WO1997025995A1

WO1997025995A1 - Methods for regenerating scarless skin and compositions used therein

Info

Publication number: WO1997025995A1
Application number: PCT/US1997/000611
Authority: WO
Inventors: Stephen M. Prouty; Laura Lawrence; Kurt S. Stenn
Original assignee: Johnson & Johnson Consumer Products, Inc.
Priority date: 1996-01-18
Filing date: 1997-01-16
Publication date: 1997-07-24
Also published as: AU1829097A

Abstract

A method for regenerating skin comprising applying to a wound or burn site a mixture of epithelial and mesenchymal cells, and a composition comprised of such epithelial and mesenchymal cells.

Description

METHODS FOR REGENERATING SCARLESS SKIN AND COMPOSITIONS USED

THEREIN

CROSS-REFERENCE TO RELATED APPLICATION

This Application claims the benefit of provisional application number 60/010,183 filed on January 18, 1996, which Is incoφorated herein by reference.

BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION

The present invention relates to a novel composition and a method for its use in wound healing. More particularly, this invention relates to methods for using novel epithelial- mesenchymal compositions for healing wounds by regenerating skin.

2. BACKGROUND OF THE INVENTION

Following injury, vertebrate tissues are faced with one of two choices: to regenerate a functional organ or repair the injury by forming less functional scar tissue. The pathway taken depends on many factors, including organ type, age, maintenance of a stem cell population and/or a molecular environment conducive to regeneration. See, e.g. 4 Wound Rep. Reg. 3-15 (1996). For example, tissues in the liver, bone, muscle, and urodele limbs can regenerate whereas that in the central nervous system, lens, and cardiac muscle form scar tissue.

The mammalian skin response to wounding uniquely undergoes a transition during development. While during embryogenes the skin is capable of regeneration without scarring, such is not possible during late embryogenesis. Several factors have been correlated with this transition. See, e.g. Hopkinson-Woolley et al., 107 J. Cell Sci. 1159-1167 (1994) (development of an adult-type inflammatory response deposition); DePalma et al., 9 Matrix 224-231 (1989)(altered extracellular matrix deposition); and Lorenz et al., 96 Plast. Reconstr. Surg. 1251-1259 (1995)(intrinsic differences between early and late fetal fibroblasts).

Adult scars are generally devoid of hair follicles, which suggests that hair follicle cells either are unable to migrate or are prevented from migrating into wounds. Moreover, the total number of hair follicles possessed by an adult typically does not increase during the postnatal period, and follicular papilla fibroblasts, which are key regulators of the hair cycle, are non-mitotic. See Wessells and Roessner, 12 Dev. Biol., 419-433 (1965). However, the de novo formation of small hair follicles in the adult does occur under pathological conditions such as cancer and certain inflammatory diseases. During studies of mid-gestational embryonic rats, differentiating hair bulbs have been found in regenerating wounds. However, wounds made during late-gestation in this model resulted in scars devoid of hair follicles, which indicates a correlation between hair follicle formation within a wound and scariess skin regeneration. See lhara et al., 110 Development 671-680 (1990).

It is well known that the interactions between epithelial cells (i.e. hair follicle keratinocytes) and mesenchymal cells (i.e. dermal cells or follicular papilla cells) (hereinafter "E-M interactions') are central to many biological processes such as the development, homeostasis, and, in some cases, tumor formations of skin. Although such E-M interactions are seen to influence cutaneous structure such as epidermal character, pigmentation, and dermal vascularity, they have been most dramatically demonstrated in the biology of hair growth. See, e.g. Hardy, M. H., 8 Trends in Genetics 55 - 61 (1992)( hair follicle morphogenesis).

Various systems have been used to analyze the EM interactions occurring in skin and hair. See, e.g., Deuchar, Cellular Interactions in Animal Development 148-178 (1975); Sengel, Oroan Culture. 379-458 (1970) (heterotypic and homotypic transplants explants in embryonic adult avian and mammalian skin preparations); Oliver, 18 J. Embryol. Exp. Morphol., 43-51 , (1967); Kollar, 55 J. Invest. Dermatol., 374-378 (1970); Pisansarakit and Moore, 94 J. Embryol. Exp. Morphol., 113-119(1986)(role of follicular papillae in hair growth using organotypic approach); Rogers et al., 89 J. Invest. Dermatol., 369-379 (1987); and Hirai et al., 69 Cell 471-481 , 1992 (use of dissociated cellular preparations). Several drawbacks exist relative to the systems described above, i.e. they do not easily lend themselves to cellular or molecular analysis, nor do they form significantly developed hair follicles. One approach for analyzing E-M interactions which overcame these cellular and molecular deficiencies was presented in Weinberg, et at., 100 Jl. Invest. Dermatol. 229 - 236 (1993)("Weinberg"), which was limited to a method for combining hair bud keratinocytes and dermal cells to induce hair growth in a wound. Lichti, et al., 101 Jl. investig. Derm. 124S- 129S (1993), further extended this method using immortalized follicular papilla cells. It would be desirable to provide a combination of epithelial and mesenchymal cells which would be useful in healing skin wounds and which preferably would do so without scarring or discoloration.

SUMMARY OF THE INVENTION In accordance with this invention, there is provided a method for regenerating skin at a cutaneous wound or bum site in an animal comprising, consisting essentially of, or consisting of: applying to a wound or a bum a composition containing hair bud cells and dermal cells in an amount effective to result in skin tissue which has the appearance and histological structure similar to that of normal skin.

