ Live steps. The proliferation, diLr~lell~ ion and migration of cells are important biological processes which underlie wound healing, which also involves fibrin clot formation, resorption W O 96/40174 PCT~US96/10006 of the clot, tissue rt-mo~lelin~, such as fibrosis, endoth~ li7~.tion and l.P1iAli7~ti~.n Wound healing involves the form~tit n of highly vasculari ed tissue that CG.~ c uu~ lvus c~rilli-.ri.os, many active fibroblasts, and ~1..."(1~"
c~ .gen fibrils, but not the form~tion of speci~li7~ skin structures.
S The process of wound healing can be inhi~t~A by Illlo.lll)o~lastin which flows out of injured cells. Thlv~nbo~lastin cOllL~;L~ plasma factor VII to form factor X a-;Li~lor, which then, with factor V and in a complex with phospholipids and c~lcillm, co~ proLl"o~bin into thrombin. Thrombin catalyzes the release of fib~ opep~ides A and B from fibrinogen to produce fibrin monomers, which ag~r~ga~e to form fibrin ~llaments. Thrombin also activates the transgl~ e, factor XIIIa, which catalyzes the formation of isopeptide bonds to covalently cross-link the fibrin fil~m~nt~. Alpha2-antiplasmin is then bound by factor XIII onto the fibrin fil~m~nt~ to thereby protect the fil~mPnt~ from degradation by plasmin (see, for example, Doolittle et al., Ann. Rev. Biochem. 53:195-229 (1984)).
When a tissue is injured, polypeptide growth factors, which exhibit an array of biological activities, are released into the wound where they play a crucial role in healing (see, e.g., Hormonal Proteins and Peptides (Li, C.H., ed.) Volume 7, ~c~ mic Press, Inc., New York, N.Y. pp. 231-277 (1979) and Brunt et al., Biotechnology 6:25-30 (1988)). These activities include recl.~iLillg cells, such as leukocytes and fibroblasts, into the injured area, and inducing cell proliferation and dirrerellLiation. Growth factors that may participate in wound healing include, but are not limited to: platelet-derived growth factors (PDGFs); insulin-binding growth factor-1 aGF-1); insulin-binding growth factor-2 (IGF-2); epidermal growth factor (EGF); Ll~rc~lll~i~g growth factor-a (TGF-oc); Ir~sr~ g growth factor-,B (TGF-~); platelet factor 4 (PF-4); and heparin binding growth factors one and two a~BGF-l and ~,~
HBGF-2, respectively). ~,PDGFs are stored in the alpha granules of circ~ ting platelets and are released at wound sites during blood clotting (see, e.g., Lynch et al., J. Clin.

_ Invest. 84:640 646 (1989)). PDGFs include: PDGF; platelet derived angiogenesis factor (PDAF); TGF4; and PF 1, which is a C~ . .O~ CI;~ . ~I for llt;ulluphils (~nighton et al., in Growth Factors ancl Other Aspects of Wound Healing: Biological and Clinical Im~lications, Alan R. Liss, Inc., New York, S New York, pp. 319-329 (1988)). PDGF is a mitogen, ~hl~m~,.ll.,.rls.,~l and a stim~ tor of protein ~y~ e~is in cells of mesenchymal origin, inr~ in~
fibroblasts and smooth muscle cells. PDGF is also a no~iLogenic ch~mo~t~ractant for endothelial cells (see, for example, Adelmann-Grill et al., Eur. J. Cell Biol. 51:322-326 (1990)).
IGF-1 acts in combination with PDGF to promote mitogenesis and protein synthesis in m~osenrllymal cells in culture. Application of either PDGF
or IGF-1 alone to skin wounds does not enh~n~e healing, but application of both factors together appears to promote connective tissue and epithelial tissuegrowth (Lynch et al., Proc. Natl. Acad. Sci. 76: 1279-1283 (1987)) .
TGF-,B is a ~~h~mo~ll . dcL~ll for macrophages and monocytes. D~endillg upon the presence or absenre of other growth factors, TGF-~ may stim~ t~ or inhibit the growth of many cell types. For example, when applied in vivo, TGF-,B increases the tensile ~ ~ of healing dermal wounds. TGF-~ also inhibits endothelial cell mitosis, and stim~ tes collagen and glycos~minoglycan synthesis by fibroblasts.
Other growth factors, such as EGF, TGF-a, the HBGFs and osteogenin are also important in wound healing. EGF, which is found in gastric se~
and saliva, and TGF-a, which is made by both normal and L-a~r~ .~ed cells, are structurally related and rnay recognize the same l~cepL~ . These lcce~ul~
m~ te proliferation of epithelial cells. Both factors accelerate reepith~ li7~rinn of skin wounds. Exogenous EGF l,loll,oLes wound healing by stimlll~ting the proliferation of keratinocytes and dermal fibroblasts (Nanney et al., J. Invest. Dermatol. 83:385-393 (1984) and Coffey et al., Nature 328:817-820 (1987)). Topical application of EGF accclc.al~s the rate of healing of partial ~ s~ wounds in humans (Schultz et al., Science 235:350-352 (1987)). Osteogenin, which has been purified from ~ "ii~-,.li7PA bone, appears to promote bone growth (see, e.g., Luyten et al., J. Biol. Chem.
264:13377 (1989)). In ~r1-1ition, platelet-derived wound healing formula, a platelet extract which is in the form of a salve or ~ for topical applir~tinn, has been ~l~s~-~ ;1 ed (see, e.g., Knighton et al., Ann. Surg. 204:322-330 (1986)).
The Heparin Binding Growth Factors (HBGFs), also known as Fibroblast Growth Factors (FGFs), which include acidic HBGF (aHBGF also known as HBFG-l or FGF-l) and basic HBGF (bHBGF also known as HBGF-2 or FGF-2), are potent mitogens for cells of mesodermal and neuroecto~l~rm~l lineages, including endothelial cells (see, e.g., Burgess et al., Ann. Rev.
Biochem. 58:575-606 (1989)). In addition, HBGF-l is chemotactic for endothelial cells and astroglial cells. Both HBGF-l and HBGF-2 bind to heparin, which ploLe~;L~ them from proteolytic degradation. The array of biological activities e~LhihiLed by the HBGFs suggests that they play an important role in wound healing.
Basic fibroblast growth factor (FGF-2) is a potent stim~ tor of angiogenesis and the migration and proliferation of fibroblasts (see, for example, Gospodalowic~ et al., Mol. Cell. Endocinol. 46: 187-204 (1986) and Gospod~ wicz et al., Endo. Rev. 8:95-114 (1985)). Acidic fibroblast growth factor (FGF-l) has been shown to be a potent angiogenic factor for endothelial cells (Burgess et al., supra, 1989). However, it has not been established if anyFGF growth factor is chemotactic for fibroblasts.
Growth factors are, therefore, potentially useful for specifically promoting wound healing and tissue repair. However, their use to promote wound healing has yielded illc~ results (see, e.g., C:arter et al., in Growth Factors and Other Aspects of Wound Healing: Biological and Clinical Implications, Alan R. Liss, Inc., New York, New York, pp. 303-317 (1988)).
For example, PDGF, IGF-l, EGF, TGF-~, TGF-~ and FGF (also known as HBGF) applied sep~lalely to ~nd~di~d skin wounds in swine had little effect WO 96/40174 PCTrUS96/10006 on the L~gP~ n of comle~ilive tissue or epith~ lm in the wounds (Lynch et al., J. Clin. Invest. 84:640-646 (1989)). Of the factors tested, TGF-,B
stim~ tecl the ,~leal~ re~onse alone. However, a c~ubi~Lion of factors, such as PDGF-bb hnmn-lim~r and IGF-l or TGF-a produced a dldll,alic increase in c~l~"ecliv~ tissue lcge~ ion and epithPli~ tirn (Id.) Tsuboi et al. have Ic~ull~d that the daily application of bFGF to an open wound stim~ teA wound healing in healing-impaired rnice but not in normal _ice (J.
Ejcp. Med. 172:245-251 (1990)). On the other hand, the application to hu~n skin wounds of crude ~r~alalions of porcine or bovine platelet Iysate, which ple;,u~ably contained growth factors, increased the rate at which the wounds closed, the ~ nbel of cells in the healing area, the growth of blood vessels, the total rate of collagen deposition and the SLI~ lI of the scar tissue (Carter et al., supra).
The reasons for such incon~i~tent results are not known, but _ight be the result of ~iffirlllty in applying grow~ factors to a wound in a llla~. l in which they can exhibit their normal array of biological activities. For example,it appears that some growth factor Iec~lols must be occupied for at least 12 hours to produce a m~xim~l biologic effect (Presta et al., Cell Regul. 2:719-726(1991) and Rusnati et al., J. Cell Pkysiol. 154:152-161 (1993)). Rec~llse of such inco~ r"l results, the role played by exogenously applied growth factors in stim~ ting wound healing is not clear. Further, a means by which growth factors m-ight be applied to wounds to produce prolonged contact between the wound and the grow~ factor(s) is not pI~i,ellLly known.

B. TSs Surgical adhesives and TSs which contain plasma ~l'oL~ S are used for sealing intPrn~l and external wounds, such as in bones and skin, to reduce blood loss and m~int~in h~most~ . Such TSs contain blood clotting factors and other blood l~roteills. FG, also called fibrin sealant, is a gel similar to a natural clot which is ~ aIed from plasma. The precise components of each W O 96/40174 PCTrUS96/10006 FG are a function of the particular plasma fraction which is used as a bLalLi"g material. Fr~cti-n~tinn of plasma components can be errecl~d by ~L~daLd protein pllrifir~tion methods, such as ethanol, polyethylene glycol, and ~mmonillm sulfate ~le~ iLdLion, ion ~xr1~ ,e, and gel filtration cl~.o",dlography. Typically FG cont~in~ a ~ Lu~e of proteins inr11l(1ing traces of albumin, fibrolle-;Li n and pl~eminogen. In C~n~ , Europe and possibly elsewhere, commercially available FG typically also contains a~roLi~ as a stabilizer.
FGs generally are ~r~al-,d from: (1) a fibrinogen conce,lL,dte, which contains fibronectin, Factor XIII, and von Willebrand factor; (2) dried human or bovine thrombin; and (3) calcium ions. Commercially p~ ,d FGs generally contain bovine components. The fibrinogen concentrate can be prepared from plasma by cryopreci~iLdtion followed by fractionation, to yield a composition that forms a sealant or clot upon mixture with thrombin and an a~;LivdLol of LLollll,ill such as calcium ions. The fibrinogen and thrombin collr~ . dL~;s are prepared in lyophili7ed form and are mixed with a solution ofcalcium chloride immrAi~t~ly prior to use. Upon mixing, the collll)ollellL~ are applied to a tissue where they coagulate on the tissue surface and form a clot that includes cross-linked fibrin. Factor XIII, which is present in the fibrinogen concentrate, catalyzes the cross-linking.
Australian Patent 75097/87 describes a one-component adhesive, which contains an aqueous solution of fibrinogen, factor XIII, a LLo"lbill inhibitor, such as allLiL~ olllbin m, l~lotlll~lllbill factors, calcium ions, and, if n.ocece~
a plasmin inhibitor. Stroetm~nn, U.S. Patent Nos. 4,427,650 and 4,427,651, describes the ple~aldLion of an enriched plasma derivative in the form of a powder or sprayable ~le~aldLion for enh~nred wound closure and healing that contains fibrinogen, thrombin and/or l"oll"o,l,bi l, and a fibrinolysis inhibitor, and may also contain other hl~,l~iiellL~, such as a platelet extract. Rose et al., U.S. Patent Nos. 4,627,879 and 4,928,603, disclose methods for plC~illg cryopreci~iLaL~d suspensions that contain fibrinogen and Factor XIII and their ~, use to pl~al~ a FG. JP 1-99565 ~ closes a kit for the ~r~ n of fibnn a&esives for wound healing. Al~lJa~ (U.S. Patent No. 4,714,457) and Morse et al. ~U.S. Patent No. 5,030,215) ~ rlose mPtho-l~ to produce autologous FG. In ~ iti~n~ ....~,uv~d FG delivery ~y~t~ s have been ~i~closetl els~;wllc;l~, (Miller et al., U.S. Patent No. 4,932,942 and Morse et al., PCT Application WO 91/09641).
IMMUNO AG (Vienna, Austria) and BEHRINGWERKE AG
(Ge. lll, lly) (Gibble et al., Tran~sion 30:741-747 (1990)) presently have FGs on the market in Europe and cls~whel-, (see, e.g., U.S. Patent Nos. 4,377,572 and 4,298,598, which are owned by IMMUNO AG). TSs are not commercially available in the U.S. However, the American National Red Cross and BAXTER/HYLAND (Los Angeles, CA) have l~cellLly co-developed a FG (ARC/BH FG) which is now in clinical studies.
The TSs which are used clinically outside of the U.S. pose certain clinical risks and have not been a~lov~d by the Food and Drug ~ tion for use in the USA. For example, the TSs available in Europe contain ~roL~s of non-human origin such as a~ Li ~i l and bovine thrombin. Since these ploL~ s are of non-human origin, people may develop allergic reactions to them. In Europe heat i~;~ivaLion is used to inactivate viruses which may be present in the collly~ L~ of the FG. However, this heat inactivation method may produce del~Lu,~,d plotei.~s in the FG which may also be allergenic. In addition, there is Collcelll that this ilLacLiv~Lion method will not inactivate prions which cause bovine spongiform encephalopathy, "mad cow disease, " which may be present in the TS due to the use of bovine proteills therein. Since this disease appears to have already crossed from sheep, in which it is called "scrapies," to cows, it is not an in.~ignifi~.~nt concern that it could infect hllm~n~ .
The ARC/BH FG has advantages over the TSs available in Europe because it does not contain bovine proteins. For example, the ARC/BH TS
contains human Lhl~Jlllbh~ instead of bovine thrombin and does not contain ~loLil~in~ Since the ARC/BH FG does not contain bovine ploLeills it should be less all~ ic in humans than those TSs available in Europe. In addition, the ARC/BH FG is virally illa.;Li~aL~d by a solvent d~L~lgcllL method which produces fewer de~Lured pr~Lcll~s and thus is less allergenic than those available in Europe. The,~Çoç~, the ARC/BH FG possesses advantages over the TSs which are now colllllle,.;idlly available in other cuullLlies.
FG is primarily formnl~t~A for clinical topical application and is used to control bleeding, m~int~in hpm()stA.~ic and promote wound healing. The clinical uses of FG have rect;lllly been reviewed (Gibble et al., Transfusion 30:741-747 (1990); Lerner et al., J. Surg. Res. 48:165-181 (1990)). By sealing tissues FG pl~v~ air or fluid leaks, induces h~omost~ , and may contribute to wound healing i ldil~;Lly by reducing or preventing events which may ~-lL~,lr~le with wound healing such as bleeding, h~".~l-.",~, infections, etc.
Although FG IllAill~ ; h~mnstAci~ and reduces blood loss, it has not yet been shown to possess true wound healing properties. Rec~nse FG is suitable for both internal and exttornAl injuries, such as bone and skin injuries, and is useful to ,,.~;.,l;.;,, h~mostA~ic, it is desirable to çl~hAI~l~e its wound healing properties.
FG with a fibrinogen conc~llLlaLion of approximately 39 g/l and a thrombin collccllLl~Lion of 200~00 U/ml has produced clots with significantly increased stress, energy absorption and elasticity values (Byrne et al., Br. J.
Surg. 78:841-843 (1991)). P~l~laL~d Teflon cylinders filled with fibrin clot (S mg/ml) and implanted subcutaneously stimnl~t~l the form~tion of gr~n--l~tinn tissue, including an increased ~,rcci~iL~Lion of collagen, when colll~ared to empty cylinders (~e~lelin et al., Eur. Surg. Res. 15:312 (1983)).

C. Bone Wounds and Their Repair The seq~en~e of bone induction was first described by Urist et al. using (1r~ll;.,..,dlized cortical bone matrix (Clin. Orthop. Rel. Res. 71:271 (1970) and Proc. Natl. Acad. Sci. USA 70:3511 (1973)). Implanted subcutaneously in allogeneic recipients, ~lemintqralized cortical bone matrix releases factors which -W O 96/40174 PCT~US96/10006 act as local ll~ilogc~ to stimnl~t~ the prolif~,r~tiQn of m~s~ y..~al cells (Rath et al., Nature a,ond.) 278:855 (1979)). New bone foTm~ti~n occurs b~,L~
12 and 18 days po~l;."l l~nt~ti-n. Ossicle development replete with llc~c~oietic lllalrvw lineage o~;ull~d by day 21 ~Reddi, A., ~ tr~7re~lu~7r Matrix Biochemistry (Piez et al., ed.) Elsevier, New York, NY, pp. 375-412 (1984))-D~ ,lli7~1 bone matrix (DBM) is a source of osteoinductive ~ Leil~s known as bone morphogenetic plo~il s (BMP), and growth factors which modulate the proliferation of progenitor bone cells (see, e.g., ~llcchk~ et al.,0 J. Biol. Chem. 261:12665-12674 (1986) and Canalis et al., J. Clin. lnvest.
81:277-281 (1988)). Eight BMPs have now been irlPntifi~A and are abblevia~d BMP-l through BMP-8. BMP-3 and BMP-7 are also known as osteogenin and osteogenic protein-1 (OP-l)"es~c~Lively.
U-lro,lu-~l~ly, DBM materials have little clinical use unless combined with particulate nl~llow autografts. There is a limit to the ~lualllily of DBM
that can be surgically placed into a recipient's bone to produce a therapeutic effect. In addition, resorption has been reported to be at least 49% (Toriumi et al., Arch. Otolaryngo. Head Neck Surg. 116:676-680 (1990)).
DBM powder and osteogenin may be washed away by tissue fluids before their osteoinductive puLcll~ial is c~-cssed. In addition, seepage of tissue fluids into DBM-packed bone cavities or soft-tissue collapse into the wound bed are two factors that may ~ignifil~ntly affect the osteoinductive p~e-lies of DBM and osteogenin. Soft-tissue collapse into the wound bed may likewise inhibit the proper migration of osteocompetent stem cells into the wound bed.
Human DBM in powder form is ~;ul~ Lly used by ~ dentists to pack jaw bone cavities created during oral surgery. However, DBM in powder form is rliffirl~lt to use.
Purified BMPs have o~L~oil- lu~ e effects in animals when delivered by a variety of means including FG (Hattori, T., Nippon. Seikeigeka. Galdcai.
Zasshi. 64:824-834 (1990); Ka~ etal., Clin. Orthop. Rel. Res. 235:302-310 (1988); Schlag et al., Clin. Orthop. Rel. Res. 227:269-285 (1988) and Schwarz et al., Clin. Orthop. Rel. Res. 238:282-287 (1989)) and whole blood clots (Wang et al., J. Cell. Biochem. 15F:Q20 Abstract (1990)). However, Schwarz et al. (supra.) ~ "-.. ,x~ ~ At' cl neither a clear positive or L~alive effect of FG on ectopic osteoinduction or BMP-dependent o~l~ol~eLclalion.
Kaw~llula et al. (supra.) found a ~yLI~ istic effect when partially purified BMP in FG was tested in an ectopic non-bony site. Th~l~,fo~, these results are inco-,~ixle-.l and co- rùsiug.
TS also can serve as a "scaffold" which cells can use to move into a wounded area to generate new tissues. However, commercially available pl~ ion~ of FG and other TSs are too dense to allow cell migration into and through them. This limits their err~cli~ ess in some in vivo uses.
In one type of bone wound, called bone nonunion defects, there is a minim~l gap above which no new bone form~tion occurs naturally. Clinically, the Llr;t~ for these ~ io--~ is bone grafting. However, the source of bone autografts is usually limited and the use of allogeneic bones involves a high risk of viral co-.~ ion. Because of this .cibl~tion, the use of cl~.-.in~lalized, virally inactivated bone powder is an attractive solution.

D. Vascular Prostheses Artificial vascular prostheses are frequently made out of polylel~ oroethylene (PTFE) and are used to replace (lice~ed blood vessels in hllm~n~ and other animals. To m~ximi7~ patency rates and ~ the thrombogenicity of vascular prostheses various techniques have been used including seeding of nonautologous endothelial cells onto the prothesis.
Various substrates which adhere both to the vascular graft and endothelial cellshave been investi~te~l as an intermerli~te substrate to increase endothelial cell see~1ing. These substrates include preclotted blood (Herring et al., Surgery 84:498-504 (1978)), FG (RoseL~ et al., J. Vasc. Surg. 2:778-784 (1985);
Schrenk et al., Thorac. Cardiovasc. Surg. 35:6-10 (1986); Koveker et al., WO 96/40174 P ~ nJS96/10006 Thorac. Cardiovasc. Surgeon 34: 49-51 (1986) and Zilla et al., Surgery 105:515-522 (1989)), rll,lulle~iLil. (see, e.g., Kesler et al., J. Vasc. Surg. 3:58-64 (1986); l~ r~k et al., J. CeU Physiol. 116.76-86 (1983) and ~m~l~njeona et al., J. Vasc. Surg. 3.264-272 (1986)), or collagen (Williams et al., J. Surg.S Res. 38:618-629 (1985)). However, one general problem with these t~lmi~e~
is that ~ f)logous cells were used for the seeding (see, e.g., Schrenk et al., supra) thus raising the possibility of tissue rejection. In addition, a confluent endothelium is usually never established and requires months to do so if it is.
As a result of this delay, there is a high occlusion rate of vascular pr~ sth~ses (see, e.g., Zilla et al., supra).

E. Ang:iogenesis Angiogenesis is the induction of new blood vessels. Certain growth factors such as HBGF-l and HBGF-2 are angiogenic. However, their in vivo ~l",i"i~l-ation ~tt~eh~l to: collagen sponges (Thompson et al., Science 241:1349-1352 (1988)); beads (Hayek etal., Bioc~em. Biop~Tys. Res. Commun.
147:876-880 (1987)); solid PTFE fibers coated with collagen arranged in a sponge-like structure (Thompson et al., Proc. Natl. Acad. Sci. USA 86:7928-7932 (1989)), or by illrUSi~ (Puumala et al., Brain Res. 534:283-286 (1990)) resulted in the ~ e~ Lion of r~nrlom, diso.gani~d blood vessels. These growth factors have not been used sllcce~r~lly to direct the growth of a new blood vessel(s) at a given site in vivo. In addition, fibrin gels (0.5-10 mg/ml) implanted subcutaneously in plexi~ s chanlbel~ induce angiogenesis within 4 days of implantation, compared to empty chambers, or chal,.bcl~ filled with sterile culture medium (Dvorak et al., Lab. Invest. 57:673 (1987)).

2~ F. Site-Directed, Locn~iz~1Dru~Delivery ,, An efficacious, site-directed, drug delivery system is greatly needed in several areas of medicine. For example, localized drug delivery is needed in the treatment of local infections, such as in periodontiti~, where the systemic W O 96/40174 PCTrUS96/10006 ?~imini~tration of ~ ial agents is in~rr~;live. The problem after sy~L~ll~ic ~ aLion usually lies in the low col-r~ .n~;on of the ,robial agent which can be aclliev~d at the target site. To raise the local c~ )n a systemic dose hl ;l.,ase may be err~iLiv~ but also may produce toxicity, microbial lesi~ e and drug hlcoLu~àLibility. To Cil~;uulv~ some of these problems, several ~11... ,.,.l;v~ methods have been devised but none are ideal. For example, collagen and/or fibrinogen dispersed in an aqueous mP(1illm as an amorphous flowable mass, and a proLeillaceous matrix composition which is capable of stable placement, have also been shown to locally deliver drugs (Luck et al., U.S. Reissue Patent 33,375; Luck et al., U.S. Patent 4,978,332).
A variety of antibiotics (AB) have been reported to be released from FG, but only at relatively low conce,lLldLions and for relatively short periods of time ranging from a few hours to a few days (Kram et al., J. Surg. Res.
50:175-178 (1991)). Most of the ABs have been in freely water soluble forms and have been added into the TS while it was being prepared. However, the incorporation of tetracycline hydrochloride tetracycline hydrochloride (TET HCl) and other freely water soluble forms of ABs into FG has i~ ,rt;,ed with fibrin polymerization during the formation of the AB-supplemPntP-(l FG
(Schlag et al., Biomaterials 4:29-32 (1983)). This hlLe~rtlc;llce limited the amount and col~ellL~alion of the TET HCl that could be achieved in the AB-FG
mixture and appeared to be AB concentration dependent. The relatively short release time of the AB from the FG may reflect the relatively short life of the AB-supplemented TS or the form and/or ~lu~lLiLy of the AB in the AB-TS.

G. Controlled DrugRelease Prom TSs For some clinical applications controlled, localized drug release is desirable. As ~ cll~se~l above, some drugs, especially ABs, have been ., incorporated into and been released from TSs such as FG. However, there is little or no control over the duration of the drug release which a~pal~llLly is at W O 96/40174 PCT~US96/10006 least partially a reflPrtion of the relatively short life of the drug-suppleln~nte~
FG. Tl~ r(J~c, a means to stabilize FG and other TSs to allow for elrt~n-lP-l loc~li7P~l drug release is desirable and n~e~lecl, as are new techniques for theinCol~old~ion and e~ten~ 1 release of other supplen-Pnt~ from TS.

SH. The Disclosed TS r~u,~ions Provide Life-Saving Emergency Trent~n~nt for Trauma Wounds Despite contin~ ad~ces in trauma care, a si~nifir~nt percentage of the population, both military and civilian, suffer fatal or severe hemorrhage every year. An ~l~rming number of f~t~liti~s are preventable since the occur 10in the plcse~ce of those who could achieve life-saving control of their woundsgiven ~ qll~tP tools and training. The availability of the herein-disclosed TS
s~ticfies the long-felt need for a advanced, easy-to-use, field-ready hemostaticala~ion, to permit not only trained mP-1iral personnel, but even untrained individuals to rapidly reduce bleeding in trauma victims. Utilization of the 15disclosed TS p,~ld~ions will result in a two-fold benefit: the reduction of trauma death, and the decreased ~ nrl upon the available blood supply.
The disclosed technology would also be available for the tre~tment of massed c~m~lties in disaster situation. When severe natural or man-made ters occur, local hospitals and clinics may be overwhelmed by the number 20of individuals requiring trauma care. Combined with the isolating effects of such disas~ s, the resulting ~iern~n~ for blood and blood products often excee~l~
the locally available supplies. In many cases, the ~ rn~n~l upon the local mtor1ir~1 personnel also exceeds the availed number of trained individuals. As a result, less seriously injured persons may be turned-away or given sub-25optimal care. The availability of the easy-to-use, self-cont~inecl TS
cpdl~ions disclosed below will permit local mtorli- ~l personnel and disaster relief w~lk~l~ to provide the injured with temporary tre~tment until dernli~ivt;

W O 96/40174 PCTrUS96/10006 care bec~.---Ps available. Mo-co~., the disclosed TS prcp~alions will permit self~ in di as~ victims, until mPt~ e can be provided.
Often the only form of mP~lir~ llllk-ll that can be applied under such ci~ es to ~ .lt death due to blood loss is pl~s~.ule dl~S~ ,S, tollrniq~letc and ~ .UlG points. U~ultul~l~ly, however, each of these L-e~ ; requires continuous molliLuli~ and attention. Since such attention is not always possible in e ~ ,e~;y or disaster citll~tir.n~, there is a clear need in the art for a simple, fast-acting, first-aid L,e~l...kl.l which can sn~cçssfillly control excessive blood loss.
The application of the disclosed TS preparations to the military is readily ~p~clll, particularly in isolated battlefield situations. The single glcdle~.~ cause of death on the battlefield is e~s.ànguilldlion~ which until nowhas accounted for up to 50% of all combat c~cu~ltiPs.

