WO1999042084A1 - Galactosaminoglycan-based extracellular matrix for wound healing - Google Patents

Abstract

The invention provides an extracellular matrix for enhancing wound healing. The extracellular matrix comprises a galactosaminoglycan, fibronectin, and a backbone matrix. The extracellular matrix facilitates wound healing by providing hemostasis and, in addition, an environment that intrinsically recruits new tissue cells to the wound site. The extracellular matrix according to the subject invention is thus used in a method for enhancing wound healing. The method comprises applying the extracellular matrix to the wound.

Description

GALACTOSA INOGLYCAN-BASED EXTRACELLULAR MATRIX FOR WOUND HEALING

The subject matter of this application was made with support from the United States Government under National Institutes of Health Grant No. AG 101143-12.

FIELD OF THE INVENTION

The subject invention is directed to an extracellular matrix for wound healing and to a method of enhancing wound healing using the extracellular matrix.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application. It is estimated that in 1992 (US), 35.2 million wounds required major therapeutic intervention (Medical Data International, Inc. 1993) . Surgical incisional wounds are performed with aseptic technique, and are closed by primary intention. Most repair and heal uneventfully. Many traumatic wounds and cancer extirpations, however, must be left open to heal by secondary intention. Furthermore, chronic wounds have significant tissue necrosis and fail to heal by secondary intention. It is estimated that 5.5 million people in the US have chronic, nonhealing wounds and that their prevalence is increasing secondary to the increase in age-related diseases, the increase in Acquired-immune Deficiency Syndrome (AIDS) , and the increase of radiation wounds secondary to cancer intervention. In the US approximately 1.5-2.5 million people have venous leg ulcers; 300,000-500,000, diabetic ulcers; and 2.5-3.5 million, pressure ulcers (Callam et al . 1987; Phillips - 2 -

and Dover 1991; Lees and Lambert 1992; Lindholm et al . 1992) . These acute and chronic open wounds require long- term care and procedures that include skin grafting and tissue flaps, debridement, frequent dressing changes and administration of pain medications. This care is costly and labor intensive. Furthermore, these wounds have a severe impact on the patients' quality of life. The chronic dermal ulcerations can cost as much as $40,000 each to heal and more disappointing is that 50% reappear within 18 months of healing. Chronic dermal ulcers are also associated with mortality. As many as 21% of patients in intermediate-care facilities with pressure ulcers die (Bergstrom et al . 1994) .

Although multiple millions of dollars have been spent on the development of numerous recombinant growth factors (Abraham and Klagsbrun 1996; Heldin and Westermark 1996; Nanney and King 1996; Roberts and Sporn 1996) and organotypic skin replacements (Boyce et al . 1995) for use in open wounds over the past decade, the evidence of cost-effective benefit is meager thus far (Brown et al . 1989; Robson et al . 1992a; Robson et al . 1992b; Phillips et al . 1993).

Many attempts have been made to produce a composition which can be used to facilitate wound repair. Many of these compositions involve collagen as a component. U.S. Patent Nos . 4,950,483 and 5,024,841 each discuss the usefulness of collagen implants as wound healing matrices. U.S. Patent No. 4,453,939 discusses a wound healing composition of collagen with a fibrinogen component and a thrombin component, and optionally fibronectin. U.S. Patent No. 4,970,298 discusses the usefulness of a biodegradable collagen matrix (of collagen, hyaluronic acid, and fibronectin) for wound healing. Yamada et al . (1995) disclose an allogeneic - 3 -