In accordance with another embodiment of this invention, there is provided a method for regenerating skin at a cutaneous wound or bum site in a human or animal comprising, consisting essentially of, or consisting of: applying to a wound or a bum a composition containing hair bud cells and follicular papilla ceils in an amount effective to result in skin tissue which has the appearance and histological structure similar to that of normal skin. In accordance with another embodiment of this invention, there is provided a method for regenerating skin at a cutaneous wound or bum site in a human or animal comprising, consisting essentially of, or consisting of: applying to a wound or a bum a composition containing hair bud cells, dermal cells, and fibroblast cells selected from the group consisting of follicular papilla cells, non-follicular papilla cells, and mixtures thereof in an amount effective to result in skin tissue which has the appearance and histological structure similar to that of normal skin.

In accordance with yet another embodiment of this invention, there is provided a method of preventing dermal melanocytosis during skin regeneration comprising, consisting essentially of, or consisting of: applying on a area in need of skin regeneration a composition containing hair bud cells, fresh dermal cells, and fibroblast cells selected from the group consisting of follicular papilla cells, non-follicular papilla cells, and mixtures thereof.

In accordance with yet another embodiment of this invention, there is provided a novel composition for the topical treatment of wound or bum sites in animal or human skin comprising, consisting essentially of, or consisting of: a) hair bud cells; b) dermal cells; and c) fibroblast cells selected from the group consisting of non-follicular papilla cells, follicular papilla ceils, and mixtures thereof. The compositions of this invention beneficially regenerate skin without scar formation and without dermal melanocytosis, and thus are especially suitable for use in healing skin wounds.

BRIEF DESCRIPTION OF THE DRAWINGS: The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. The invention will be more fully understood and further advantages will become apparent when reference is made to the following detailed description of the invention and the accompanying drawings in which:

FIG. 1 illustrates a healthy terminal human hair follicle, and relevant components. FIG. 2 is a representation which illustrates a sagittal section of the dorsal skin of a neonatal mouse showing the papillary dermis containing hair buds.

FIG. 3A is a color representation which illustrates a dorsal view of a wound site four weeks after the implantation of only hair buds to the wound site.

FIG. 3B is a color representation which illustrates a histological view of the wound site of FIG. 3A

FIG. 3C. is a color representation which illustrates a dorsal view of a wound site four weeks after the implantation of hair buds and fresh dermal cells.

FIG. 3D is a color representation which illustrates the histology of the wound site of FIG. 3C. FIG. 4A is a color representation which illustrates an elastin stain of a wound site implanted only with hair buds.

FIG. 4B is a color representation which illustrates an elastin stain of a wound site implanted with hair buds plus fresh dermal cells.

FIG. 4C is a color representation which illustrates an elastin stain of a wound site implanted with hair buds and ts TFP fibroblasts.

FIG. 5A is a color representation which illustrates a dorsal view of a wound site which was implanted with a mixture of ts TFP cells and hair buds.

FIG. 5B is a is a color representation which illustrates a lateral view of a wound site which was implanted with a mixture of ts TFP cells and hair buds. FIG. 5C is a color representation which illustrates the histology of the graft site of

FIG. 5A.

FIG. 6A is a color representation which illustrates a dorsal view of a wound site implanted with non-FP fibroblasts (NIH3T3) and hair buds.

FIG. 6B is a color representation which illustrates a histological view of the graft site of FIG. 6A.

FIG. 6C is a color representation which illustrates the histological view of the graft site of FIG. 6A when viewed at a magnitude 5 times that used in FIG. 6B.

FIG. 7A is a color representation which illustrates a dorsal view of a wound site implanted with hair buds, NIH3T3 fibroblasts, and fresh dermal cells. FIG. 7B is a color representation which is the histological view of the wound site of

FIG. 7A.

FIG. 7C is a color representation which illustrates the dorsal view of a wound site implanted with hair buds, NIH3T3 fibroblasts, and fresh dermal cells which were passaged about 3 times with a split of 1 :5/passage. FIG. 8 illustrates a graph of tyrosinase induction as measured by area (μM²) versus conditioned media added (ml).

DESCRIPTION OF THE PREFERRED EMBODIMENTS The epidermal component of the composition of the present invention is preferably a hair bud which may be obtained from any animal. As used herein, "animal" shall mean "any member of the kingdom that is generally characterized by the power of voluntary motion, specialized sense organs that provide rapid motor response to stimuli, limited capacity for regenerative growth, the lack of rigid cell walls, and the inability to manufacture nutrients from inorganic substances,' as defined in Academic Press Dictionary of Science and

Technology 117 (1992), and includes but is not limited to humans and mammals. By "hair bud^* it is meant the stem cell keratinocytes of hair follicles that are formed during hair follicle development and as illustrated by the arrows in FIG. 2.

The epidermis may be separated from the der is layer of isolated skin samples by methods well known in the art such as via floating the skin in an enzyme capable of proteolyzing the junction between the epidermis and the dermis. Such methods are described in, for example, Freshney, Culture of Animal Cells 310-312, (3rd Ed. 1994) ("Freshney"), which is incoφorated by reference. Examples of suitable enzymes include a dispase or a trypsin-containing solution such as a trypsin /saline solution without ethylenediaminetetraacetic acid (EDTA) as disclosed in Weinberg, which is incorporated by reference herein in its entirety. Trypsin-containing solutions are preferred.