Summary of the Invention In one embodiment, t'nis invention provides a composition of matter, co~ g a TS, wll~l~ill the sealant does not inhibit full-ll.;rL .. ~ skin wound healing.
In another embodiment, this invention provides a colll~osilion of matter, coll~lisillg: a TS, whclei~l the total protein concell~ldlion of the sealant is less than 30 mg/ml.
In another embodiment, this invention provides a composition of matter comprising a supplemented TS wheleill the total protein concentration is less than 30 mg/ml and the supplement is a growth factor(s) and/or a drug(s).
In another embodiment, this invention provides a composition of rnatter col--~lisillg a supplpmf~ntp-d TS wl-~ll;i-- the total protein conce--~l~Lion is greater than 30 mg/ml and the supplement is a growth factor(s) andior a drug(s).
In another emb~limPnt this invention provides a composition of matter that promotes the directed migration of animal cells, COlll~liSill~,; a TS; and an WO 96140174 PCT~US96/10006 t;rr~;Live co~r~ m of at least one growth factor, Wh~ ll the collceLlLlaLion of the growth factor is ~rr~live in ~)lU IuLillg the directed migration of the animal cells.
In a~uLllel embo~limPnt~ the present invention provides a co~ osiLion of matter that promotes wound healing, co~ l;si~lg: a TS; and an t rr~liv~
conce~ lio~ of at least one growth factor, wherein the col~cellll~tion is effective in promoting wound healing.
In another embo-1im~nt the present invention provides a composition of matter that promotes the endothPli~li7~tion of a vascular l)ro~lhPsis~
comprising: a TS; and an trr~liv~ collcenLl~Lion of at least one growth factor, wherein the concellLl~lion is effective in promoting the endothPli~li7~ti~ n of a vascular ~lo~lllesis.
In another embo~limpnt~ the present invention provides a composition of matter that ~lullluL~S the proliferation and/or dirrel~ Liation of animal cells, colll~lisillg: a TS; and an eLr~;Live col.~ dLion of at least one growth factor,wherein the concentration is effective in ~rollloLillg proliferation and/or dirrer~llLiation of animal cells.
In another embodiment, the present invention provides a composition of matter that promotes the localized delivery of at least one drug, CollllJliSillg:
a TS; and at least one drug.
In another embo-1imPnt, the present invention provides a composition of matter that promotes the localized delivery of at least one growth factor, comprising: a TS; and at least one growth factor.
In another embodiment, the present invention provides a process for promoting the healing of wounds, COlll~liSillg applying to the wound, a composition that contains a TS and an erre~;Liv~ concellLlaLion of at least one growth factor, wh~c;ill the co~ is err~,;Live to promote wound healing.
In another embodiment, the present invention provides a process for l~rulllo~ g the endothP~ li7~tion of a vascular prosthesis, collll,lisillg applying to the vascular prosthesis a composition that contains a TS and an effective conrent~titm of at least one growth factor, W~ cill the collcellLldLion is crre~;livc to promote the endoth~ li7~tion of a vascular prothesis.
In anoLll~r embo~1imrnt~ the present invention provides a process for promoting the proliferation and/or dirr~"lLiaLion of animal cells, colllyli~ing S placing the cells in ~.. rrir~ yro~Lill~ily to a TS which contains an errcetivc concentration of at least one growth factor, wll~,lelll t_e collcellLlaLion is effective in promoting the proliferation and/or dirr~rellLiaLion of the cells.
In a further embotlim~nt~ the present invention provides a process for the localized delivery of at least one drug to a tissue, colll~lisillg applying to the tissue a TS which contains at least one drug.
In anot_er embo-1imrnt the present invention provides a process for t-h-e localized delivery of at least one growth factor to a tissue, COlll~liSillg applying to the tissue a TS which contains at least one growth factor.
In another embodimrnt, this invention provides a process for producing the directed migration of animal cells, comprising: placing in sufficient yLO~ill~iLy to the cells, a TS which cGIlL~ins an erree~iv~ collcel,Ll~tion of at least one gro-wth factor, wh~r~ the c~ r~ ;on is effective to produce the desired directed migration of said cells.
In another embo~limrnt this invention provides a simple to use, fast acting, field-ready fibrin bandage for applying a tissue sealing composition to wounded tissue in a patient, COlllyli~ g an occlusive backing, aff~ed to which is a layer of dry materials cc,Lu~lising an err~c~ive amount, in colllbi~aLion, of (a) dry, virally-inactivated, purified fibrinogen, (b) dry, virally-inactivated,purified thrombin, and as n.ocess~ry (c) effective amounts of calcium and/or Factor XIII to produce a tissue-sealing fibrin clot upon hydration.
In a further emboriim~nt this invention provides a method of treating wounded tissue in a patient by applying to said wound a fibrin bandage, com~ ing: (1) a occlusive backing, affixed to which is a layer of dry materials CUll~ illg an ~rr~Liv~ amount, in combination, of (a) dry, virally-inactivated, purified fibrinogen, (b) dry, virally-inactivated, purified thrombin, and as , W ~ 96140174 PCTrUS96/10006 nPcec~Ary (c) errG~;livG A.l..~....lx of cAlrjl~m and/or Factor XIII to produce a tissue-sealing fibrin clot upon hyt1r~tinn In yet another embo~1imP-nt~ this invention provides a simple to use, fast acting, field-ready fibrin drGssiLIg for treating wounded tissue in a patient, is S form~ tt~rl as an ~ AI~lA~le foam COlll~ lllg an GCr~;Live ~monnt, in combination, of (1) virally-il,a~;livatGd, purified fibrinogen, (2) virally-inactivated, purified llllol~lbin, and as n~A. y (3) cAlr-illm and/or Factor XIII;
wh~,leill said composition does not signifir~ntly inhibit fUll-t~irknPss skin wound healing.
While in a further embo-limPnt this invention provides a method of Ll~dl.illg wounded tissue in a patient by applying to said wound a tissue sealant ~xl,A.,~lAhle foam dressing, col~lplisillg an GrrG~:~ivG Am~llnt in colllbillation, of (1) virally-inactivated, purified fibrinogen, (2) virally-inactivated, puriffed thrombin, and as ntocç~sAry (3) calcium and/or Factor XIII; wl.cLGi.l said composition does not .~i nifirAntly inhibit full-thir'rn~ss skin wound healing.
In the embodiments of this invention, the TS may be FG.
In the various embodiments of the invention FG may be made from the mixing of topical fibrinogen complex (TFC), human thrombin and calcium chloride. Varying the co.,r~ .alion of the TFC has the most .~ig~irir,A~I effectupon the density of the final FG matrix. Varying the concGl~ lion of the .Jmb~ll has an in~ig~;rirAIll effect upon the total protein concGnll_lion of thefinal FG, but has a profound effect upon the time required for the polymeri_ation of the fiblil1ogell component of the TFC into fibrin. While this effect is well known, it is not generally a~leciatGd that it may be used to mA~imi7~ the effectiveness of the FG, when it is used alone or supplennPntP~
Because of this effect one can alter the time bGIweGll the mixing of the FG
components and the setting of the FG. Thus, one can allow the FG to flow more freely into deep crevices in a wound, ~ g it to fill the wound completely before the FG sets. ~ AIivGly, one can allow the FG to set quickly enough to ~lGvel,L it from exiting the wound site, especially if the wound is leaking fluid under pl~S~ul~, (i.e., blood, lymph, intercellular fluid,etc). This ~>lu?c.ly is also i)oll~l to keep the FG from clogging delivery devices with long p~s,.~s, i.e., ci1~h~lr~ ~, Pnrioscopes, etc., which is i~u~
to allow the application of t_e FG or suppl~ PA FG to sites in the body that S are only ~rcessible by surgery. This effect is also important in keeping the insoluble supplements in ~ e~l~ioll and ~lC~ ~g them from settling in the applicator or in the tissue site.
As used herein, TFC is a lyophili7Pd ~--~ of human plasma ,uroL~ lls which have been purified and virally inactivated. When reco~ u~ed TFC
contains: Total Protein: 100-130mg/rnl Fibrinogen: (as clottable protein) 80% of total protein Albumin (Human): 5-25 mg/ml pl~minngen: S mg/ml Factor XIII: 1040 Units/ml Polysorbate-80: 0.3% (m~xim-lm pH: 7. 1-7.5.
The reco-.~ TFC may also contain trace amounts of fiblolleclill.
As used herein, human thrombin is a lyophiii7~ lul~ of human plasma proteins, which have been purified and virally inactivated. When recol~ l it contains:
Thrombin Potency: 300 ~t 50 International Units/ml Albumin (~nm~n): S mg/ml Glycine: 0.3 M i~ .05 M
pH: 6.5-7.1.
Calcium chloride is added in sufficient collc~ aLion to activate the Lhlolllbill. As long as there is ~urrlciel.~ calcillln, its concentration is notimportant.
In the compositions of this invention cont~ining a growth factor, the composition may contain an inhibiting colll~u~llld(s) and/or po~e,.l;~i-.g WO 96~4al74 PCTAUS96/10006 c~,lll~o-L~d(s), whtlei~ the inhil)ili~g compound(s) inhibit the activities of the sealant that illL~r~lG with any of the biological activities of the growth factor, the polP..~ compound(s) poLt;llLiate, mP~ te or enh~nre any of the biological ~;LiviLi~s of the growth factor, and wllcl~eill the co~cellLl~Lion of the S ir~ ing or ~u~ ;n~ co~ uulld is effective for achieving the inhibition, potentiation, mp~i~tiQn or e.-h~ t The growth factor-supplP~mpntp~1 TSs of this invention are useful for promoting the healing of w~ ds, especially those that do not readily heal, such as skin ulcers in diabetic individuals, and for delivering growth factors including, but not lirnited to, FGF-1, FGF-2, FGF4, PDGFs, EGFs, IGFs, PDGF-bb, BMP-1, BMP-2, OP-l, TGF-~, cartilage-inrll-cing factor-A
(CIF-A), cartilage-inducing factor-B (C~IF-B), osteoid-inducing factor (OIF), angiogenin(s), endothelins, hepatocyte growth factor and keratinocyte growth factor, and providing a m~ m for prolonged contact between a wound site and the growth factor(s). The growth factor-supplern~nted TS may be used to treat burns and other skin wounds and may comprise a TS and, in addition to the growth factor(s), an antibiotic(s) and/or an analgesic(s), etc. The growth factor-supplem~nted TS may be used to aid in the engr~ftment of a natural or artificial graft, such as skin to a skin wound. They may also be used cosmPti~lly, for example in hair transplants, where the TS might contain FGF, EGF, antibiotics and minoxidil, as well as other compounds. An additional cosmetic use for the co~ osiLions of this invention is to treat wrinkles and scars instead of using silicone or other compounds to do so. In this embo-lim~nt for example, the TS may contain FGF-l, FGF4, and/or PDGFs, and fat cells.
The growth factor-suppleIn~nt,ocl TSs may be applied to surgical wounds, broken bones or gastric ulcers and other such internal wounds in order to promote healing thereof. The TSs of this invention may be used to aid the hlL~;~;ldLion of a graft, whether artificial or natural, into an animal's body as for example when the graft is composed of natural tissue. The TSs of this invention can be used to combat some of the major problems associated with certain con~iitinn~ such as periodontitis, namely l~clsi~7lclll infection, bone resorption, loss of li~ and ~'lclll~Ul~, re-epithP~ tion of the dental pocket.
In another embo~limlont~ this invention provides a ll~i~Lulc of FG, DBM
S and/or pllrifi~ BMP's. This mixture provides a matrix that allows the cellular colll~ollcllLs of the body to migrate into it and thus produce osteoinrl~lcti~ nwhere needed. The matrix colll~osilion in terms of ploLcills (such as fibrinogenand Factor XIII), enzymes (such as thrombin and plasmin), BMPs, growth factors and DBM and their conccllLldlions are ~deqll~t~ly form~ t~d to ~,~Lh~ e the longevity of this temporal scaffolding structure and the osteo;~ "~;Linn which needs to occur. All the FG components are biodegradable but during osteogenesis the mixture provides a non-collapsible scaffold that candetermine the shape and location of the newly formed bone. Soft tissue collapse into the bony nonunion defect, which is a problem in bone reconstructive surgery, will thus be avoided. The use of TS supplem~nt~od with growth factors such as CIF-A and CIF-B, infra, which promote cartilage development, will be useful in the reconstruction of lost or damaged cartilage and/or damaged bone.
In a preferred embodiment, an effective concellLlaLion of HBGF-l is added to a FG in order to provide a growt~h factor-supplem~nte-l TS that possesses the ability to promote wound healing. In another pler~llc;d embodiment, an effective amount of a platelet-derived extract is added to a FG.
In other pler~ d embo~im~ontc, an err~ e concellLlalion of a n~ixLule of at least two growth factors are added to FG and an effective amount of the growth factor(s)-supplemented FG is applied to the wounded tissue.
In addition to growth factors, drugs, polyclonal and monoclonal antibodies and other compounds, in~ lrling, but not limited to, DBM and BMPs may be added to the TS. They accelerate wound healing, combat infection, neoplasia, and/or other disease processes, m~Ai~t~ or f . Ih~ the activity of the growth factor in the TS, and/or ~lLtlr~re with TS components which inhibit the W O 96J40~74 PCT~US96/10006 activities of the grov-~th factor in the TS. These drugs may inrh~ , but are notlimited to: antibiotics, such as ~L,a~,ycline and ciproflo~r~cin;
antiprolir~Livelcyloto~ic drugs, such as S-fluorouracil (S-FU), taxol andlor taxotere; antivirals, such as gd~;y~lovir, ziduvlldi le, ~mAnti-lin~, viddl~bi~,ribaravin, Llinulidi~c, acyclovir, dideoxyuridine and antibodies to viral cûl~o~ or gene products; cytokines, such as a- or ~- or y-I,ll~.f,ç~n, a-or ~B-tumor necrosis factor, and interleukins; colony stimlllAting factors;
~yll~i opoietin; ~ ;r~ l.c, such as ~liflllr~n, ketaconizole and lly~,L~Lill;
antiparasitic agents, such as p~"~;""irlin.o; anti-infl;t""llAtoly agents, such as a-1-anti-trypsin and a-1-~lLicllyl"c L~ypS~l; steroids; ~ irs~; ~n~l~sics; and hormones. Other compounds which may be added to the TS include, but are not limited to: viLal~ ls and other nutritional supplements; hormones;
glyco~luLeills; fibronectin; peptides and p~ùL~ S; carbohy~a~s (both simple and/or complex); proteoglycans; A-,liAn~ingenins; antigens; oligonucleotides (sense and/or ;-"li~e~ e DNA and/or RNA); BMPs; DBM; antibodies (for example, to infectious agents, tumors, drugs or hormones); and gene therapy reagents. Gen~ti~lly altered cells and/or other cells may also be included in the TSs of this invention. The o~L~oi"ductive compounds which can be used in pr~ctiring this invention inr.~ e, but are not limited tû: osteogenin (BMP3);BMP-2; OP-1; BMP-2A, -2B, and -7; TGF-~, HBGF-1 and -2; and FGF-1 and 4. In addition, a"yll il,g which does not destroy the TS can be added to the TSs of this invention.
The studies reported herein llnrYrect~r1ly ~ dLe that the inclusion of compounds such as the free base TET or ciproflc Y~r.in (CIP) HCl, in FG or the lle~l."~"l of FG thc.c~wiLIl confers eytpn(lecl longevity to the supplemrnte~
FG. This phenomenon can be exploited to increase the duration of a drug's release from the TS. ~lL~ iv~ly~ this phenomenon can be exploited to modulate the release of another drug(s) other than the compound used to stabilize the FG, which is (are) also inwl~ led into the TET-FG, and/or to cause the FG to persist for a greater period in vivo or in vitro.

CA 02223889 l997-l2-0~
W 0 96/40174 PCTtUS96tlO006 In gellPr~l, poorly water soluble forms of a drug, such as the free base of TET, i~ ase the delivery of the drug from the TS more than freely water soluble forms thereof. The.~r~ " the drug may be bound to an insoluble carrier, such as fibrinogen or acliv~d charcoal, within the TS to prolong the delivery of the drug from the supplpnlpnte-d TS.
In another embot~ the supplu ..~ ~ TS can be used in organoids and could contain, for example, growth factors such as FGF-1, FGF-2, FGF~
and OP-l.
In another embodimPnt this invention provides a composition that promotes the localized delivery of a poorly water soluble form of an antibiotic(s), such as the free base form of TET, and other drug(s), CO~ )liSlllg a TS and an effective concentration of at least one poorly water soluble form of an antibiotic. Similar delivery methods are also applied to other drugs, antibodies, oligonucleotides, cytotoxins, cell proliferation h~l~ilo.~, osteogenic or cartilage inducing colllpoullds, growth factors or other supplements herein disclosed.
The present invention has several advantages over the previously used TS compositions and mt~thotlc. The first advantage is that the growth factor-and/or drug-supplPmPntPcl TSs of the present invention have many of the charact~rictirs of an ideal biodegradable carrier, namely: they can be form~ t~l to contain only human proteins thus eli.,.;,.~t;.,g or ...i,~;...;~;.-g immlmngenicity problems and foreign-body reac~ions; their ~-l"lilli~!,dlion is versatile; and their removal from the host's tissues is not required because they are degraded by the host's own natural fibrinolytic system.
A second advantage is that the present invention provides a good way to effectively deliver growth factors and/or drugs for a prolonged period of time to an internal or extern~l wound. It appears that some growth factor receptors must be occupied for at least 12 hours to produce a m~xim~l biological effect. Previously, there was no way to do this. The present invention allows for prolonged contact between the growth factor and itc W O 96~4~174 PCTrUS96/10006 l~C~ Ol~ to occur, and thus allows for the production of strong biological effects.
A third advantage of the present invention is that animal cells can migrate into and through, and grow in the TSs of the present invention. This aids en~lAr~ of the cells to neighboring tissues and prostheses. Based on the composition of the TSs which are available in Europe, it is e~pectt-(l that this is not possible with these formulations. Tn~t--~(l, a~mal cells must migrate around or digest commercially available TS. Since the importation into the U.S. of commercially available TSs from Europe is illegal (their use in the USA has not been approved by the U.S. FDA).
A fourth advantage is that because of its initial liquid nature, the TS of the present invention can cover surfaces more thoroughly and completely than many previously available delivery sy~Lellls. This is especially important for the use of the present invention in coating biomaterials and in the endothelialization of vascular prostheses because the growth factor-supplemented FG will coat the interior, exterior and pores of the vascular prosthesis. As a result of this, plus the ability of endothelial cells to migrate into and through the TS, engl~rL-~ent of autologous endothelial cells will occuralong the whole length of the vascular prosthesis, thereby decreasing its thrombogenicily and anti~e,lici~y. With previously used TSs, engr~ nt started at the ends of the vascular prosthesis and proceef1e-1, if at all, into the interior of the same, thus allowing a longer period for thrombogenicity and antigenicity to develop. Previously used TSs for vascular prostheses also primarily were seeded with n~n~lltologous cells which could be rejected by the body and could be easily washed off by the shearing force of blood passing through the prosthesis.
A fifth advantage is that the supplem~teci and unsupplem~nt~l TS of this invention can be molded and thus can be custom made into almost any desired shape. For example, TS such as FG can be supplemented with BMPs and/or DBM and can be custom made into the needed shape to most r~liaL~ly treat a bone wound. This carmot be done with DBM ~uwdc.
alone because DBM powder will not .~ ill its shape.
A sixth advantage is that the AB-supplem~ntecl FG of this invention, such as TET-FG, has Ill~rYl~lr~lly increased the longt;vily and stability of theS FG cu~ ed to that of the unsupplPmpnte(l FG. This increased stability corltim~es even after appreciable qll~ntiti~s of the AB are no longer reTn~inin~in the FG. For example, soaking a newly formed FG clot in a s~ aled solution of TET produced from free bâse TET, or in a solution of CIP HCl, produces a FG clot which is stable and ~l~,S~ ved even after ~lb~ lly all the TET or CIP has left the FG clot. While not wishing to be bound by any theory as to how this effect is produced, it is believed that the AB, such as TET or CIP, inhibits plasminogen which is in the TFC and breaks down the FG. Once the plasminogen is inhibited, its continnp~d inhibition does not appear to depend on a~ ciable qn~ntiti.os of the TET or CIP reTn~ining in the FG. As a result of this stabili_ing effect, one can expect an increased storage shelf life of the TS, and possibly an increased persistence in vivo.
The seventh advantage of the present invention is a direct result of the prolonged longevity and stability of the TS. As a result of this unexpected increase in stability of the TS, AB-supplelll~ P~l FG can be used to produce loc~li7Pcl, long term delivery of a drug(s) and/or a growth factor(s). This delivery will continue even after the stabilizing drug, such as TET or CIP, has ly left the TS. Inclusion of a solid form, preferably a poorly water soluble form of a drug such as free base, into a TS that has been stabilized by,for example, TET or CIP, then allows the stabilized TS to deliver that drug (or growth factor) locally for an extPn-lecl period of time. Some forms of drugs, such as free base TET, allow for both stabili7~ti~ n of the TS and for prolongeddrug delivery. Other drugs may do one or the other but not both. A compound used for the stabilization of a TS to produce prolonged, loc~li7P~ drug deliveryis not previously known in the art.

wo 96r40174 PCTrUS96/10006 AI1 eighth advantage of the present invention is that it allows site-directed angiogenesis to occur in vivo. While others have ~ ..Yl~d loc~li7e~ non-b~c.,irlc angiogenesis, supra, no one else has used a TS to L.lolllo~ site-directed angiogenesis.
A ninth advantage of the present invention is that because the components of the TS can be form~ t~l into several forms of simple to use, fast-acting field dlessillgs, it is now possible to control bleeding from hemorrh~in~ trauma wounds, thereby saving llulller~us lives that previously wouId have been lost. Although life-saving methods of treating such wounds are possible by trained mrtlir~l personal or in fully-equipped clinics and hospitals, the present invention s~ti~fi~s society's long-felt need for an easy-to-use, first-aid (or even self-applied) L.e~ that will, in ~ lley or disaster situations, allow an untrained individual to treat L~aulllatic injuries to control hemorrhage until m.--1ic~l a~si~t~nre is available.

Brief Description of the Figures Fig. 1 shows Western blots of gels on which samples co~ g HBGF-l~B had been inr~lb~tr~ with 250 U/ml thrombin in the presence of incl~asillg eo~re~ ions of heparin. Solutions col~ i..illg HBGF-l,B (10 ,ug/ml), Il~ lllbi (250 ~4g/ml), and in~:lcasillg concentrations of heparin (0, 0.5, 5, 10, 20, and50 units/ml) were i~ ~ at 37~C for 72 hours. Aliquots were periodically removed from each of the inrub~ting ll~i~ules and were loaded onto 8% SDS
polyacrylamide gels that were prepared and run as described by T.~rnm1i a~ature 227:680 (1970)). The gel was then electroblotted onto nitrocellulose and the band corresponding to HBGF-l,B was i~e~ntifif -l using an affinity-purified polyclonal rabbit antiserum to HBGF-1~.
The collc~;llL,d~ions of heparin in the inrllb~tin~ UleS were: panel A) O units/ml; panel B) 0.5 U/ml; Panel C) S U/ml; panel D) 10 U/rnl; panel E) 20 U/ml; and panel F) 50 U/ml. In the gels pictured in each of panels A-F, W O 96/40174 PCTrUS96/10006 each lane contains the following: lane 1 co"l~",s SDS-PAGE low molecular weight ~l~dalds; lane 2 co~ ls biotinylated ~l~dalds; lane 3 colll~ s 10 ,ug/ml HBGF-l,B; lane 4 CO~ ~llS 250 U/ml l~ulll~in; and lanes 5-13 contain ~les l~,~o~ed from the ;..~ .a~ mi~ s at times 0, 1, 2,4,6,8,24,48, and 72 hours.
Fig. 2 shows the i~cul~oldlion of 3H-thymidine as a fim-~tir~n of relative HBGF-l,B co~llllalion. Samples of the HBGF-l~ were i~ , as descrihed in Figure 1 and Example 2, in the presence of 250 U/ml ~olllbil~ and S U/ml heparin for 0, 24 or 72 hours. Dilutions of these samples were then added to NIH 3T3 cells, which were plated as described in Example 3. CPM is plotted v. HBGF-l concellllalion.
Fig. 3. Typical pattern of human umbilical vein endothelial cells after 7 days' growth on FG supplçm~nted with 100 ng/ml of active, wild-type FGF-l. Note the large number of cells and their elongated shape. Compare with the paucity of cells grown on unsuppl~ment~d FG (Figure 5).
Fig. 4. Typical pattern of human umbilical vein endothelial cells after 7 days' growth on FG suppl~on~nt~l with lO ng/ml of active~ wild-type FGF-l.
Note the large number of cells and their elongated shape. Compare with the paucity of cells grown on unsupplem~nt~d FG (Figure 5).
Fig. 5. Typical pattern of human umbilical vein endothelial cells after 7 days' growth on unsupplem~nttod FG. Note the small number of cells, compared to the number of cells in Figures 3 and 4, which in~lic~tes a slower proliferation rate.
Fig. 6. Typical pattern of human umbilical vein endothelial cells after 7 days' growth on FG suppl~on~t?nttod with lO0 ng/ml of inactive, mutant FGF-l.
Note the small number of cells, cu,lll.ared to the number of cells in Figures 3 and 4, which in~ t~s a slower proliferation rate.
Fig. 7. Typical pattern of human umbilical endothelial cells 24 hours after having been çmhedcled in FG at a concel~ Lion of 105 cells per ml of FG.
The protein and 11ll ul~lbin collce~ ions of the FG were 4 mg/ml and 0.6 NIH

W O 96t4~174 PCTAJS96/10006 units/ml"~,~e~;Lively. Note, their çlon~t~oA multipodial llwl~hology and that they formed a cellular llelwulh where they came in contact with each other.
- Compare with the cobblestone shape of similar cells grown in r~ ollecLi~ (Fig.
9-) S Fig. 8. Typical pattern of human umbilical enrloth~lial cells 48 hours after having been embedded in FG at a col-r~ on of 105 cells per ml of FG.
The culture conditions were as described in Fig. 7. Note the further ~r~e.~ tt~rl, elt)n~t~l and multipodial morphology and increasing development of cellular ncLwolh~. Compare with the cobblestone shaped cells grown in fibronectin (Fig. 10) and note the lack of a cellular network in the latter.
Fig. 9. Typical pattern of human umbilical endothelial cells 24 hours after having been cultured on a surface coated with r,l"ulle-;L"~. Note the cobblestone shape of the cells and the lack of cellular networks. Compare to Figure 7.
Fig. 10. Typical pattern of human umbilical endothelial cells 48 hours after having been cultured in a commonly used f~ of fib~vnecLill. Note the cobblestone shape of the cells and the lack of cellular networks. Compare to Figure 8.
Fig. 11. Micrographs of cross sections of PTFE vascular grafts that were explanted from dogs after 7 days (panels A, C, E) or 28 days (panels B, D, F). Prior to implantation, the grafts were either ullLlca~ed (A and B), coated with FG alone (C and D), or coated with FG supplem~ontP~I with heparin and HBGF-1 (E and F).
Untreated controls (A & B) showed minim~ os~nrl~yll~al tissue ingrowth, with both their interstices filled with, and their Illmin~l surfaces coated with fibrin coagulum. The FG-treated grafts showed mesenchymal tissue ingrowth in only the outer half of the grafts' hlL~ lices, with the rest being filled with fibrin co~gllhlm Very few illL.,.~LiLial capillaries were present. In contrast, the grafts treated with FG cc",l;.i";,.~ FGF-l showed moreabundant i"L~ lilial ingrowth and by 28 days showed llumelvus capillaries, WO 96/40174 PCT~US96/10006 myofbroblasts and macrophages, with inner capsllles co~ of several layers of ll.yorlbloblasts beneath conflll~nt endothelial cell layers. Results of similar grafts after 128 days of implantation were similar, with greater . .~ l h .
of capillaries in the FG + FGF-1 group (data not shown).
S Fig. 12. Sc~nnin~ electron micrographs of the inner lining of t_e vascular grafts cleserihe~l in Figure 11 after 28 days of implantation. The grafts were either ullL~ d (G), coated with FG alone (H), or coated with FG
supplemented with h~parin and HBGF-1 (I). Untreated control grafts (G) showed sparse areas of endothelial cell coverage amidst areas of thrombus c- .. 1;1;ni.,g red blood cells, platelets, and areas of exposed PTFE graft material (visible in the center and top of the picture). Grafts coated with FG alone (H) showed islands of endothelial cells arnidst areas of fibrin coagulum. In contrast, grafts treated with FG + HBGF-1 (I) showed confluent endothelial cells oriented along the direction of blood flow.
Fig. 13. Graph showing the inhibition of smooth muscle cell proliferation by the release of tributyrin from supplemented fibrin sealant.
Unsupplementto~l fibrin sealant = (O); tributyrin-supplern~nted fibrin sealant = (--)-Fig. 14. P,ey~ion of disc-shaped implants 1 rnrn thick and 8 mm in di~m~ter yl~parcd using an ~l.. ",;""". mold.
Fig. 15. Diagrarn illu~d~illg intr~mllsc~ r bioassay for the inrlllcti~n of bone formation in rats by DBM alone, by FG implants or by DBM-FG.
Fig. 16. Diagram illustrating the induction of bone formation in calvarial irnplants by DBM-FG.
Fig. 17. Radio-opacity data at 28 days yo:jlopc.dliv~: from intr~mllscul~r implants of DBM-FG, DBM or FG.
Fig. 18. Radio-opacity data from DBM-FG (30 mg/ml) calvarial implants at 28 days, 3 months and 4 months postoperative.
Fig. 19. Figure l9A is a photograph of a craniotomy site at 28 days post surgery in a treated animal. Figure l9B is a photograph of the calvarial W O 96~40~74 PCT~US96/10006 wound from an ullLl~aLt;d control at 28 days ~o~LJ~ dLiv~. Note that only fibrous co~ecliv~ tissue has developed across the craniotomy wound.
Fig. 20. Photograph from the craniotomy wounds of ~nim~l~ which were treated with DBM particles only.
S Fig. 21. Photograph of new bone formed in the craniotomy site in response to DBM-FG (15 mg/ml).
Fig. 22. Photograph of new bone formed in the craniotomy site in response to DBM-FG (15 mg/ml). Note that typically more bone lllalll:)W
formed in craniotomy wounds that had been implanted with DBM-FG disks than with DBM implants alone.
Fig. 23. The release of TET from 3 x 6 mm ~ m~ter disks of FG at 37~C. The concentration of the released TET was measured ~e~;L,u~huLu,,.~Llically in 2 ml of PBS ~U~ l that had been replaced daily.
Two of these "static" in vitro e~ lents were carried out with identir~l results. The results of one of them is shown here.
Fig. 24. The release of TET from 3 x 6 mm ~ m~ter disks of FG at 37~C. The disks contained 100 mg/ml of TET and were placed in closed vessels filled with 2ml of PBS. The TET concellLl~tion was measured s~e~;Llu~hotometrically in the PBS effluent which had been continuously exrlu.. gerl at a rate of 3 mVday. The volume of the PBS ~ i1t;~ had been kept constant at approximately 2 ml. The data are the average of two experiments.
Fig. 25. The release of TET into saliva from 3 x 6 mm ~ m~t~r disks cont~ining 50 or 100 TET mg/ml FG at 37~C. The TET concellL dLion was measured spectrophntnmt~trically in 0.75 ml of saliva sup~ l.l that had been replaced daily. The saliva used in these expeli~ L~ had been pooled from ten donors, cenlliruged, filtered and kept at 4~C.
Fig. 26. The stability of TET-supplemPnt~d FG was increased compared to that of control FG. Photographs of 3 x 6 mm tli~m~t~r FG
matrixes without TET and with 50 and 100 mg/ml TET over a period of 15 W O 96/40174 PCT~US96/10006 days. The disks had been kept in 0.75 ml of saliva which had been çh~
daily. The saliva had been pooled from 10 donors. It had then been Cf ntrif~lgffl, filtered and stored at 4~C before use in this ~ l Note thatat nine days, the FG matrix which did not contain TET had de~y~d more than the ,.. ,.~ es which co.. l;~ill~ either 50 or 100 mg/ml of TET Also note that at 15 days, the FG matrix which did not contain TET had almost totally decayed, whereas the FG ~.llices which co-~ PA 50 or 100 mg/ml of TET were almost l...r~ ged Th~ l~folG, the inclusion of 50 or 100 mg/ml of TET dr~m~tir~lly prolonged the longevity of FG matrices in saliva in vitro.
Fig. 27. Antih~ctf ri~l activity of TET released from TET-supplf mf ntPA
FG. Two ml PBS ~Ull~Ull~li~ the 3 X 6 mm TET-su~lf ~ d FG disks was replaced daily. For testing the ~ obial activity of the released TET, 6 mm paper disks ull~lG~l~led with the collected eluates were in- llb~te~l for 18 hours at 37~C on agar plates co..l~il-i..g E. coli. Then the r1i~m~t~r of the zone of inhibition was measured.
Fig. 28. The release of ciprofloxacin, amoxicillin and mGLlu.~idazole from FG matrices. Individual 3 X 6 mm ~ mt~er disks cont~ining 100 mg/ml of the respective antibiotics were i.--.--- ~e~l in 2 ml of phnsph~t~-buffered saline at 37~C. The ~U~ was replaced daily and the antibiotic concentration was measured spectrophotometrically at 275, 274 and 320 nm, respectively.
Fig. 29. The release of TET from TET-supplemented FG disks was proportional to the temperature of the PBS bathing the TET-FG disks.
Fig. 30. The effect of FG protein collcellL.~lion on the release of TET
from TET-FG. Note that higher FG protein concentrations resulted in a slower TET release rate from the TET-FG.
Fig. 31A. Graph showing the elution profile of in vitro release of antibiotic from a supplem~nt~(1 fibrin sealant disks.
Fig. 31B. Graph showing the elution profile of in vitro vs. in vivo release of tetracycline from supplemented fibrin sealant disks.

W O 9614~74 PCrnUS96~0006 Fig. 31C. Graph showing the inhibition of b~cLlial growth by LcLla~iyclille supI~lp-mlontecl fibr~n sealant disks as co~ d to unsuppl~m~nt~d hbrin sealant disks and culture media alone.
Fig. 32. The release of 5-FU from 5-FU-supple...... k~-lPA FG was prolonged by the use of solid forms of 5-FU.
Fig. 33. Graph shuwi~g the effect over time of ~u~ !i from taxol-suppletn~nt~d fibrin sealant composition on rapidly prolirc~aLing human ovarian caicillol,la cells (OVCAR).
Fig. 34. Dose-response relationship of the chemotactic les~onse of NIH
3T3 fibroblasts to Fiblullc~;Lill. A step gradient of increasing col~ccl~lldtions of Fil~ro.lecLill was added to the lower wells of the modified Boyden's çh~mlters.
The data are expressed as means +/- S.E. of migrated cells per high power field and demonstrate that, as a function of dose, fiblollecLi,l in~ ced the chemotaxis of NIH 3T3 cells toward it.
Fig. 35. Dose-lc~pollse relationship of the chemotactic lc~unse of NIH
3T3 fibroblasts to FGF-l. A step gradient of increasing collcenL-aLions of FGF-l was added to the lower wells of the modified Boyden's ch~.--heL~ in the plei,c,lce of heparin. The data are expressed as the means +/- S.E. of migr~t~
cells per high power field and demonstrate that, as a function of dose, FGF-l inrl~lce~ the chemotaxis of fibroblasts toward it.
Fig. 36. Dose-response relationship of the chemotactic re~OllSe of NIH
3T3 fibroblasts to FGF-2. A step gradient of increasing collce,lLldLions of FGF-2 was added to the lower wells of the modified Boyden's chambers. The data are e~ ssed as the means +/- S.E. of migrated cells per high power field and demonstrate that, as a function of dose, FGF-2 in~llrer1 the chemotaxis of fibroblasts toward it.
Fig. 37. Dose-response relationship of the chemotactic l~onse of NIH
3T3 fibroblasts to FGF4. A step gradient of increasing collcellLlaLions of FGF4 was added to the lower wells of the mntlifi~l Boyden's challlbe~s in the ~lcsellce of hl~p~rin The data are ~ ,essed as the means +/- S.E. of migrated W O 96/40174 PCT~US96/10006 cells per high power field and ll~orno"~ dle that, as a function of dose, FGF-4 intlllre~l the ch~ xi.c of fibroblasts toward it.
Fig. 38. Dose-response rel~ti~n~hir of the chemotactic rcs~onse of human dermal fibroblasts (HDFs) to FGF-l. A step gradient of increasing col-r~-~", ~;olLS of FGF-1 was added to the lower wells of the mnrlifiPd Boyden's eJX in the pr~3e"ce of heparin. The data are e,~lessed as the means +/-S.E. of migrated cells per high power field and demonstrate that, as a filnrtionof dose, FGF-1 intl~cecl the chemotaxis of HDFs toward it.
Fig. 39. Dose-~ ,onse relationship of the chemotactic response of HDFs to FGF-2. A step gradient of increasing conce,llldlions of FGF-2 was added to the lower wells of the modified Boyden's chambers. The data are expressed as the means +/- S.E. of migrated cells per high power field and demonstrate that, as a function of dose, FGF-2 in~ ced the chemotaxis of HDFs toward it.
Fig. 40. Dose-response relationship of the chemotactic response of HDFs to FGF-4. A step gradient of increasing concentrations of FGF-4 was added to the lower wells of the modified Boyden's ch~nnhers. The data are e~,t;;ssed as the means +/- S.E. of migrated cells per high power field and demonstrate that, as a function of dose, FGF4 in~ ce~ the chemotaxis of HDFs toward it.
Fig. 41. Dose-r~ollse relationship of the chemotactic response of HDFs to FGF-4 in solution and in FG. FGF-4 was incol~o,dted into FG and placed in the bottom well of chemotaxis rh~mhers. The amount of FGF in the FG was sufficient to result in the intiir~tr(l concellLIdLions when evenly distributed tbroughout the FG and mPrlillm in the lower rh~mher. Negative controls included mP~ m alone and FG without FGF. Medium co~ g FGF-4 at a conce~ d~ion of 10 ng/ml in the lower chamber was utilized as a positive control. The data are expressed as the means +/- S.E. of migr~t~-1 cells per high power field and demonstrate that, as a function of dose, FGF4 released from FG in(ll~ce~l the chemotaxis of HDFs toward the FG.

WO 96/~0174 PCTAJS96/10006 Fig. 42. Diagram of a self-conL~i~ed TS Wound Drts~il~g.

Descripfion of the Preferred Embodiments Defini~ions Unless defined ~ cl~ e, all lP~ ;c~l and sciPntific terms used herein have the same m~ning as is commnnly understood by one of skill in the art to which this invention belongs. All patents and publications mPnti~nlod herein areincorporated by lcrc.ellce.
As used herein, a wound inrl~ldes damage to any tissue in a living ol~lli.clll The tissue may be an intPrn~l tissue, such as the st--m~rh lining ora bone, or an external tissue, such as the skin. As such a wound may include, but is not limited to, a ga~ s~ l tract ulcer, a broken bone, a neoplasia, and cut or abraided skin. A wound may be in a soft tissue, such as the spleen, or in a hard tissue, such as bone. The wound may have been caused by any agent, including L~ l ic injury, infection or surgical i~ lion.
1~ As used herein, TS is a ~llb~ re or composition that, upon application to a wound, seals the wound, ~elel,y reducing blood loss and Ill~ illg hPmnst~ . As used herein, FG is a composition, ~r~aç~d from recombinant or plasma ~loL~hls, that upon application to a wound forms a clot, thereby sealing the wound, reducing blood loss and m~int~inin~ hemostasis. FG, supra, is a form of TS.
As used herein, supplemPnt~cl TS inrllldPs any TS that, without substantial modification, can serve as a carrier vehicle for the delivery of a growth factor, drug or other colll~oulld, or ll~i~LulcS thereof, and that, by virtue of its adhesive or adsorptive prupe.~ies, can m~int~in contact with the site fora time sufficient for the supplemPnte~l TS to produce its desired effect, for example to promote wound healing.