cultured dermal substitute that is prepared by plating fibroblasts onto a spongy collagen matrix and then culturing for 7 to 10 days. Devries et al . (1995) disclose a collagen/alpha-elastin hydrolysate matrix that can be seeded with a stromal-vascular-fraction of adipose tissue. Lamme et al . (1996) disclose a dermal matrix substitute of collagen coated with elastin hydrolysate. U.S. Patent No. 5,489,304 and Ellis and Yannas (1996) each disclose a collagen-glycosaminoglycan matrix. There are also numerous compositions which involve hyaluronic acid (HA) as a component. Ortonne (1996), Borgognoni et al . (1996), and Nakamura et al . (1997) each discuss the usefulness of HA for wound healing. In Nakamura et al . (1997), the HA was combined with chondroitin sulfate in one series of experiments. In U.S. Patent No. 5,604,200, medical grade HA and tissue culture grade plasma fibronectin were used in combination with calcium, phosphate, uric acid, urea, sodium, potassium, chloride and magnesium to create a moist healing environment that simulates the fetal in utero wound healing matrix. U.S. Patent No. 5,631,011 discloses a composition of HA and fibrin or fibrinogen. Various other compositions have also been explored for their wound healing capabilities. Kratz et al . (1997) used a gel of heparin ionically linked to chitosan. Bartold and Raben (1996) studied platelet- derived growth factor (PDGF) . Henke et al . (1996) disclosed that chondroitin sulfate proteoglycan mediated cell migration on fibrinogen and invasion into a fibrin matrix, while Nakamura et al . (1997) concluded that chondroitin sulfate did not affect wound closure in a corneal epithelial wound. Henke et al . (1996) also disclosed that an anti-CD44 antibody blocked endothelial cell migration on fibrinogen. U.S. Patent No. 5,641,483 discloses topical gel and cream formulations containing human plasma fibronectin for healing of cutaneous wounds. Schultz et al . (1992) disclose a composition of epidermal growth factor (EGF) , fibronectin, a synthetic collagenase inhibitor, and Aprotinin.

Various studies involving fibronectin (FN) and/or particular fibronectin peptides and wound healing have also been reported. Many of these studies involve the RGD sequence, part of the cell binding domain of FN (see Schor et al . 1996; Steed et al . 1995; Sponsel et al . 1994; Kartha and Toback 1992; Kishida et al . 1992). Schor et al . (1996) disclose that only the gelatin binding domain of FN (GBD) stimulates fibroblast migration into a 3-D matrix of native type I collagen fibrils at femtomolar concentrations; whereas peptides of the other FN functional domains do not stimulate fibroblast migration in this assay at femtomolar to nanomolar concentrations. Schor et al . (1996) also disclose that the RGDS-containing cell binding domain of FN does, however, stimulate fibroblast migration in the transmembrane (or "Boyden chamber") assay. Steed et al . (1995) disclose that the RGD peptide matrix (known as Argidene Gel™ or as Telio-Derm Gel™) promoted wound healing. On the contrary, Sponsel et al . (1994) disclose that an RGD peptide impaired healing of a mechanical wound made in a confluent monolayer of one epithelial cell line. Kartha and Toback (1992) also concluded that an RGDS peptide completely inhibited cell migration into a wound area. Kishida et al . (1992), however, disclose that an RGD-albumin conjugate adsorbed onto a polyurethane sponge exhibited tissue ingrowth-promoting activity.

Other portions of FN have also been studied for wound healing activity. U.S. Patent No. 5,198,423 studied the effects of a polypeptide containing a cell binding domain and a heparin binding domain of FN on wound healing. U.S. Patent No. 4,589,881 studied the effects of a 108 aa polypeptide fragment of FN on wound healing, as well as a biologically active fragment thereof. Sponsel et al . (1994) studied the effect of the tetrapeptide REDV and the peptide LDVPS on wound healing.

The severity of the problem of chronic, nonhealing wounds dictates that continual efforts be made to define new and more effective matrices and methods for facilitating wound healing.

SUMMARY OF THE INVENTION

This need is met by the subject invention which provides an extracellular matrix for enhancing wound healing. The extracellular matrix comprises a galactosaminoglycan, fibronectin, and a backbone matrix. The extracellular matrix facilitates wound healing by providing an environment that intrinsically recruits new tissue cells to the wound site.