The separated epidermides (containing hair buds) are then dispersed in a physiological fluid and chopped into pieces. Suitable physiological fluids include any liquid which can support hair bud growth and viability such as phosphate buffered saline (PBS), Eagle's Minimal Essential Media/fetal calf serum (EMEM/FCS) available from

GibcoBRL("Gibco"), or mixtures thereof, with EMEM/FCS being preferred. In a preferred embodiment, the EMES/FCS mixture contains, based upon the total volume of the EMES/FCS mixture, from about 10 percent to about 20 percent of FCS. More preferably the EMES/FCS is extracted with a divalent cation chelator such as paired iminodiacetate ions coupled to a styrene divinylbenzene support, which is available from Bio-Rad Laboratories, under the tradename, "Chelex." Suitable amounts of chelator may range from about 3 g to about 20 g, and preferably from about 4 g to about 6 g., based upon 100 ml of the total volume of physiological fluid and chelator. Although the amount of physiological fluid will depend upon the amount of epidermides, it is preferable to use a fluid:epidermis volumetric ratio of from about 1 :1 to about 5:1 , and preferably from about 1 :1 to about 3:1.

The epidermides may be chopped by any suitable means known in the art such as with multiple knifes and preferably with scissors until the resulting pieces are from about 1 mm² to about 5 mm², and preferably from about 1 mm² to about 2 mm² in area. The epidermide-liquid mixture is then filtered via any conventional filtering devices known in the art capable of removing large skin pieces from the hair buds, such as, for example, by pouring the mixture through cheesecloth or preferably through a plastic or metal filter mesh. The openings of the filter mesh are small enough to let only about a single hair bud through, and typically may range in size from about 50 μm to about 100 μm, and preferably from about 75 μm to about 125 μm.

The hair bud filtrate is then placed in a conventional centrifuge device, preferably a Beckman GS-6R tabletop centrifuge, and spun for about 2 minutes to about 5 minutes at a speed of about 200 φm to about 500 φ , and preferably about 250 φm to about 400 φ . In a preferred embodiment, the resulting, centrifuged hair buds pellets are then placed in another container and subjected to density gradient centrifugation processing ("DGC") at least once and more preferably at least twice. Details of DGC are well known in the art and described in, for example, Griffith, Techniques of Preparative. Zonal, and Continuous flow Ultracentrifuαation 3-18 (1986) which is incoφorated herein by reference. In DGC, a density gradient former such as a neutral, highly branched, hydrophilic polymer of sucrose, i.e. the compound available from Pharmacia, Inc. under the tradename, "Ficoll," is added to the container containing the hair bud pellets before centrifugation. Although the amount of gradient former used to suspend the hair buds is not critical and will depend upon the amount of hair buds and the size of the container used, it is preferable that the volumetric ratio of gradient formergradient former/hair bud suspension is from about 1 :1 to about 1 :5, and preferably 1 :1 to 1 :3. During DGC, the hair bud suspension/gradient former mixture is centrifuged for about 4 to about 6 minutes at a speed of from about 300 φm to about 500 φm.

After aspirating the upper layers of the resulting gradient in the centrifuge at the conclusion of centrifuging or DGC, the spun hair bud suspensions are preferably washed at least once, and more preferably from about 1 to about 5 times, with any of the above- mentioned physiological fluids in a volumetric ratio of hair buds:fluid of from about 1:1 to about 1 :100, and preferably from about 1 :50 to 1 :100, via conventional methods well known in the art. The physiological fluid is preferably EMEM/FCS, and more preferably EMEM/FCS supplemented with CaCI₂ in an amount to yield a final fluid concentration of 1.3 mM CaCI₂. The preferably washed, isolated hair buds are resuspended in any of the above- mentioned physiological fluids at a volumetric ratio of hair bud:fluid of from about 1 : 1 to about 1:50, preferably 1: 10 to about 1:40, and more preferably 1 :30 to about 1:35.

Although the amount of hair buds required for regenerating skin will depend upon the surface area of the wound or bum site (collectively hereinafter "wound site¹) and the total volume of the grafting chamber, one of ordinary skill in the art could readily optimize the amount of hair buds necessary without undue experimentation to achieve the desired tissue having the appearance and structure of normal skin. Typically, the preferred ratio is from about 1000 to about 4000 hair buds 300 ml fluid/ 3.5 cm² of wound site surface area. The second component in the composition may be comprised of follicular papilla cells, non-follicular papilla cells, or mixtures thereof. As used herein, "follicular papilla cells" (FP) are dermal fibroblasts obtained from any mammal that are capable of hair induction and are enclosed by hair bulb kerotinocytes as illustrated in FIG. 1 , while "non-follicular papilla cells" (non-FP) are all other dermal fibroblasts which are not FP.

FP may be dissected from the hair follicles in hair-bearing skin samples via any conventional method known in the art such as that described in Williams, et al., 130 Br. Jl. Dermatol. 290 -297 (1994)("Williams').

In a preferred embodiment, the FP are immortalized by any conventional methods known in the art such as those described, for example, Filsell, et al., 107 J. Cell Sci., 1761- 1772, 1994 (use of polyoma large T antigen) ("Filsell") and Bayley, et al., 642 Ann. N.Y. Acad. Sci., 439-441 , 1991(use of adenovirus E1A oncogene)("Bayley").