W O 96/40174 PCT~US96/10006 As used herein, a growth factor-supp~ d TS is a TS to which at least one growth factor has been added at a co~-r~ ion that is errG~iLivG for its stated ~ se. The growth factor can, for eY~mpl~, ~C~ , promote or illlL)lovG wound healing, or tissue (re)g~ The growth factor-suppl~ ed TSs may also contain additional co~o~ellL~, including drugs, antibodies, antico~ nte and other co~l)c,ullds that: 1) poL~,.ILidLG, stim~ t~
or ,,,t~A;..I~: the biological activity of the growth factor(s) in the TS; 2) decrease the activities of components of the growth factor-supplrm~nt~ TS which would inhibit or destroy the biological activities of the growth factor(s) in the sealant;
or 3) allow prolonged delivery of the supplement from the TS; 4) possess other desirable properties.
As used herein, a po~e~ colll~uulld is a col~o~ d that l-~r~ rs or oLh~l~ise increases the biological activity of a growth factor in the TS.
Heparin is an example of a co~l~ou"d that poLe~ s the biological activity of HBGF-l.
As used herein, an inhibiting compound is a compound that inhibits, hll~.rGlGs with, or otherwise destroys a deleterious activity of a component of the TS that would illLGlr~le with or inhibit the biological activity of a growthfactor or factors in the TS. IllhibiLillg compounds may exert their effect by ~ LG~;Lhlg the growth factor from degradation. An inhibiting compound does not, however, inhibit any activities that are e~eenti~l for the desired ~r~clLies, such as, for example, wound healing of the growth factor-supplr.m~ntr(l TS.
An example of an inhibiting compound is heparin.
As used herein, a growth factor inrl~ rs any soluble factor that reg~ t~s or m~ trs cell proliferation, cell dirr~,lG~ n, tissue regellGldLion, cell attraction, wound repair and/or any developmrnt~l or proliferative process.The growth factor may be produced by any a~lopliàLG means inrlll~ling extraction from natural sources, production through ~Y11L11GliC r.hrmi~try, production ~rough the use of recombinant DNA techniques and any other techniques, including virally inacLi~aLGd, growth factor(s)-rich platelet releasate, W O 96/40174 PCT~US96~1~006 which are known to those of skill in the art. The term growth factor is meant to include any ~ ;Ul:jUl~ ;, dc~ivalives~ or ûther forms thereof which possess similar biological acLiv-ily(ies), or a subset thereof, to those of the growth factor from which it is derived or otherwise related.
S As used herein, EIBGF-l, which is also known to those of skill in the art by ~lL. ~ ..i.liv~ names, such as endothelial cell growth factor (ECGF) and FGF-l, refers to any biologica~y active form of HBGF-l,inrlll~ingHBGF~
which is the precursor of HBGF-loc and other llullcat~d forms, such as FGF.
U.S. Patent No. 4,868,113 to Jaye et al., herein incorporated by r~r~lc.lce, sets forth the amino acid sequences of each form of HBGF. HBGF-l thus includes any biologically active peptide, inrlnrling precursors, ~ r~ or other modified forms, or ~ thereof that exhibit the biological activities, or a subset thereof, of HBGF-l.
Other growth factors may also be known to those of skill in the art by iVt; llnlll~.~rl~hlre. Acco.~ ly, ~c:re~nce herein to a particular growth factor by one name also includes any other names by which the factor is known to those of skill in the art and also inr.lll(les any biologically active derivatives or ~lc~ui~ors, trllnr~trd mllt~nt, or otherwise modified forms thereof.
As used herein, biological activity refers to one or all of the activities that are associated with a particular growth factor in vivo and/or in vitro.
Generally, a growth factor exhibits several activities, inr.l~ ing rnitogenic activity (the ability to induce or sustain cellular proliferation) and also non-rnitogenic activities, including the ability to induce or sustain dirrt;~ tion and/or development. In addition, growth factors are able to recruit or attract particular cells from which the proliferative and developmel.ktl processes proceed. For example, under a~-~-iate conditions HBGF-l can recruit endothelial cells and direct the formation of vessels thel~L~lll. By virtue of this activity, growth factor-suppl~ d TS may thereby provide a means to enhance blood flow and ~uLIie.l~ to specific sites.

W 096/40174 PCTrUS96/10006 As used herein, exten~led longevity means at least a two fold i~ ease in the visually observable, useful in vitro lifesp~n of a TS.
As used herein, ~ alized bone matrix (DBM) means the organic matrix of bone that re llaills after bone is decalcified with Lydlochloric or another acid.
As used herein, bone morphogenetic ~roleills(BMPs) mean a group of related pl~o~iuS originally idPntifip~ by their presence in bone-~.~u~;livt; extracts ofDBM. At least 8 related mémbers have been irlentifi~ and are rle~ign~t~
BMP-1 through BMP-8. The BMPs are also known by other names. BMP-2 is also known as BMP-2A.BMP~is also known as BMP-2B.BMP-3is also known as osteogenin. BMP-6is also known as Vgr-l. BMP-7is also known as OP-l. Bone morphogenetic p,oleills is meant to include, but is not limited to BMP-1 through BMP-8.
As used herein, ~ngmP~t~tion means using a supplPmP-ntP~l or 1~ unsupplemP-ntPtl TS to change the internal or çxtern~l surface contour of a component of an animal's body.
As used herein, a damaged bone is a bone which is broken, fractured, missing a portion thereof, or otherwise not healthy, normal bone.
As used herein, a deficient bone is a bone which has an in~t1PqU~tP
shape or volume to perform its function.
As used herein, bone or DBM which is to be used to supplement a TS
can be in the form of powder, suspension, strips or blocks or other forms as nPcess~ry to ~,c;,rO,lll its desired function.
As used herein, organoid means a structure that may be composed of natural, artificial, or a combination of natural and artificial el~mPntc, that wholly or in part, replaces the fimrtion of a natural organ. An example would be an artificial ~dl~ as collsi~Lillg of a 11CLWO1k of capillaries surrounded by W O 96~40574 PCT~US96/~0006 cells I~A~;r~~d with an ~r~ssion vector co..l~;..;,l~ the gene for insulin.
Such an organoid would fimrtion to release insulin into the blood~ of a patient with Type I Diabetes.

r~ c,~ion of Suppl~ente~ TS
As a first step when ~ ;,,g any of the embor1im~ont~ of the invention disclosed herein, the supplement and TS must be selPcte~1 The supplement and TS may be pl~aled by methods known to those of skill in the art, may be ~urcllased from a supplier thereof, or may be pr~ared acco,dillg to the methods of this applir~tion In a ~l~r~lr~d embodiment, growth factor, drug-or DBM-supplemPnt~cl FG is ~ d.
In any of the embotlim~ntc of tbe present invention the supplement may be added to the fibrinogen, the ~ ombill, the c~lrillm and/or the water component(s) before they are mixed to form the TS. ~Itern~tively, the supplement(s) can be added to the COlll~)Ol~llki as they are being mixed to formthe TS.
In embo~i",~ i of the present invention, the calcium and/or IlliO~
may be supplied endogenously from body fluids as, for example, those in a wound.

Preparation of TSs In certain emborlim~-nt~ of this invention such as, but not limited to, vascular prostheses, and in bone and cartilage al1gm~nt~tiQn, TS which allows cells to rnigrate into and/or through it may preferably be used.
Any TS, such as c~ll elcially available FG, may be used in some embo-iimPnt~ of this invention. For example, FGs which are well known to those of skill in the art (see, e.g., U.S. Patent Nos.: 4,627,879; 4,377,572; and 4,298,598, all herein incorporated by ,er.,l~,ce) may be purchased from a supplier or m~mlf~rtllrer thereof, such as IMMUNO AG (Vierma, Austria) and BEHRINGWERKE AG (Ge~ .,y). For these uses, such as localized drug CA 02223889 l997-l2-05 delivery, the particular composition of the selPctPA TS is not critical as long as it r....~ as desired. Cc,~.-;ially available FGs may be ~u~l~ with growth factors, antibiotics and/or other drugs for use in the embo~limPnt~ of this invention inrlll(lin~, but not limited to: in vitro cellular pr~ lifer~tion and/or dirrcrel~lid~ion; drug delivery; growth factor delivery, etc.
For the c~L~c~ ,lLs exemplified herein, FG was prc~d,ed from c,y~~ iLdLe from fresh frozen plasma. The components of the FG that were used included: fibrinogen coL~ce"L,dte; thrombin; and calcium ions.
In a lJlcfellcd embo(limPnt of this invention, the total protein conccllL,a~ion in the ~ aled FG is from about 0.01 to 500 mg/ml of FG. In a more plcrc~lcd embo~limPnt the total protein concentration in the ~l~cd FG is from about 1 to 120 mg/ml FG. In the most ~rerc~cd embotlimPnt, the total protein conctl~d~ion in the prepared FG is from about 4 to 30 mg/ml FG.
In a ~crcllcd embodiment of this invention, the fibrinogen concentration used to p,~ale the FG is from about 0.009 to 450 mg/ml of solution. In a more pLer~l.cd embo~limPnt the fibrinogen co~lcclll~dLion in thispreparatory solution is from about 0.9 to 110 mg/ml. In the most ~,cf~"ed embo-lim~nt, the fibrinogen conce~L~dLion in this pl~lJaldLoly solution is from about 3 to 30 mg/ml.
In a IJlcr~llcd embodiment, the LILL'OnIbiI1 conccllLIdLion used to p,~e the FG is 0.01-350 U/ml. In a more prefel~cd embo-limPnt, the thrombin collce,lL dLion is 1-175 U/ml. In the most prereilcd embodiment, the thrombin concentration is 2-4 U/ml.
It is important that the calcium ion conce~ dLion be sllfFicient to allow for activation of the thrombin. In a l~lcrellcd embo~limPnt, the USP calcium chloride concentration is 0-100 mM. In a more p~er~l,cd embo-limPnt, the USP calcium chloride conce,lL,d~ion is 1-40 mM. In the most p~c;r~lled embodiment, the USP calcium chloride collce,lL,dLion is 24 mM. In some embo.l;...~ of this invention, the calcium may be supplied by the tissue or body fluids as, for example, in the wound dressing embo-limPnt In pl~ep~mg the TS, sterile water for injection should be used.
Although the collce~ ion(s) of growth factor(s), drugs and other compounds will vary d~e~ g on the desired objective, the c.~..r~ lions must be great enough to allow them to be err~cLivc to accomplish their stated S purpose. In a ~Cft,llCd embodiment of this invention, the growth factor concellLation is from about 1 ng/ml to 1 mg/ml of FG. In a more ~lcr~ cd embodiment, the growth factor co-lcellL~ation is from about 1 ,ug/ml to 100 f4g/ml of FG. In the most p.c~.led embodiment, the growth factor concentration is from about S ~g/ml to 20 ,ug/rnl of FG. In a ~l~r~ d embodiment of this invention the TET or CIP collce~L,~Lion is from 0.01 to 300 mg/ml FG. In a more ~lcfell~ emborlim~nt of this invention the TET or CIP
concentration is 0.01-200 mg/ml. In the most preferred embodiment of this invention the TET or CIP conce.-ll~lion is 1-150 mg/ml. The amount of the supplements to be added can be empirically determined by one of skill in the art by testing various col~cellllalions and selecting that which is ~Lr~c~ivt; for the intended purpose and the site of application.

Preparation of Growth Factors The growth factor(s), or ll~ixlulc: thereof, may be ~ all;d by any method known to those of skill in the art or may be purchased cornmercially.
Any growth factor may be selçcted inrlu-ling, but not limited to, for example, growth factors that stim~ te the proliferation and/or attraction of certain celltypes, such as endothelial cells, fibroblasts, epithelial cells, smooth muscle cells, hepatocytes, and keratinocytes, and/or growth factors which inhibit the growth of the same cell types and smooth muscle cells. Such selection rnay be dependent upon the particular tissue site for which the growth factor-supplemented TS will be applied and/or the type of effect desired. For example, an EGF-supplemPntp~l TS may be pl~r~ ,d for application to wounds in the eye and for ~ gastric ulcers while an osteogenin-supplçmPntP~ TS

W O 96140174 PCTrUS96/10006 may be ~ led for applir~tion to bone fractures and bone breaks in order to plomoLe healing thereof.
In another plef~ d embodiment HBGF-l,B was pl~cd and added to FG. HBGF-l~, or HBGF-la, or any other active form of HBGF-1, can be S purified from natural sources, from gçn.otir~lly e.~ elcd cells tbat express HBGF-l or a de~ dlive thereof, or by any method known to those of skill in the art.
HBGF-l~ has been plc~alcd using lccu~ ~lL DNA m~th~lology (Jaye etal., U.S. Patent No. 4,868,113; Jaye et al., J. Biol. Chem. 262:16612-16617 (1987)). Briefly, DNA encoding HBGF-l~ was cloned mto a prokaryotic ~"~ression vector, a pUC9 derivative, and e~lcssed intracellularly in E. coli.
The e~lessed peptide was then released from the cells by ~/lC:i~iUlC, using a cell disrupter operated on high c~,lu~l~,ssion-dec~lu~lession cycles. After disruption, cell debris was removed by filtration and HBGF-l~ was purified from the ~upe~ lll using standard methods of protein purification including affinity chlolllalography on heparin Sepharose~ followed by ion-exch~nge chromatography on CM-SepharosesY.
In addition to HBGF-l, described above, other growth factors that may be added to the FG include, but are not limited to, HBGF-2, IGF-l, EGF, TGF-,B, TGF-a, any platelet-derived growth factor or extract, BMPs, and Ul~,S of any growth factors. For example, platelet-derived extracts, which serve as rich sources of growth factors, may be added to the TS in addition to or in place of other growth factors, such as HBGF-l.
In a i,rer~llcd embo~lim~nt~ a platelet-derived extract, prepared by any method known to those of skill in the art, is added to a TS. Such an extract hasbeen ~r~alcd from plasma derived platelets for use with FG.
Platelet-Derived Wound ~e~ling Factor (PDWHF) may be p~cd and added to FG (Knighton et al., Ann. Surg. 204:322-330 (1986)). Briefiy, to prepare PDWHF, blood is drawn into anticoagulant solution and platelet-rich plasma is prepared by refi iger~ted centrifugation. The platelets are isolated and W o 96~4~174 PcTnusg6~

stim~ ted with Lh~ bill, which releases the co~ Ls of the alpha granule contents. The platelets are removed and an erfecLive co~re~ ;on of the .i,.i"~ extract is added to a TS.

Additional Components of Growth Factor-Suppl~n1en~ TS
Since they are essP-nti~lly plasma fir~-~ti~nc, the TSs c~ .lated for use with growth factors contain numerous components, some of which may iuL~,r.,~c with the biological activity of the selected growth factor(s). For example, thrombin, which is an essG.~ component of FG, can act as a proteolytic enzyme and specifically cleave HBGF-l~. Therefore, it may be ~-P~.y to include ~-lition~l co~ ou,lds, such as p.. )L~,ase or other il~hibitul~, that protect the selected growth factor(s) from the action of other components in the TS which h~ re~c with or destroy the biological activity of the growth factor(s).
Selection of the particular inhibiting compound(s) may be e~ ilically deLe~llPilled by using methods, ~i~c-ccetl below, that assess the biological activity of the growth factor(s) in the TS. Methods to assess biological activity are known to those of skill in the art.
In addition, in order for certain growth factors to exhibit their biological activities, it _ay be l~e~ y to include compounds that potentiate or mP~ te the desired activity. For example, heparin pul~ S the biological activity of HBGF-l in vivo (see, e.g., Burgess et al., Annu. Rev. Biochem. 58:575-606 (1989)).
The supplemPntPcl TS of the present invention may contain compounds such as drugs, other chemicals, and proteins. These may include, but are not limited to: antibiotics such as TET, ciprofloxacin, amoxicillin, or llleLlo~ 7ole, anticoagulants, such as activated protein C, heparin, prostracyclin (PGI2), prost~gl~n-linc, leuk(JL,iehes, ~lLiLlll~olllbin m, ADPase, and pl~cminngen acLivdLul, steroids, such as dex~mPthasone, inhibitors of prostacyclin, prost~ .c, leuku~Lielles and/or kinins to inhibit infl~mm~tion;

cardiovascular drugs, such as ~tlril-m channel blockers; cl-~ ct~tntc; local s such as bupivacaine; and antipro~ a~ tntiblmnr drugs such as 5-fluo~ aeil (5-FU), taxol and/or ~XOt~ ,. These suppl~m~nt~tl com~ ds rnay also include polyclonal, I l lnnnc lo~ or c~ elic antibodies, or fimrti~n~tl delivaLives or r.~g.. ~.. l.~; thereof. They may be antibodies which, for example, inhibit smooth muscle proliferation, such as antibodies to PDGF, and/or TGF-~, or the proliferation of other ulldesilable cell types within and about the area treated with the TS. These antibodies can also be useful in sitll~tionc where anti-cancer, anti-platelet or anti-infl~ toly activity is ntoede~l. In general, any antibody whose efficacy would be improved by site-directed delivery may benefit from being used with this TS delivery system.

Assays for Asse~ g the Wound Healing Propeffies of a Growth Factor-Suppleme~lte~l TS
In order to ascertain whether a particular growth factor-supplemPnte~l TS ~lul,lo~s wound healing and to select optimal co,lcel,ll~ions of the growth factor(s) to do the same, the composition may be tested by any means known to those of skill in the art (see, e.g., Tsuboi et al., J. Exp. Med. 172:245-251(1990); Ksander et al., J. Am. Acad. Dermatol. 22:781-791 (1990); and Greenh,tlgh et al., Am. J. Path. 136:1235 (1990)). Any method inrlllfling both in vivo and in vitro assays, by which the activity of the selPcfç(l growth factor(s) in the TS composition can be ~sç~se~ may be used. For example, the activity of HBGF-l,B has been a~sessed using two independent in vitro assays. In the first, the proliferation of endothelial cells that had been suspended in a shallow fluid layer covering a plastic surface which had been impregn~ttçd with growth factor-supplrnttontrd FG was ",ea~ d. In the second, the incorporation of 3H-thymidine in cultured fibroblasts in the p,~sel.ce of HBGF-1 was measured.
In an in vivo assay, FG that had been supplem~ltted with HBGF-l~ has been tested for its ability to promote healing in vivo using mice âS a model system. In this method iflrntir~tl punch biopsies were made in the dorsal region W ~96/4~174 PCTAUS96/10006 of the mice, ~1vhich were then se~ d into test, treated control and ullLI~Led control groups. The wounds in the mice in the test group were treated with the growth factor-supp~ l TS. The wounds in the mice in the treated control group were treated with unsuppl~-r~r~l TS. The wuullds in the u~ dled S group were not treated with TS. After a time s~ ienf for ~letect~hle wound healing to ~r~ceed, gen~ lly a week to ten days, the mice were sacrificed and the wound tissue was lllie,usco~ically eY~TninP,d tû histologically assess the extent of wound repair in each group.
The ability of the growth factor-supplementPd TS to induce cell proliferation and to recruit cells may also be assessed by in vitro methods known to those of skill in the art. For example, the in vitro assays described above for mP~llring the biological activity of growth factors and described in detail in the Examples, rnay be used to test the activity of the growth factor in the TS composition. In addition, the effects of adding inhibiting and/or pu~ iaLillg compounds can also be ~sessed Generally, the necessity for adding inhibiting and/or potenti~tin~
compounds can be empirically tlP~ pd For example, in the experiments described below, the HBGF-l~ in HBGF-l-supple~P~tPd FG was specifically cleaved in a stochastic ~ , suggesting that a component of the FG
,u~ 'alion~ most likely ~ U1111Jin, was lc~ol~ible. Heparin, which is known to bind to HBGF-l and protect it from certain proteolytic activities, was added to the HBGF-l-supplemented FG. The addition of relatively low conc~ dlions of hPp~rin ~luL~l~d HBGF-l~ from cleavage that would destroy its biological activity in the FG. Therefore, TS compositions that include HBGF-l may include hPp~rin or some other ~lbsl~lce that inhibits the cleavage of HBGF-l by LlllUllll~ill or other proteolytic components of the FG.
Similarly, the ability of a selected inhibitor to protect a growth factor from degradation by TS Colllpullt;lll:~ may be ~es~ed by any method known to those of skill in the art. For example, heparin has been tested for its ability to inhibit cleavage of HBGF-l by ~olllbm, w_ich is an es~Prlti~l component of FG. To do so, ~ cs of various conre~ll . a~ s of heparin and HBGF~
supplemP-nts~l FG have been p~ cd, and inr~lb~tP(1 for various times. The biological activity of HBGF-l in the ~ e has been tested and the illL~ liLy of the HBGF-1 has been asc~ ai~ed using wc~L~ll blots of SDS gels.
Relatively low co~ . aLiorls7 about a 1:1 molar ratio of hPp~rin ~RGF-1, are sllffiri~nt to protect HBGF-l from degradation in FG.
It can also be c~ ically ~,t~ PA wll~Ll~ a particular compound can be used to polelllia~, mPtli~t~ or enh~nre the biological activity of a growth factor(s) in TS.

0 Topical or Internal Application of the Growth Factor-Supplemer~te(~ TS to an Internal or External Wound Prior to clinical use, the growth factor and TS, or the growth factor-supplemrnt~-l TS is pasteurized or otherwise treated to inactivate any pathogenic co~ therein, such as viruses. Methods for inactivating 1~ c~-l-l;.. ill;.lll~i are well-~own to those of skill in the art and include, but are not limited to, solvent-deL~ lL trP tmPnt and heat trr~ttnPnt (see, e.g., Tabor et al., Thrombosis Res. 22:233-238 (1981) and Piszkiewicz et al., Transfusion 28:198-199 (1988)).
The supplpnnrnt~(l TS is applied directly to the wound, other tissue or other desired location. Typically for eYt~rn~l wounds it can be applied directlyby any means, inrlll-ling spraying on top of the wound. It can also be applied intPrn~lly, such as during a surgical procedure. When it is applied int~rn~lly, such as to bones, the clot gradually dissolves over time.

Self-Con~ained Applica~ions of the Supplernented or Unsupplemented TS for lnternal or External Wounds The TSs may be fotmnl~tsd as a self-c-nt~inrd wound dressing, or fibrin sealant bandage, which contains the n.ocess~ry thrombin and fibrinogen components of the FG. The self-contained dl~S~lllg or bandage is easy-to-use, WO g61~al74 PCT/US96/10006 ~5-'~'l";~ in~ no adv~ced l~l".i.~l hluwl~dge or skill to operate. It can even be self-~ ;l as an ~ cy first aid ~asule to preserve life until mrrlir~ re becnmrs available.
The self~ TS wound dleD~i~ or fibrin sealant b~n~ e is an S adv~ r~ r~l1 over the current technûlogy in that the field-ready pr~A.~ -- can be stored for long periods, and be used to provide rapid TS Ll~ --l ûf a h~ ging wound without the time delay associated with solubili7~tion and mixing of the colllpoll~llL~. These characteristics make it ideal for use in field applications, such as in trauma packs for soldiers, rescue wu~ 7, ambulance/paramedic teams, firemen, and in early trauma and first aid tre~tm~nt by em~l~el,~;y room personnel in hûspitals and clinics, particularly in disaster .cihl~ti~lnc. A small version may also have utility in first aid kits for use by the general public or by mPrlir~l practitioIlers.
The self-co~t~in~ TS wound dressing or fibrin sealant bandage co~ ises a tissue sealing composition Culll~liDillg a tissue sealant or fibrin complex of the type previously described. For example, the composition may be comprised of purified fibrinogen, thrombin and calcium chloride with sufficient Factor XIII to produce a fibrin clot. In one embodirnent the fibrinogen and Factor XIII components are suRlied in the form of topical fibrinogen complex (TFC).
When used on human patients, the colll~ol~llL~ are most preferably pathogen-inactivated, purified components derived from human sources. In particular, the components of the present invention, inr~ ing additives thereto,are treated with a d~l~.gc;llL/solvent, and/or otherwise treated, e.g., by pa~L~ul~Lion or ultrafiltration to inactivate any pathogenic co~
therein, such as viruses. Methods for i~cLivaLulg c~.-.l;..-,i,.,...l~ are well-known to those of skill in the art and include, but are not limited to, solvent-d~L,r~e treatmrnt and heat Lle~l.-.~..l Solvent-detclg~ Lle~ is particularly advantageous in that the ~roL~Iaceous components are not exposed to irreversible heat-d~ ula~ion.

~ CA 02223889 1997-12-05 The c~lril-m and/or Factor XlII colll~oll~llL~ may be c- nt~inP-cl in either the Lh.~olllbi-l and/or the fibrinogen cc~ on.,~l(s), and/or absorbed from the patient's endogenous c~lrillm present in the fluids ~sca~ g from the wound.
Thr~...bi~ may also be supplied endogenously. Either or both of the ll..o.l.bL.
or fibrinogen components can be, but does not have to be, supplemPntPd in each of the following embo~limPnt~ with one or more growth factors, drugs, il~ilJili--g co~ oullds (to inhibit the activities of the sealant that may illL~lÇel~
with any of t_e biological activities of the growth factor or drug), and ;.,g compounds (to ~tr-~ P, mediate or P~h~l~re any of the biological activities of the growth factor or drug), compounds which inhibit the breakdown of the fibrin clot, or dyes.
The growth factor may include, e.g., fbroblast growth factor-l, fibroblast growth factor-2 and fibroblast growth factor~; platelet-derived growth factor; insulin-binding growth factor-l; insulin-binding growth factor-2;epi-lerm~l growth factor; L.~Ç(.l,.,;l-g growth factor-oc; Lla~ ll g growth factor-~; cartilage-inducing factors -A and -B; osteoid-inducing factor;
osteogenin and other bone growth factors; collagen growth factor; heparin-binding growth factor-l; heparin-binding growth factor-2; and/or their biologically active de-ivaLivt;s.
The drug may be an analgesic, a.. Lis~Lic, antibiotic or other drug(s), such as antiproliferative drugs which can inhibit infection, promote wound healing and/or inhibit scar form~ti~m More th~n one drug may be added to the composition, to be released ~imlllt~nPously, or the drug may be released in pre~etPrmin~-cl time-release manner. Such drugs may include, for example, taxol, tetracycline free base, tetracycline hydrochloride, ciprofloxacin hydrochloride or 5-fluorouracil. The addition of taxol to the fibrin sealant complex may be particularly advantageous. Further, the drug may be a vasoconstrictor, e.g., epinephrine; or the drug may be added to stabilize the tissue sealant or fibrin clot, e.g., al?-olil~ . The supplement(s) is at a concellL aLion in the TS such that it will be effective for its intPn(1Pfl purpose, wo 96/40174 PCTAUS96/10006 e.g., an ~ ,io!ir, will inhibit the growth of ~c obes, an ~n~l~P~ir will relievepain, etc.
Dyes, lllal~. or tracers may be added, for e~mrl~, to intlir~tP the extent to which the fibrin clot may have entered the wound, or to measure the subsequent resorption of the fibrin clot, or the dye may be released from the tissue sealant in a pred- t~ A, time-release manner for ~ nnstir ,uul~OSeS.
The dyes, ...;.,1~ or tracers must be physiologically compatible, and may be selected from colored dyes, inr1~1-1in~ water soluble dyes, such as toluidine blue, and r~ r-tive or fluùl~sce~ . or tracers which are known in the art. The dyes, llla.h~.~. or tracers may also be compounds which may be cllPmic~lly coupled to one or more components of the tissue sealant. In addition, the marker may be selected from among ~loL~illaceous materials which are known in the art, which upon exposure to proteolytic degradation, such as would occur upon exposure to proteases escaping from wounded tissue, change color or develop a color, the inlcllsiLy of which can be q l~ntifi~d Moreover, when the TS is used to replace or repair wounded or damaged bone or ossified tissue, the composition may also be supplemPnted with t;r~ ivc~ ~lluullL~. of ~ ,;,,. .,.li7PA bone matrix and/or bone morphogenic plo~ s, and/or their biologically co".~ le derivatives.
The cuncellLldLion of the rlb~ cll and/or Llllol~hl components of the self-contained TS wound dl~ ssillg or fibrin sealant bandage may have a signifir~nt effect on the density and clotting speed of the final fibrin matrix.This principle may be used to satisfy specific uses of the self-contained TS
wound dressing or fibrin sealant bandage in specialized situations. For 2~ example, the tre~tmPnt of an arterial wound may require the fibrin clot to set very rapidly and with s~ff~iPnt integrity to Wi~ les~--- ;~P-d blood flow.
On the other hand, when filling deep crevices in a wound, ~ lPIII may require the components to fill the wound completely before the fibrin clot sets.
T*e Gel Pack Embocliment~

In the gel pack emboflim~nt of the self~J..IA;.~ dlcssillg, the L}llùml~
and fibrinogen cc,~ )ol~cllL~ are individually CO..IA;..~ in intlepçntlent quick-cva~ul_Lillg gel layers (e.g., methylr~ ose/alcohol/water), WllClCill the two gel layers are sepA. "t~ A from each other by an ;" ~ . Ahle ...- -..l .. Al ~f', and the pair are covered with an outer, p~oLe-;livc, second ;.. pe~.-.PAble membrane.
The bandage may be coated on the surface that is in contact with the gel in order to insure that the gel pad lt ~l~S in place during use. (See Figure 42).
In use, the ~c~ e Sc~ Lillg the two gel layers is removed, allowing the two components to mix. The outer membrane is then removed and the bandage is applied to the wound site. The action of the thrombin and other col~oLlcllLs of the fibrinogen ~l~A, Al io~ cause the conversion of the fibrinogen to fibrin, in the ~ lleL previously disclosed for other FS applications. This results in a natural inhibition of blood and fluid loss from the wound, and establishes a natural barrier to infection.
In a similar gel pack embo~lim~nt~ both the thrombin component, and the plastic film sepal~Lillg the Ll,ro"~bill gel and the fibrinogen gel, may be omitted. In operation, the outer impervious plastic film is removed and the bandage applied, as previously described, directly to the wound site. The tbrombin and calcium naturally present at the wound site then induce the conversion of fibrinogen to fibrin and inhibit blood and fluid loss from the wound as above.
This ~ . .",livc embodiment of the gel pack has the advantage of being simpler, cheaper, and easier to produce. However, there may be c..~;~".,~ çs in which a patient's wounds have in~ iPnt ll..ul~ ill to cLrccLi~rely L~ rùllll the fibrinogen gel into a fibrin tissue sealant. In those cases, the thrombin cùllllJullcllL must be exogenously supplied, as in the earlier~escribed gel packembodiment of the invention.

The F'ibrin Sealant Bandage Embo(~ nt~

WO 96/40174 PCT~US96/10006 A fibrin sealant b~ntl~ge embo~1im~nt is form~ t~1 for applying a tissue sealing co.-l~o~ilion to wounded tissue in a patient, wlle~ the b~ntl~ge ~ cu.. ~ es, in order: (1) an occlu~iv~ b~f"Li~-~,; (2) a physiologically-acceptable a&esive layer on the wound-facing surface of the b~rL ;.~; and (3) a layer of S dry materials COlll~lliSlng an ~rr~cLive amount, in combination, of (a) dry, virally-inactivated, purified fibrinogen complex, (b) dry, virally-inactivated, purified ~rombin, and as ,-rcess~.y (c) effective alou.lLs of c~lr~ m and/or Factor XIII to produce a tissue-sealing fibrin clot upon hydration, wllclcill the layer of dry materials is affixed to the wound-facing surface of the adhesive layer. In one embo~lim~ont the occlusive backing and the physiologically-acceptable adhesive layer are one and the same, if the backing layer is sufficiently adhesive to errt;~lively bind the layer of dry materials.
In ancLllel embo-lim~nt a removable, w~L~ ûof, l,role~;Li~e film is placed over the layer of dry materials and the exposed adhesive surface of the bandage for long-term stable storage. In operation the waLel~,oof, ~roL~ -;Liv~
film is removed prior to the application of the bandage over the wounded tissue.
The tissue sealant colll~ol~llL of the bandage in one embodiment is activated at the time the bandage is applied to the wounded tissue to form a tissue sealing fibrin clot by the patient's endogenous fluids escaping from the hemorrh~ging wound. Preferably, the tissue sealant is hydlaLed and fluid loss from the wound will be ~igni~lr~ntly ~iiminiche~l within ...i..,~s of application of the bandage to the wounded tissue. Although the speed with which the fibrin clot forms and sets may be to some degree ~lirt~te~ by the application, e.g., 2~ rapid setting for arterial wounds and h~lllo~ ging tissue damage, slower setting for Ll.,"~ l of wuullds to bony tissue, preferably the fibrin clot will form within twenty ...i..~es after applir~ticn- More preferably, this effect will be evident within ten mimltes after application of the b~n~l~ge. Most preferably, the fibrin clot will form within two to five mimltes after applir~tion In the embodiment CU~ li5illg the most rapidly forming fibrin clot, the tissue seal will be ~ lly formed within 1-2 "~i"~ s, more preferably within 1 minute, and most preferably within 30 secon(lc after applir~ti(m It may be ~PcesC~y to use ~lcs~ule in aRlying the fibrin sealant b~n~i~ge until the tissue sealing fibrin clot has formed over the wound site.
S In the all~-."~live, in si~tionc where fluid loss from the wound is ;"~,.rri.~i~ .,l to provide ~lP~ te hydration of the dry tissue sealant m~t~Pri~lc, or where time is of the essence, as in a life-ll~r~ .,i"g ci1ll~tion, the tissue sealant components are hydrated by a suitable, physiologically-acceptable liquid prior to application of the bandage to the wounded tissue.
To construct the bandage, the dry materials may be obtained, for example, by lyophili7~tion or free~-drying, or suitable, c~ l~l~cially-availablem~tPri~lc may be lltili7P~ Anhydrous CaCl2 may also be added to the dry TS
components to accelerate the speed of fibrin form~tion upon hydration of the fibrin sealant bandage. The binding of the dry materials to the adhesive or backing layer may be Pl~h~ ~l by adding a binder, preferably a water soluble binder, to the dry components.
The backing of the fibrin sealant bandage may be of collv~ )n~l, non-resorbable materials, e.g., a silicone patch or plastic material; or it may be of biocompatible, resorbable materials. The b~cl~i..g material may act as more than a delivery device. Its pl~rellc~d composition is cl~l~.. i.. ~cl by the desired application of the fibrin sealant bandage. For example, a non-resorbable backing is al)propiiat~ for many external uses, where it provides strength and protection for the fibrin clot. In an ~llrl ~liv~ embo~limPnt the non-resorbablebacking is reil~lced, e.g., with fibers, to provide extra strength and durability for the protective covering over the fibrin clot.
Suksequ~nt removal of the clot with the backing is acceptable in many situations, such as when the fbrin sealant bandage is used as a first aid ~lle~uuntil m.o.rlit ~l a~Si~t~nre becollles available. In such a situation, the clot will have served its purpose to pl~v~llL life Illic l~ ;llg loss of fluid, and it will be wo 96r40174 P ~ nUS96/IU006 de~ilal)l~ to remove the clot without causing ad~liti~n~l tissue damage to permit proper ll~ or surgical repair of the wound.
In the ~ "~ e, the non-resorbable baclrin~ may be used to provide strength to the tissue sealing fibrin clot during its formation, e.g., when the S hclllo~ in~ fluids are esca~i~ under plcs~urc~ as in an arterial wound. Yet, if such a wound is int~rn~l, it is advantageous to remove the backing from the fibrin clot without disturbing the tissue seal. Therefore, a fibrin sealant b~n~ e is provided in which the adhesive layer is of a material having a lower shear strength than that of the fibrin clot, pclll~iLIillg removal of the backing without damage to the fibrin clot or the tissue surrounding the wound.
By cou,l)alison~ certain inttorn~l applications m~nll~t~ the use of a resorbable backing to elimin~t~ the need for subsequent removal of the dressing. A resorbable m~tori~l is one which is broken down s~olll~eously or by the body into C~,lll~ull~llLs which are consumed or elimin~t~o(l in such a manner as to not .~ignifi~ntly h~ r~-~ with healing and/or tissue regeneration or fimrtion, and without causing any other metabolic di~,Lulballce. Homeostasis is preserved. Materials suitable for prel)~illg the biodegradable backing include p,oL~ aceous ~ub~L~lces, e.g., fibrin, collagen, keratin and gelatin, orcarbohydrate derived sub~l~ces, e.g., chitin, chi~osall, carbo~Lylll~ll.ylc~ llcse or cellulose, and/or their biologically co",~aLible derivatives.
The adhesive layer, if separate from the occlusive backing layer, is sel~ct~l on the basis of the int~-le~l application of the fibrin sealant bandage, and may comprise conventional adhesive materials. Antiseptic may be added to the adhesive layer.
If the tissue sealing fibrin clot is to be removed from tlle wound with the occlusive backing, such as prior to surgery, the a&esive must be sufficient to affix the dry material layer to the occlusive b~cl~ing, and to m~int~in an a&esive capability after hydration which is greater than the sheer strength of fibrin.