The extracellular matrix according to the subject invention is thus used in a method for enhancing wound healing. The method comprises applying the extracellular matrix to the wound.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features and advantages of this invention will be evident from the following detailed description of preferred embodiments when read in conjunction with the accompanying drawings in which:

Fig. 1 illustrates the in vitro model for assaying cell transmigration from a collagen gel into a fibrin gel; Fig. 2 illustrates the effect on cell migration of β-xyloside and -xyloside;

Fig. 3 illustrates the effect on cell migration of chondroitinase ABC and chondroitinase AC; Fig. 4 illustrates the effect on cell migration of increasing concentrations of chondroitin-4 -sulfate;

Fig. 5 illustrates the effect on cell migration of increasing concentrations of dermatan sulfate;

Fig. 6 illustrates the effect on cell migration of increasing concentrations of chondroitin-6-sulfate ; and

Fig. 7 illustrates the effect on cell migration when fibrinogen with or without fibronectin is added to varying concentrations of dermatan sulfate.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides an extracellular matrix for wound healing comprising a galactosaminoglycan, fibronectin, and a backbone matrix. As used herein, an "extracellular matrix" refers to a scaffold in the cell's external environment with which the cells may interact via specific cell surface receptors. As further used herein, a "wound" is intended to include both acute and chronic dermal wounds including, for example, surgical incisional wounds, traumatic wounds, cancer extirpations, radiation wounds, venous leg ulcers, diabetic ulcers, and pressure ulcers.

The extracellular matrix according to the subject invention comprises a galactosaminoglycan, fibronectin, and a backbone matrix. These components are necessary for the subject extracellular matrix to enhance (e.g. improve, increase) wound repair, although additional components may also be included in the extracellular matrix. These additional components, such as platelet - derived growth factor as discussed below, may further enhance the beneficial effects of the extracellular matrix on wound healing.

Enhancement (e.g. improvement, increasing) of wound healing refers to the traditional sense of wound healing where clean closure of the wound occurs. Since naturally occurring wound healing involves the movement of fibroblasts into the wound site, enhancement of wound healing can be assayed in vitro using the model for cell transmigration provided in copending, co-assigned U.S. Serial No. 08/723,789, filed September 30, 1996 (the contents of which are incorporated by reference herein) . Briefly, the model provides a contracted collagen gel containing fibroblasts surrounded by a fibrin gel (see Fig. 1) . When the extracellular matrix of the subject invention replaces or is added to the fibrin gel, fibroblast movement from the collagen gel into the extracellular matrix or modified fibrin gel is enhanced compared to movement into the "gold standard" fibrin gel. The extracellular matrix of the subject invention comprises a galactosaminoglycan, fibronectin, and a backbone matrix. Galactosaminoglycans are commercially available for use in the subject invention. Galactosaminoglycans include chondroitin-4- sulfate, chondroitin-6-sulfate, and dermatan sulfate. As used herein, "galactosaminoglycan" is intended to include the various forms of galactosaminoglycans known in the art . These various forms include galactosaminoglycans chemically modified (such as by cross-linking) to vary their resorbtion capacity and/or their ability to be degraded. Optimal galactosaminoglycan formulations will be resorbable in a few days to a week.

Fibronectin is also commercially available as a dry (for example, lyophilized) powder, and can be reconstituted to a fibronectin solution (in accordance with manufacturer's suggestions) for use in the subject invention. Preferably, the stock fibronectin solution is prepared with one milligram of dry fibronectin per milliliter of fibronectin reconstituting solution (such as, for example, sterile distilled water) . The final concentration of fibronectin in the matrix is preferably about 10 micrograms to about 100 micrograms of fibronectin per milliliter of matrix. More preferably, the final concentration is about 30 micrograms of fibronectin per milliliter of matrix.

As used herein, a "backbone matrix" refers to natural extracellular matrices as well as biocompatible synthetic polymers. These backbone matrices provide the scaffold of the extracellular matrix and when the galactosaminoglycan and fibronectin are mixed with the backbone matrix, cells can move around on the scaffold.