The FP cells are cultured in any suitable container possessing a suitable tissue culture medium known in the art. Examples of suitable tissue culture medium include, but are not limited to DMEM/FCS (Dulbecco's Modified Eagle Medium/fetal calf serum), which is available from Gibco, and preferably amniocytic media available from Irvine Scientific under the tradename, 'Chang's Media.' The culturing of FP cells occurs under conditions of ambient pressure and a temperature ranging from about 30° C to about 40 °C, and from about 30^* C to about 35 °C if from about 10 units to about 200 units of gamma interferon derived from any mouse per ml of tissue culture media is added thereto. As used herein,

"unit" shall mean the concentration of gamma interferon in unit/ml that inhibits proliferation of WEHI-279 mouse lymphoma cells by 50%. The FP cells are cultured under the above temperature and pressure conditions for a period of time until a confluence in the container of from about 50% to about 95%, and preferably from about 75% to about 85% is reached. Typically, the time required for such a confluence may range from about 3 to about 5 days. In a preferred embodiment, the FP are dissociated from the surface of the first culture container by methods well known in the art such as that described in Freshney, 153 - 156 (passaging via enzymatic digestion; or via agitation or via scraping). As used herein, a "passage" means the transfer of less than all of the cell-media mixture in one container to at least two other containers in order to enable the cells to maintain their logarithmic growth phase. Any enzymatic solution suitable for passaging tissue culture cells may be used, preferably trypsin or PBS, and more preferably a solution, based upon the total volume of PBS, of from about 0.5 mM to about 1.5 mM EDTA and from about 0.1% to about 0.4% trypsin in PBS. Preferably, the suspension of FP in enzymatic solution is passaged under ambient conditions at from about a 1 :3 to about a 1 : 10 split, and preferably from about a 1 :4 to about a 1 :6 split into other containers containing tissue growing media as described above. Preferably, from about 2 to about 4 passages of the initial cell-media mixture occur.

FP are harvested from the culturing containers via harvesting methods well known in the art such as that set forth in Freshney, then the harvested FP suspensions are recentrifuged for a period of from about 1 minute to about 5 minutes at a speed of from about 600 φm to about 1000 φm in a conventional centrifuge device, preferably a Beckman GS- 6R Tabletop Centrifuge. The resulting FP pellets are resuspended in the above-mentioned tissue culture medium, preferably Chang's Media, to the concentration desired. Typically, the volumetric ratio of FP:medium may range from about 1 :1 to about 1:10, and preferably from about 1 :3 to about 1 :7.

The FP in tissue culture medium solution are counted according to methods well known in the art such as those described in Tissue Dissociation Guide, 1993, pp. 9-10, Worthmgton Biochemical Corporation ("Worthington").

Non-FP are commercially available or may be obtained from any mammal via conventional methods well known in the art. Examples of suitable methods include those described in, for example, Todaro, et al., 17 Jl. Cell. Bio. 299 - 313 (1963), which is incorporated herein by reference.

Non-FP may be cultured, harvested, and counted as described above for FP, with the exception that the culturing temperature may range from about 35 "C to about 39 ^βC in the absence of gamma-interferon.

The third component in the composition of the present invention is the fresh dermal cells. As used herein, "fresh dermal cells" means the total dissociated cell preparation of the dermis layer of skin which likely includes, but is not limited to, at least one of the following cells: FP, non-FP, endothelial cells, muscle cells, adipocytes, and mixtures thereof. Fresh dermal cells may obtained from a dermis isolated according to methods known in the art such as that described in Weinberg. If a reduction in the hair-inducing ability of the dermal preparation to be grafted is desired, it is preferable to culture the fresh dermal cells according to {he culturing and passaging procedure and conditions set forth above for FP, with the exception that the preferred range for passaging the dermal cells may range from about 1:2 to about 1 :10, and more preferably from about 1 :3 to about 1:6.

Although the amount of fresh dermal cells, FP, and non-FP required for grafting may depend upon the surface area of the wound, it is preferable to use from about greater than about 0 to about 5 x 10⁷ cells, more preferably from about 4 x 10⁶ cells to about 16 x 10 ⁶ cells, and most preferably from about 7 x 10⁶ cells to about 9 x 10⁶ cells per 3.5 cm² of graft site for each respective cell-type.

Amounts of the first component and second component and/or third component within the above-prescribed ranges are combined under temperature conditions ranging from about greater than 0 *C to about 25 °C, and preferably from about 2 ^βC to about 10 'C and centrifuged for a period of from about 3 to about 10 minutes at a speed of from about 500 φm to about 1000 φm, and preferably from about 700 φm to about 900 rpm in order to further concentrate the cell mixture composition.

A conventional grafting chamber is inserted into a wound via methods well-known in the art such as those described in Strickland, Workshop on In Vivo and In Vitro Models of Cellular Transformation. (AACR Meeting, 1992). Samples of the cell mixture composition are placed into the grafting chamber by any means known in the art such as via pipeting. While the volume of the desired cell mixture depends upon the volume of the grafting chamber and the amount of desired cells depends upon the surface area of the wound, one skilled in the art can readily determine the amount of cell mixture and size of grafting chamber to be used without undue experimentation, but typically the amount of cells used may range from about 5 percent to about 95% of the volume of the grafting chamber, and typically a chamber volume of from about 50 μl to about 500 μl is suitable for a wound having a surface area of from about 1 cm² to about 5 cm².

The grafting chamber is removed from the wound after at least about 1 day, and preferably after about 1 week subsequent to the implantation of the cell mixture therein. During this time, the cell mixture is infiltrated by blood vessels and mesenchymal cells from normal tissue in the wound site of the host. Between about 1 and 2 weeks after the removal of the chamber from the wound site, a scab typically forms on the wound.