W O 96/40174 PCT~US96/10006 If the tissue sealing fibrin clot is to remain in position over the wound, but the occlusive ba~kil,g must be l~,.lluvc;d after appli~tion, the adhesive must be sllfficiçntly sticky to aff~x the dry material layer to the occlusive b~r~in~, but yet have an adhesive capability after hydration which is less than the sheer~LIe~ of the fibrin clot. In the ~ liv~, the adhesive layer may be of a material which becomes solubilized or less sticky during hydration of the dry materials, pe....;~ g removal of the backing from the fibrin clot. In the allc;ld~ive for such purposes, the dry material layer may be affLlced directly to the occlusive bandage.
In another embo~limlo~t~ the adhesive layer comprises two different adhesives to permit removal after hydration of the occlusive layer without di~lulbillg the tissue sealing fibrin clot. Typically, in such a situation the dry, tissue-sealant component materials are affixed to a specific region of the backing, the "inner region," e.g., the center, with an ~ e.ll ll.llhel~d area ofadhesive ~x~e,-~ -g beyond the area of dry material, the "outer region. "
The outer region of adhesive is affixed directly to the skin or tissue surrounding or ~ çnt to the wound in such a way that the dry m~ttq~ri~l region of the bandage forms a fibrin clot directly over the wound. The a&esive layer on the region of backing which is not covered by the dry material layer of the bandage is s--ffi~ i~nt to affix the fibrin sealant b~n(l~ge to the tissue ~ullou~
the wound until its physical removal. The adhesive on the outer region must be sufficient to hold the bandage in place, even if fluids are hf~.llo. . l.~gi..g from the wound under pres~url;:, e.g., an arterial wound.
The inner region of adhesive is sufficiently sticky to affix the dry m~teri~l layer to the occlusive backing, but yet have an a&esive capability after hydration which is less than the sheer ~lle~lll of the fibrin clot. In the altelna~iv~, the inner region of adhesive is of a material which becomes solubilized or less sticky during hydration of the dry materials, ~e....... il~;.. g t removal of the backing from the fibrin clot. In the alL~,dliv~ for such ~ oses, the dry m~t~ri~1 layer may be affixed in the inner region di-~;Lly to the occlusive bandage, with an adhesive layer added only to the outer layer.
Thus, in the two adhesive embo~linlpnt~ the b~rlring of the fibrin sealant b~n~ G~illS in place affixed to the tissue ~u~ uildil~g the wound until the S b~n~l~ge is physically l-"lwv~d. But upon removal, the b~r~in~ se~ s from the tissue sealing fibrin clot without di~Lu billg the tissue seal.

The Dual~ rsu~te~ Embo~ nt~ of the Fibrin Sealant Rn~ ge In yet another embodiment of the fibrin sealant bandage, an independent hydrating layer colnyli~illg an erre~;Live amount of carbonated water or physiologically-acceptable buffered hydrating agent, such as PBS, or comparable gel, is contained within a rupturable, liquid-i",l~r~ "~ble COIl~
The rupturable, liquid-hll~ eable cont~inpr ç~ p~ ting the hydldLillg layer is affixed directly to the above-described occlusive bandage layer or to the above~escribed adhesive layer ~dj~c~o-nt to the occlusive bandage. Affixed to the exposed side (the side which is not ~tt~rllP~i to the backing or adhesive layer) of the rupturable, liquid-impçrmP~ble container encapsulating the hydrating layer is a dry layer of finely-ground, powdered fibrin components, as described above. The layer of dry ccm~ull~ inr!ll-les powdered fibrinogen or fibrinogen complex, Lll-~--.bin, and as nPcçcc~ry sufficient ç~lcinm and/or Factor XIII to, upon hydration, form a fibrin clot.
The dual layers (the dry layer and the hydrating layer) are together covered on all sllrf~res not in contact with the occlusive backing or adhesive m~tPri~l ~ffixing the layers to the occlusive backing, with an outer, protective, second impermeable membrane. Thus, in this dual-layer embodiment, the contents are entirely enca~ulated within an i~ leable co.l~ , wherein one side is the occlusive backing material and the other side and all edges are formed by the outer, l,loL~ )e, second ull~t;lllleable membrane.
In operation, the inner liquid-impermeable container ~r~ ting the hydrating layer is physically ruptured to release the hydl~lLulg m~tçri~l W O 96/40174 PCT~US96/10006 couLd~ed therein into the dry fibrin COLU~)OI~e11~ layer, rçsllltin~ in a fully-hydrated tissue sealing fibrin clot to inhibit blood and fluid loss from the wound, and to provide a natural barrier to infection The outer, second i...l,~. ..~F~ble Lu~nll,~ e retains the released hyflldLiug m~ri~l in contact with the dry cfJlu~ollc.lL~. until a m~llP~hle fibrin complex forms, at which time the outer ~U~;1111J1aneiS physically removed and the b~nfl~ge placed over the wound to form a tissue sealant.
In the ~ ive, the outer melLl,ldlle may be physically removed, and the dual layers forcefully applied to the wound area in a manner which, u~Lu c~sthe inner liquid-iu,~ll,leable f f)--~ P. and releases the hydld~ g agent into the dry fibrin components so that the tissue sealing fibrin clot is formed directly on the wounded tissue.
As in other embo~ of the fibrin sealant bandage, the selectef3 adhesives and backing m~tPri~l~ may be de-lf Ill;llF~(l by the intPnflPfl application of the b~n~ e. The backil~g may be removable or resorbable, and the adhesive may have the intf nrl.od purpose upon removal of the bandage of removing the tissue sealant from the wound, or of leaving the tissue sealing fibrin clot llnfli~tllrbed. The adhesive may be a se~dldlt:ly bound layer, or the backing may itself act as an adhesive to affix the dry fibrin components.
The L1L~U1U1~ c~lcillm and Factor XIII CO1U~OUC;~ which are nf C~ l y to form the fibrin complex may be affixed as dry material(s) in the dry m~tPri~
layer, or they may be inrlllrlf d in liquid or gel form in the hydrating layer.
Moreover, they may be divided between the two layers, so long as all of the nPcçcs~ry fibrin-forming components are present, and the dry layer remains non-hydrated until the bandage is used. In addition, additives, such as the previously disclosed growth factors, antibiotics, antiseptics, antiproliferativedrugs, etc. may also be in~l~ldfd in this embodiment of the fibrin sealant bandage.
If the hyLdLillg layer couldius a liquid ..u~tlsaluld~d with gas, the dry material layer will be hydrated as an ~ hle, foaming, fibrin tissue sealant.

W O 96140S74 PCT~US96/100~6 ,~ In the all~ ~ " ~~iv~, the dry m~teri~l layer may be suppl~ nt~d with m~teri~l~
which produce gas, and hence foaming, upon contact with the hydlaL~ll~ agent.
If the Ly~dLul~, layer is in the form of a gel, such as a quick~v~7lal;u~~7 gel layers (e.g., methylc~ lose/alcohol/water), the rupture ofthe ~.UllOllll~
impelllleable barrier permits the drv material fibrin components to dLI,clly contact the hy&aLillg layer as disclosed above to produce the tissue sealing fibrin clot. The gel layer, in the manner described for a liquid hy~aLill~, layer, may conl~lise any one, or all, of the thrombin, calcium or Factor Xm el~-m~on of the fibrin complex, and/or any one of the above-disclosed additives.
In an alternate dual layer embodiment, the tissue sealant is delivered as a wound sealing dressing, which need not be affixed to a b~cl~ing. The components are ol~u~i~d ess~nti~lly as a capsule within a capsule, wherein the term capsule is used to define a broad concept, rather than a material. The above-described e~ te(l hy~dLillg layer is itself contained within a second enc~ ting unit, which contains both the dry fibrin co~ oll~llL m~t~ri~l~ and the encapsulated hydrdlillg layer.
In operation, the inner, liquid~ elllleable container e~r~ ting the hydrating layer is physically rlptured to release the hydrating m~teri~l contained therein into the dry fibrin component layer, both of which remain completely contained within the outer, second encapsulating unit. The integrity of the outer, second enr~rsnl~ting unit is not broken when the inner container encapsulating the hydrating layer is physically ruptured.
The mixing of the hydldLulg layer with the dry fibrin c~.lll~3oll~uL~7 within the outer encapsnl~ting unit results in a fully-lly~dL~d tissue sealing fibrin clot, which is then released or expelled onto wounded tissue to form a tissue seal.
To release the fibrin mass, the outer en~pslll~ting unit is physically cut or torn, either randomly or at a specific location on the surface, e.g., to form a pour spout to direct the flow of the m~ ble fibrin mass onto the wound site.
If the hy&dli~ layer is a agent supelsalulal~d with gas, the mixing of the hydrating agent with the dry fibrin components results in an expan~l~ble W O 96/40174 PCT~US96/10006 foaming llli~lUl'C, which is then applied to the wounded tissue. The rOa~
may, in the A1~ I;ve, be achieved by hydration of the dry COU1~0l~C~11L layer.

The Self-Foan7ing F~brin .Sen~n~t Embo~i-ne~t~
S A self-~oall~illg fibrin sealant dl~ssi~g embodiment for treating wuuLlded tissue in a patient is formlllAt~l as an e~ tlAhle foam coll~ isiug a fibrin-~lllLUlg effective amount, in combination, of (1) virally-inactivated, purified fibrinogen, (2) virally-inactivated, purified lllolubill, and as l-~ocess~.~ (3)calcium and/or Factor XIII; whelei~l said composition does not si~nifirAntly inhibit full-thickn~oss skin wound healing. The previously described TS
col~ollenL~ are stored in a ca~ r or tank with a pless... ;,.~1 propellant, so that the components are delivered to the wound site as an expantlAble foam, which will within minllf~s form a fibrin seal.
Acceptable formulations of the expandable foam embodiment provide the hydrated c~ onel,~ of a fibrin clot, which in operation expand up to twenty-fold. The extent of ~xp~n~it n of the tissue sealing fibrin clot, however, is determined by its intended application.
For example, use of the Pxp~n-l~hle foam fibrin sealant dressing within the abdomen provides a fibrin tissue sealant to signifi~ntly ~l;lll;ll,~h or prevent blood or fluid loss from injured internal tissues organs or blood vessels, whilealso providing a barrier to infection However, at the same time the Ç"l~
of the foam must be controlled to prevent h~rmfill ~res~ulc on llnrl~m~ed tissue, organs or blood vessels. Such a situation may w~ldlll the use of an expandable foam dressing in which the expansion is limited to only 1- or 2-fold, and not more than 5-10 fold.
By colll~aLison, use of the exr~ntl~hle foam fibrin sealant dressing to fill gaps within bone, may Wall~ the use of material which çxp~ntl~ at a much greater rate to produce a tight and firm seal over the wounded area. Arterial wounds may also respond well to a highly ~l~s~ul~ed foam tissue sealant dressing. The extent of the eXp~n~inn of such material may be in the range of W O 96140174 PCT~US96/10006 above 20-fold, ~lthol-gh preferably 10-20 fold, or more preferably 5-10 fold.
An eY~ ic-ll of less than 5-fold, inr~ lin~ 1- to 2-fold may also be applicable t to repair of blood vessels or injured bone, for eY~mple in small areas, such as the inner ear.
Like the e~r~n~ion rate, the set-up time for the forrnation of the fibrin seal using the ~xp~ntlAhle foam fibrin dressi~g is also related to its inf~n~ledapplication. In certain sihl~tion~ loss of life may be i""~ t such as in a patient who has suffered arterial wounds or damaged heart tissue. In such a ~it l~ti~n the fibrin dr~sJl~ must expand very rapidly and form the fibrin tissue seal as quickly as possible, n~ces~rily before exsanguination. Preferably the seal will set-up and significantly ~imini~h the patient's fluid loss within 2 mimltes or less, more preferably in 1-2 mimlt~s, and most preferably in less than 1 minute.
On the other hand, not all wounds are im m~ t~ly life Ill,e~ ,.g. For example, the ~ ;n~ lh of the tissue sealant repair of bony tissue is more important than a rapid set-up time. In such situations, the composition of the tissue sealing fibrin clot may be modified to permit greater cross-linking or thi~kening of the fibrin fibrils, or to permit delivery of a more dilute composition which will continue to expand for a longer period of time. Such fornml~tions may either permit or require a slightly longer time to set-up the tissue sealing fibrin clot. Although a set-up time of under 1 minute is a~ro~liate for such applications, set-up times of 1-2 ",;..~les, or up to 5 ",i""les would be acceptable. In ci,~ es recognizable to one of ol-li~y skill in the art, a long set-up time of 5-10 ~l~hlu~es, or even up to twenty Ill;ll~lt;s, may be acceptable innon-life Illl~ lri~ g situations.
The delivery devices, e.g., cani~L~r, tank, etc., may be developed especially for the present application, or they may be commercially available.
The canister may c~ "l~,ise either a single or multiple reservoirs. Separate ,~sc;, ~ , although more expensive, will advantageously permit the hydrated CA 02223889 l997-l2-05 W O 96/40174 PCT~US96/10006 colll~oll~l~Ls to remain seL,al~lèd and stable until they are mixed upon appli-~tion The propellant must be physiologically acceptable, suitable for ph~rm~-~ological applir~tionc~ and may include collv~ n~lly recognized propellants, for example, CO2, N2, air or inert gas, such as freon, under pleia~iu~ In the ~ ;v~, the dry fibrin co"l~o~le~lL~ may be supple~nt~-l with m~t~ri~l(s) which produce gas, and hence rO~,,-,,g, upon contact with the hydld~ g agent.
Since delivery pl~ .Ul'~ of the ~x~ hle foam fibrin dressing from the delivery device, when combined with the composition of the fibrin clot itself and its set-up time, .i~ .oc the extent of expanci~n of the dressing, the delivery plcs~uleis~el~ by the nature of the wound being treated. As described above, certain wounds require i~""~ formation of the tissue sealing fibrin clot to prevent loss of life, while others wounds require slow delivery or time to form e~Lellsive cross-links to str~ngth~on the tissue sealing composition. Therefore, delivery p,~,s~u,c may ideally be situation specific.
Pressure of 1 atmosphere, or less (14.7 lbs/inch2) will provide a low level of expansion and a slower rate of delivery. However, certain life LLe~ -i-lg situations may wal~ a delivery ~ u,~ of 1-5 atmospheres, or more. In most cases, the delivery pres~ure chosen co~lesl!o~ds to that of commercially available cani..L~,. devices. As an addition factor, the delivery pressure may be important to keep the tissue sealant material from clogging delivery lines or devices.

Combined Embodiments of the Self-Contained Wound Dressing and Fibrin Sealant Bandage Finally, certain Ll~ ic injuries will be best treated by combining several embo-lim~ontc of the self~ont~in~1 fibrin sealant dl~s~i lg. For example, in serious car accidents or injuries caused by ~Li~ onnel-mines or explosives, the wounds may be not only life-thleale Ig but e~LellSive, involving large, WO 96/40174 PCrnJS96/10006 _59_ jagged open-llg~ in tissue or bone with ~ignifi.~nt inttorn~l clAmA~e, often with acc~a-,yillg serious burns. Such wounds may present uu~u~vu~. severed arteries and blood vessels in ~ ition to ~ ~.ivt; areas of wounded tissue. In such wounds, it may be adv~ntA~eollc to first liberally apply a rapidly setting ~ Ahle fibrin foam dres~i~ to quickly control hemorrh~gin~, and then to wrap the entire area in an embodiment of the fibrin sealant bandage to ~.u~po, and protect the wounded area and seal slow fluid loss from, for ~mpl~, burned tissue, until the victim can be L~ olled to a m~-lirAl facility, or untilpror~inn~l mrr1ir~1 a~ ç can A~ rl ed. In most in~fAnrPs~ ~rl~litir~n~l formulations of the fibrin sealant dressing will then be applied by the trained personnel for the long-term repair, 1l~ lll and protection of the injured tissue.
The following examples are inr~ rd for illustrative purposes only and are not inten-lPcl to limit the scope of the inventian.

Example I
Preparation of HBGF-I For Supplementafion of FG

An 800 ml culture of recombil~L E. coli ContAining a plasmid that included DNA .onro(ling HBGF-l~ was prepared. After in~ rtil n and c~lt~ring for 24 hours at 37~C, the cells were centrifuged and the supern~t~nt was discarded. The cell pellet was le~u~l~ellded in 25 mls of 20 mM phosphate buffer, cont~ining 0.15 M NaCl, pH 7.3. The suspended cells were di~ d with a cell disrupter and the cell debris was se~ala~ed from the res~ in~
solution by cçntrifilgAtion at 5000 g for 20 min.
The pellet was discarded and the ~P~ lll cont~ining the solubilized HBGF-l~ and other bacterial ~rolei- s was loaded onto a 2.6 cm ~1;A~ Irl by 10 cm high column of Heparin-Sepharosen' (Pharmacia Fine Ch~ ir~l~, Upsala, Sweden). The column was washed with 5 column volumes of 0.15 M NaCl in W O 96/40174 PCT~US96/10006 ~0-20 mM ph--SE~h~tto buffer, pH 7.3, and then was eluted with a 0.15 M NaCl in 20 mM phosph~te- buffer to 2.0 M NaCl gr~ nt The eluate was lllom~olcd by W absorption at 280 nm. Three peaks of W abso~ , m~t~ri~1 eluted and were analyzed by SDS polyacrylamide gel S elecL,o~holci,is. Peak llulllbeL three clecll~horesed as a single band at about 17,400 daltons and co~ subst~nti~lly pure HBGF-l~.
In order to further insure that the HBGF-l~ was free of co-~ ;l.g bacterial p,- lchls, peak l~ bei three, which contained the growth factor activity, was dialyzed overnight against 20 mM histidine, 0.15 M NaCl, pH
7.5. Two mg of protein was loaded onto a 1 ml CM-SepharosesH (Pharmacia, Upsala, Sweden) ion IqY~h~nge column. The column was washed with 10 bed volumes (0.5 ml/min) of 20 mM hi.ctirlin~, 0.15 M NaCl, pH 7.5 and eluted with a gradient of 0.15 M NaCl to 1.0 M NaCl in 20 mM hi.~ti(line, pH 7.5.
The eluate was monitored by W absorption at 280 nm and HBGF-l,B was i-lentifi~cl by SDS polyacrylamide gel electrophoresis.
This purified HBGF-l was used to supplement FG in subseq~lPnt examples.

Example 2 Stability of HBGF-l It was nl~ces~ry to add an ingredient to the FG that would inhibit or prevent the digestion of HBGF-l~ by ~ olllbill (Lobb, Biochem. 27:2572-2578 (1988)), which is a component of FG. Heparin, which adsorbs to HBGF-l, was selected and tested to determine whether it could protect HBGF-l from digestion by thrombin and any other proteolytic components of the FG. The stability of HBGF-l in the ~lcsellce of increasing collcellildlions of heparin was ~sesse,l Solutions c~ g HBGF-l~ (10 ~g/ml), thrombin (250 U/ml), and increasing concentrations of heparin (0, 0.5, 5, 10, 20, and 50 U/ml) were wo 96r40~74 PCT/US96/10006 inMlb~t~rl at 37~C. Aliquots were periodically removed from the i~ bal;-~
solutions and were frozen and stored at -70~C for further testing.
After the inrllhatinn was co",r,l~t~, the samples were thawed and s~al~Led on 15 % SDS polyacrylamide gels under re~lllcin~ cont1ition~
according to the method of T ~mmli (Nature 227:680 (1970)). The gel was then electroblotted onto nitrocellulose and the band corresponding to HBGF-1 was i~letltifip-A using an affinity-purified polyclonal rabbit ~lListlu~ll to HBGF-l.
The Western blots are shown in Fig. 1 on which the HBGF-l,B band at 17,400 mw can be seen. The results inAir~t~A that in the presence of collc~ d~ions of heparin as low as 5 U/ml, HBGF-l~ was ~lote~;~ed from digestion by thrombin. In ~lAition, as described in Example 3, its biological activity was not altered.

Example 3 The Biological Activity of HBGF-l~ after Incubation in the Presence of Heparin and Thrombin The biological activity of HBGF-1 in the incubation ~ LLulc that contained 5 U/r~ of heparin, and was described in Example 2, was ll,ea~u,ed using an 3H-thymidine incc"~o,~Lion assay with NIH 3T3 cells.
NIH 3T3 cells were introduced into 96 well plates and were inr~b~t~A
at 37~C under starvation contlition~ in Dulbecco's Modified MrAillm (DMEM;
GIBCO, Grand Island, New York) with 0.5% fetal bovine serum (BCS;
GIBCO, Grand Island, New York) until the cells reached 30 to 50%
confll1~nre. Two days later, varying dilutions of HBGF-l from the saInples l,l~cd in Example 2 were added to each well without rll~n~ing the mPAillm Diluent (inrllb~tion buffer) was added in place of growth factor for the negative controls and DMEM with 10% BCS, which contains growth factors needed for growth, was added in place of the HBGF-l sample for the positive controls.

W O 96/40174 PCT~US96/10006 After i"~-~,ba~;on at 37~C for 18 hours, 0.25 ~Ci of 3H-thymidine, specific activity 6.7 ~Ci/mol, was added to each well and the i.,~ l~b~lion was contiml~cl at 37~C for an ~cl-lition~l 4 hours. The plates were rinsed with rphosphate-buffered saline (PBS) and fixed with 0.5 ml cold 10% tric~lor~e~tir acid (TCA) for 15 min at 4~C. The TCA was removed, the plates were rinsed with PBS and the acid-precipitable material was solubilized with 0.5 ml/well of 0.1 N sodium hydroxide for 1 hour at room Ir~ e. The samples were Ll~f~llGd to scintillation vials and 10 ml of scintill~tit)n fluid (New Fngl~ntlNuclear, Aquasure~) was added per vial.
The results, which are shown in Figure 2, demol~LIdLGd that HBGF-1, which had been i~ b~lr(1 in the ~;lcsGnce of Ll~rulllbill and hPp~r~n, retained its biological activity. The observed co~cellLldLion depen(l~nre of thymidine incorporation was independent of inrllhation time and was typical of that expected for the deprn~ nre of the proliferation of cells as a fimrtion of growth factor concentration. Growth factors typically exhibit an optimal col~ce.lllaLion at which cell proliferation is m~xim~l.
The biological activity of HBGF-l in the ~lesellce of Lhrolllbill and heparin was also measured by obse.vi~ endothelial cell proliferation. The surfaces of petri dishes were i~ reg~Led with the HBGF-l supplemrnt~l FG.
A shallow layer of endothelial cells was added and the number of cells was measured. Over time the number of cells increased. In ?~ ition, the cells appeared to be olg~ illg into vessels.
TherGfulG, HBGF-l retains its biological activities in FG that includes heparin, which ploLecL~ HBGF-l from the degradative activity of thrombin and may also potentiate the biological activity of the HBGF-1 in the growth factor-supplemented FG.

W O 96/40174 PCT~US96/10006 Example 4 HBGF-I Diffusion from a FG Clot AFG clot was fonned in a 5 rnl plastic test tube by mixing 0.3 ml of the fibrinogen complex C~ nt~ining 10 U/ml heparin and thrombin and 40 rnM
S CaCl2. Four test tubes were set up as follows:
(A) 0.5 U/ml ~clllbilland 10,ug/ml HBGF-l;
(B) 0.5 Ulml l~ llbi~ and 50 ~g/ml HBGF-l;
(C) S U/ml thrombin and 10 ~ug/ml HBGF-l; and (D) 5 U/ml thrombin and 50 ~Lg/ml HBGF-l.
Each clot was covered with 0.2 M hictit1inP buffer, pH 7.3. Thirty ,ul samples of the overlying buffer were removed from each tube every t~vo hours and were run on a western blot.
The results of the expe~ llL demonstrated that HBGF-l diffusion out of the clot is a function of time and its concentration in the clot, and that the collc~ dLion of ~.. ulllbill in the clot does not affect the rate at which HBGF-l is released from the clot.

Example 5 The Behavior of Human Umbilical Vein Endothelial Cells in Growth Factor-Supplemented FG: The Effect of Wild Type and Mutant FGF-I

To study the in vitro effects of acidic fibroblast growth factor (FGF-l)-supplemented FG on human endothelial cells, suspensions of these cells were added to 10 cm ~ meter petri dishes that contained evenly spread layers of 2.5 ml of FGco~ g appr )xim~tely 9 mg of fibrinogen per ml and 0.25 NIH
units of thrombin per ml. The FG was supplemented in the following ways:
(A) No added growth factor;
(B) Supple~n~ntto~l with 100 ng/ml of active, wild-type FGF-l;

W O96/40174 PCT~US96/10006 (C) Suppl~ rA with 100 ng/ml of inactive, mutant FGF-1; or (D) Suppl~n ~nt~l with 10 ng/ml of active, wild-type FGF-l.
The cells seeded onto the FG layer were ...~i--li.i-.~l for 7 days in DMEM
cc,..~i,.;,.~ 10% fetal bovine serum (FBS).
S The cells became elon~tPA and proliferated errcielllly when in contact with FG suppl~otn~nt~(1 with biologically active FGF-1 (Figures 3 and 4). In contact with unsuppl~m~nt~l FG (Figure S) or with FG suppl~ ontPd with biologically inactive mutant FGF-1 (Figure 6), the cells become elon~tP-l but proliferated relatively slowly.

0 Example 6 The Behavior of Human Umbilical Vein Endothelial Cells in FGP-I- Supplemented FG

To study their growth, human umbilical endothelial cells, 105 or more cells per ml, were embedded in FG, the protein concellllaLion of which was 4 mg/ml. The collcell~d~ion of thrombin in the FG was adjusted to 0.6 NIH
U/ml. The culture m~ lrn used in all of the e~eLilllents was M199 (Sigma Ch~ ir~l Co., St. Louis, MO) suppl~ nt~d with 10% fetal bovine serum, 10 ,ug/ml ~ ,pL(Jlllycill, 100 U/ml penicillin, 1 ng/ml FGF-1 and 10 U/ml heparin.
Within 24 hours in FG the cells became elo~g~t~o~l, multipodial and formed a cellular llclwu-h when they came in contact with each other (Fig. 7).
This growth continued for at least 5 days. Figure 8 shows this situation at 48 hours.
As a control, an ~ ntir~l cell suspension was cultured on a surface coated with fil)ronec~Lll at 10 ~g/cm2. Control cells acquired a cobblestone shape and m~int~inPcl this morphology for at least S days. Figures 9 and 10 show this ~itn~tinn at 24 and 48 hours, lc~l-eclivc;ly.

Example 7 CA 02223889 l997-l2-05 W O 96140174 PCTflUS96/l~a~6 ~5-The Behavior of PMEXNEO-3T3-2.2 Cells In PG

PMEXNE0-3T3-2.2 cells are r~ ubl&.~.l cells that contain a m~ ified ..... o with the yoLtllL;al to eY~.ress ge~ lly cllg;i-~el~ed proteins (l:orough et al., J. Biol. Chem. 268:2960-2968 (1993)). To ~ P the behavior of these cells in FG, 105 cells per well were cultured under three con.1iti~n~:
(1) embedded in FG; (2) on the surface of FG; and (3) in the absence of FG
(controls). The expe~ lL~. were carried out in duplicate in 24-well plates in DMEM media (Sigma Ch~mi~l Co., St. Louis, M0) supplem~nt~l with 10%
FBS. The FG protein col~cellLI~lion was 4 mg/ml. In iAI~ntir~ y~.ull~llL~. the m~ n was suppl~TnPnt~l with 1.5% FBS was used as negaliv~ controls.
In the ylesence of media supplement~A with 10% FP.S, the cells in all 3 groups grew and became conflnent In the l~egalive control e~y~l Ullt;llL~ in which the media was supplem~nt~A. with 1.5 % FBS, the cells grew and survived for at least five days in the ~lese~ce of FG, but not without it.
However, their growth was faster in FG supple-m~nte~l with 10% FBS than in that supplem~nt~A with 1.5% FBS. In the absence of FG, in the media supplemPnt~ with 1.5% FBS, the cells died within 48 hours. The criteria for survival was the ability of the tested cells to proliferate upon Llal~.rt;l to fresh media supp]em~nt~A with 10% FP.S.

Example 8 The Endothelinli~fi~n of Expanded PTFE Vascular Grafts by HBGF-I r~ ,ea~nent Two studies llem~ ed that yl~eLl~e~ ll of blood-cont~eting bio~L~lials with endothelial cell (EC) mitogens enh~nre-l endoth~ tion.
The first study eY~min~-~l the in vivo washout characteristics of HBGF-1-supplemlont~l FG ~u~yension applied to eYp~nAeA PTFE grafts implanted into W O 96/40174 PCT~US96/10006 rabbit aortas. In the second study similar grafts were implanted into the aortaileac position in dogs. HBGF-l, an angiogenic factor, was used in studies. Other growth factors such as a FGF, FGF-4 and/or OP-1 can also be used as a supple~ lll(s) for the vascular grafts.

S A. Washout Study In general, the modified FG was sterilely ~le~aled by adding approximately 1 ng/cm2 area of the inner and outer graft surfaces of human reCO~ Lld~ll2sI-HBGF-l,20 ~g/cm2 porcine illle~l;ll~l mucosal heparin, and 2.86 mg/cm2 fibrinogen to 2.86 x 10~2U/cm 2recon~tit~lt~, commercially available, human thrombin (1000 U/ml) to induce polymerization.
The '25I-HBGF-1 was *,eciri~lly prepared as follows. Fibrinogen was reco~ d by adding 500 mg of fibrinogen into 25 ml of PBS to produce a fibrinogen concellLlalion of 20 mg/ml of PBS. Three ml of this solution which contained 60 mg fibrinogen were placed into 12 Eppendorf plastic tubes and Il.~ od at -70~C. Each of these aliquots was used individually.
The Lll.ulllbh~ was recon~ lecl by diluting a commercially available ~l~aldLion thereof (Armour Pl.~ Co., K~nk~k~e, IL) at a concellL,dlion of 1000 U/ml by a factor of 1:10 in sterile solution to produce aconcellL.dlion of 100 U/ml. This thrombin solution was again diluted 1:10 to produce a solution of 10 U/ml.
The bovine heparin (Upjohn, K~ m,lr~Qo, MI) was reconstituted by diluting the ~l~dlion at a concentration of 1000 U/ml by a factor of 1:1000 using normal saline.
One and 48/100 (1.48) ml of the reco~.~lil..lt~ fibrinogen, 63 ,uL of the l~col.~lil.lled heparin, plus 15.66,uL of l25I-HBGF-1 were mixed in a glass scintill~tinn tube. This mixture was then aspirated into a 3 ml plastic syringe.Five ml of the recoll~lilllled thrombin was placed into a glass scintill~tion tube.
One end of the exp~n~lP(l PTFE graft was placed over a plastic 3-way stopcock nozzle and was secured there with a 2-0 silk tie. The PTFE was then W O 96140~74 PCTAUS96/10006 encircled with a 3 x 3 cm square of Parafi1m~ which was then e~ e~ there with a straight h~most~t to establish a watertight seal. A second 2-0 silk tie was poCitio~ over the parafilm ~lj~nt to the slolJcoc~ to form another seal.
A str~ ht hemostat was then used to clamp the distal 2 mm of the S PTFE/parafilm to seal this end.
Equal volumes of fibrinogen and lllolllbin solution ~r~a,cd as described above were rnixed and allowed to react for approximately 30 seeon-lc which is when poly- ~ Ir.~ 1 ion occurs. The 1l~ UlLlbil~pOlyl I If ;~ fibrin is then opaque. (This time factor is appr~-Yim~tP~ and varies from one thrombin lot to another. The a~roplial~ length of time to polymerization can be ~let~
by viewing the opacity of the ~ lulc). The fibrin/thrombin ~ c was aspirated into a one cc syringe. (NOTE: The volume of this graft was 0.42 ml.
For a graft with a larger volume one needs to use a larger syringe.) The syringe was at~rhP~l to the ~tu~cocl; and the mixture was inJected by hand over a period of 5 seconds until the liquid was seen to "sweat" through the PTFE
interstices and filled the space between the PTFE and the Parafilmm. The 3-way stopcock was closed to the PTFE graft for 3 ..,;..~es and a scalpel blade was used to cut the ligature at the end of the PTFE over the stopcock. The PTFE graft/p~rafilm was removed from the stopcock and a hPmost~t was used to remove the PTFE from the parafilm envelope. To clear residual growth factor-supplem~nt~cl FG from the graft lumen, a number 3 embohPctomy catheter was passed through the graft five times until the graft lumen was completely clear. The growth factor-supplp-mpnttod FG-treated PTFE graft was allowed to dry overnight for about 12 hours under a laminar flow hood. The treated graft was then ready for implantation.
ely, this HBGF-supplr~ FG was pres~ule perfused into a 34 mm (24 mm ~ 5 mm at each end) x 4 mm (internal ~ t ) thin-walled, PYp~ntlPA PrFE graft thereby coating the graft's luminal surface and r~ x~ri~-liu~
through the nodes to the graft's outer surfaces. The lumen of the graft was cleared as stated above. These grafts were then interposed into the i~ nal -~KIrmin~l aortas of 24, 3-5 Kg New 7P~l~nt1 white rabbits. In the first study, the animals were ~~ ;rir~xl and s~ were eYl.l~ rA at 0 time (to correct for losses due to surgical m~nirll~ti-~n) and after 5, 30, and 60 min, and 1, 7,14, and 30 days. E2rsi~ l r~rli~ ~rl;vily was ~ ..;--PA by gamrna COull~illg.
S 1~ 25I-HBGF-l, coll~;l~d for ~ulll~ c decay, is eA~l~s~ed as a ~e..;~llktge of the zero time value.
The w~L~u~ of l25I-HBGF-l followed classic kinetics with a rapid initial losswiththereestabli~l....- ,lofcirculation(%/~n=-24.1 between5and 60 ~illu~t;s) followed by a slow loss after 1 hr (%/in = -0-03) with 13.4% i 0 6.9 % l~ -g after 1 week and 3.8 % it 1.1 % rçm~ining after 30 days.

B. In Vivo F~ot~2eljn~ en Study The second study evaluated the effects of the applied HBGF-l-supplemented FG suspension on: the rate of endothPli~li7~tiQn of widely exranll~P~ 60 ,um internodal ~ t~nre exp~ntle~l PTFE grafts implanted into canine aorta-iliac positions; the proliferative activity of these endothelial cells as a function of time; and the relative contributions of the HBGF-1 and the FG
in stimnl~ting the observed endothelial cell proliferation. Three groups of 50 x 4 mm non-leillrurced exp~n~lP(l PTFE grafts were implanted in the aortailiac position of 12 dogs. Group 1 (n = 6) contained 20 ,ug/cm2 heparin, 2.86 mg/cm2 fibrinogen and 2.86 X 10-2 U/cm2 of human LL~ bLl plus 1 ng/cm2 of HBGF-1. Group 2 (n = 3) co..~ ~l the same FG without HBGF-1. Group 3 (n = 3) col-c;~ of i-l~Pntir~l but u~lleal~d control grafts.
Tritiated thymidine (3H-TdR; 0.5 ,uCi/kg) was injected in 10 hours before explantation. Grafts were explanted at 7 and 28 days for light and electron ll~iClOSCO~y, Factor vm immlln~hi~torllr.. i~!.y, and en face autoradiography for çnrlothrli~l cell proliferation in random high power fields. Each graft was viewed by three observers who did not know from which l~ lllrlll group the graft came. Dirr~ nces in endothelial cell proliferation were st~ti~tir~lly analyzed by two-way ANOVA and independent t-tests.