There are numerous examples of backbone matrices suitable for use in the subject invention. These examples include fibrin, hyaluronic acid, polyethylene glycol , poly-L-glycol , and poly-L-lactate . Presently preferred backbone matrices include fibrin and hyaluronic acid. Fibrin is provided, preferably, at about 300 μg to about 300 mg, more preferably at 300 μg to 3 mg . The optimal fibrin: fibronectin molar ratio is 1:10. Therefore, if the fibrin is provided as 300 μg, the fibronectin is provided as 30 μg . Hyaluronic acid is another suitable backbone matrix, and is commercially available as a dry (for example, lyophilized) powder. The dry powder can be reconstituted to a hyaluronic acid gel (in accordance with manufacturer's suggestions) for use in the subject invention. Depending upon the viscosity desired, a hyaluronic acid gel having about 5 milligrams to about 50 milligrams of hyaluronic acid per milliliter of reconstituting solution can be used. At 5 - 9 -

milligrams/milliliter, the hyaluronic acid gel will be more liquid, and at 50 milligrams/milliliter the hyaluronic acid gel will become more viscous and less easy to manipulate. Preferably, the hyaluronic acid gel is provided as a gel having about 20 milligrams of dry hyaluronic acid per milliliter of reconstituting solution. Suitable reconstituting solutions include, for example, sterile distilled water, sterile phosphate buffered saline (PBS) , or a cell culture medium.

As used herein, "hyaluronic acid" is intended to include the various forms of hyaluronic acid (HA) known in the art . These various forms include HA chemically modified (such as by cross-linking) to vary its resorbtion capacity and/or its ability to be degraded. Optimal HA formulations will be resorbable in a few days to a week.

In a further embodiment of the subject invention, the extracellular matrix further includes platelet- derived growth factor (PDGF) . The PDGF may be provided at a final concentration of about 1 nanogram to about 100 nanograms of PDGF per milliliter of matrix; more preferably, at a final concentration of about 30 nanograms of PDGF per milliliter of matrix. The invention further provides a method of enhancing wound healing. The method comprises applying the extracellular matrix (as described above) to a wound. As discussed above, the method of applying the extracellular matrix to the wound may vary depending on the type and location of the wound as well as the viscosity of the extracellular matrix. If the extracellular matrix can be "poured" into and contained in a wound area, then a more liquid form of the matrix will be satisfactory. If the extracellular matrix is "spread" over and/or into a wound - 10 -

area, then a more viscous form of the matrix will be desirable. In either case, a dressing of some form will often cover the applied extracellular matrix to help prevent contamination and infection of the wound. It should be readily apparent that the extracellular matrix itself (and each of its components) must be sterile (free of biological and/or chemical contamination) to also prevent contamination and infection of the wound. Preferably, the extracellular matrix is viscous enough to be "spread" over the wound and will not run off after application.

MATERIALS AND METHODS

Normal human dermal fibroblasts Primary cultures of human adult dermal fibroblasts, acquired from Marcia Simon (Living Skin Bank, SUNY at Stony Brook) , the ATCC (Bethesda, MD) , or the NIA (Bethesda, MD) , are cultured in Dulbecco ' s modified Eagle's medium (DMEM, Life Technologies) containing 42 mM sodium bicarbonate and supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS, HyClone, Logan, Utah) , at 37°C and 5% C0₂/95% air in a humidified atmosphere. The cells are used between passages 4 and 12.

Fibroblast migration assays: transmigration from orqanotypic collagen gel constructs into fibrin/fibronectin gels or outmigration over protein coated surfaces Preparation of floating, contracted collagen gels

Fibroblast cultures at 80% confluence are harvested by treatment with 0.05% trypsin/0.01% EDTA. Trypsin is inactivated by addition of soy bean trypsin inhibitor in PBS containing 0.2% BSA. The cells are washed twice with - 11 -

DMEM + 2% BSA and resuspended at a concentration of 1 x 10⁶ cells/ml . The fibroblasts are mixed with neutralized collagen (Vitrogen 100, Celtrix Labs., Santa Clara, CA) , 2% BSA, 30 ng/ml PDGF-BB, 30 μg/ml fibronectin, and concentrated DMEM so that the final concentration of DMEM and sodium bicarbonate is lx. 600 μl of the cell mixture is added to the wells of a 24-well tissue culture plate, which has been precoated with 2% BSA. The collagen is allowed to polymerize at 37°C. The final concentration of collagen is 1.8 mg/ml and each gel contains 6 x 10⁴ cells. After two hours incubation, the gels are gently detached from the plastic surface to allow contraction with the addition of 0.5 ml DMEM + 2% BSA and 30 ng/ml PDGF-BB per well. The gels are incubated overnight at 37°C in 100% humidity, 5% C0₂ and 95% air.