In one embodiment, when samples of fresh dermal cells and hair buds in a hair bud:dermal cell numeric ratio of about 1 :1000 to about 1 :20,000, preferably from about 1 :2000 to about 1 :10000, and more preferably from about 1 :6000 to about 1 :9000 were combined and implanted into a wound, we have not only found hair growth, but also unexpectedly discovered the presence of skin regeneration having normal pigmentation at the wound site as shown in FIG. 3C. As used herein "skin regeneration^* means the reformation of the original cellular structures present in normal, unwounded skin and without the visual appearance of a scar. As illustrated in FIG. 3D, the eumelanin is located in the hair shaft as identified by the arrowhead or in the hair bulb as identified by the arrow. Thus, in skin regeneration, the majority of the melanocytes is located properly in the hair follicle bulb.

In yet another embodiment, when samples of hair buds and FP were combined in a hair bud.FP numeric ratio of about 1 :1000 to about 1 :20,000, preferably from about 1 :2000 to about 1 :10000, and more preferably from about 1 :6000 to about 1 :9000, and implanted into a wound, we have not only found hair growth and skin regeneration at the site, but also unexpectedly discovered the presence of dermal melanocytosis, similar to human common blue nevus, which causes the skin to appear deep blue-black as shown in FIGS. 5A and B. FIG. 5C shows that the melanocytes, as identified by the arrow, and the melanophages, as identified by the arrowhead, are located in the dermis, in addition to their noπnal location in the hair bulb as shown in FIG. 3D. Similariy, when samples of hair buds and non-FP were combined in a hair bud:non-FP numeric ratio of about 1 :1000 to about 1 :20,000, preferably from about 1 :2000 to about 1 :10000, and more preferably from about 1 :6000 to about 1 :9000, and implanted into a wound, we have found the presence of dermal melanocytosis but the absence of both hair growth and skin regeneration as shown in FIGS. 6A - 6C. As used herein, 'dermal melanocytosis" shall mean the ectopic accumulation of melanocytes in the dermis. While not wishing to be bound to this theory, we believe that the FP are important to hair growth and skin regeneration, and FP and/or non-FP, in isolation from other dermal cells, appear to significantly contribute to dermal melanocytosis. This belief is further supported by FIG. 5C as well as FIGS. 6B and C which show that the melanocytes are located in the dermis, in addition to their normal location in the hair bulb as shown in FIG. 3D. These Figures suggest that the FP cells appear to contribute to the ectopic accumulation of melanocytes in the dermis which results in blue skin pigmentation. In yet another embodiment, when samples of fresh dermal cells, hair buds, and fibroblast cells selected from the group consisting of non-FP, FP, and mixtures thereof, in a volumetric ratio of from about 1 :1000:1000 to about 1 :20,000:20,000, preferably from about 1 :2000:2000 to about 1:10000:10000, and more preferably from about 1 :6000:6000 to about 1 : 9000: 9000, were combined and implanted into a wound site, we have not only found the presence of hair growth and complete skin regeneration, but also the absence of any significant visual hypeφigmentation as illustrated in FIGS. 7A and B. While not wishing to be bound by any theory, we believe that the fresh dermal cells act as a eumelanogenic inhibitor to balance the eumelanin-stimulatory effect of the FP and non-FP cells and thus contribute to the regenerated skin of normal pigmentation. This belief is further supported by the finding that conditioned media from the fresh dermal cells contains a factor that inhibits pigmentation of melanoma cells in vitro, as seen in FIG. 8.

We have further unexpectedly found that the inhibitory effect of the fresh dermal cells towards hypeφigmentation is likely due to the presence of the non-FP component contained therein since the passaging of fresh dermal cells only reduced the quantity of hair formation without significantly affecting their ability to inhibit hypeφigmentation as shown in FIG. 7C.

The invention illustratively disclosed herein suitably may be practiced in the absence of any component, ingredient, or step which is not specifically disclosed herein. Several examples are set forth below to further illustrate the nature of the invention and the manner of carrying it out. However, the invention should not be considered as being limited to the details thereof.

EXAMPLES

All tissue culture materials used in the following examples were obtained from Gibco (Gaithersburg, MD) and all chemicals were obtained from Sigma Chem. Co. (St. Louis, MO), unless otherwise specified. In the following examples, all centrifugations were performed using a Beckman GS-6R Tabletop centrifuge. The following tests are used in the Examples that follow:

1) Hematoxylin and Eosiπ fH&E) staining of histological sections: This procedure is described in Williams. Skin section samples were analyzed after staining as follows: Scars have a flat dermal-epidermal junction, no skin appendages such as hair follicles or sweat glands, and dense collagen fibers oriented parallel to the epidermis, whereas normal skin contains rete ridges at the dermal-epidermal junction, abundant appendages, and a loose, reticular organization of dermal collagen. See Martin, 32 Mechanisms of Wound Healing in the Embryo and Fetus, Current Topics in Developmental Biology 175-203, (1996); Stocum, 4 Wound Rep Reg 3-15 (1996).

2) Elastin-stainina of histological sections: This procedure is described in Kligman, 3 Am. J. Deπmatopathol. 199-200 (1981). Skin samples were analyzed after staining as follows: Elastin fibers are abundant in the papillary dermis of normal skin, however elastogenesis in cutaneous wounds is greatly retarded and has an abnormal distribution, which is typical of scarring. See Davidson, et al., Wound Healing: Biochemical and Clinical Aspects 223 - 236 (1992).