CA 02223889 l997-l2-05 W O96/40174 PCTAUS96~1aO~6 At 7 days 33% of both the FG and HBGF-l-suppl~mPntçcl FG grafts demo..~l"1led non-contiguous foci of endothelial cells (Fig. 11). The surface of the control grafts ~ A a fibrin co~ lm At 28 days, every HBGF-l-suppl~n~nt~l FG showed e~lc~ivc capillary i~gruwlll and confluent endothrli~li7~d blood co.,l;.. -l;.-g s ~ res, which were not seen in any ~
of the other two groups (Figs. 11 and 12). Figure 12 cl~ .dl~s that u~ dlcd grafts at 28 days had few visible endothelial cells on their surface (Panel G). Grafts treated with FG alone had about 33% of their surface covered with endothelial cells in-lir,~ting that FG tre~tn-~nt alone encouraged some reendotheli~li7~tion (Panel H). However, grafts treated with FG
suppl~mrntrd with HBGF-l (Panel I) a~eall,d to be completely (>95%) covered with endothelial cells which display the cl~l~ r cobblestone morphology of endothelial cells. Thus, the c~ ~Lion of growth factors delivered by FG was able to encourage e~nti~lly the complP-te covering of the vascular graft with a non-thrombogenic endothelial cell lining. En face autoradiography revealed a st~ti~tir~lly .~ ";l;r5"1 increase (p < .05) in 3H-TdR incorporation into the DNA of endothelial cells in the HBGF-l-supplemPntrd FG grafts at 28 days vs. all other groups both as a function of time and of graft tre~tmrnt These data demo~ dle that pl~s~ule perfusion of an HBGF-l-supplemPnt.od FG ~ùs~ sion into 60 ~ intern~ t~nre exp~n~ PTFE
grafts promotes endotheli~li7~tinn via capillary ingrowth and increased endothelial cell proliferation.
These studies demol~lLdte enh~nred ~u~ eous re-endotheli~1i7~tion of small ~ mrter vascular grafts, and also a method for stimlll~ting a more rapid confluence of transplanted endothelial cells.

Example 9 Delivery of Tribuly~,.from ~ib~in Sealant The inA--rtion of PnAothP~ i7~tion of artificial vascular grafts by FGF-l deli~ d in fibrin sealant ~ ,elll~ an i~olL~l ~1.F.a~ ir application of the use of suppl~--..k~.fP~l fibrin sealant as a delivery vehicle. Hy~ liferation ofsmooth muscle cells in arterial walls is a ~ip.. ;rir~ colll~ollelll of arte~iosrlerosis, and in l.,..L~-.-r~ following angioplasty (Cercek et al., Amer.
J. Cardiol. 68:24C (1991)). Thel~r~lc;, delivery of an anti-prolire.dlive or dirr~ l;AI;"~ agent suitable for intravascular L~ lllrl-l from a supplP~mPntPA
fibrin sealant deli~.y system was coIl~iAPred to prevent or treat this condition.
10In choosing an agent to prevent smooth muscle cell hyperproliferation, a drug with extremely low toxicity was selecte~l as it was important not to induce cell damage that might exacerbate the underlying condition. Butyric acid has been shown to plc;vt~ the hype-~-oliferation of retinoblastoma cells (Kyritsis et al., Anticancer Res. 6:465 (1986)), Swiss 3T3 cells (Toscani et al., 15J. Biol. Chem. 265:5722 (1990)) and other cell types (Prasad et al., Life Sci.27:1351 (1980)) by inducing a dirr~l~,llliaLion program.
An inA~ l preven~ion of hy~ 'oliferation also has been achieved in smooth muscle cells by a related colll~ulld, llibulyliul. This effect on smooth muscle cells requires a concelllldlion of tlibulylill that is close to saturation, 20making systemic therapy Aiffirlllt Therefore, the following experiment was conA--cte~l to demo~Lldle the efficacy of delivering tributyrin directly to the lesion from a supplemPnt~A fibrin sealant composition.
Tributyrin was mixed with thrombin, which was then mixed with fibrinogen to form a fibrin sealant matrix. The supphPmPntPA fibrin sealant was 25placed into 24-well culture plates. Culture mPAillm (2 ml) was then placed in wells cont~ining the tributyrin supplPmpnte~l fibrin sealant, and these were b~eA at 37~C. The mPAillm from a new set of three wells was harvested daily, and che ~u~ used to culture proliferating smooth muscle cells (10,000 rat or rabbit smooth muscle cells per well, which had been allowed to WO 96~4~174 PCr~US96~I0006 attach overnight). After i~ b~lion for two days (48 hours), the ll.--llb~l of cells in each smooth muscle cell culture was l-l~ur~d using the MTS assay (a bio~ ;ol~ of the tetrazolium cu,ll~vu,ld MTS (P,ul,le~;~, Madison, WI) into a soluble r~ .. cl~v~Lv~hoie ~l~tP~ctpd by s~ecLlv~hoLvllleLl~ at 490 nm.) As shown in Figure 13, the ",~.li.. ", l~ L~d from wells co--~
fibrin sealant alone ~ ~olL~d the growth of the smooth muscle cells, while the m.o-linm from wells with fibrin sealant cont~ining tributyrin .~ignifi~ntly inhibited smooth muscle cell pr~lif~r~til~n As the llulllbel of days of llibulylh diffusion into the mP.1;..." i~cl~ased, the degree of inhibition il~ ased. Theseresults in~ te(l that a cell regulatory drug, tributyrin, can be delivered from fibrin sealant for exten~1e(~ periods and that it retains the sllst~in~d ability to inhibit the proliferation of a specific cell type.

Example 10 Formulation and Delive-y of TGF-~2 from Fibrin Sealant Fibrinogen and ~ lllb~.l were prepared per instruction of the Alllelic Red Cross, Rockville, Maryland. Upon recol~Lilulion, the protein conr~ n of the Topical Fibrinogen Complex, (TFC) was 120 mg/ml (the standard formlll~tion for hemostasis). The human ll~lulllbill was reco,,~
with 40 mM CaCl2 to yield a solution at 300 units/ml.
To evaluate the compatibility of ll~rOll~ g growth factor ~2 (TGF-~2) in Topical Fibrinogen Complex, TGF-~2 (purified recombinant human protein provided by Genzyme Corp., Fl,....;.-~ .--, MA) was spiked into TFC
at 10 and 1 ,ug/ml. Samples were inrub~tPd for two weeks at 2-8~C. TGF-~2 was extracted for analysis by passing the gel-like m~t.ori~l through a narrow bore stopcock cv~ ;Lecl to two syringes. The ELISA data in~1ir~t~
lccû~ y of TGF-~2 from the TFC. Analysis in the in vitro bio-assay in~iic~ted that the extract was bioactive.
-W O 96/40174 PCT~US96/10006 TGF-~2 was then spiked into the TFC solution at a co..~-e~.l ntion of 1 ,llg/ml or 100 ng/ml. 50~1 aliquots were placed into sterile test tubes and 50 ~1 of the ll"c,~bin solution was added to form the fibrin clot. Clot ru~ l;r,l-occurred within a few secon-lc. These samples were allowed to sit u~ L
S at 2-8~C. Test sample t~lbes were then overlaid with 400 ~1 of PBS/0.1%
human serum albumin pH 7.0, with or without 10 ~g/ml plasmin. The test samples were inrllb~tP(l for two days at 37~C to evaluate the release and lec~ y of the TGF-~2. Complete resolution of the clot was observed in the plasmin treated samples. The clot rPm~inP<l intact in the non-plasmin treated samples. The diffusion ~uy~ "~ was analyzed by ELISA. The data are bllllll~ ed in Table 1.
-Table 1 TGF-~2 C~ lion in % Recv~ in D~ ioll Su~ l (by ELISA) Fibrin Clot With Plas~ , Without Plasmin 500 ng/ml 100 % 2.5 %
50 ng/ml 100% (not ~etPct~ble) Theoretical concentrations of components in the final clot based on dillltion TFC protein = 60 mg/ml; ll~o,llbi-l activity = 150 units/ml; TGF-,B2 =
500ng/ml or 50ng/ml.
The data inrlir~tP not only that TGF-,B2 is stable in TFC, but that the delivery of TGF-,B2 from fibrin sealant by diffusion can be sllst~inP~l in low amounts. Moreover, the release of TGF-~2 from fibrin sealant requires dissolution of the fibrin clot by pl~min inrlir~tin,~ that in vivo delivery of TGF-~2 from the supplpmpntprl tissue sealant composition would be mPrli~t~Pcl by resohltion of the fibrin clot. Thus, the . "~rhA";~"~ of delivery from the TGF-~2 supplPmPntPd tissue sealant composition is readily di~Lill~uished from simple diffusion kinPtics.

, Example 11 The Prepara~on of a Platelet-D~,~"e.l Extract for Use wi~h ~G

Plasma l~,duced platelets were pl-,~a~cd and p~ ot~d The s~lJe~
Splasma was l~,~uv~d. The pelleted plsltf~let~ were washed, ~ ed in buffer co--l;.;..;--~ 50 mM hi~ti-lin~ and 0.15 M sodium chlûri-l~ at pH 6.5, and treated with bovine lh~Ollll~ . After l~ l, the ~u~L..~ was cûllected by cçn1Tifi~tinn and aliquots were frozen at -80~C. The extract was thawed and mixed with FG or other TSs.
10The platelet extract obtained in this manner was biologically active since it i~ ~sed the i~COl~OlaliOll of r~Aioactive labeled thymidine into the DNA of proliferating NIH3T3 fibroblasts cOlll~al~d to the controls.
To evaluate the effect of platelet extract on wound h~lin~, r~
irlPntir~l to those carried out below in Example 12 with HBGF-l~ were carried 15out with platelet extract in diabetic mice. From the results of these e~illlell~
is clear that, given the low concellL.aLion of growth factors in the platelet extract, a dose larger than 100 ~g of platelet extract protein per wound needs tû be used to promote wound healing.

Example 12 The Effect of FG on S~in Wound Healing ln Vivo A. unsuppl~m~n~e~1 FG
An~
S Female C57BL/KsJ-db/db mice were obtained from Jackson Labolalolies (Bar Harbor, ME) and were 8 to 12 weeks old at the start of the expelilnell~. They were housed in s~aldle cages after surgery in an animal care facility.
These mice are used as a model of impaired wound healing in diabetic hllm~n~ because the metabolic abnorm~liti~s seen in these mice are similar to those found in human ~ hetirs. In addition, the healing imp~irmtont characterized by markedly delayed cellular infiltration, gr~nlll~tion tissue formation, and time required for wound closure suggest that healing in this mouse model may be relevant to the healing impairmPnt seen in human diabetes.

Fibrin Sealant The Collc~llLla~d topical fibrinogen complex (TFC) used in this study was produced from fresh frozen pooled human plasma. The TFC product (American Red Cross--Baxter Hyland Division, Los Angeles, CA) was supplied in Iyophili7~d form. After l~cn~ ion with 3.3 ml of sterile water, the protein characteristics of t_e TFC solution used in this study were: total protein, 120 mg/ml; fibrinogen, 90 mg/ml; rlb~ ecLi l, 13.5 mg/ml; Factor XIII, 17 U/ml; and plasminogen, 2.2 ~g/ml.
Topical bovine l.l~olllbill (5000-unit vial, Armour Ph~rm~eutir~l Co., K~nk~k~e, IL) was reco~ lerl with S ml sterile water and was serially diluted in 80 mM calcium chloride solution (~ Reagent Laboldtolies, S_irley, NY) to a concentration of 15 U/ml.
Equal volumes of TFC and reco~ d Illl~lllbin were mixed to produce FG. In order to fill a round 6-mm-diameter full thi~ wound, WO 96~40174 rC~r~US96~IaOa6 0.015 ml of TFC was mixed with 0.015 ml of Ill.o~ in~ The FG that was produced had a protein col~r~ m of a~rr)xi"~ ly 60 mg/ml.
A diluted FG with a protein c~ dLion of a~l~xi...~ly 1 mg/ml was also used.

SSurgery The mice were ~ .~ with a mLxture CO~ of 7 ml kr~ P
Lydr~cllloride (100 mg/ml; K~ot~cet, Aveco Co., Inc., Fort Dodge, LA), 3 ml xylazine (20 mg/ml; Pct)~nr-n, Mobey Corp., Shawnee, KA), and 20 ml physiological saline, at a dose of 0.1 ml per 100 g body wt, ~ lel~d illL,~ s~ rly. The dorsal hair was clipped, and the skin was washed with povidone-iodine solution and wiped with 70% alcohol solution. Two full-thicknto~s, round surgical w~ullds (6 mm tli~mPter) were made on the lower back of the mouse, one on each side, e~ ..l from the midline. The medial edges of the two wounds were S~al~L~d by a margin of at least 1.5 cm of u~w~ullded skin.
TmmP~ t~ly after the wounding had been pclrollued, FG and/or a dl~ssing was placed over the dP~ign~t~ wound. The dlCS~llg was a llL s~ cable adhesive polyul~ll~le dressing (Opsite, Smith and Nephew, M~illon, OH). Tincture of Benzoin co~ uulld (Paddock Laboratories, ~innP~polis, MN) was applied at the periphery of the wound area prior to application of the dlCbSillg. There was a margin of at least 0.5 cm of skin ~ull~ullding the wound edge over which no lill~;lul~, of be~oill was applied to avoid the possible ;Il~ ly effects of be~oi,l on the raw wound. No further L~ x were applied to the wound for the duration of the ~elilllelll.

CA 02223889 1997-12-0~

Trenfnl~nt Groups The mice were divided into 4 L1~A~ groups, with each mouse serving as its own control: , Group I: The wound on one side of the animal was treated with FG (60 S mg/ml) while the contralateral wound received no 1~ lll Both wounds were covered with Opsite .
Group II: Diluted FG (1.0 mg/ml) was topically applied to the wound on one side while the contralateral wound received no ll~aL~ L. Both wounds were covered with Opsite .
Group m: FG (60 mg/ml) was topically applied over both wounds.
The wound on one side was left uncovered while the contral~teral wound was covered with Opsite .
Group IV: No topical tre~tm~t was applied over the wounds. The wound on one side of the animal was left uncovered while the wound on the contralateral side was covered with Opsite~.

Wound Analysis The animals were ~ 1 on Day 9 of the e~ The wounds were excised down to the muscle layer, including a margin of 0.5 mm of unwounded skin, and were placed in buffered 10% formalin solution. The specimens were ~ublluu~d to a histology labold~l y for processing. Specimens were embedded in p~drr-~, and the midportion of the wound was cut in 5-~m sections. The slides were stained with hematoxylin and eosin, or with Masson's trichrome for histologic analysis.
Each slide was given a histological score ranging from 1 to 15, with 1 corresponding to no healing and 15 corresponding to a scar with olg~ .oA
collagen fibers (Table 2). The scoring scale was based on scales used by previous investigators. The criteria used previously were modified and were further defined to more precisely reflect the extent of: reepitheli~li7~ti~ n, W ~ 96/40174 PCT~US96/10006 degree of cellular invasion, gr~mll~tion tissue folmation, collagen deposition, vascularity, and wound cv~ clion. The histologic score was ~si~nPA

W O96/40174 PCT~US96/10006 Table 2 Criteria for Sco ~ing OI n;~ X;~ Se. ~ ;."- -Score Criteria 1-3 Epith~ li7~ti-)n None to very minim~l Cellular content None to very minim~l (mainly i"n ~ ul~ cells) (~r~n~ tinn tissue None to sparse amount at wound edges Collagen deposition None Vascularity None 4-6 Epith~ 1i7~tion Minimal (less than half of wûund diolllelel) to moderate (more than half of wound Cellular content Predo,.~ lly i~ll*~ ul y cells, few fibroblasts C~Tr~nnl~tion tissue None to thin layer at wound center, thicker at wound edges Collagen deposition Few collagen fibers Vascularity Few capillaries 7-9 Epith~ 1i7~tion Completely epith~ li7e(1; thin layer Cellular content More fibroblasts, still with infl*.
cells Granulation tissue 7, sparse at wound center, mainly adipose tissue nn-l~rnP~th epithelium 8, thin layer at wound center; few collagen fibers 9, thicker layer; more collagen (Contimled on next page) W O 96/40174 PCTAUS96~10006 Table 2 C~t~a for Sco~ ng of T'~ r Secffons Score Criteria 10-12 E~ li7~tirn Thicker ~iLllelial layer Cellular content Pre~ .,lly fibroblasts Gr~mll~ti~n tissue Uniformly thick Collagen deposition MoA~r~tP to ~ nsive collagen deposited, but less mature when c~ a-~d to collagen of u~lwuullded skin margin V~ul~rity Moderate to e~LL.~sive neovascularization 13-15 Epith~ i7~tiQn Thick epitheliurn Cellular content Fewer number of fibroblasts in dermis Gr~n~ ti--~ tissue Uniformly thick Dense, o~ d oriented collagen fibers Few well-defLned capillary systems St:~aL~:ly by at least three analysts. The code desclibi.lg the wound tre~mrnt was broken after the scoring was completed by all obselvt;l~.

S .~t~h~ a~ Analysis The values of the histological scores of the analysts were averaged and were expressed as the mean ~t standard error of the mean.
The paired t test was used for col,l~ Oll of paired means in the dirr~lellL tre~tmlont groups. The analyses were performed using the RS/1 Release 3.0 st~ti~tir~l software p~rk~ge (BBN Software Products Corporation).
The sample mean dilr~lc;llces were tested for analysis of variance USiIlg the St~ti~tir~l Analysis Software (SAS) System.

W O 96/40174 PCT~US96/10006 Results The Effect of PG on Wound Closure (Group I) In Group I both wuullds on each mouse were covered with Opsite .
Under these con~ition~, the topical application of FG with a protein S col~cellLl~Lion of 60 mg/ml to only one side of the animal resulted in ~ lly lower mean histological scores (3.06) for the FG side cOl~alcd to the u~lLI~L~d wounds (5.26) (P<0.005) ~rable 3).
Table 3 The Effect of FG (60 mg/ml) on Wound Closure (Group I) T-edLl.c.ll Histologic score N
FG + Opsite~ 3.06 i 0.7 15 Opsite~ alone 5.26 i 2.21 15 The Effect of Dilute FG on Wound Closure (Group II) In this group, both paired wounds which were covered with Opsite~, topical application of dilute FG (protein concellLlalion of 1 mg/ml) resulted ina mean histological score (4.0) that was not st~ti~tir~lly dirr~,~nL from that for ullLl~aled wounds (4.36) (P=0.17) (Table 4).
Table 4 The Effect of Dilute FG (1 mg/ml) on Woumd Closure (Group II) Trcd~ .lL Histologic score N
Dilute FG + Opsite~ 4.00 _ 0.77 11 Opsite~ alone 4.36 _ 0.67 11 The Effect of Opsite~ on FG-Treated Wounds (Group IIIJ
In this group of paired wounds both treated with FG with a protein cul~ce~Ll~Lion of 60 mg/ml, the application of Opsite to one side resulted in a mean histological score (4.2) which was not 5~ lly dirrer~llL from that for wounds which were left uncovered (4.93) (P=0.11) (Table 5).

W ~96~4~174 P ~ nUS96/100~6 Table S
The Effect of Opsite" on Paired Wounds Treated wit:h ~G (Group III) Tl~a~ Histologic score N
Opsite~ + FG 4.20 ~ 1.93 15 FG but no Opsite~ 4.93 ~t 1.09 15 Effect of Opsiten' on the Closure of Paired Untreated Wounds (Group IV) In this group of paired wounds which did not receive topical tl~a~
of FG, application of Opsite~ to one side resulted in a signifi~ ntly lower meanhistological score (4.92), as cOlll~al~d to that for wounds which were left uncovered (6.31) OE<0.0005) (Table 6).
ANOVA of the Ir~ effects on sample mean ~lirr~ ces was significant at < 0.0001.
Table 6 The Effect of Opsite~ on the Closure of Paired U,~ aled Open Wounds (Group IV) T~ lLHistologic score N
Opsite~ (no FG)4.92 ~: 1.26 13 NoOpsite~(noFG)6.31 :~ 1.25 13 Discussion The results of this study in~ te~l that in mice (1) when applied over open wounds, FG at a concentration forrn~ te~l for h~most~.~is (60 mg/ml) resulted in lower histological scores at Day 9 which in(lic~t~d slower rates of J wound healing coll,~,d to that of ullLI~aLed wounds; (2) tlihltiC)n of the FG
protein concellll~ion to 1 mg/ml resulted in a higher histological score at Day 9 which in~lic~te~l a faster rate of wound healing; and (3) application of a W O 96/40174 PCT~US96/10006 s~ hle dl~..siug (Opsite ) per se ~ignifir~ntly retarded wound closure in this animal model by itself.
The total protein collceuLlaLion of FG is an irnportant variable when co.u~alLug the results of studies using FG. Beneficial effects of fibrin in promoting wound healing and tissue repair have been reported, but lower couccuLlaLions of fibrinogen have been used in the present studies than is co uonly found in commercial ~lcpala~iorls.
FG at a concentration of 60 mg/ml delayed wound closure (Group I).
The total protein concellLlaLion of FG which is commercially available in Europe, after lllix.Lulc of the fibrinogen and thrombin components, is 37.5 to 57.5 mg/ml. These data in-lic~te that FG as presently form~ ted for h~mnst~tir and adhesive intlir~ti~ns retards healing when applied to open skin wounds. This effect may be due to (1) ~,.or.h~",l ~1 obstruction to the migration or proliferation of cellular elemPnt~ that actively participate in the wound healing process, (2) m.orh~nir~l inhibition of wound contraction or (3) a chemir.~l inhibitory effect of one or more FG components on wound healing.
~erh~nir~l obstruction and inhibition of wound closure may be the more likely explanation, since at Day 9 there is p~ Lt nce of a solid fibrinogen-based clot on the wound surface.
In order to help rlr~lllillr if this was the cause, the total protein collcellLlaLion of FG was diluted to 1 mg/ml. Topical application of this diluteFG resulted in a histological score that was not ~ignifr~ntly dirr~ lll from that for ullLleated wounds (Group II), suggesting t_at lower total protein concellLl~ions do not ~ignifir~ntly inhibit the wound healing process.
It is also worth noting that the mean histological score for covered wounds treated with the same conceuLla~ion of FG (60 mg/ml) but belonging to dirrel~llL Lle~ l,l groups (Groups I and m) had ~ignifr~ntly dirrel~uL
values (3.06 for Group I vs. 4.2 for Group m). These data demo~Llated that animal to animal variation makes it rliffirlllt to derive derllliLi~/e conclusions from dirr~ellL animals subjected to the same treatment variables because some W O g6~40174 PCTnJS96/10006 slnim~l.c may heal faster or slower than the others despite ~~ceiv,Lg the same L~ l This is reflected in the range of ~L~d errors for the mean scores.
For this reason each animal served as its own control, e.g. wounds in the same animal were co l~a.ed to each other. By having the control wounds in the same animal as the test wounds, the effects of illt,.d~ ual variability was ~.i"i"-i,~. These data also show that an adhesive dl~ssiug such as Opsite~
signifir.~ntly delayed wound closure. It should be noted, however, that in partial Ihi~sL ~IP~ skin wounds in pigs the protein concelll,dLion of the FG does not appear to be related to the rate of wound healing.

0 B. Growth Factor-SupplPn1ente~ ~G on Wound Healing In Vivo.
The effect of HBGF-lB growth factor-supplen~lont~l FG on the rate of wound repair in diabetic mice was ~sess~P~l. The methods used in this experiment were similar to those just described above. Two 6 mm full-thirl nPss skin biopsies on the dorsal part of each of 6 test mice were filled with FG to which 5 ,ug of HBGF-l~ had been added. Jd~Pntir~l biopsies in six mice were left ullLIc~ed, and in six control mice were filled with unsupplemPnt~
FG. After 9 days, all of the mice were sacrificed and histological pl~Llions of 5 micron thick slices from each of the wounds and su~loullding skin were ~lcpa,~d and stained with hematoxylin and eosin.
The extent of wound repair in each sample, which was not itlentifie(l as to the tre~tmPnt group from which it came, was "blindly" evaluated by each of three trained analysts, who ~essecl collagen deposition, reepitheli~li7~tion, thicknr~,c of the gr~nnl~tinn tissue and the density of infl~ y cells, fibroblasts and capillaries. Each sample was scored from 1 to 15, ranging from no to complete repair. The samples from the wounds treated with unsupplemented FG were co,l~ le"lly given the lowest scores and those from the ullLlcaLcd wounds or wounds treated with the growth factor-supplemPnt~r FG were given the highest scores.

Example 13 FG as a Delive~ Vehicle of Osteoinductive Substances In Vivo Fibrin Sealant CcllcellL aLed human TFC (13axter Hyland Division, San Pedro, CA) and human Llllolllbill (Baxter Hyland Division, Glendale, CA) were produced for the American Red Cross from screened fresh frozen pooled human plasma.
Both components underwent viral inactivation using the solvent de~e,~
method (New York Blood Center) during their production and were supplied in lyophili7Pcl form. After l~,co"~ lion with 3.3 ml of sterile water, the protein characteristics of the TFC solution were: total protein = 120 mg/ml;
fibrinogen = 90 mg/ml; fil)rone-;Lil- = 13.5 mg/ml; Factor XIII = 17 U/ml;
and pl~sminogen = 2.2 ~bg/l.
Human Llllolllbill (1000 U vial) was reco,.~ with 3.3 ml sterile water, and was serially diluted in 40 mM calcium chloride solution (American Regent Laboratories, Shirley, NY) to a concellL,dLion of 15 U/ml. Human Llllulllbill was used for ~l~p~illg disks implanted which were onto calvarial defects.
Topical bovine thrombin (5000 U vial, Armour Pl~ e~lti~l Co., K~nk~kee, IL) was leco.. ~ d with 5 ml sterile water, and was serially diluted in 40 mM calcium chloride solution to a concentration of 15 U/ml.
Bovine thrombin was used for pl~illg implants for illLl~ c~ r bioassay.
In practicing this embodiment of this invention the fibrinogen should be present at a concentration of 1 to 120 mg/ml FG, more preferably from 3 to 60 mg/ml FG, most preferably from 10 to 30 mg/ml FG. DBM should be present at an approximate co~ce~ ion of about 1 to 1000 mg/ml FG, more preferably from 50 to 500 mg/ml FG, most preferably from 300-500 mg/ml FG. The particle size of rlf~.";n. l~lized bone powder should be from 0.01 to 1000 microns, preferably from 20-500 microns and most preferably from 70-250 W O 96~40174 PCTAUS96/10006 llliC;lUllS. The o~La~i~Iu~;livt; grow~ factor(s) or BMPs should be present at aconcc;n~ ion(s) of about 1 to 100 ~g/ml wherein the collcellLIdtion(s) is ,~ err~iv~ to accomplish its desired ~u.~ose. Growth factors which may be used as o~L~oi.-~ v~ ~Ub~lilllr-~s in l~is ~ bod ~1ll include, but are not limited to:
S osteogenin (BMP3); BMP-2; OP-1; HBGF-l; HBGF-2; BMP 2A, 2B and 7;
FGF-1; FGF-4; and TGF-~. In ~ ti()n~ drugs, such as antibiotics, can be used to supplement the TS for use in bone repair.

~mplant Preparation Rat DBM was p~ cd as follows. The epiphyses of the long bones of rats were removed leaving only the diaphyses behind. The diaphyses were split, if n~cess~ry, and the bone l~llow was then thoroughly flushed with ~leinni7,o~l water (Milli-Q Water Purification Systemn', Millipore Corporation, Bedford, MA). The diaphyses were then washed at room le~ dture. At 4~C, 1000 mls of deionized water was added to 100 g of bone. The llli~LUlC
was stirred for 30 .. li.. ~lrs and the water was Aec~nted This step was repeated for two hours.
At 4~C, one litre of cold absolute ethanol (Quantum Ch~mic ~l Corporation, U.S.I. Division, Tuscola, IL) was added for every 100 g of bone.
After stirring for lS ..-;.~ s, the ethanol was ~lec~nted This was repeated four times for a total of one hour's duration.
Under a fume hood, 500 ml of diethyl ether (M~llinc~rodt Speciality Chemic~, Paris, KY) was added to the bone to cover it. This was stirred gently for 15 I-~;--,-lrs and the ether was then dec~nted An ~d(1ition~l 500 mlsof ether was added to the bone and the mixture was stirred for 15 min~ltes. The ether was again ~lec~nt~l. The bone was left under the fume hood for the lion of the ether to occur. Defatted bone can be stored in-lPfinitely in an ultralow freezer (-135~C).
The bone was then milled to make bone powder. The powder was sieved and 74 to 420 micron size particles were collected.

W O96/40174 PCT~US96/10006 Ten gram aliquots of the bone powder were placed in 250 ml c~-ntrifilge bottles. Eighty rnls of 0.5 N HCl was added to each bottle slowly in order to avoid IioLllillg. The collLcllL~ of each bottle were then stirred gently. After 15 s, an ~ tion~l 100 mls more of 0.5 N HCl was added to each bottle over the course of 10 l~ s. The bottles were then stirred gently for an ~ flition~l 35 ".i....les. The total time that the powder was in the HCl did notexceed one hour.
Each u~ixlul~ was then spun in a centrifuge at 3000 rpm at 4~C for 15 ~ . .i - .. .~s. The pH of the ~ul,c- . .~ . .l was then ch~oçk~d If the pH was greater than 2, the :jU~Clll~ was poured down a ~ h~mi~ l sink without di~lùubulg the pellet(s). If the pH of the supe~ was less than 2, the ~ul!c-l~L~L was poured off into a hazardous waste collL~u~l. If the pellet(s) were loose, the centrifuge time was increased to 30 ...i..~ s. These steps were repeated until the pH of the su~ellldL~ was equal to O.S N HCI.
The pellets were then washed with 180 mls of deionized water by stirring to produce an even suspension. The suspension was then centrifuged for an additional 15 min~ltes. The ~u~ll~lll was then tlec~nt~l as before.
The washing was l~ed~ed until the pH of the ~iU~)~llla~ equaled the pH of the deionized water.
The pellets were then frozen at -180~C in a freezer. They were then lyophilized using standard procedures.
Disk-shaped implants 1 mm thick and 8 mm in ~ m~t~r were produced using a 4-piece al.~ ... mold (Figure 14). Twenty-five mg of rat DBM
powder was added into the mold chamber. Thirty ~1 of TFC was then pipetted onto the DBM and mixed until the DBM had absorbed all of the solution. The concentrations of TFC which were used were 10, 20, 40, 80, or 120 mg/ml.
Thirty ~Ll of ~ o~ul~ill solution (15 U/ml in 40 mM calcium chloride solution) was then added to the DBM-TFC complex, was mixed, and was c~ lc;ssed into a disk-shape using a piston-shaped lid. It was del~ iuled that 25 mg of W 09614~74 PCTAUS96/10006 DBM ~owd~,. had a volume of 20 ~l. After DBM had been added to the FG, the final protein co~ a~ions were as follows:

Table 7 TFC Thrombin DBM FG, ToW
S (mg/ml) (~/ml) (mg) ~ t~conc.
(mg/ml) Disk implants composed of DBM alone or FG alone (4, 8, 15 and 45 mg/ml total protein col-rel.l . alions) were likewise made using the same mold.
Fifty mg of DBM was poured into an ~l-....;..--..- mold, to which 60 ~1 of TFC was then added to the DBM and mixed until fully absorbed. Sixty ~l of llllol~lll was then added to the DBM-TFC complex, mixed and colll~lGssed into a disk-shape with a ~ mrter of 1 cm and a thirL~n.oss of 2 mm using a piston-shaped lid. The disk was then cut m~ml~lly into the desired shape (triangle, square or donut).
For the intr~mll~cnl~r bioassay exl-~, ;...~..I, impl~nt~ were placed in a sterile nylon bag having a mesh size of 70 ll~lCl'OllS and l l l~ g 1 cm x 1 cm.
A rti~?n7~
Male Long-Evans rats were obtained from Charles River Laboratories (Wihllil~ , MA). For the ~ ",.~ r bioassay, 28 to 35 day old rats were used. Three month old rats were used for the craniotomy e~

Surge~y The ~nimAl~ were ~-P!ill.. 1;~1 with a Il~ixLulc CO~ ;..g of 10 ml k~l;....;.-~ hydlo~ ide (Vetalar, 100 mg/ml, Parke-Davis, Morris Plains, NJ), 5 ml xylazine (P~-)mrln, 20 mg/ml, Mobay Corporation, Shawnee, KN), and 1 ml physiologic saline (0.9% NaCl), at a dose of 0.1 ml per 100 gm body weight, A.~ ,.ed illLl~ s~ rly. The O~aLivt: site of the animal was prepped with 70% alcohol solution, followed by povidone-iodine solution. The surgical procedure was then ~t;lÇoll-led using aseptic t~cllni~ e.
Ir~lr~ sculnr Rion~ y. A rnidline ventral incision was made and a space was created between the pectoralis muscles with blunt ~i~section. A
nylon envelope cont~ining the ~le~ignAt~i e~elullellL~l material was inserted into the ulLlA~ ;ulAr space and secured with a 3-0 Dexon suture (Figure 15).
The same procedure was then l~eaL~d at the contralateral side. The skin was then closed with staples. The implants were hal v~ d after four weeks, were x-rayed and were prepared for histology.
Disk-shaped implants were placed randomly and consisted of the following: DBM alone (n = 12); FG alone at dirr~re,~L concellllations (4 mg/ml, n = 14; 8 mg/ml, n = 3; 15 mg/ml, n = 3; and 45 mg/ml, n = 12), and DBM-FG complex (4 mg/ml, n = 12; 8 mg/ml, n = 12; 15 mg/rnl, n = 12; and 45 mg/ml, n = 12). There were four each of the square-, triangle- and donut-shaped implants.
Craniotomy Procedure. A linear incision was made from the nasal bone to the mid-sagittal crest. Soft tissues were reflected gently and the periostellrn was ~ sectecl from the craniotomy site (occipital, frontal, parietal bones). An 8-mm craniotomy was prepared with a trephine in a slow-speed rotary handpiece using copious saline irrigation as needed. The calvarial disk was secte~l free while avoiding dural ~ çolA-Lions and superior sagittal sinus intrusion. The 8-mm calvarial defect was either left ul.L.~ d as control or filled with a 1 x 8-mm DBM or DBM-FG disk (Figure 16). The skin was then closed with skin staples.