Preparation of three-dimensional transmigration model

For preparation of "gold standard" transmigration assays containing a dermal organotypic construct surrounded by a fibrin clot as previously described (Greiling and Clark 1997) , dried fibrin fibril-coated dishes are washed once with PBS and fibroblast-contracted collagen gels are placed on the surface. Fibrinogen, at a final concentration of 300 μg/ml, is mixed with DMEM and 1.0 U/ml thrombin, added to the wells so that the solution is level with the top of the collagen gel, and allowed to clot at room temperature for 30 min. When needed, other supplements such as 30 ng/ml PDGF-BB are added to the mixture. For galactosaminoglycan (GAG) 3- dimensional transmigration, wells are coated overnight at 37°C with fibrin fibrils as above. The next day a fibroblast-contracted collagen gel is placed on the well and the GAG plus fibrinogen, fibronectin and thrombin in DMEM, with or without 30 ng/ml PDGF-BB, is added so that the solution is level with the top of the collagen gels. - 12 -

All migration assays are quantified after a 24 hour incubation at 37°C in 100% humidity, 5% C0₂ and 95% air. Evaluation of cell migration

The number of migrated cells was quantified under a Nikon inverted phase microscope by visually counting identifiable cell nuclei located outside of the contracted collagen gel in the fibrin gel (transmigration assay) . Within a given experiment each condition was run in triplicate and means ± SD calculated. All experiments were repeated at least three times. Statistical differences among conditions can be determined by ANOVA.

EXAMPLE I Assay of Wound Healing The extracellular matrix of the present invention was tested by use of the in vitro model as described in U.S. Patent Application Serial No. 08/723,789, which is hereby incorporated by reference. The basis of the in vitro model is a contracted collagen gel containing fibroblasts which acquire a tissue-like phenotype within the collagen matrix. Surrounding the collagen gel, or dermal equivalent, with a fibrin clot produces a simple inside-outside model of the early cutaneous wound (Fig. 1) . Without an added stimulus, no more than a few of the normal adult human dermal fibroblasts within the collagen gel would migrate into the fibrin gel. However, the transmigration of fibroblasts from the collagen gel into the fibrin gel is enhanced by the replacement of the fibrin gel with the extracellular matrix of the subject invention or by the addition of the extracellular matrix to the fibrin gel, since the extracellular matrix facilitates cell movement thereby enhancing wound healing. - 13 -

EXAMPLE II

Using the 3 -dimensional transmigration assay described above, the matrix of the subject invention was modeled. These experiments led to the conclusion that galactosaminoglycan can enhance human dermal fibroblast movement from a collagen gel into a fibrin/fibronectin gel. Initially, experiments were run to determine whether chondroitin sulfate proteoglycans were required for fibroblast transmigration. Preincubation of fibroblasts with β-xyloside, an inhibitor of chondroitin sulfate addition to proteoglycan core proteins, abrogates the ability of the cells to transmigrate; while α- xyloside, an inactive epimer, had no effect (Fig. 2) . Treatment of cells with chondroitinase ABC, a mixture of endoglycosidases that remove all galactosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate) from their proteoglycan core proteins also abrogates cell transmigration; while chondroitinase AC, which removes chondroitin-4 -sulfate and chondroitin- 6-sulfate but not dermatan sulfate, did not abrogate cell movement (Fig. 3) .

In the next experiments, the effect of various galactosaminoglycans (GAGs) on cell migration into fibrin/FN gels was determined. The galactosaminoglycans chondroitin-4 -sulfate (Fig. 4), dermatan sulfate (Fig.

5), and chondroitin-6-sulfate (Fig. 6) each enhanced cell movement at some concentration. Dermatan sulfate (Fig. 5) was especially effective at enhancement of cell movement . The next experiments examined whether fibronectin in fibrin composites containing dermatan sulfate was necessary for cell movement. Therefore, dermatan sulfate was added to fibrin clots in the absence or presence of fibronectin (Fig. 7). Clearly the optimal fibrin - 14 -

composite for cell migration contains both fibronectin and dermatan sulfate.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

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REFERENCES

Abraham, J.A., and Klagsbrun, M., in "The Molecular and Cellular Biology of Wound Repair", 2d edition, Clark, R.A.F., ed, Plenum Press, New York, NY (1996) .