3) Assessment of common blue nevus (CBN): As used herein, "common blue nevus" (CBN) means an accumulation of dermal melanocytes containing a variable number of fibrocytes and melanophages, sometimes associated with dermal fibrosis. See Pinkus and Mehregan, Ch. 32 A Guide to Dermatohistopathology 362 (1981)("Pinkus"). Skin samples were stained in accordance with H&E staining, then analyzed for common blue nevus: 1) via the histogical assessment set forth in Pinkus, as well as 2) via visual inspection of the skin for the appearance of a deep blue color. See Mosher et al., 1(12) Dermatology in General Medicine 978 (1993).

Example 1 : Generation of Hair Buds:

Hair buds were partially purified from about 40 newborn (postnatal day 0-2) Black Swiss mice (Taconic Farms Inc., Germantown, NY) by floating their isolated skin ovemight at 4°C on 0.25% trypsin/saline without EDTA. The separated epidermides were washed briefly with media A, which was comprised of 10 ml CaCI₂-free EMEM (obtained from Biowhittaker, Inc.) , 1 ml of 10% Chelex (obtained from Bio-Rad Laboratories, lnc.)-treated fetal calf serum, 1.3 mM CaCI₂, 2 M l-glutamine, and 50 units/ml penicillin/streptomycin obtained from Biowhittaker, Inc. The washed epidermides were pooled in 40 ml of media A and chopped finely with scissors into pieces having an area of about 1 mm².

After the chopped epidenmide solution was gravity filtered through a 100 μM nylon mesh (Martin Supply Co., Baltimore, MD), and centrifuged at about 300 φm for about 3 minutes, the resulting pellet was suspended in 30 ml of media A then recentrifuged under similar conditions.

The resulting pellet was suspended in about 16 ml of a mixture of 4.5% Ficoll (Pharmacia, lnc.)/media A, 8 ml of which were introduced independently into two respective 15 ml conical tubes, each tube containing 5 ml of 9.0% Ficoll/media A. After centrifuging the resultant gradient suspensions for about 5 minutes at 400 φm in a centrifuge, the pellets from each respective tube were independently resuspended in 8 ml of 4.5% Ficoll/media A. Each suspension was then independently layered into one of two tubes containing 5 ml of 9.0% Ficoll media A, then the suspensions in each tube were independently recentrifuged under similar conditions as set forth above.

After suspending both of the resulting pellets in about 30 ml of media A, the resulting suspension was centrifuged at about 300 φm for about 3 minutes. This washing step was repeated two additional times.

After the third wash, the pellet was resuspended in a container containing 20 ml of media A so that the final volume in the container represented 2 mouse skin equivalents (i.e. about 2000 hair buds) per 1 ml of media A. As used herein, a "mouse skin equivalent" represents the total number of hair buds from the entire skin of one mouse pup. The compositions resulting from this procedure were identified as partially-purified preparation containing primarily hair bud organoids.

Example 2: Generation of Fresh Dermal Cells

Fresh dermal cells from five newborn Swiss Webster albino mice (obtained from Taconic Farms, Inc.) were derived from their whole dermis layer which was isolated from trypsin-separated skin prepared in accordance with the procedure set forth in Example 1.

The isolated dermides were rinsed in about 10 ml of media A as formulated in Example 1 , then transferred to a container containing a solution of about 10 ml of media A and, based upon the total volume of the solution, 0.35% collagenase type 1 obtained from Worthington Biochemical Co., such that the volume of resulting suspension was about 2 ml collagenase-media A solution/dermis.

After the dermal tissue was coarsely cut into pieces ranging in size from about 5 mm² to about 1 cm² with scissors and triturated briefly with a 10 ml pipette, the resulting suspension was incubated at 37°C in a Precision Scientific Model 50 reciprocal shaking waterbath agitated at about 100 φm. After 25 minutes of agitation, DNAse I (Worthington Biochemical Co., Freehold, NJ) was injected via pipette thereto at 400 units/ml of the suspension (or "digest"). The resulting suspension was incubated under the above-described conditions for an additional 5 minutes.

The resulting dermal solution was diluted 5 fold volumetrically with media A, filtered through a 100 μM nylon mesh, then centrifuged for about 5 minutes at a speed of about 800 φ in order to generate supernatant A and pellet A. Supernatant A was transferred to a new tube while pellet A was resuspended in about 50 ml of media A. Supernatant A was recentrifuged for about 5 minutes at a speed of about 1400 φm in order to generate supernatant B and pellet B. Pellet B was resuspended in 8 ml of media A, while the Pellet A suspension was centrifuged for about 3 minutes at a speed of about 300 φm to generate supernatant C and pellet C. The Pellet B suspension and supernatant C were centrifuged independently for about 5 minutes at a speed of about 800 φm in order to generate supernatants and pellets D and E, respectively. After Pellets D and E were independently resuspended in about 10 ml of media A, the resulting solutions were combined and centrifuged for about 3 minutes at a speed of about 800 φm. The resulting pellet was then resuspended in about 30 ml of media A, then recentrifuged for about 3 minutes at 800 φm; this step was repeated. After resuspending the resulting pellet in about 40 ml of media A, the resulting suspension was filtered and the cells of the filtrate were then counted as set forth in Worthington. This procedure resulted in a composition comprised of a single-cell suspension of total dermal cells, termed "fresh dermal cells," lacking large anagen hair follicles.