W O96140174 PCTAUS96~10~6 Following surgery, each rat was ir1PntifiPA by ear ~u~lches and L~,l.. k~1 to its cage where they were ambulatory within 2-3 hours.
The first set of calvarial impl~nt~ consisted of DBM alone (25 mg, n = 3) or DBM in a FG matrix (15 mg/ml, n = 2; 30 mg/ml, n = 3; and 45 mg/ml, n = 3), and were ~ ved after 28 days. The second set of calvarial i~l~L~. co~-e;~ d of 25 mg DBM in a 30 mg/ml FG matrix and were ~ itved at dirr~ l po~l~c.aLive times (28 days, n = 10; 3 months, n = 9; and 4 months, n = 5).

Retrieval of Implants At the infliratPd times, the rats were ~ Pd in a carbon dioxide chamber. A skin incision was made around the expe~ le~lL~l recipient bed (i.e., pectoralis major or calvaria) and the soft tissues were reflectto-1 from the recipient beds. In orthotopic sites, tne craniotomies with 34 mm contiguous bone were l~co~ d from the fronto-occipito-parietal complex. In heterotopic sites, sharp and blunt ~ section was used to recover the implanted nylon envelopes.

Radiography The implants were radiographed using X-OMATL high contrast Kodak x-ray flm (F~tm~n Kodak Coll~ ly, I~ Pst~r, NY) in a Minishot Benchtop Cabinet x-ray system (TFI Corporation, West Haven, CT) at 30 kvp, 3 Ma, and 10 seconds. Gray-level ~len~iti~s of hlLlA..,..cc~ r and craniotomy site radiographs were analyzed using a Cambridge 920 Image Analysis System (Cambridge Instruments T imittqd Cambridge, Fn~l~nrl).

Histological Analysis All retrieved s~ n~ (soft and hard tissues) were immto~i~t~ly placed into al~ro~liately labeled vials co~ g pLcscl~/aLive solution and were submitted to a histology laboratory for processing. Histologic specimens were W O 96/40174 PCTrUS96/10006 4.5 icl~e~.-thick s~ through the coronal ~ . ". ~. . . For each ~
site, one section was l,.~aled with h~ ylin and eosin stain (for phOI~Jll~iclu~ lly and ~xi~ l;nn of cell and stromal detail) and the other section was p-~al~,d with a von Kossa stain.

s Results Radio~ hy of Ir~lr .,~ nr Plants All DBM disks displayed radio-opaque images. Forty-five out of 48 implanted DBM-FG disks (93.75%) were radio-opaque. All DBM-FG disks, regardless of protein concellLl~ion (4-45 mg/ml) intl~lce~ radio-opacity (Figure17). Radio-opacity lllea~ lGllL~ of some DBM disks (Figure 17) were higher than DBM-FG disks but the other llleasu~ llcllL~ were well within the range of mea~u.c,llc;~L~ for DBM-FG disks. Thirty out of 32 FG disks which were not supplemente~l with DBM (93.75%) did not develop radio-opacity.
DBM-FG disks in the form of squares, triangles or donuts were also m~rk~tlly radio-opaque as colll~cd to FG disks which were not supplemPnto-l with DBM. The original shapes of the implants were generally retained.

Histology of Ir~lr~ "~ nr ~n1rln~t~
The hlL~ l ls~ r bioassay was posiLiv~ for DBM and DBM-FG
implants, as evidenced by form~ti- n of ossicles with a central cavity filled with lll~llOW and resorption of previously implanted DBM particles.

Radiography of Calvarial Implants X-rays showed DBM implants in a FG matrix to be generally more radio-opaque than DBM implants alone or ullLle~L~:d controls. There was no m~rkecl discernible dirr~ ce belweell dirr~ L collcellL,~tions of FG used to deliver DBM. The radiographs of untreated 8-mm ~ ",~ calvaria defects showed a negligible amount of radio-opacity.

W O 96Ç~74 PCTAUS96n~006 The second set of calvarial imrl~n~ using DBM in 30 mg/ml FG matrix showed m~rkf~ly illcl~as~i radio-opacity within tbe craniotomy wounds of 3 or 4 month-old calvaria over 28 day calvaria (Figure 18).

Histology of Calvarial 7~rl~7-1t~
Non-treated 8 mm craniotomy wounds showed only fibrous CQI.. ,f~ v~
tissue developing across the craniotomy wound (Figures l9A and B).
Histology of DBM imrl~nt~ showed DBM particles to be scattered all over the field. Some DBM particles migrated over and under the edges of host bone (Figure 20). Most DBM particles were, however, within the confines of the craniotomy wound and were ~ulloullded by loose co~ f.li~/e tissue that was well v~c~ ri7~1 Active resorption of DBM by osteoclasts was noted. A lot of DBM particles were also noted to be populated by live cells. New osteoid and bone laid down by osteoblasts were quite evident.
The histology of DBM imrl~ntc in a FG matrix showed DBM particles localized within the craniotomy wound, ~ul~ullded by much denser and more cellular colllle.;Live tissue (Figures 19 and 20). Osteoid matrix and bony trabeculae formation were quite evident. More bone ~low was noted to have formed in craniotomy wounds implanted with DBM-FG disks than with DBM
impl~nt~ alone. There was also greater neov~c~ ri7~tinn with DBM-PG disks than with DBM implants alone or ul~ ,aLed controls. Osteorege.. f.i.lion was evident at all concf llLlations of FG used to deliver DBM.

Discl~ion The natural biocompatibility and biodegradability of FG are characteristics that make it an ideal delivery vehicle for DBM and BMPs. FG
facilitated the shaping of DBM into the desired form to fill bony defects, IIIZ~;III; il~f'~l DBM within the defect, and may have been ~ynel~i~Lic with DBM.
Furthermore, soft tissue prolapse did not occur and bony contour was m~int~inf~l DBM-supplemented FG pos~e~cer~ an a~lopli Luicl~Jalc~ r~LII~c, biodegradation profile and release kinPtirs to ~.u~u,~
osteoblast l~ iLLu~ l and o..Ls;o~cge~e~lion.
Overall, the data inflir~t~d that DBM delivered in FG at any of the tested FG protein co~rS~ rn~ in~ue~d as much bone folm~tion as the DBM
S did alone. Moreover, when DBM was confi~lred with FG to a particular pre-opel~Live form, the in(1~lced bone closely retained the original shape po~.lop~l dliv~ly .
Since the shape of the DBM-FG matrix ~ (l the morphology of the newly formed bone, when possible, the DBM-FG matrix should be made of a predeL~ led shape. However, the DBM-FG matrix in liquid form can be delivered or injected into an irregularly shaped defect where it will poly.l.t.,~e and encourage bone formation in the DBM-FG-filled area.

Example 14 The Release of Antibiotzcs (AB) Prom PG and Increased Longevity of the AB- Supplemented FG

A. Prepara~ion of the AB-FG
1. TET Free Base Three-and-one-half ml of water for injection was injected into a vial of lyophili7lod human topical fibrinogen conce"L,al~ (TFC), supplied by The American Red Cross. The protein collcellLlalion of the resllltin~ solution was approximately 120 mg/ml.
Freeze-dried thrombin concentrate, supplied by The American Red Cross/Baxter-Hyland, Inc., Glendale, CA, was reco,.~Li~ d with 3.5 ml of a 40 mM solution of calcium chloride ~lc~ d in water for injection. T_e reslllting solution contained a~ aLt;ly 250 U/ml.
TET-FG was forrmll~tPIl by mixing the desired weight of TET with 1 rnl of reco~.LiLu~ed TFC solution and with 1 ml of rec-J"~ d thrombin solution --CA 02223889 l997-l2-05 W O 9614~174 PCTfUS96/10006 in the ~ ,~e of iniectirm quality ç~lrinm chloride (L~ul~hased from ~IIIF. ;I'A~I
~2~Zlgt'.nt, Shirley, NY). The TET was in the free base form and was ~u~
from Sigma ChP.mir-~l COLUPaUY (St. Louis, MO). The TET-FG was formed by mixing TFC and l1I1ULU~LU through a Duoflon' di*)~ se~ m~irS, CA) S onto a Millipore UC1~1~e in a 12 mm ~ m~tlor Millipore culture plate (Millipore Cu~ dLion, Bedford, MA). The mixture was allowed to set for one hour at 22~C. Six mm d;~ lel~ disks cont~ining the TET-FG and the Millipore lneL~br~le were cut from the latter using a 6 mm punch biopsy. The TET-FG-c(J-I~ g disks were used for the TET release studies.
The release of TET from the TET-FG into phosphate burrGl~d saline (PBS) or saliva was measured using 24-well cell culture plates (Corning Glass Works, Corning, NY) under two dirr~.Glll sets of con~liti~n~. In one con-lition,the static mode, 2 ml of PBS or 0.75 ml of saliva was replaced daily in the 24-well cell culture plates. In the other condition, the continuous ~rll~nge mode, 15 TET release from the TET-FG was Luca~ulGd with PBS having been e~ch~
at a rate of appro~im~tely 3 ml per day. The samples were stored at -20DC
until analyzed. The saliva had been collected from 10 dirr~len~ people, had been pooled, and clarified by cellkirugation at 5000g. It was then filtered through a 0.45 ~m pore sized Illr.llhl~ and was stored at 4~C for daily use.
In order to llle~ulc the c~nr~ n and biological activity of the TET
which had been released from the TET-FG disks, the eluted TET was thawed and was analyzed ~e~kopholonlGkically at 320 nm and/or biologically by the inhibition of E. coli growth on agar plates. To c~ r~t~ these assays, standard curves covering TET concenL.~tions of from 0 to 50 and 0 to 500 ,ug/ml, 25 respectively, were used.

2. Ciprofloxacin HCI (CIP)-, ~no~ieillin (AMO)- and Metronida~ole (MET) Supplemente~ FG.
FG colll;1i";"g CIP HCl, AMO or MET were ~l~aled as before for TET. To monitor the release of these AB from the corresponding AB-FG into 30 the immediate ellvilullluent, the AB-FG disks were placed in individual wells W O 96/40174 PCT~US96/10006 in a 24-well cell culture plate and were covered with 2 ml of PBS that was collected, replaced daily and stored at -20~C as before, until analyzed. The conce..L dLions of CIP, AMO and MET in the eluates were measured D~ecLlu~hol~ ;r-Ally at 275, 274 and 320 nm, ,~,~e.;Lively, and were cO.~al~d to ~L~dald curves C(~ A;II;II~ O to 50 ug/ml of the colle~olldillg AB.

B. Structural Integri~;y of AB-FG
The ~..A;..Ir,~A..re of the structural integrity of the FG and the TET-FG
disks was e~li.--AIrA by visual obse.~Lion and physical inspection by "poking"
the disks with a fine spatula. The porous membrane which had been cut out while m~lcing the disks lC;IIIA;IIf'Cl Att~r.hP~ to the TET-FG and was used to help position the disks during the evaluation of their structural hlL~glily~ Picturesof top and lateral views of the disks were also taken and were used in the evaluation.
The structural hlL~ y of FG and TET-FG were measured under both sterile and non-sterile conditions. For the non-sterile ek~elilllellL~, the PBS and saliva were stored fro~n until analyzed. For the sterile e~elh..~..L~, the same procedure was used except that the entire process was run under sterile conditions. The sterility of the system was tested by i-.~ ba~;-.g 0.2 ml of sample and 2 ml of broth at 37~C and the turbidity of the broth was monitored for 48 hours. Lack of lullJidiLy i~ iCAl~d sterility of the system. The stability of the CIP-, AMO-, and MET-FG were studied as above but under non-sterile conditions only.

C. In Vitro ~ntimicrobial Achvity of AB Releasedfrom AB-FG
The ~ntimi~robial activity of the AB released from the AB-FG was e~li,.. A~t;cl by measuring the ~ of the zones of inhibition gene.~Led by the eluate from the 6mm .li .",~l~, disks from the daily collected PBS or saliva~u~ ullding the AB-FG. The eluates from unsupplem~nt~l FG served as controls. AB solutions of known con~llLldlion were used as standards. E. coli WO 96/40~74 pcTAus96/la~o6 cultured on agar plates were used to l,lea~ the AB activity of the released TET, CIP and MET. To make the culture plates, 100 ~1 of the bacterial cell s~ cion, c~ ;..g d~ro~ y 108 cells/ml, was miAed with 3 ml of top agar at 50~C and im m~ tr~y poured onto the plate hard, bottom agar to make a ulurullll layer of cells. The plates were inr~lb~t~d at 37~C for 18 hours.

Results A. TET
1. TET Release Data The release of TET from TET-FG disks into the ~ull~ull~ulg PBS in the "static" eA~ illltllL~ was llleasul~,d spectrophot~ ;c~lly by del~.llil,i"g the TET conrçntr~ti- n achieved in the 2 ml of PBS which was replaced daily. The TET collcellLldLions which were obtained for dirr~,lellL amounts of TET that hadbeen inc~l~uldLcd into TET-FG are shown in Figure 23. At TET
concentrations in the TET-FG of less than 50 mg/ml, the release of TET was completed in five days or less. However, the release of TET from TET-FG
disks which collLdilled TET co~r~ f)nS of 100 and 200 mg/ml occurred for approximately two weeks, and more than three weeks, lc~ecLi~ely. The structural hlL~liLy of the TET-FG disks was plcselvcd for three to five weeks.
These results demol~Llal~d that the TET release was independent of the FG
degradation and that the rate of TET release depen~Pd on the amount of TET
which rem~in~o~l in the TET-FG disks.
The spectrophoto~ ;c data which were collected in the continuous exchange experiment are shown in Figure 24. These data in~lic~te that a continuous TET release from a TET-FG disk which originally co..l;.;..PA a TET
collcel~Lldtion of 100 mg/ml FG occurred over a two week period. The FG disk retained its structural integrity during this two week period, infra. The TET
release data obtained in the continuous mode eA~ ;Illrlll also in-lir~trA that the rate of TET release oppullulliLy depended on the concellLldLion of TET which rrn~inr-l in the TET-FG disk.

W O 96/40174 PCTrUS96/10006 While not wislli~g to be bound by theory, it is believed that the initial ,~
high TET c--.r~ iQns observed in these e,~l,e~ lents were probably a con~Pql~enr~ of the diffusion of TET from at or near the disk's surface. That is, as the TET "Ll~ed" at these locations was ~x~ , the rate of solubili7~tion and/or r~ ion decl~,ased in a fashion that was most probably ...;..PC1 by the TET c- .~r~ n gradient and by the shape or con~ r~tion of the FG.
Temperature and FG protein concellLI~lion also played a role in rlrl~. ~II;IIil~ the TET ~ sion rate from the TET-FG disks (see Examples 13 and 14), but these two ~al~lleL~l~ were kept CC?n~t~nt in these e~elilllents.
The release of TET into saliva from TET-FG c~nt~inin,~ 50 and 100 mg/ml of TET was measured in static e~ by r1r~.. Ill;lli~g the TET
collct;~LIdlion in 0.75 ml of saliva that was replaced daily. These results (Fig.
25) are similar to those obtained in PBS except that the concellLldtion of TET
was higher, most probably reflecting the smaller volume of saliva which was used to collect the released TET. In addition, the presence of TET in the FG
matrix again une~e(;Ledly prolonged the structural hllegliLy of the TET-FG
IlldLlices for at least 15 days compared to that for the control FG disks which had begun to decay by 9 days and were almost completely decayed by 15 days (Figure 26).

2. TET~ .robial Data The antimicrobial effects on E. coli growth of several TET
collcellLIaLions in PBS are shown in Figure 27. The lowest TET concentration detectable by this method was approximately 5 ~4g/ml. These results clearly inrlicate that the released TET has ~ iclvbial activity. These TET data corroborate those obtained by s~e-;Llo~hotolll~LIy and inrlir~te that the amountof TET incorporated into the FG ~ete~min~s the TET concellll~lion in the solution ~ullvulldi~g the TET-FG. These data also ~lemnnctrate that the amount of TET in the FG can be tailored to m~int~in the desired TET concentration in W O 96/40174 PCTrUS96/10006 the m~ lm ~ullvu~di~g the TET-FG at or above the ~ desired TET
co~ o~.

3. TET-FG Matrix Lor~g~v;ly The l~cvily of control FG and AB-FG disks was evaluated by visual ~c~s~;.,.~.. / of the disks. The porous membrane, cut during the making of the disks" ~ A ~tt~ch~o~ to the FG and helped to position the disks during their i~lL~liLy ev~ ti()n Top views of disks cont~inin~ no TET (controls), and 50 or 100 mg of TET per rnl of FG are shown in Figure 26 at days 0, 9 and 15.
This figure shows typical results, namely, the FG control disks were degraded within two weeks whereas the TET-FG disks r~ in~ intact, or nearly so, for 15 days. In a l~ition~1 eA~. ;",~ TET-FG disks reln~in~q(l intact or nearly so for at least five weeks (date not shown). No si~nifir~nt change in the FG
longevity was observed between sterile and non-sterile TET release e~clllllents .

B. CIP, AMO and METData 1. CIP, AMO and MET R~lense Data The antibiotic released from CIP-, AMO- and MET-FG is shown in Figure 28. CIP was released at an a~alcl~L constant rate for approxim~tely 4 weeks and then the rate decreased gradually for approximately one more week.
The release of AMO and MET was complete within 3 days.

2. CIP and METAntibacterial Actzvi~y The anti" iclobial activity of released CIP and MET (data not shown) parallels the profiles ~leL~ l spectrophoL~.l.eL--cally for i~lentir~ FG
disks.

~ 25 3. Supple~nfcq~-PG Matrix Lur~v~ly The results for CIP-FG were similar to those for TET-FG. The results for AMO- and MET-FG were similar to those obtained for the FG control. No ~j;g"i l~r. . ~ ~l change in the FG lo~ evily was obs~- ved l)CLW~ sterile and non-sterile e~

S Di.. ~ n The results demol~LldL~d that poorly water soluble forms of CIP and TET provide a combination of factors that increase .signifi~ ~ntly the m~ximllm AB load, release period and longevity of the FG matrix into which they are mixed. ~l~r~ iv~ly, the FG disks can be stabilized by h-~ g them in solutions of AB such as TET or CIP.
The results also clearly showed that the AB delivered by AB-FG
~resel ved its ~ ul)ial activity as ~l~,mon~trated by the inhibitioIl of E. coligrowth. These results demol~LlaL~d that TET and CIP suppl.om~nt~tion of FG
and other TS can o~/ercullle the degradation of FG as a limiting factor in drug delivery thel~rlolll. That is these ABs stabilized the FG so that their release periods and the released AB collcellllalions can be controlled using AB
collcellL.alions in the FG. Using these ~.oce.lures TET and CIP can be loaded into FG and their release can be controlled for a period of days or weeks at c;rreclive antimicrobial cullce-.llalions.
The TET- and CIP-in~ cecl FG stabilization can be exploited for controlling the total release time not only for these ABs, but also for other drugs or "supplem~nt~" added to FG whose release rate and/or total release duration depends on the hllegli~y of the FG matrix.
These results have clinical applications in periodontal and other conditions where FG can serve as a localized drug delivery system. The TET-or CIP- in-lnce~l FG stabilization can be exploited for controlling the total release time of TET, CIP and other drugs or supplements which have been added to the TET-FG or CIP-FG matrices.

W~ 96/4U174 Pcrnuss6~oo6 Example 15 Effect of Temper~ur~ on the T ~ET Rele~e Rate from TET-Supplemented PG

FG was supple~-lr~ r,d with 50 mg/ml of TET free base and was shaped as 6 x 2.5 mm disks for this study. The protein co~c~--LLaLion of FG was :~rljll.~tP~ to 60 mg/ml. The disks were placed in 2 ml of PBS, pH 7.3 and were allowed to stand at 4, 23 and 37~C. To wash the disks, the PBS was replaced every 10 ~ s, 6 times, with 2 ml of fresh PBS. Thel~,ar~ the PBS was replaced every hour for 4 hours. The TET concentrations in the collected samples were d~te~ ed ~e~;Ll~lu~ L ically against a sL;~dald curve as before.
The results d~ 1 that the rate of TET release was propollional to the temperature (Figure 29).

Example 16 Effect of FG Protein Conce~ lion on t*e TET Release Rate from TET-Supplemented FG

FG supplpm~ntp~l with 1 mg/ml of TET HCl solution was prepared and was shaped as 6 x 2.5 mm disks for this study. The protein collcellL.aLion of the FG was adjusted to 60, 30 and 15 mg/m~. Each disk was placed in 3 ml of l~ ti~ water. The water was replaced with the same volume of water every 10 mimltes for a total of one hour. The TET concentration in the collected samples was ~ l..in~ spectrophotom~trit~lly against a standard curve as before.
The data (Figure 30) show that the TET release rate was highest from the FG with the lowest total protein collcellLldLion and vice versa. That is, the TET release rate was inversely proportional to the FG protein cc,llcellLld~ion.

W O 96/40174 PCT~US96/10006 Example 17 In Vivo Antimicrobial Activi.fy of AB Releasedfrom AB-Supplemented FG

To test the ~ robial activity of TET and CIP released from TET-S and CIP-FG, the ea~a~;ily of these AB-suppl~,tl -.le~ FGs to protect mice from in~ ed peliLuniLis was ev~ tP~l E~ ly~ at day 1, each one of 5 animals per group were injected iulLla~eliLolleally with 0.5 ml PBS (Group-I), FG (Group-lI), TET-FG (Group-m) or CIP-FG (Group IV). FG and AB-FG
was ~lmini~tered using a ~m~e-lirs dis~ellser co.ll;-i--i-~ 0.25 ml of TFC at 120 mg/ml and 0.25 ml of human Llllul~ at 250 U/ml. In the case of TET-and CIP-FG, the tl-- olllbill solution contained 50 mg of the re-,pecLiv~ AB. Atday 2, all the animals were injected hlLl~;eliLo"eally with 2X108 (E~e,iLue"L
1) or 4X108 (Experiment 2) colony forming units (cfu) of S. aureus 202A.
Results: (E~ lellL 1, Experiment 2. Animals ~,u~ ~iviu~g at 48 hours after infection): Group I, 0 and 1 ~Ul~/iVO1~7; Group II, 3 and 1; Group m, 3 and 5;
and Group IV, S and 4 survivors. Most survivors lived through the duration of the ~ ent (2 weeks) but some died or were int~ntiOr~ y killed because they were sick.
These data de~ al~d that TET-FG and CIP-FG protected mice from death caused by S. aureaus 202A for at least 48 hours after the a~lmini.~trationof the AB-supplempnt~d FG.

Example 18 T*erapeutic Applications of Supplemented Pibrin Sealant Compositions The development of ultrathin microfiberoptic endoscopes has offered the laryngologist unique access to the limited spaces of the temporal bone and skull W O 96~4~74 PCTAUS96/10006 base. While diagnostic middle ear endoscopy is well ~io~ p~ (FAel~t~in~
D.R. et al., Am. J. Oto. 15:50-55 (1994); Poe, D.S. et al., Laryngoscope 102:993-996 (1992); Poe, D.S. et al., Am. J. Oto.13:529-533 (1992); BaLkany & Fradis, Am. J. Oto. 12:46-48 (1991)), thera~eu~ic microPn-loscopy offers the S exciting advantages to the patient of minim~l invasi~ ess, reduced patient morbidity and lower hospital cost. Microendoscopes of Col~ y ~11rinking ~1;A~ t~ 7 yield images of good quality and resolution. Coupled to a laser and fibrin sealant applicator, several new surgical ~pplications in the middle ear and skull base are now feasible. Potential therapeutic applications were derived from the fibrin sealant's ~pcl~ c~l properties in soft tissue repair and use as a s~l~t~into~l delivery vehicle for ph~rm,.ceutit~ and biologic growth factors.
Possibilities include ototopical aminoglycoside therapy, using for example ge~ llly.;hl for the l1G;'IIII~ of Ménière's disease, tr~n~eUst~r~ n CSF leak prophylaxis and Iy~llpa~lic membrane repair.
Preli,lli~l~y antibiotic "release profiles" were obtained using pooled fibrin sealant (American Red Cross, Rockville, Md.), and either amoxicillin and metronidazole as "water soluble" agents, or tetracycline and ciprofloxacin in the "low solubility" category. For this procedure, four human head specimens were preserved and underwent latex vascular injection using the fresh tissue cadaver protocol actively in progress in the Naval Medical Center San Diego, San Diego, CA. (The fresh tissue cadaver protocol is advantageous in preserving the specimens without loss of "fresh tissue" qualities.) Both fiberoptic and rigid systems were used as provided by Xomed Corporation (Jacksonville, FL). The Alphascope 8 model was a flexible microfiberoptic endoscope with an outside t1i~m~t~r of 0.8 mm and a 115 degree flexible tip which provides a field of view of 65 ~ with 1.5-15 mm depth of observation. The fiberoptic cable was composed of 3,000 pixels and provides 10 cm of insertion length. The Alphascope 12A model was a rigid endoscope with an outside ~ lf ~ of 1.2 mm and an obliquely angled shaft of 25~ and tip of 45~ which provided a field of view of 65~ with 2-20 mm W O 96t40174 PCT~US96/10006 depth of obs~ valion. The fibe.~u~Lic cable was composed of 6,000 pixels and provided 8 cm of insertion length. A 0.28 mrn I~TP laser (T ~C~c~e, Palo Alto, CA) was used for all laser applications.
T .imit~l-sink conrliti~ n~ were created using 6 x 3 mm fibrin sealant discs mixed with a set co~lcellLlaLion of antibiotic. Conrf~ nl ion.~ in tbe eluate were measured on a daily basis (~g/ml) and evaluated over time to develop the "release profile" in vitro.
A duo-flow c~ Lc. was clesign~l specifically to facilitate endoscopic application of fibrin sealant, having a 0.75 mm inner c~nn~ with a 1.5 mm outer c~nn~ The 1.5 mrn outer ~ m~ter allowed coupling to a ucl~orl~L~u~lic endoscope for access to the middle ear space, ellct~rhi~n tube and cranial cavity. A "coaxial," recessed tip allowed continuous tissue sealant application under visual guidance without clotting of the delivery ports.

Microendoscopic and Laser Te~h~liql~es Initial procedures were pc.rolllled on human temporal bone specimens to floc~m~nt the feasibility of microendoscopic work within the middle ear and ~ ,lal bone. Both LI~Ly~ anic as well as Ll,~ rlli~n tube routes were used to access the middle ear. All surgery in the posterior cranial fossa was perforrned through "keyhole" incisions in the posterior fossa dura through a suboccipital approach. Procedures utilized standard otologic e(luil~ulc;lll.
Coupled with the KTP laser, surgical manipulation was safely achieved around the oval window, to include lysis of adhesions and stapedotomy.
Through a "keyhole" retrosigmoid approach, the flexible endoscope was introduced into the posterior cranial fossa with ready identification of the 7-8nerve complex. When a comfortable level of teç~lnir~l colu~.,Lellce WâS
reached, the KTP laser was s -cce~sfi-lly employed for vestibular nerve section in 6 cadaver s~e~;illlells without structural damage to neighboring ll~uluvdscular structures. Although ~liffiyll~ was encountered in g~llging the depth of v~pol~alion in the first two specimens with damage a~al~lll to the anteriorly W o 96/40174 PCT~US96/10006 located facial nerve, ~he problem was resolved with ,~r~ of the ttochni~Iul?
and a change in the laser angle. The duo-flow ç,.lllrl~ was ~rh~o~1 to the endoscope when using the KTP laser to suction laser plume.

Fibrin ,Se~71n~t Delive~y Coupled to a microfiberoptic endoscope, the Duo-Flow c~
e~ s Corp., Malibu, CA) was used to deliver ;~ ~bial col.,~o~i~ll-supplr~ A fibrin sealant under direct view to the eust~ n tube and middle ear space. Several routes of delivery were used in~hl~ing Ll~LyLl~aLic~
tran~e l~t~r~ n tube and L~ toid through the facial recess. Sncces~fi-l "sealing" of the rniddle ear cavity, t;U~ .. tube and rnastoid cavities was achieved with each method of delivery. Fibrin sealant was noted to persist in these "static" speci.l~ s for over one week following application.
Tetracycline release profiles from the fibrin sealant disks showed a prolonged decay pattern in excess of three weeks. Co,~re~ i()ns above th~ uLic Minimllm Inhibitory CollcenLlalions (MICs) r~ in~l for up to 42 days. Fibrinogen conce,lLl~Lions ldllging from 20-76 mg/ml had little effect on the release profile of ciprofloxacin.
This demonstration of a suct~in~l-release capacity of fibrin sealant demol~Ll~Led the great potential of the sup~ rA fibrin sealant composition as a ll~ uLic delivery system. On the ~ vbial level, topical application of fibrin sealant allows long-term delivery of antibiotic doses at many times the current minim~l inhibitory collce.lLl~tion, often avoiding side effects observedin a systemic therapy. In particular, when coupled with the laser, microendoscopic surgery using a fibrin sealant localized-release "bioreservoir"
offers great potential in the tre~tm~nt of a broad spectrum of otolaryngic disorders ranging from oLoLo~ical amino-glycoside tre~tm~nt of Ménière's disease to laser nerve section and topical al Li~iclobial therapy of acute and chronic sinusitis and otitis.

W O 96/40174 PCT~US96/10006 Frn~~I~le 19 Sl~sf~7;ne~ R~len~e of ~lnfi~icrobi~l C~"~ilions From Fibrin Seo~nnt Fibrin sealant (F;S) disks were made by the e~y~Lic Cull~ DiOll of fihrinn~rl to fibrin by 11~ l, and s.ll,se~ y cross-linked by Factor Xm. Briefly, 100 mg of human Topical S Fibrinogen Comr!PY (TFC, ~ n Red Cross, Rockville, MD), co. .l~;. .;.. ~ 76% fibrinogen and Factor XIII, was collll ~ed with 10 mg human IlllO~l~ Red Cross, Rockville, MD) and 0.9 ml 40 mM c~lri~lm rhlori de sollltion The cro~slinking fibrin clot was quickly placed into a 20 x 10 x 3 mm mold and pressed to form a slab. FS disks were then punched from the slab using a 6 mm biopsy punch. Following the same procedure, antibiotics were mixed with the Iyophili7P-~l TFC and Ih.~ prior to hydration to form antibiotic-~l~Led FS (AB-FS) disks. Telldcy~ e free-base, ampicillin free-acid and ciprofloxicin hydrochloride (Sigma ChPmir~l Co., St. Louis, MO) were added se~a~a~ely as 345 mg to the TFC and thrombin prior to c~lrjllm clllori-lP a(l~lition (final fibrin CO- ~r~ - dlion was 76 mg/ml;
final antibiotic c~,,r~ ion was 50 mg/disk).
Antibiotic release was 1-~ as~d in vitro under two ~Ll~.lle con~lition~, ~limited sink"
and "infinite sinlc." Under limited sink c~n-liti~n.~, FS and AB-FS disks were placed individually imo wells of a 24-well tissue culture plate with two rnl of phosph~tP buffered saline (PBS, pH 7.4). Tissue culture plates were left at 37~C willlo~L~ ~git~tion. The total volume of PBS was exeh~ngecl daily and the eluates evaluated for antibiotic content. Under infinite sink conditions, FS and AB-FS disks were placed individually into 50 rnl conical centrifi-ge tubes with 4~ ml PBS and ~it~tPd by inversion (20 times/min). All tubes were Illi.illl;l;llf~ at 37~C. The total volume of PBS was ~xeh~ ed daily and the eluates evaluated for antibiotic content.
Antibiotic collce~ L~ions were r~lr~ tP~1 from linear ~daL-l curves of optical density versus collceL~dlion (0-200 ug/ml). Tetracycline samples were evaluated spectrophoL(.~ 'r~lly at 340 mn. Ampicillin was ~Lea~uled by first reacting 0.1 rnl of the eluate sample with 2.9 ml BCA reagent (Pierce ChPmir~l Co., Rockford, IL) for 30 min at W O ~6~4~74 PCTAUS96n~006 -lOS-37~C. The resllhin~ colored ~ U~il was mu~ulcd at 560 rm. Ciproflt-xirin samples were evaluated dilc~;lly at 340 nm.
To evaluate ~LilJiu~ release in vivo, t~_Lla~;;liuf (TET)-~u~J~k ---f!nlr~1 FS disks were imrl~ntPJl into _ice attwo ~l;rr~ l kx~ . Male BALBIc mice (20-25 g) were ~.f ~
for the ~ o~s (s.c.) or i~ltla~ lf ~1 (i.p.) impl~nt~tif)n of disks. Incision sites were closed with resorbable sutures and ~Isl;l~l~ss steel clips. Disks were lc,,u~ved at 2, 7, 14, 21 or 28 days post imrl~ 1;hl~ and ~y...~ lly .1i~stf~sl with 0.1% trypsin/0.4 ~ EDTA at 37~C for 4-7 days. TET Col-~ tiull~c of the lysates were measured as above to rlPte~ P
the rnass of TET lf ~..~;..;.~ in disks after in vivo inruhation.
To assess the bioavailability of the antibiotic in TET-FS disks, TET-FS disks were placed into test tubes cr~--l;~;.-i.~ a log phase culture of S. aureus (1x107 CFUfml). Cultures with FS disks cont~inin~ no antibiotics served as controls. All cultures were inrub~te~l at 37~C for 10 hr. R~tt~ l s~mpl~s (0.1 ml) were serially diluted and plated onto ..~ ;P~,I agar to ~k;l~ ...i..P the viable bact~ri~l count during the in~ it)n with the disks. An culture was also monitored for Ct.. p~ on.
The elution profiles for the three antibiotics evaluated under limited sink con~ition~ are pl~sellLt;d in Figure 31A. After an initial burst of antibiotic release, the freely water soluble ampicillin eluted comrlPtely from the supplt~mPntPd FS matrix within 7 days. This contrasts the elution profile of tetracycline free-base which demol~Llat~d a slowly decreasing, steady release over 42 days. Tetracycline elution at day 42 was a ~ ; ;llPrl anti-microbially err~;Liv~
amount, 0.03-0.04 mg/ml. The release kinPtirs for ciprofloxicin parallelled those of tetracycline; although, data were only collecte~l for 14 days. The elution profile for infinite sink conditions demonstrated an e~ nr~l release of antibiotics during the first 7 days for all three antibiotics culll~aled with limited sink conditions. O~erwise the elution profiles paralleled those observed for the limited sink conditions.
Release of tetracycline in vivo was measured by c~lr~ ting the antibiotic rem~inin~ in AB-FS disks after 2, 7, 14, 21 or 28 days of in vivo implantion. The data are presented in Figure 31B (culll~ined with in vitro data) and show that the elution profile for TET-FS disks parallels the elution profile of the limited sink model in vitro. After 14 days in vivo, TET-FS
disks still co~t~inP~l 50% of the starting col~rP~ ion with no dirr,.e.lce observed between the two sites (~20% i.p. at day 28). These data ~ o~ that both the s.c. and the i.p. sites f~r.ili~t~d the long-term delivery of TET from the TET-FS disks, and that the in vitro e~ were highly ~ ti~, of the ~ --- h.~i~-aled in vivo ~ ;r effect.
.t~ activity was ~ PA by the ability of TET-FS disks to inhibit growth of a S. ~-lreus culture in vitro (Figure 31C). TET-FS disks si~.. ;r~ ly inhibited b~
growth in the 10 hr of study as cO~alcd with FS disks alone. Release of tetracycline and ciprofloxicin from FS disks was long term in both in.vitro models l1pmn~ àtillg the coll~laLion bcLwcen the long term delivery of antibiotics and solubility. Antibiotics of relatively lower solllbility were co.~ r~"l~y released over longer time periods than highly soluble ~,e~alalions. The delivery kin~tirs in vivo resemble those of the lirnited sink model suggesting a limited flow of body fluids at the s.c. and i.p. sites of delivery. Suppl~
FS disks were shown to provide long-term delivery of col-r~-",dlions of all~iùlic s-lfficiPnt to err~livc;ly inhibit b~ J;~l growth, ~l~mo~ tillg that FS is an ideal, biocompatible, resorbable delivery system capable of rel~ing efficacious localized doses of antibiotic over an e~t~n-lP~ period of time.