Bartold, P.M., and Raben, A., J Periodontal Research 31 (3) :205-216 (1996) .

Bergstrom, N. , et al . , "Treatment of Pressure Ulcers", U.S. Department of Health and Human Services, Clinical Practice Guideline, Vol. 15, Rockville, Maryland (1994).

Borgognoni, L., et al . , Euro J Dermatology 6(2) :127-131 (1996) .

Boyce, S.T., et al . , Ann Surg 222:743-752 (1995) .

Brown, G.L., et al . , N Eng J Med 321 : 76-79 (1989) . Callam, M.J., et al . , Br med J 294:1389-1391 (1987) .

Devries, H.J.C., et al . , Laboratory Investigation 73 (4) :532-540 (1995) . Ellis, D.L., and Yannas , I. v., Biomaterials 17 (3) : 291-299 (1996) .

Greiling, D., and Clark, R.A.F., J Cell Sci 110:861-870 (1997) .

Heldin, C.-H., and Westermark, B., in "The Molecular and Cellular Biology of Wound Repair", 2d edition, Clark, R.A.F., ed, Plenum Press, New York, NY, pp 249-274 (1996) .

Henke, C.A., et al . , J Clin Investigation 97(11) :2541- 2552 (1996) .

Kartha, S., and Toback, F.G., J Clinical Investigation 90 (1) :288-292 (1992) .

Kishida, A., et al . , Biomaterials 13 (13 ) : 924-930 (1992) .

Kratz, G. , et al . , Scandinavian J of Plastic and Reconstructive Surgery and Hand Surgery 31(2) :119-123 (June 1997) .

Lamme, E.N., et al . , J Histochemistry and Cytochemistry 44 (11) :1311-1322 (1996) . - 16 -

Lees, T.A., and Lambert, D., Br J Surg 79:1032-1034 (1992) .

Lindholm, C, et al . , Acta Derm Venereol (Stockh) 72:227- 230 (1992) .

Medical Data International, Inc., "Wound Card in the US: Emerging trends, management and new product development" (1993) .

Nakamura, M., et al . , Experimental Eye Research 64 (6) :1043-1050 (1997) .

Nanney, L.B., and King, L.E., in "The Molecular and Cellular Biology of Wound Repair" , 2d edition, Clark, R.A.F., ed, Plenum Press, New York, NY, pp 171-194 (1996) .

Ortonne, J.P., J Dermatological Treatment 7(2) :75-81 (1996) .

Phillips, L.G., et al . , Ann Plast Surg 31:331-334 (1993) .

Phillips, T.J., and Dover, J.S., J Am Acad Dermatol 25:965-987 (1991) .

Roberts, A.B., and Sporn, M.B., in "The Molecular and Cellular Biology of Wound Repair", 2d edition, Clark, R.A.F., ed, Plenum Press, New York, NY, pp 275-310 (1996) .

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Claims

- 17 -What Is Claimed Is:

1. An extracellular matrix for wound healing comprising a galactosaminoglycan, fibronectin, and a backbone matrix.

2. The extracellular matrix of claim 1 wherein the galactosaminoglycan is chondroitin-4-sulfate .

3. The extracellular matrix of claim 1 wherein the galactosaminoglycan is chondroitin-6-sulfate .

4. The extracellular matrix of claim 1 wherein the galactosaminoglycan is dermatan sulfate.

5. The extracellular matrix of claim 1 wherein the backbone matrix is fibrin.

6. The extracellular matrix of claim 1 wherein the backbone matrix is hyaluronic acid.

7. The extracellular matrix of claim 1 wherein the backbone matrix is polyethylene glycol .

8. The extracellular matrix of claim 1 wherein the backbone matrix is poly-L-glycol .

9. The extracellular matrix of claim 1 wherein the backbone matrix is poly-L-lactate .

10. The extracellular matrix of claim 1 further comprising platelet-derived growth factor.

11. A method of enhancing wound healing which comprises applying the extracellular matrix of claim 1 to a wound .