Example 3: Generation of FP cell lines

Immortal FP fibroblast cell lines were dissected from an adult H-2/ΛtsA58 transgenic mouse obtained from Charles River Labs under the name, "Immortomouse" which contained temperature-sensitive T antigen in all cells under control of the MHC I promoter, H-2lt. Unlike other immortalization methods known in the art, i.e. Filsell and Bayley, which disadvantageously suffer from variable clonal cell lines with respect to integration site, oncogene expression level, and permanence of oncogene expression, such drawbacks were eliminated by using cells from this mouse.

Mouse vibrissae follicular papilla fibroblasts were obtained using the dissection procedure of Williams: After dissecting adult mouse vibrissae from the surrounding tissue, about 10 vibrissae were placed in 10 ml of Chang's media supplemented with 1% bovine serum albumin. Using a Leitz stereo dissecting microscope, the follicular papillae were gently removed from their respective matrix keratinocytes. Five papillae were pipetted into one well (35 mm diameter) of a Falcon 6-well plate. The well contained about 5 ml of Chang's media and 100 units/ml Chang's media of mouse gamma interferon. After two weeks of growth at 33 °C, the resulting follicular papilla cells were passaged at a 1 :1 split into a larger dish (3.5 cm diameter) containing 5 ml of Chang's media supplemented with gamma interferon.

After diluting the follicular papillae cells with Chang's media at a volumetric ratio of about 1 :10000, the resulting dilution was evenly distributed into about 5 dishes (10 cm diameter). 10 μl of a 100,000 unit/ml gamma interferon solution was added to the solution in each dish. After two weeks of exposure to a 95%0₂/5% C0₂ environment in a Forma Scientific incubator maintained at 33°C, the follicular papilla cell lines were then generated by ring cloning as described in Freshney at. 169-171. The resultant cloned cell lines, designated tsT FP followed by the clone number, were further passaged at 80% confluence using a 1 :5 split in Chang's Media with gamma interferon in the above incubator in accordance with the processing method and conditions set forth in Freshney. The media in each dish was replaced every 3 days in culture.

During the last passage (which was less than the 30th passage) prior to a grafting experiment, tsT FP fibroblasts were grown to near confluence and harvested using 0.05% trypsin/0.5 mM EDTA/PBS. Harvested cells were then resuspended in Chang s media without gamma interferon in a volumetric ratio of about 1 :5, then counted as set forth in Example 2.

This procedure resulted in a composition comprising clonal FP cell lines that were capable of inducing hair growth.

Example 4: Non-FP fibroblast cell line

The immortalized fibroblast cell line, NIH3T3 (obtained from John Sedivy at Brown University) was grown and cultured in accordance with the procedures set forth in Example 3, but with the exception that the splits were 1 :10, the culture temperature was 37 *C, and the media used was Dulbecco's Modified Eagle's Medium/10% calf serum (Biowhittaker, Inc.).

On the last passage prior to a grafting experiment, NIH3T3 fibroblasts were harvested and counted in accordance with the procedure set forth in Example 3, but with the exception that the cells were resuspended in an appropriate volume of DMEM/10% CS.

Example S: Implantation of Hair Buds or Fresh Dermal Cells

One ml of the hair buds of Example 1 were pipetted into a 15 ml conical tube and centrifuged at 4 °C at 800 φm for 5 minutes. The resulting supernatant was then aspirated such that only a small amount of film was overlying the pellet. This pellet was then pipetted into a silicone grafting chamber (about 2 cm diameter) obtained from Renner, Gmbh, which was previously inserted and clipped into a 3.5 cm² full thickness excisional wound on the dorsum of an adult male NCR nude mouse (Taconic Farms, Inc., Germantown, NY). The mouse having the chamber inserted therein was housed in a microisolator obtained from Lab Products Inc. for a total of five weeks.

After the first week post-grafting, the chamber was removed from the mouse. We found that a scab began to form during the second and third week post-grafting. The scab fell off at about the third week post-grafting. At the fourth or fifth week post-grafting, the tissue in the graft site was excised and processed for histological analysis by removing the tissue with scissors, blocking the tissue in the sagittal plane, and immersing the blocked tissue in buffered formalin. The tissue was removed from the formalin, embedded in paraffin, and cut into sections having a thickness of about 6 μ . Five sections were stained via H&E, and 5 other sections were stained for elastin.

FIG 3A shows a graft of only hair buds, which resulted in a non-pigmented scar. FIG 3B shows the host-graft junction as indicated by the arrow. Host tissue on left has pilosebaceous structures whereas graft tissue on right has scar moφhology consisting of a flat dermal-epidermal junction, a lack of hair follicles, and thick collagen bundles oriented parallel to the epidermal surface. FIG 4A illustrates that the host tissue on the left side contains hair follicles and abundant elastin in the papillary dermis as indicated by the arrows, whereas the graft tissue on the right side of FIG. 4A shows no hair follicles and no elastin, which is indicative of scarring. This result was repeated in 11 of 11 animals tested, each of which contained a single graft, in 10 independent experiments. This example was repeated but the hair buds were substituted with about 8 million fresh dermal cells of Example 2. We have also found that when only fresh dermal cells were similarly implanted into a wound site, the results were the same. Thus, we have found in this example that when a wound site was implanted exclusively with hair buds (or exclusively with fresh dermal cells), a scar was produced. Further, these results indicate that neither the hair buds or fresh dermal cells, when singularly added to a wound, could regenerate skin.