Example 20 Long Term Site-Directed Delivery of Cytotoxic/Antiproliferative Drugs from FG

The rlbl-uogell was solubilized with sterile water or, for one group with water sdLula~d with 5-FIJ at a co~rr~ inn of 17 mg/ml. Tlnulllbin solutions were made with sterile water, and then were diluted in 40 mM CaCI2 to a co~ce.lL~dLion of 15 U/ml, or Thrombin was dissolved in 40 mM CaCl2 saturated with 5-F U in a co.~rel,l,dlion of 17 mg/ml.
Control FG clots did not contain 5-FU and were produced by mixing 200 ,ul of TFCsolution (at 60 mg/ml) with 200 ~1 of TLullll~in solution (at 15 U/ml) and allowing 20 mimlteS
to polylll~,~,,c;. These clots were made in 12 by 75 mm test tubes and then were placed in 10 mls of 0.05 M ~i~tiriinP, 0.15 M NaCl, pH 7.3 (Buffer).
FG clots co"l;~;ll;l~g sdlul~ted levels of liquid S-F U were produced by mixing 200 ,ul of TFC (60 mg/ml ~ 17 mg/ml S-FU) with 200 ~Ll Tl~olllbill solution (15 U/ml + 17 mg/ml -WO g6/40174 PCTtUS96rlO006 S-FU) and allowing 20 ~ c for the clots to fully poly~ c. The ~ iti(~n of ~ /~n~lteA
levels of S-FU in both the TFC and T~ulu~ill solu~inn~ sol~ .l~t altered clot ru~ ;n~
J- ~ a clot that was 1- ~ 1 as c~ l~'edtO the control FG clots which were quite opaque. The clots that were formed were physically the same as those made with FG alone 5except in color. Clots were formed in 12 by 75 mm test tubes and then placed in 10 ml of buffer.
A second group of FG clots were made that co~ an ~Tn~llnt of solid anhydrous 5-FU equal to the amount inrln~ed in clots formed with Salu ated solutions of S-FU. These clots were formed by the ~ tion of 7 mg of solid a~ydlc us S-FU to 200 ~l of TFC (60 10mg/ml)and200~1OfTlllu~l/ill(15U/ml). SevenmgofS-FUwasplacedina 12by75mm test tube. Two hundred ~l of TFC was then added followed by 2ûO ~l of Thrombin. The 3 components were then rnixed by ~ h~g back and forth until a hc,~uogclluus ~lulc was observed and further mixing was inhibited due to the clotting ~ I Clots were then placed in 10 ml of hicti-linr buffer.
15The final group c~ d S0 mg of solid ~hydluus S-FU per clot. Due to the illclcased mass of S-FU (50 mg instead of 7 mg) the previously used mf~th(t~ did not work.
Instead of producing a homogenous clot, a clot was formed with the majority of the S-FU
having settled to the bottom of the test tube. To avoid this problem the bottom of the test tube was first coated with 100 ~1 of TFC (60 mg/ml) and 100 ~Ll of Thrombin (15 U/ml). This 20formed a clot which covered the concave bottom of the test tube. Next, 50 mg of solid a~hydluus S-FU was added to the surface of the 200 ,ul clot. Following this, 100 ~1 of TFC
was added along with 100 ,ul of Throllll,ill. The two soll~tion~ were mixed using an ~ ",~I ;r UOl until the protein started to gel. When this occurred, the ~i~el~illg was ended and the clot was allowed to poly,~ e for 20 .,.;..~ s. The final product was a clot that cont~inPd 25a dense core of a~lo~ ly S0 mg of S-FU. As with the other clots, these were then placed in 10 mls of buffer. The final total protein co~ ation of the FG in all groups was 30 mg/ml.
Each group co..~ 10 replicates. Each ~ plir~t~ was inrllb~t~ at 37~C in lO rnls of buffer. Buffer was e~rh~l.~d for lO mls of fresh solution at 5, 10, 22, 33, 52, 75 and 114 30hours. Aliquots of the eluate buffer were then ~min~l in a s~e~lloyhotometer at a W O 96/40174 PCTrUS96/10006 wa~lc~gth setting of 260 mn. Previous f~ had fi~..... ~.~l.,.~fA that 5-FU absu,~cd sL,ollgly at this Wa~ 511-, while eluates from control FG clots did not.
The results are shown in Figure 32. Control clots co.~ no 5-FU gave no ir~ cadi~ 7. Clots made with 7 mg of 5-FU either in the form of ~,;.I---i.l~A snll~tinn~
S of 5-FU or an equivalent amount of solid 7~hydl. us 5-FU cr~ ,lf ~ d their deli~Ly of S-FU
ell 5 to 10 hours, while the clots co..~ 50 mg of solid a~yL~,us S-FU cr...l;..~e~l to deliver 5-FU for at least 75 hours. Peak levels in all cases oc~;ul,~,d at the 5 hour time point.
While not wi..l.i..g to be bound by theory, it is believed that tbe duration of 5-FU
delivery a~talcd to be a fimrtion of the mass of 5-FU loaded into the gel. As a result, the amount of 5-FU deliverable from the clots co..l;.;..;..~ 5-FU in solution was lirnited by the solubility of the drug. Thus the inrlu~ion of al~luuuLs of solid anhydnous form equal to the amount present in the clots formed from liquid s~ l~ with 5-FU resulted in nearly i~lrntir~l delivery kinetics, while the inrll-~inn of greater ~ ; of 5-FU in the solid form than were possible using the liquid form, resulted in a tripling of the total ~ rAtion of delivery, and typically a 10-fold i~ asf in the duration of delivery of a given concc~LlaLiou of the drug.
It would be ~xrectPd that the inclllcion of still greater Amo-lnt~ of the solid anhydrous 5-FU
would also result in even greater delivery times. In other e~ , it has been found that the amount of 5-FU inrlnd~l in the clots can be i~ ~cd at least 5-fold and probably higher, and that the 5-FU-FG ~ Lule can also be form~ t~od into an i~ ilable form (data not shown).
It would further be ~ e~ cl that the use of an analog or other forrn of 5-FU that was less soluble in the ~ull~Julldillg aqueous ",f-li.~., than the anhydrous form, and/or had a slower dissolution rate, would result in a further in~ilease in delivery times.
The result of this process is a ~ ;"~hle delivery of the antiproliL., ~ ;v~/-;yLc,to~ic drug 5-FU from fibrin clots for at least 10 times longer t~an is possible using the drug in the aqueous forrn. This trrllnnlogy (i.e., the use of a solid form of the drug, preferably one with a low solubility and/or dissolution rate? should be generally applicable regardless of the matrix in which the drug particles are sn~ren~1e~l or the drug itself.

Example 21 -WO 96/40I74 PC~r~US96nO~06 Delivery of Taxolfrom Fibrin ~~n~nnt { Based upon the s.~cre.~r~l controlled deli~.y of 5-FU from a .. U~l~]~ A fibrin sealant matrix, pl~ locols were d~ ,lo~ed to collsidc~ the delivery of other che~ull.f .~ ;r CO~ U~ Recently, ~C~ P1 or taxol has been l~cû~cd as a very ~ro...;.~;n,~ agent for S the L1~ JII of ovarian and breast cancers (Nicoleffl et al., Ann. of Oncology 2:151 (1993)).
One problem with ~ n;~;xt~ taxol, sy~lr~ y iS that it is highly insoluble in aqueous ~.y~ ~s. This has ..Pc~ s~ the use of a systemic delivery vehicle co~ of an oil and alcohol u~i~Llure (Rose, W., Anti-cancerDrugs 3:311 (1992)). Ullru~Lu~ y~ this systemic delivery vehicle causes severe re~rtion~ in rnany patients, and current t~la~euLic applications call forpre-.,.P~]k~.~ion tû .. ;.. ;.. ;, f them (Weiss, etal., J. ~lin. Oncol. 8:1263(1990); Arbuck et al., Seminars in Oncol. 20:31(1993). The m~ n~nries for which taxol is Cull~llly under clinical use are generally slow-growing, suggcsLing that an ~xten~lPfl exposure to taxol from supplemPntP~l fibrin sealant would be desirable. Additionally, since the lesion produced by these ~ es is often ~cce~ihle clinir~lly through l.elcutd,leuus biopsy or l~dloscopic prûcedures, the prolonged delivery of effective local concentrations of taxol from a fibrin sealant matrix a~eal~ed ~ a~c~ rally feasible.
The kinPtirs of taxol delivery from fibrin sealant were initially ev~ tPd, by incorporating taxol (0.26 mg), either as an anlly~ous solid or dissolved in ethanol, into a 400 ~1 fibrin sealant cc~osiLion. The reslllting supplemPntP~l fibrin matrices were then placed in 2 ml hi.~ti~linP buffer, and ;~ b .l~ at 37~C. The buffer was e~rrh~nged after two days, and again ten days later. The relative co~r~ . aLion of taxol in the rP~lllting eluates ~ie~ P~l by m~o~cllrin~ their ability to inhibit the growth of a human ovarian cal~;.uunla cell (OVCAR) in vitro (MacPhee et al., In Current Trends in Surgical Tissue Adhesives: Proceedings of the First Internat.on~l Sym~osium on Surgical Adhesives, R. Saltz and D. Sierra, eds. Springer-Verlag).
Briefly, 1000 OVCAR cells in.l00 ,ul of growth m.-~lillm were plated into each well of a 96 well culture plate and inr~hatr(l for 24 hours. A 100 ,ul volume of various dilutions of the eluates was then placed into the wells (10 wells per ~lillltio~), and the plates inrllb~te~i at 37~C. After five days, tne number of cells in each well was measured using the MTT assay W O 96/40174 PCT~US96/10006 -1 1~

(R~dp;~lL et al., American Journal of Clinical Pathology 97:84 (1992)). In this assay, the effect of an anti-prolifi--o~ agent is seen as a d~wsc in the ~ of cells in the final cultures, and ev..~r~ y, as a dcelwse in the amount of MTT that is coll~.,,~d into a cL~ rhnle. The ~ cl~ hnre is AP!tt~rt~l by s~LIu~holc~ y at 570 nm. The results of the e~ and the source of each eluate is provided in Figure 33. (p < .001 elali~, to the .,.~A;.. control (Dunns test)).
The controls inrlnr1yl an in tial (cellular) activity control (IAC) showing the ~mollnt of ~ub~ aLe produced by the OVCAR cells at the time of ~ ition of the eluates, and the ".r(l;...., control, ~huwing the ...~x;..-~ amount of ~L.ale produced after 5 days in culture.
The eluates from unsuppl~ cl fibrin sealant alone did not affect this growth.
The results obtained using taxol in solution in ethanol showed that the taxol was cc...plt,lely delivered for up to 85 days. When taxol was hlcol~laled into the fibrin sealant in the solid ~hyd~ u~ form, the OVCAR cells were signifir~ntly inhibited for up to 85 days.
Subsequent eluates leco~ ,d after an additional 10 days in culture (day 12 eluates) also signifir~nt1y inhibited the growth of OVCAR cells equally well at dilutions from 1:200 to 1:20,000. This intlir~tPd that when the fibrin matrix is supplem~nt~1 with the solid form of taxol, delivery was ~ ;.;..r(l beyond the initial 2 day period, and that the amount of taxol delivered in the period from day 2 to day 12 ç~rree~1çd that which was delivered in the first 48 hours.
These ~A~~' ;1ll- 11'; showed that long term delivery of taxol from a supplrrn~nt~l fibrin sealant coll,~o~iLion can be accomplished by loading a mass of drug that excee-ls its solubility in the matrix volume. This was possible both by illcol~o~aLing the taxol in its solid form, as well as by dissolving it in ethanol prior to incorporation. This is because the molecular weight of ethanol is much lower than that of ta~col. As a result, ethanol will rapidly diffuse from the 2~ matrix leaving the higbly water insoluble taxol behind to preci~ into solid form within the matri~c.

Example 22 Fibroblast Chemotaxis in Response to Fibroblast Growth Factor-Supplemented FG and Fibronectin -WO 96~40174 PCr/ JS96~10006 Dulbecco's Mo~ifi~l Eagle's M~illm (DMEI~ was ~LI1~5~ from Sigma C~h~
Co., St. Louis, MO. A~ LIuywLic solution was ~u.~ d from GlBCO (Grand Island, N.Y.). R~ r~ obl~L growth factor-l (FGF-1) and 4 (FGF4) were a kind gift of R~ogin~ Kidd, Plasma D~i~ Li~ Labol~lu~ - Red Cross, Rockville, MD, andt'~notirsT~ ... h~ e,MA),-~i.y~Li-_ly. R~o~l)i~uLr~ oblai,Lgrowthfactor-2 (FGF-2, alsû known as basic FGF or bFGF) was yurcllased from Upstate I~io!~ rh"nlngy, Inc.
(Lake Placid, NY) All plastic ware l~u~ for sterile prop~ tinn of cultures as well as the r -~ xic assays were yulchdsed from Fisher Sc~ (Newark, DE). Milli~ll-PCF (12.0 ~m) inserts were ~ulcllased from Millipore, Inc. (Bedford, MA). ~Pp~rin was o~ ed from the UpJohn Co ~ y ~l~msl7Oo, M~.
NIH/3T3 fibroblasts at passage 126 were purchased from the ~...f . ;r~-~ Type Culture Collection, Rockville, MD. Cultures from pa~gPs 129-133 were used in the ~ wla~is assays. Cultures were prop~tr-~l in DMEM supplPmPntrd with 10% Calf serum and approximately 1% antibiotic &~lLilllycotic solution. Human dermal fibroblasts (HDFs) were ~ulcl1ased from Clonetics, Inc. (San Diego, CA) at passage 2. Cultures from p~s~s 3-5 were used in the chemotaxis assays. Cultures were cultivated in DMEM supple...~ l with 20% FBS (Hyclone LabGl~Lulies, Inc., Logan, UT) and â~l~rlJx;lll~t~ly 1% antibiotic all~illlycolic solution (Gibco, Grand Island, N.Y.).

CeU Ch~n~ofn~i~ Assays The procedure used to (~ r~ I l l;l lP cellular rh~.. o!;.x;e was a combil,aLion of two known methodologies. A m-ltlihr~tion of Boyden's cl~bel was used as follows: MiIlicell-PCF
(Millipore, Inc., Bedford, MA) (12.0 ~m) 12.0 mm ~li~-,-- t~ inserts were placed in individual wells of 24 well plates to create the upper and lower chemotaxis Cha~ S. Chemotaxis results were arrived at by pelr,lll~illg ch~.bodld analysis for every colll~i~Lion of cells and growth factors. Co.~ rln~ ranging from .1, 1, 10, 100 ng/ml with/wi~ou~ added heparin (10 U/ml) were used for FGF-1, FGF-2 (no heparin) and FGF-4 with all the cell types m.ontiont~l in the materials section. Briefly, cultures were lly~ (l and placed in DMEM
+ 0.1% Bovine Serum Albumin (BSA) (Sigma ~h~omir~l Co., St. Louis, MO) for W O 96/40174 PCT~US96/10006 a~ At~ ly one hour at 37~C in a 5 % CO2 ~ ;r;~A r.l~_. nl~,f.. . Two to 2.5 x 105 cells in 50 ~1 were added per insert to the upper rl~ c- of the setup of the 24 well plates.
Tl~ ; were added as m~ntionP~ above. The assay was kept at 37~C in a 5% C02 l.. ;.l;r;~l ch ~-.. l~- for 4 hours. All ~ k;.. ,.1;. l~ tested were ~ - r ~- ---rYl in t~irlir~tP At the S end of 4 hours, the plates were L~ O~d from the ;~ lnl and the filters were stained following the protocol for sl;~ inrll~d~PA with the ~illir~ pcF inserts. Briefly, the fluid ~Illl'U"~ , the inside and outside of the ~illirPll-PCF inserts was ~ .,d. Three percent ~lllt~ralrlPhyde (Sigma ~h~mir.~l CO., St. Louis, MO) was added to the outside and inside of the inserts for approxim~tely 20 ...;.---l- ~. Following removal of the 3.0% glutaraldehyde, 0.5% Triton X-100 (E.M. Science, Cherry Hill, N.J.) was added for 5-7 .. ;.. --IPS. On removal of the 0.5% Triton X-100, Fisher's ~~ lo~cylin Solution Gill's Formnl~tion (Fisher Sri, -.~ r, Newark, DE) No.l was added for about 10 I--;..l~rs. This sollltion was washed off in l~ i.Ctill~ water for about 5 ",;..~ ,. Using a cotton swab the upper side of the filter was swabbed to remove cells which had not mi~S~tf~Cl Filters were mounted lower side facing up on slides in Crystal Mount (13iomP~, Inc., Foster City, CA) solution and 10 r~nrlrJm fields were counted per slide both visually at 400 x and at 200 x using an Image Analyzing System to ~I~lO~ the e~ alion of the cells on the underside of the filters.

Check~,bou,~ Analysis As required, rhP~L~.~Gald analysis was carried out to t1~ k ran-lom migration, and po~iLive and ~e~;aliv~ ch .. !i~xi~. Growth factors were added to the upper and/or lower cl~b~l~ to observe whether cells mif~r~t~cl towards the GF alone (rh~mot~xi~)~ whclller _igration was random i~ ec~ of whether the growth factor was added to the upper or lower well (chPmokin~ ) or W~ lel cell migration was against the chP-mot~rtir gradient (lle~aliv~ chemotaxis).

2~ Cell Migration Assay to FGF RPIeCI ~e~ From FG
Chrmot~xi~ rl.~.,.hel~ and cells were utilized as rle~rri~e~l above. Fifty ~1 of 8 mg/ml Topical Fibrinogen Complex (TFC, A~ ic~ Red Cross, Rockville, MD) was added to the bottom of 24 well plates. For~ l of test growth factor +/- heparin at a final collce,lllaLion CA 02223889 l997-l2-05 W O 96S4~74 P ~ ~US96~0006 of 10 U/ml (FGF-1, FGF4 with h~ n, FGF-2 alone) was added to the TFC and Illoluu~ly mixed. Ten ,ul of bovine Thr~ ..I,i.l (Armour r~ r~ -l;r~l C0~ y~ K~nks~k~ L) was added and mixed l~luu~ly. The Culu~)o~ were allowed to gel at room le~ for a~lu~ ,ly 30 ..~ ,s Total volume in the lower S and upper c~ was made up to 0.5 ml each with DMEM + 0.1% BSA. The co..r~ on ûf the FGF's added to the TFC was adjusted to ~roduce the desired overall collce~ ioll as d~ d by:

Overall FGF Co~ n = ~p of FGF added to TFC
Volume of liquid in upper cl ~mher +
Volume of FG & liquid in lower ~h~.. h~l The assay was ~-elrolll,ed at 37~C in a 5%CO2 hllmiAifi~cl chamber for apprn~im~tely 24 hours. At the end of 24 hours, the filters were removed, fixed and stained and the number of cells on the underside of the filter was ~ .. ~,il~rd as described above.

Results C'np~ for Migra~con of Fibroblasts The ability of NIH 3T3 fibrobl~ts to migrate tow~ds various well known chemotactic agents was ~ fd to ensure that the cells used in this assay retained this ca~aeiLy.
Fibro~e~ l was the most effective ~ . ..J~ ;r agent tested for both NIH 3T3 and HDFs with m~xim~l le,~o~es oc~ . . ;..g at 20 ,uglml (Figure 34, Table 8). Thc~earlel, fil~lolle~;lin at 20 ,ug/ml was used as the ~osilive control for migration.

Chemotaxis of NIH 3T3 Fi~roblasts Towards FGF-I
~ raximnm stim~ tion of migratinn of NIH 3T3 fibroblasts by FGF-l was observed at 10 nglml in the ~r~ e of 10 Ulml of heparin (Figure 35). ChPcl~rboard analysis revealed that FGF-l was chemotactic for NIH 3T3 cells (Table 9).

-1 1~

Chemotaxis of NIH 3T3 Fibrobl~zsts Towards FGF-2 M~;... ~;;",.. l.,t;~" of ~ of N~I 3T3 rll,r~bl~ by FGF-2 w~ obsc.~d at 1 ng/ml of FGF-2 (Figure 36). ~h~. L ll~GaL-l analysis showed that FGF-2 was ~.h~."ol .r.tir for NIH 3T3 cells (data not shown).

S Chemotaxis of NIH 3T3 Fi~.~Dbla~ Towards FGF4 ~r~ximnm stimnl~tion of mi~tion of NIH 3T3 fibroblasts by FGF4 was obs~lved at 10 ng/ml (Figure 37). C~h~L~ ~boal~l analysis revealed that FGF4 was r~ J!~c~ for NIH 3T3 cells (data not shown).

~he~10tnris of HDFs Towards FGF-I
M; xi----l-,l sfimlll~tinn of migr~tion of HDFs by FGF-l was observed from 1 to 10 ng/ml (Figure 38). ~h~ oald analysis showed that FGF-l was chemotactic for HDFs (Table 10).

Ch~ .ol~ of HDFs Towards FGF-2 Maximum stim~ tion of migration of HDFs by FGF-2 was observed at 10 ng/ml (Figure 39). Che~ oal-l analysis revealed that FGF-2 was ch~mot~eti~~ for HDFs (data not shown).

C.~h~ Otnric~ of HDFs Towards FGF4 M~ximllm stimlll~tion of migration of HDFs by FGF4 was observed at 10 ng/ml (Figure 40). (~h~ .lJoald analysis showed that FGF-4 was chemotactic for HDFs (data not shown).

n~z Dermal Fibroblast Migration to FGF-l, -2 and 4 Inc~r~Futed in FG
M~im~l llligldtOIy lc;~ollse to FGF released ~om FG was elicited at an incorporated and total collcellLIalion of FGF4 in FG of 1 ng/ml (Figure 41). Similar results were also found when FGF-1 and FGF-2 were incol~ola~d into the FG (data not shown) except that the Collc~lLlaLion of FGF-2 that elicited the peak chemotactic les~onse was 0.01 mg/ml.

W O 96/40174 PCT~US96/10006 Table 8 C~ r ~ C~r ~ ~of ril~u~c~,Lill In Upper of r;l.~ f. ~i"
In Lower C~
O ~g/ml10 ~g/ml 20 ,ug/ml 50 ~g/ml O ~glml 48.53 +/-62.3 +/- 69.6 +/- 62.0 +/-4.695 3.269 12.25 2.616 lO~g/ml 68.03 +/-47.53 +1-64.86 +1- 74.66 +1-10.793 5.605 7.961 3.946 20 ~g/ml 90.53 +/-88.73 +/-56.9 +t- 76.23 +/-5.203 4.152 3.289 1.8190 50 f~g/ml 72.43 +/-91.3 +/-63.26 +/- 57.46 +/-8.276 1.003 3.835 2.287 Table 9 C~ G~ ûf FGF-I In Upper Cu~
of FGF-1 In Lower ~'~""I' '' I'''' "I
O ng/ml 1 ng/ml 5 ng/ml 10 ng/ml Ong/ml 32.1 +/-53.93 +/-27.27 +/- 25.96 +/-6.328 4.152 3.873 4.151 1 ng/ml 59.46 +/-36.9 +/- ~.l +/- 35.86 +/-6.89 5.728 9.232 2.074 S ng/ml 64.867 +/-41.44 +/-24.84 +/- 41.6 +/-1.75 1.866 4.337 6.717 10 ng/ml 70.83 +/-39.73 +/-39.73 +/- 41.83 +/-2.752 2.428 2.428 6.879 CA 02223889 l997-l2-05 W O 96/40174 PCT~US96/10006 ~+ ~+ ~ + ~ +
D . X ~ ~ ~ ~ ~
8 ~ ~~ ~ ~ ~

_ +
+ ~ + X + ~ + ~ ~o _ + 1~ + ~ + 1_ + ~ + ~

4~
o o q~ o E~ a ~3 + ~ + ~o + ~+ _, + 00 a a Oo~

--+
+ ~ + ~_ +
~; ~o o ~ o t-- o bO ~ ~D ~ ~ O t-- ~t ~ ~ O.
a ~ ~ ~ o ~ ~ cr~ ~ ~
o ~oa~ o~ ~ ~ ~
-40~ -- a'~
c 3 c ~ ~~ ~ c c ~ p~ c o c~ o ~ o _, W O 96J40174 PCT~US96/10006 Discussion The FGFs produced a ~roruulld chemotactic lc~onse in HDFs. For every chemotactic assay pclrolllled with HDFs, a very good r~ ll was o~L~ ed b~tw~;n the ~eg~Livt: control and the ~"r -..I.,.I;~n of FGF which elicited a ..~
S migratory- response: 18, 12 and 10 fold in lc~(,llse to FGF-l, -2 and 4, l~s~ecLiv~ly.
The stim~ tinn of ch~ xi~ by growth factors was not as high for NIH 3T3 cells as it was for HDFs, possibly due to the high passage Llulllber of the available stock cultures of the NIH 3T3 cells as co~ led to the HDFs.
FGF-1, FGF-2 and FGF4 were found to be potent stiml]l~fors of fibroblast chemotaxis. Directed migration of fibroblasts by one or a combination of the above growth factors could result in fibroblast ~lcs~llce in the site of injury, thereby leading to fibroplasia and the laying down of collagen and an extr~cçlllll~r matrLx. Thus, aside from it's well recognized angiogenic ~lopelLies, FGF's may have a role in wound healing, acting either alone or in a colllbillaLion with PDGF,IGF-I,TGF-~
and/or other factors.
Previous studies into the use of FGF's to speed wound healing have not yielded ~ignifir~nt results (Carter et al., 1988). This may be due to a re~ clllellL
for the prolonged exposure of cells to the factors in vivo for a m~xim~l response (Presta et al., Cell Regul. 2:719-726 (1991) and Rusnati et al., J. Cell. Pkysiol.
154:152-161 (1993)). U~ ~Lely, it is ~1iffirlllt to deliver growth factors to wounds for such long time periods under conditions that would not ~ r~ with the healing process.
The present invention of incol~laLillgFGFs into FG allows for the prolonged exposure of cells to the FGFs and can be applied to a wound. The res-llting fibrin coating mimics the natural le*)onse to tissue injury, while delivering the growth factor directly to the wound site. In a previous study by the present ill~r~lllul~,FG
which co-.~ 1 FGF-l was used to line artificial vascular grafts (Example 8, herein).
When these grafts were placed into the vessels of rabbits, the FGF-l was released for a period of up to 28 days. ~ further studies involving canine grafts, the effect of the incorporation of FGF-1 into the graft walls was the total endoth~ tion of the CA 02223889 l997-l2-05 W O 96/40174 PCTrUS96/10006 artificial grafts within the same period (Greisler et al., Surge)y 112:244 255 (1992)).
Thus, this form of application elicits a p.ofu-u~d biological effect in vivo. The fibroblasts are ~ttr~t~ ~wald~ FGF l~,leased from FG. This ~r~ly will be useful in Ll~,aLiLIg wounds with GF-supplpmpnt~(l TS.

Example 23 Site-Directed Angiogenesis Using TS to Deliver Angiogenic Substances This embodiment permits the directed gene~dlion of new blood vessels in a controlled manner witbin the body. In this embo~lim~nt the TS c~ and delivers angiogenic subsldllces, such as Fibroblast Growth Factor-l (FGF-1), in an amountsuch that its conc~llL,~Iion which is released from the suppleJnt-nt~l TS is err~-;liv~
to induce angiogenesis.
This embodiment is used in a controlled llla~ el to rev~cul~ri7e body areas which have been deprived of an acleqll~te blood supply such as cardiac, brain and muscle tissue, and the retina. This embodiment is used to restore or improve circulation to implanted organs or re-~tt~h~l limbs. This embodiment can be usedto generate a vascular llc;Lwoll~ or "vascular bed" for: the ~ t;ne,dLion of artificial organs or organoids, the delivery and/or loc~li7~tion of and/or nouri~hm~nt of cells used in gene therapy, or as a target of gene therapy, for the lluvl ;.~l""~ont and/or loc~li7~tion of cells for tissue ~ nPnt~tion. This embodiment also precludes thenecessity of implantation of a device or substance which may induce a foreign body or other excessive infl*"",.~loly reaction which could colll~lom,se the blood vessel formation or the function of the underlying organ(s).
The invention eonsi~ of a formlll~tiorl of fibrinogen, (suitable for the 2~ formation of fibrin) with or without fibl.. lle~;Li,l and/or collagen, into which is placed an ~plopliate co,lcellL.~ion of an angiogenic substance, such as FGF-l. The fibrinogen may also contain stabili_ers to protect against the proteolytic activity of Thrombin. In the case of FGF-1, heparin sulfate (1-1000 U/ml) may be used as the W O ~6~4~574 PCT~US96/10006 stabilizer in the range of c~ nn of from 1 ng/ml to 1 mg/ml. ~ ;vcly the angiogenic ~ub~L~ce is c~ ;..Fd, in an a~r~lial~ coll~cllLlalion, in the thrombin, c~ lm, or water components. This form~tinn is then mixed with Lh~ubil~ and rapidly applied within the body in a line con~ the desired sites, or to a single site. The rll,l"lo,~c,l-LIl~ ll ill mix then poly".~ s to form FG. T~he FGF-l, or other angiogenic s..l.sl;.,~e, l~nalllS trapped in the FG matrix, either as a free form or bound to the stabilizer or another component of the ll~i~lul'~. In one embo~limPnt, the c~ e~ ,.lion of the FGF-l in the TS should be from 0.1 ng/ml to1 mg/ml, more preferably from 1 ng/ml to 100 ,ug/ml, most preferably from 100 ng/rnl to 10 ,ug/ml. The FGF-l, or other angiogenic substance, will induce bloodvessel formation within the body of the deposited FG. The FG will be n~hlr~lly biodegMded leaving the intact blood vessel(s).

Example 24 Site-Directed Cartilage In~ fi~n This embodiment perrnits the controlled ge~ alion of new cartilage as well as the guided reg~nelaLion of t1~m~geA cartilage within the body. In this t;lllbodi.llent the TS contains and delivers a cartilage ~rollwlillg factor(s), such as cartilage-inducing factors-A and/or -B (CIF-A and CIF-B, l~c~;Liv~;ly, which are also known as TGF-BI and TGF-B2, re~.~ecLively) and/or another, factor(s) such as Osteoid-Inducing Factor (OIF) in an amount such that the co~e~ on of the inducing factor(s) which is released from the supplemente~l TS is ~rr~.;tiv~ to induce cartilage formation. In one embodiment the concentration of the int1uc.ing factors should be 0.1 ng/ml to 1 mg/ml, more preferably from 1 ng/ml to 500 ng/ml, most preferablyfrom 100 to 250 ng/ml. This embodiment may also contain dru~s, such as antibiotics, and other growth factors, such as EGF, PDGF, and bFGF in the TS. The e cartilage inducing substance is co~ ed in an a~L~lia~e concell~ld~ion in the fibrinogen or thrombin or calcium or water component(s) which are used to plc:~ale the TS.

W O 96/40174 PCTrUS96/10006 The supp!t~-mlont~od TS can either be pre-shaped to the desired final cartilage form prior to impl~nt~tinn or it can be implanted into the body of the recipient in the liquid form as the TS is mi~ed and poly~ s. The reSlllting form may then be sclllpted as desired to ~l~luce the required shape of cartilage ntoe~l~l The Cartilage Tl.. ll.çi,~g TS (CI-TS) ~xLule can also be used to precoat a collv~ ;on~l impl~nt with the result being a collv~ ;on~l implant with a coating of living cartilage.
Using any of the terhni~ çs desclil ed above, the CI-TS is then implanted into the body of the l.,~ iellL. This impl~nt~tic n can be I~L.,l~lo~.ic or orthotopic. After an ~r~liate interval, the CI-TS is be replaced by living cartilage with the form of the original CI-TS implant.
Such implants can be used to replace ~l~m~ed or lost caTtilage, or to ~ro~/e the tissue illL~ lalion and/or fimrtion of an artificial implant. Examples of such uses include the repl~eçmr-nt or reconstruction of nasal or ear tissue, the gene.dLion of a functional joint surface on a bone implant grown in vivo, or the gell~,laLion of a similar surface on an artificial implant. The repair of cartilage damaged by disease, such as rhrllm~toid arthritis, can also be accomplished using the CI-TS to produce a new and smooth cartilage surface to the arthus. Tmr!~ntc inten~le(l for space filling applications in Plastic/Reco~.Llu-;Live surgery can also be either formed from CI-TS, or coated with CI-TS to e,~ nre tissue illL~laLion and reduce foreign body re~ctic)n~.
Since current technology does not permit the guided l~gel~.,.alion of cartilage,this invention is an adv~.~r~ because it permits the gell~lalion of cartil~ginmls tissue which is required to fully mimic the body's natural make-up. This results in improved joint repair, artificial joints and other implants, both for orthopedic and other applications.
For example, this embodiment can be used: to produce hlll.rov~d orthopedic implants or irnproved plastic/r~onslluctive implants: for joint repair for Llill.",i11;r, congel~i~l or pathologically damaged or dy~.rul~lional cartilage; to produce coatings of p~Pmslker implants and wires to increase their tissue integration and to reduce foreign body reactions. Similar co~tingc could also be applied to any implantable device for the similar puTposes.

CA 02223889 l997-l2-05 WO 96~40174 P ~ ~US96/10006 E~u"~le 25 Supplemented TS as a Sur~ace Coa~ing for Bioma~erials ,~
This embodiment uses suppl~m~nteA TS as a coating for the surfaces of orthopedic devices and other bi..~ which are to be imrl~nt~1 into an anirnal's S body. Examples of these devices are urinary ~ s, inLLd~rds~;ular c~
sutures, vascular plv~ ses, in~oc~ r lenses, contact lenses, heart valves, chmlltle.r/
elbow/hip/knee repl~r~ devices, total artificial hearts, etc. U~olLu,ldLely, these biom~t~r-i~l.c may become sites for bacterial a&esion and coloni7~tion, which eventually may lead to clinical infection that will endanger the life of the animal. To ~ this problem, the biomdtclial is coated with a supplem~nt~l TS.
In this embodiment the TS can be supplem~nt~d with: a growth factor(s); a drug(s), such as an antibiotic; BMP; and/or cultured cells, etc. Examples of antibiotics that may be incorporated into the TS include, but are not limited to: the penicillins; cephalosporins; tetracyclines; chloramphenicols; .l-ello-~idazoles; and aminoglycosides. Examples of grow~ factors which may be incorporated into the TS include but are not limited to FGF, PDGF, TGF-,B. Examples of BMPs which may be incorporated into the TS include, but are not limited to, BMP 1 through 8.
DBM can also be added to the TS. Examples of cultured cells which may be incorporated into the TS include, but are not limited to, endothelial cells, osteoblasts, fibroblasts, etc.
The supplement(s) may be contained in either the thrombin, fibrinogen, calcium or water colll~ollcllL(s). The concentration of the supplement in the TS is adequate such that it will be crrcc~ivc for its intPn-le-l purpose, e.g., an antibiotic will inhibit the growth of microbes on the biolnalc,ial, a growth factor will induce the growth of the desired cell type(s) in the TS and/or on the surface of the biolllalclial.
This invention is an improvement for exi~ p bioll~lclial products, which include I il;.,.iu.,- and lili1llill.,~ alloy devices (such as fixation plates, shoulder/elbow/
hip/knee repl~c~om~t devices, osseohll~grated dental implants, etc), solid silicone products (such as Silastic nasal implants, liquid and/or gel silicone products (such as W O 96/40174 PCT~US96/10006 breast implants and testirll1Ar implants), and natural or synthetic polymers used as collvr~ Al m~t~.ri~l.e in healing a wound site, which may have various forms, such as monofil~m~nt~, fibrous assemblies (such as cotton, paper, nonwoven fabrics), film.~, sponges, bags, etc.
S FG is produced from 3 colll~oll~llLs: fibrinogen (for example as TFC); and thrombin, both of which may be in the lyophilized form; as well as calcium. The lyophilized fibrinogen is reco~ e~l with sterile water, while the l~ulllbih component is l~co.~ lt;(l with calcium chloride solution. A supplement may be added to any of the three cul~ol~llL, prior to mixing. Ap~loplidt~ volumes of the fibrinogen and thrombin col-~ ;llg calcium are mixed to produce the FG. The FG
is then applied to the biomaterial's surface as a coating thereof as, for example, by s~layillg, ~a;ll~ g, etc. ~ ;vt:ly, the implant is dipped in the FG while it is still liquid. A supplement may also be added to the FG before or after it has been coated on a bi~ 1 surface. For i,~ "~e, a FG-coated implant is soaked in an antibiotic solution for a specified period of time so that the antibiotic will diffuse into the TS.
Another example is coating a device with TS after which cultured cells are seeded onto the fibrin coating. Coating the surface of biollla~.ials, which will be implanted into an animal, with suppl~lllr-"l~d TS will serve several purposes, including: the inhibition of bacterial ~tlh~cjon to the bi~ 1; the inhibition of growth of bacteria adhered to the biomaterial; local immlm~ stimlll~tinn and/or norm~li7~ti-~n; thepromotion of would healing; and the promotion of engr~ nt of the biomaterial to the ~ul~oullding tissue.

Example 26 Self-Contained, TS Wound Dressing This embodiment is a self-contained TS wound dressing, or bandage, which contains both the thrombin and fibrinogen components of the FG. The calcium is co~ i"~A in either the ~ .n~ and/or the fibrinogen component(s). Either or both of the thrombin or ~lbrinogen components can be, but does not have to be, suppl~ ~ with a growth factor(s), such as a FGF or bFGF, or a drug(s) such as, an ~n~ ocir, ~li~ ic or other drug(s), which can inhibit infecti-)n, ~l~u~ woundhealing and/or inhibit scar f~rrn~tion The ~U~lJl~ (s) is at a co..r~ on in the TS such that it will be ~r~;livc~ for its int~ntled ~ulpose, e.g., an ~LI~ioLc will inhibit the growth of microbes, an ~n~1~sic will relieve pain.
The Ll~oml~ and fihrinl g~n are se~aLed from each other by an i...l~ ...f~ble l~e, and the pair are covered with another such ~ lnl~l~e. The Ll ~ , and fibrinogen are co..l;1in~l in a quick e~ra~ol~ lg gel (e.g., methylcellulose/alcohol/
water). The bandage may be coated on the surface that is in contact with the gel in order to insure that the gel pad remains in place during use. (See Figure 42).
In operation, the ~ aLIe se~aLllg the two components is removed, allowing the two co~ onellL~ to mix. The outer mc;~lll,la~e is then removed and the bandage is applied to the wound site. The action of the thrombin and other components of the fibrinogen pl~alaLion cause the collv~l~ion of the fibrinogen to fibrin, just as they do with any application of FS. This results in a natural inhibition of blood and fluid loss from the wound, and the establichm~nt of a natural barrier to infection.
In a similar embo~1im~nt the thrombin co-ll~ollellL and the plastic film ~epalaLillg the Thrombin gel and the Fibrinogen gel may be omittPd The e~lril~m that was previously in the Th~ l)in gel may or may not be included in the Fibrinogen gel as desired. In operation, the outer impervious plastic film is removed and the bandage applied, as previously described, directly to the wound site. The Tl~r~ ill and c~lrinm n~t~lr~lly present at the wound site then induce the coll~ ion of fibrinogen to fibrin and inhibit blood and fluid loss from the wound as above.
This embodiment has the advantage of being simpler, cL~a~eL, and easier to produce.
However, there may be c.,.;~ re,c in which a patient's wounds have insufficient thrombin. In those cases, the previous embodiment of the invention should be used.
This embodiment is an ad~ rPl~ over the current technology as it p[~
the rapid application of TS to a wound without the time delay associated with solubilization and mixing of the components. It also requires no l~r~ ir~l knowledge WO 96/40174 PCT~US96/10006 or skill to operate. These c~ t~ A ;~I;r-s make it ideal for use in field ~rp1iration~, such as in trauma packs for soldiers, rescue w~ , ambulance/~e~lic teams, fil~.llcll, in first aid kits for the general public, and by e~l~cu~;y room ~ o hospitals. A small version may also be useful for use by the general public.

Ela~,~le 27 ~ih~nal Self-Contained, TS Wound Dressings The TSs may be form~ t~l as a self-contained wound dressing, or fibrin sealant bandage, which contains the n~ces~. y thrombin and fibrinogen componentsof the FG. The self-co~ ed ~CS~illg or bandage is easy-to-use, reqllirin~ no advanced teçhniral knowledge or skill to operate.

T*e F~bnn Sealant Rn~rzge The present illvclllols have p,~a,ed a fibrin sealant bandage for applying a tissue sealing composition to wounded tissue in a patient, wherein the bandage co,l,~,ises, in order: (1) an occlusive b~e~ing; (2) a ph~rm~rologically-acceptable adhesive layer on the wound-facing surface of the b~el~in~; and (3) a layer of dry materials co~ is,llg an ~rr~;Livc amount, in colllbinalion, of (a) dry, virally-inactivated, purified tissue fibrinogen complex, (b) dry, virally-i~cLi~aled, purified IllloL~ ill, affixed to the wound-facing surface of the adhesive layer or backing, and (c) calcium chloride. A removable, wal~ Oof, soft plastic, ~r~ Icclive film was placed over the layer of dry materials and the exposed adhesive surface of the bandage for stable storage purposes. In operation the w~le~roof, I,rotecLive film is removed prior to the application of the bandage over the wounded tissue. The bandage was applied with plc~ulc until the TS has formed over the target area.
The fibrin sealant bandage was tested using a cc"~ lional, adhesive silicone patch measuring 6 cm X 5 cm, having a total area of 30 cm2. The dry components were placed over the adhesive patch to a depth of l/z cm, so that the total volume of fibrin formed by the TS upon hydration equaled 15 cc (30 cm2 x l/2 cm). The W 096/40174 PCT~US96/10006 mAt~riAlc used were: 360 mg of topical fibrinrgen complex (TFC), desc~ d previously; ap~,o~ullldL~ly 340 U Ll~ ul)ill, also described previously; and 88 mg CaC12 (40 mM).
The binding cal ~iLy of the bandage for the dry material layer was, in part, S ~le~e~ upon applying the dry mAt~riAlc as a u~irul~ly-ground, fine ~uw~le.. The c~le~ m chloride was ground to a fine powder and mixed with the finely ground lyophili7ed TFC and thrombin, and applied as a powder to the adhesive side of the silicone patch and allowed to adhere to form the fibrin sealant patch. In ~d~ition~l versions of the fibrin sealant bandage, the dry materials were mixed and ground together.
Signifi~Antly more of the finely ground powder adhered to the silicone patch when ~les~ ; was applied. However, the ~lu~lLiLy of dry mA~riAl added to the fibrin sealant bandage was ~ A..liriAhle~ It was found, for example, in one application using the silicone patch backing that an area, 2 X 1 cm2, when completely covered by the dry fibrin components increased in weight by 30 mg. This mea~ was extrapolated to a dry fibrin colll~ollellL mass per area covered on the backing of 15 mg/cm2.
The fibrin sealant patch was applied to a damp celllllc)se sponge, represellLa~ of a tissue wound, so that the fibrin sealant colll~oll~llL was A~ljaC~ont to the surface of the sponge. The sponge had been previously ~ pe-~tsd with room-temperature distilled H2O.
Fibrin formAtion began to develop within 30 secon-l.c of application. Within three mimltes of applirAtion, a fibrin gel had formed ~ffixing the tissue sealing fibrin clot to the sponge. This first patch hyL_L~d by the endogenously available liquid was labeled FSB#l .
The previous steps were lcpeated to ~lcpale patches FSB#2 through FSB#5, however, prior to placing the fibrin sealant bAn~lAge against the ~1AIII1~ cellulose sponge, 8 ml of warm PBS were applied to the dry fibrin colll~ullellls affixed to the patch. T.,.-~b~lion of applied patches FSB#2 through FSB#5 was at 37~C rather than W O 96/40174 PCT~US96/10006 o ~ ~ ~ ~
~o ~: d o .5 ~~

~ C ~ .~ ~ ,_ o ~ o o E~ i-,.$ C ,c o E ~ ~~ u-~ o ~_ ~ c ,c ," ,~

o ~~ .5 .!3 ~ ,c 3~ 'e .5 .5 ~

c ~ ~
~ .5 .Ei .~

W O 96J4~74 P ~ nUS96/10006 room ~ c. The results, set forth in Table 11, eyrmrlify the an applir~ti-~n of the fibrin sealant b~ a~,f, _-..ho~ 1 ~L,.~,~ the dry m~t.~ri~l~ are exog~lluu~ly lly~d~ed prior to applir~tit~n Patch FSB#3 was ~ ,d the same as FSB#l, but absent the ll~u~ )in S Co~ olw~L. Patch FSB#4 was pl'~)dlCii the same as FSB#l, but absent the TFC
CO~O~ l. Patch FSB#5 was ~ ,d the same as FSB#l, but absent the c~lril~m chloride co .~u.,~llL. The results of each test were evaluated over time. As shown below in Table 11, a clotted gel formed when the fibrin components were lly&dl~dwith PBS, but ~ d in solution when either the fibrinogen or Llllulllbih components were deleted from fibrin sealant b~ntl~ge composition. Similarly, although a weak, watery- gel was formed after 30 ~ es when the calcium CO~ u~ was deleted from the fibrin sealant b~ e and from the hy~d~ g fluid, the composition was unable tû develop intû a tissue sealing fibrin clot.
To more clearly visualize the formation of the fibrin clot and the extend to which it bound to ~ r~ r~ces, a small amount toluidine blue was ground into the pûwdered fibrin co,l,~o~ell~ as a color ~ AI~1.
In practice, with ~!,rr;ri .l hydration the silicone patch was easily removed from the fibrin clût after hydration of the drv, fibrin cûmpûnent layer.
The fibrin sealant b~ e, f~ t~l on silicone patches as ~ r.rihetl above, were also found to t;L~;liv~ly form fibrin seals when tested on gelatin sllrf~r-çs and in vivo on rat tissue. Based on the ~.-cc~s~r ~1 form~tic)n ûf the fibrin seal to a variety of materials and tc~ cs, inrl~ in~ basic in vivo testing on an u~ j~cd rat, animal studies will be contlllr-t~l as described in the previous Examples ev~ ting the TS
composition to opLi~ c the h~...osl;.l;r utility of the fibrin sealant bandage, and to establish delivery kinPtics of supplL.. k~ y cu.. l.o~ s to be added, e.g., growth ho~ olles, drugs, antibiotics, ~nti~eptirs, etc.

Ihe Self-Foa~ning Fibrin Sealant The present illvcllLul~ have ~oucd a self-fo~illg fibrin sealant dl~s~ for applying a tissue sealing composition to wounded tissue in a patient, wL~eihl the W O 96/40174 PCTrUS96/10006 dlcssil~g is applied as an e~ Able foam co.,.l,l;ii..g an ~;rr~;live ~ in co~ i~n, of (1) virally-illa-lival~d, purified fibrinogen complex, (2) virally-~acliv~t~d, purified ll-.u~lJiu, (3) cA1ri-lm~ and (4) a physiologically acceptable Ly~]laLion agent; wl,~,~,.u said composition does not .c;~..;r~ y inhibit fu11-l1.;~ L "~
skin wound healing. In p,a~;lice, the previously described TS components will bestored in a ca~Ot" or tank with a ~ 1 propellant, so that the co~ o~ulo are delivered to the wound site ~ an r~ n~l~ble foam, which will within minute(s) form a fibrin seal.
A bench model test system is ~ al~d from standard Amicon pl~,S~ulc;
~~h~.-.hc. ~ to r1ele~ optimal particle size. Particle size h~ proven to be i~ u~
Prel;.-~ e,~e, ;Ill~ have revealed that a reduction in particle size of the TFC,fibrin and cA1~-inm coul~ol~uLO results in a ci~..;r.~.., reduction in the time required to hydrate the reagents.
Testing is also relevant to clel~- ...i"i--g the feasibility of combining all of the reagents within a single l~,sel~.)il, or whether it is more advantageous to m~intAin each component in a sepdl~le ~csc.~()u until application. Although probably moree~enOiv~, the latter c~ lo~oLy~e (having multiple sepa~dl~ reservoirs) may prove advantageous, in terms of stability and long-term storage.
The test system COlloioLo of one or two pll;ooUl'e vessels driven by a ~ 70lll ;~r~1 l~selvoil c~ the lJh~llllA~"Ii~Al1y acceptable hydldLillg agent (e.g., water or PBS), and pressuli~ed co,ui)lessed gas cylinders. The reagents are placed into the ai~U1U~1ia~ Ch~ e1(S) and the 1~Se1~Ui1 charged with hy~ldLulg agent saL~d~d with the propellant at the desired ~ o~iUle. Mixing of water and the reagents in their lcsel~uilO is accomplished by o~eL~ill~ co.~ g valves. The output is dir~cLed into either a single line, or in the case in which the components remain s~;~ ~ ~l~l into the joining piece of a ~em.o-1ics Fibrin Sealant DiOpensel.
In the present case, the TFC was l~,hy~dL~d with 3 cc dH2O, and warmed to 37~C to the c~ el.l.~ n~ shown in Table 12. The Llllolubiu was rehydrated with 0.5 cc CaCl2 solution (100 mM) to the concellLldlions shown in Table 12. The lly~lldLt;dcoLu~oll~LlL~ were mixed and c~bo~dL~d water (10 cc) was added to produce WO 96~40-74 PCT~US96/10006 the volumes shown in Table 12. The reslllting fOa~ ~P~LU1~, was placed in a vacuum jar to illcl~,ase the rO~g. Vacuum ~ was applied until the foam dried. The result was a ~ t .,~ lrJl, foamy mass of fibrin, which e~
a~prok;~ ly 5-fold, and which was both self-adherent and adherent to a~
S l~lulcd ~UlrhCes.
The foam was also ~ VGly ~u.~,d in r~1ihr~t~ plastic beakers. After two ~ s, the volume of the foam was llle~ ed and the mass was gently probed to rlc~-."i~ that it had set. The ~lU~ ivG Lue&~uleLuents of the e~ iol- of the self-r~ g fibrin sealant is i"~ in Table 12. Once set, the exp~n~l~kle foam was no longer adhesive to new ~ulraces.
Based on the ~cces~rl~l fonn~tion of the self-fo~illg fibrin dressing, animal studies will be co~ as ~lescrihe~l in the previous Examples ev~ ting the TS
colll~o~ilion to c~l illli~ the hemnst~tir utility of the self-foam fibrin sealant d~ess~g, and to establish delivery kin~tirs of supple~k~ y Colll~)ol~ to be added, e.g., growth hormones, drugs, antibiotics, etc.
Other embo~ of the invention will be ~ l to those of skill in the art from a consideration of this specifir~tion or ~-a;~ice of the invention disclosed herein. Since mo~1ifir~tinns will be ~al~lll to those of skill in the art, it is intentle-l that this invention be limited only by the scope of the appended claims.

Claims (96)

What is claimed is:

1. A fibrin sealant bandage for dressing wounded tissue in a patient, said bandage comprising: (i) an occlusive backing, and (ii) a component layer comprising fibrinogen in an amount which is capable of forming a fibrin matrix in the presence of Factor XIII, thrombin and Ca++.

2. The fibrin sealant bandage of claim 1, wherein said component layer further comprises at least one component selected from the group consisting of Factor XIII, thrombin and Ca++.

3. The fibrin sealant bandage of claim 1, wherein said component layer further comprises at least two components selected from the group consisting of Factor XIII, thrombin and Ca++.

4. The fibrin sealant bandage of claim 1, wherein said component layer further comprises Factor XIII, thrombin and Ca++.

5. The fibrin sealant bandage of claim 1 which further comprises a physiologically-acceptable adhesive layer.

6. The fibrin sealant bandage of claim 5, wherein said component layer is affixed to the wound-facing surface of the adhesive layer.

7. The fibrin sealant bandage of claim 6, wherein said adhesive layer is of at least one material having a lower shear or tensile strength than that of the fibrin matrix formed from fibrinogen in the presence of Factor XIII, thrombin and Ca++,thereby permitting removal of the backing without damage to the fibrin matrix or to tissue surrounding the wound.

8. The fibrin sealant bandage of claim 7, which further comprises a physiologically-acceptable adhesive layer affixed to a specific region of the backing.

9. The fibrin sealant bandage of claim 8, wherein the adhesive layer extends beyond the component layer so that upon application of said bandage to the patient, the unencumbered adhesive layer is affixed directly to tissue adjacent to the wound, placing the component layer over the wound.

10. The fibrin sealant bandage of claim 7, wherein said adhesive layer becomes solubilized or less sticky upon application, thereby permitting removal of the backing from the fibrin matrix.

11. The fibrin sealant bandage of claim 1, wherein the backing also functions as a physiologically-acceptable adhesive layer to which the component layer is affixed on the wound-facing surface.

12. The fibrin sealant bandage of claim 1, further comprising a removable, waterproof, protective film over the component layer and the exposed surface of said adhesive, wherein said film is removed prior to application of said bandage.

13. The fibrin sealant bandage of claim 1, wherein at least one component of the component layer of the bandage is dry.

14. The fibrin sealant bandage of claim 13, wherein the dry component(s) of the component layer of the bandage are hydrated by a physiologically-acceptable hydrating agent prior to application to wounded tissue.

15. The fibrin sealant bandage of claim 13, wherein the dry component(s) of the component layer of the bandage are hydrated when said bandage is applied to wounded tissue by endogenous fluids escaping from said wound.

16. The fibrin sealant bandage of claim 13, wherein the dry component(s) of the component layer of the bandage are hydrated when said bandage is applied to wounded tissue by a physiologically-acceptable hydrating agent containing within a separate layer of said bandage.

17. The fibrin sealant bandage of claim 1, wherein at least one component of the component layer of the bandage is a gel.

18. The fibrin sealant bandage of claim 1, wherein said component layer further comprises at least one compound selected from the group consisting of the following supplements: analgesics, antimicrobial compositions, antibodies, anticoagulants, anti-inflammatory compositions, antiproliferatives, cytokines, cytotoxins, chemotherapeutic drugs, growth factors, hormones, interferons, lipids, oligonucleotides, osteoinducers, polymers, polysaccharides, proteoglycans, polypeptides, protease inhibitors, steroids, vasoconstrictors, vasodilators, vitamins, minerals and stabilizers.

19. The fibrin sealant bandage of claim 18, wherein said component layer comprises at least one antimicrobial composition.

20. The fibrin sealant bandage of claim 18, wherein said component layer comprises at least one growth factor.

21. The fibrin sealant bandage of claim 20, wherein said growth factor is selected from the group consisting of: fibroblast growth factors, including fibroblast growth factor-l, fibroblast growth factor-2 and fibroblast growth factor-4; platelet-derived growth factor; insulin-binding growth factors, including insulin-bindinggrowth factor-l and insulin-binding growth factor-2; epidermal growth factor;
transforming growth factors, including transforming growth factor-a and transforming growth factor-b; cartilage-including factors, including cartilage-inducing factor-A and cartilage-inducing factor-B; osteoid-inducing factor; osteogenin and other bone growth factors; bone morphogenetic growth factors; collagen growth factor; heparin-binding growth factors, including heparin-binding growth factor-1 and heparin-binding growth factor-2; cytokines; interferons; hormones and biologically active derivatives of said growth factors.

22. The fibrin sealant bandage of claim 21, wherein said component layer further comprises at least one compound selected from the group consisting of aneffective amount of one or more inhibiting compounds, one or more potentiating compounds, and biologically compatible derivatives thereof, wherein said inhibiting compounds inhibit biochemical activities of factors interfering with a biological function of said growth factor, while said potentiating compounds potentiate and/or mediate biological activity of said growth factor.

23. The supplemented tissue sealant composition of claim 22, wherein said regulatory compound potentiates and/or mediates the biological activity of said growth factor, while also inhibiting the biological activity of factors interfering with the activity of said growth factor.

24. The fibrin sealant bandage of claims 20 or 22, wherein said matrix further comprises at least one antibody and/or antimicrobial composition.

25. The fibrin sealant bandage of claim 20 or 22, wherein said matrix further comprises at least one cytotoxin or cell proliferation inhibitor composition.

26. The fibrin sealant bandage of claim 18, wherein said matrix comprises at least one cytotoxin or cell proliferation inhibitor composition.

27. The fibrin sealant bandage of claim 26, wherein said cytotoxic or cell proliferation inhibiting composition comprises at least one composition used in chemotherapy selected from the group consisting of alkylating agents, enzyme inhibitors, proliferation inhibitors lytic agents, DNA synthesis inhibitros, membrane permeability modifiers, DNA intercalators, metabolites, mustard derivatives, protein production inhibitors, ribosome inhibitors, inducers of apoptosis, and neurotoxins.

28. The fibrin sealant bandage of claim 26, wherein said cytotoxic or cell proliferation inhibiting composition comprises at least one drug selected from the group consisting of 5-fluorouracil, actinomycin D, adriamycin, azaribine, bleomycin, busulfan, carmustine, chlorambucil, cisplatin, cytarabine, cytarabine, dacarbazine, estrogen, hormone analogs, insulins, hydoxyurea, L-asparaginase, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin C, prednisilone, prednisone, procarbazine, steroids, streptozotocin, testosterone, thioguanine, thiotepa, vinblastine, vincristine, taxol, taxotere, gentamycin, carboplatin, cyclophosphamide, ifosphamide, maphosphamide, ricin, diptheria toxoid, venoms and functionally equivalent analogs thereof.

29. The fibrin sealant bandage of claim 26, wherein said matrix further comprises at least one antibody and/or antimicrobial composition.

30. The fibrin sealant bandage of claim 18, wherein said compound is released long term.

31. The fibrin sealant bandage of claim 30, wherein said compound is in solid form.

32. The fibrin sealant bandage of claim 31, wherein upon application said compound is introduced into said matrix in solution in a carrier, said carrier having a higher rate of dissolution than said composition contained therein, so that the composition is deposited within the matrix as a solid precipitate.

33. The fibrin sealant bandage of claim 30, wherein said compound interacts with said fibrin matrix.

34. The fibrin sealant bandage of claim 30, wherein said compound is of sufficiently low solubility to permit localized, sustained-release from said fibrin matrix.

35. The fibrin sealant bandage of claim 30, wherein the mass of said compound exceeds an amount which is soluble in the volume of said fibrin matrix,thereby permitting localized, sustained-release from said fibrin matrix.

36. The fibrin sealant bandage of claim 35, wherein upon application said compound is introduced into said matrix as an emulsion.

37. The fibrin sealant bandage of claim 1, wherein said matrix further comprises at least one component selected from the group consisting of:
demineralized bone matrix, including human demineralized bone matrix; bone morphogenetic proteins 1 to 8; and biologically compatible derivatives of said components.

38. The fibrin sealant bandage of claim 1, wherein said component layer further comprises at least one component selected from the group consisting of fibrin, collagen, gelatin, chitin, chitosan and derivatives thereof.

39. The fibrin sealant bandage of claim 1, wherein the backing comprises either resorbable or non-resorbable material.

40. The fibrin sealant bandage of claim 36, wherein the resorbable backing is selected from the group consisting of: fibrin, collagen, gelatin, chitin, chitosan and derivatives thereof.

41. The method of treating wounded tissue in a patient by applying the fibrin sealant bandage of claims 1-5 to said wounded tissue.

42. The method of treating wounded tissue in a patient by applying the fibrin sealant bandage of claims 7-11 to said wounded tissue.

43. The method of treating wounded tissue in a patient using the fibrin sealant bandage of claim 12, said method comprising removing the film and applying the fibrin sealant bandage to the wounded tissue.

44. The method of treating wounded tissue in a patient by applying the fibrin sealant bandage of claims 13-18 to said wounded tissue.

45. The method of treating wounded tissue in a patient by applying the fibrin sealant bandage of claims 30-38 to said wounded tissue.

46. A method of preparing a fibrin sealant bandage comprising layering (i) an occlusive backing, and (ii) a component layer comprising fibrinogen in anamount which is capable of forming a fibrin matrix in the presence of Factor XIII, thrombin and Ca++.

47. The method of preparing the fibrin sealant bandage of claim 46, further comprising a physiologically-acceptable adhesive layer.

48. The method of preparing the fibrin sealant bandage of claim 46, wherein at least one component of the component layer of the bandage is dry.

49. The method of preparing the fibrin sealant bandage of claim 46, wherein at least one component of the component layer of the bandage is a gel.

50. The method of preparing the fibrin sealant bandage of claim 46, further comprising a removable, waterproof, protective film over said component layer and exposed surface of said adhesive; wherein said film is removed prior to application of said bandage.

51. The supplemented fibrin sealant matrix which is formed upon application of the fibrin sealant bandage of claim 18.

52. A fibrin sealant dressing for treating wounded tissue in a patient, which is applied as an expendable foam comprising fibrinogen in an amount which is capable of forming a fibrin matrix in the presence of Factor XIII, thrombin and Ca++.

53. The fibrin sealant dressing of claim 52, wherein said dressing further comprises at least one component selected from the group consisting of Factor XIII, thrombin and Ca++.

54. The fibrin sealant dressing of claim 52, wherein said dressing further comprises at least two components selected from the group consisting of Factor XIII, thrombin and Ca++.

55. The fibrin sealant dressing of claim 52, wherein said dressing further comprises Factor XIII, thrombin and Ca++.

56. The fibrin sealant dressing of claim 52, wherein said dressing further comprises at least one compound selected from the group consisting of the following supplements: analgesics, antimicrobial compositions, antibodies, anticoagulants,antiinflammatory compounds, antiproliferatives, cytokines, cytotoxins, chemotherapeutic drugs, growth factors, hormones, interferons, lipids, oligonucleotides, osteoinducers, polymers, polysaccharides, proteoglycans, polypeptides, protease inhibitors, steroids, vasoconstrictors, vasodilators, vitamins, minerals and stabilizers.

57. The fibrin sealant dressing of claim 56, wherein said matrix comprises at least one antibody and/or at least one antimicrobial composition.

58. The fibrin sealant dressing of claim 56, wherein said matrix comprises at least one growth factor.

59. The fibrin sealant dressing of claim 58, wherein said growth factor is selected from the group consisting of: fibroblast growth factor-1, fibroblast growth factor-2 and fibroblast growth factor-4; platelet-derived growth factor; insulin-binding growth factor-1; insulin-binding growth factor-2; epidermal growth factor;
transforming growth factor-.alpha.; transforming growth factor-.beta.; cartilage-inducing factors -A and -B; osteoid-inducing factor; osteogenin and other bone growth factors;
bone morphogenetic growth factors; collagen growth factor; heparin-binding growth factor-1; heparin-binding growth factor-2; cytokines; interferons; hormones and biologically active derivatives of said growth factors.

60. The fibrin sealant dressing of claim 58, wherein said matrix further comprises at least one compound selected from the group consisting of an effective amount of one or more inhibiting compounds, one or more potentiating compounds, and biologically compatible derivatives thereof, wherein inhibiting compounds inhibit biochemical activities of factors that interfere with a biological function of said growth factor, while potentiating compounds potentiate and/or mediate a biological activity of said growth factor.

61. The supplemented tissue sealant composition of claim 60, wherein said regulatory compound potentiates and/or mediates the biological activity of said growth factor, while also inhibiting the biological activity of factors interfering with the activity of said growth factor.

62. The fibrin sealant dressing of claims 58 or 60, wherein said matrix further comprises at least one antibody and/or antimicrobial composition.

63. The fibrin sealant dressing of claims 58 or 60, wherein said matrix further comprises at least one cytotoxic or cell proliferation inhibitor composition.

64. The fibrin sealant dressing of claim 56, wherein said matrix comprises at least one cytotoxic or cell proliferation inhibitor composition.

65. The fibrin sealant dressing of claim 64, wherein said cytotoxic or cell proliferation inhibiting composition comprises at least one composition used in chemotherapy selected from the group consisting of alkylating agents, enzyme inhibitors, proliferation inhibitors, lytic agents, DNA synthesis inhibitors, membrane permeability modifiers, DNA intercalators, metabolites, mustard derivatives, protein production inhibitors, ribosome inhibitors, inducers of apoptosis, and neurotoxins.

66. The fibrin sealant dressing of claim 64, wherein said cytotoxic or cell proliferation inhibiting composition comprises at least one drug selected from the group consisting of 5-fluorouracil, actinomycin D, adriamycin, azaribine, bleomycin, busulfan, carmustine chlorambucil, cisplatin, cytarabine, cytarabine, dacarbazine, estrogen, hormone analogs, insulins, hydoxyurea, L-asparaginase, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin C, prednisilone, prednisone, procarbazine, steroids, streptozotocin, testosterone, thioguanine, thiotepa, vinblastine, vincristine, taxol, taxotere, gentamycin, carboplatin, cyclophosphamide, ifosphamide, maphosphamide, ricin, diphtheria toxoid, venoms and functionally equivalent analogs thereof.

67. The fibrin sealant dressing of claim 64, wherein said matrix further comprises at least one antibody and/or antimicrobial composition.

68. The fibrin sealant dressing of claim 56, wherein said compound is released long term.

69. The fibrin sealant dressing of claim 68, wherein said compound interacts with said fibrin matrix.

70. The fibrin sealant dressing of claim 68, wherein said compound is of sufficiently low solubility to permit localized, sustained-release from said fibrin matrix.

71. The fibrin sealant dressing of claim 68, wherein said compound is in solid form.

72. The fibrin sealant dressing of claim 71, said compound is introduced into said matrix in solution in a carrier, said carrier having a higher rate of dissolution than said composition contained therein, so that the composition is deposited within the matrix as a solid precipitate.

73. The fibrin sealant dressing of claim 68, wherein the mass of said compound exceeds an amount which is soluble in the volume of said fibrin matrix,thereby permitting localized, sustained-release from said fibrin matrix.

74. The fibrin sealant dressing of claim 73, wherein upon application said compound is introduced into said matrix as an emulsion.

75. The fibrin sealant dressing of claim 52, wherein said matrix further comprises at least one component selected from the group consisting of:

demineralized bone matrix, including, human demineralized bone matrix; bone morphogenetic proteins 1 to 8; and biologically compatible derivatives of said components.

76. The fibrin sealant dressing of claim 52, wherein said matrix further comprises at least one component selected from the group consisting of: fibrin, collagen, chitin, chitosan and derivatives thereof.

77. The fibrin sealant dressing of claim 52, further comprising a chemical foaming agent.

78. The method of preparing the fibrin sealant dressing of claim 52, wherein said components are stored within one or more compartments of a foam-forming device.

79. The method of preparing the fibrin sealant dressing of claim 52, wherein at least one component is dry.

80. The method of preparing the fibrin sealant dressing of claim 79, wherein at least one dry component is supplemented with at least one material which produces gas upon hydration.

81. The method of preparing the fibrin sealant dressing of claim 79, wherein upon hydration of the component(s), gas is produced.

82. The method of preparing the fibrin sealant dressing of claim 79, wherein at least one dry component is supplemented with at least one material which produces gas sufficient to produce a foam upon hydration.

83. The method of preparing the fibrin sealant dressing of claim 79, further comprising a hydrating agent.

84. The method of preparing the fibrin sealant dressing of claim 82, wherein said hydrating agent is supersaturated with gas, which upon activation or release causes the fibrin-forming components to foam.

85. The method of preparing the fibrin sealant dressing of claim 52, wherein at least one component is a gel.

86. The method of preparing the fibrin sealant dressing of claim 52, wherein a propellant causes the fibrin-forming components to foam upon activation or release.

87. The method of preparing the fibrin sealant dressing of claim 52, wherein at least one matrix-forming material is stored within said foam-forming device in hydrated form.

88. The method of preparing the fibrin sealant dressing of claim 52, wherein said components are extruded from a foam-forming device by gas pressure exceeding that of the environment.

89. The method of treating wounded tissue in a patient by applying to said wound the fibrin sealant dressing of claims 52-56.

90. The method of treating wounded tissue in a patient by applying to said wound the fibrin sealant dressing of claims 68-76.

91. A fibrin sealant dressing for treating wounded tissue in a patient, which is applied dry, said dressing comprising fibrinogen in an amount which is capable of forming a fibrin matrix in the presence of Factor XIII, thrombin and Ca++.

92. The dressing of claim 91 wherein said dressing comprises a dry, sprayable powder.

93. The method of treating wounded tissue in a patient, wherein said dry dressing of claim 91 is hydrated upon delivery by a physiologically acceptable hydrating agent.

94. The method of treating wounded tissue in a patient, wherein said hydrating agent of the fibrin sealant dressing of claim 93 produces an effectiveamount of gas to produce a foam.

95. The method of preparing the fibrin sealant dressing of claim 91-94, wherein said components are extruded from a delivery device by gas pressure exceeding that of the environment.

96. The fibrin matrix which is formed upon application of the fibrin sealant dressing of claim 56.