Example 6: Preparation and Implementation of Hair Buds and Fresh Dermal Cells

1 ml of hair buds of Example 1 and about 8 million fresh dermal cells of Example 2 were combined, centrifuged, and prepared into a pellet in accordance with Example 5. After this pellet was implanted into a wound, the tissue was harvested and processed, all in accordance with the procedures set forth in Example 5. FIG 3C shows that a hair bud/ fresh dermal cell- graft resulted in skin having dense, pigmented hair follicles. The histology illustrated in FIG 3D further indicates that such a graft site contains regenerated, normal skin structure and pilosebaceous unit development. Eumelanin in the hair cortex (as shown by the arrowhead) and the matrix region (as shown by the arrow) indicated the presence of induced donor-derived hair follicles. We have also found that the eumelanin was confined to hair follicles, which not only is the normal distribution in NIH Black Swiss mice but also further evidenced that normal cellular interactions had occurred.

As illustrated in FIG. 4B, hair follicles were present in the dermis and elastin fibers were present in the papillary dermis, both of which were indicative of skin regeneration. These results were repeated, with minor variability, in 19 of 19 animals in 12 independent experiments.

In this Example, we have found that the combination of fresh dermal cells and hair buds, when implanted into a wound, lead to skin regeneration, a result that correlates with the formation of hair follicles in the graft. The former was indicated by the normal appearance of the skin structure at the wound site, as well as the presence of elastin in the papillary dermis at the wound site. These results, in conjunction with those from Example 5, suggested that the dermal component of the grafting composition was a significant contributor to skin regeneration. Furthermore, the normal distribution of the melanocytes in the wound site indicated that the normal mechanisms for melanocyte development and function were operative.

Example 7: Preparation and Implementation of FP and Hair Buds

1 ml of hair buds of Example 1 and about 8 million ts TFP cells of Example 3 were combined, centrifuged, and prepared into a pellet in accordance with Example 5. After this pellet was implanted into a wound, the tissue was harvested and processed, all in accordance with the procedures set-forth in Example 5

As illustrated in FIG 5A, the skin of the resulting graft site possesses eumelanin- containing hair follicles and is blue. As illustrated in FIG 5B, the pigmented hair follicles are clearly evident at the wound site, which is indicative of significant hair growth generated by the follicular papilla cells. FIG 5C illustrates that the skin of the graft site is regenerated and possesses an accumulation of melanocytes (as shown by the arrow) and melanophages (as shown by the arrowhead) in the mid-dermis. FIG. 4C shows that elastin was regenerated in the papillary dermis in the graft site. These results were repeated with minor variations in 17 of 17 animals over 6 independent experiments, using two independent ts TFP cell lines. We have found that it was necessary to use follicular papilla cells, in combination with hair bud cells, to regenerate skin, a response that correlates with hair follicle formation. The former was indicated by the normal skin structure and the presence of elastin in the wound site. We further believe that the presence of FP cells contained within the dermal cell/FP mixture of Example 6 were responsible for initiating the skin regeneration response. We also believe that: 1) the presence of FP cells induced ectopic accumulation of eumelanin- containing melanocytes, a condition that resembles human common blue nevus; 2) ts TFP cells, and perhaps FP cells in general, produced a factor that promotes melanocyte survival and or eumelanin production; and 3) cells within the fresh dermal cell preparation were capable of inhibiting ectopic melanocyte survival and/or eumelanin production since the grafting of a hair buds dermal cells mixture of Example 6 did not result in ectopic accumulation of melanocytes.

Example 8: Preparation and Implementation of non-FP and Hair Buds

1 ml of hair buds of Example 1 and about 8 million NIH3T3 fibroblasts of Example 4 were combined, centrifuged, and prepared into a pellet in accordance with Example 5. After this pellet was implanted into a wound, the tissue was harvested and processed, all in accordance with the procedures set forth in Example 5.

FIG 6A shows that the hair buds- NIH3T3 fibroblast graft resulted in a scar with dark blue skin color. FIG 6B illustrates that a NIH3T3 fibroblast-graft possessed not only skin structure typical of a scar, but also a massive accumulation of eumelanin-containing melanocytes in the dermis which is indicative of common blue nevus. FIG 6C, which represents a high power magnification of a similar section to FIG 6B, shows the presence of eumelanin granules within the melanocytes, which evidenced that the pigmented cells in the dermis were melanocytes. These results were repeated in 8 of 8 animals over 5 independent experiments. In this Example, we have found that a fibroblast cell line unrelated to FP cells, i.e. the NIH 3T3 non-FP cells, was incapable of inducing hair, but was able to induce accumulation of melanocytes in the dermis. The results of Examples 7 and 8 suggest that a factor which promotes dermal melanocyte accumulation was unrelated to the factor(s) that were required to produce hair. Additional support for this belief was based upon our observation that some ts TFP cell lines did not form hair, yet did induce CBN-like moφhology.

Example 9: Preparation and Implementation of Hair Buds. Fresh Dermal Cells, and NIH3T3

1 ml of hair buds of Example 1 , about 8 million fresh dermal cells of Example 2, and about 8 million NIH3T3 of Example 4 were combined, centrifuged, and prepared into a pellet in accordance with Example 5. After this pellet was implanted into a wound, the tissue was harvested and processed, all in accordance with the procedures set forth in Example 5.

FIG 7A illustrates that the resulting graft contained eumelanin-containing hair follicles but lacked blue skin color. FIG 7B further shows that the graft possessed regenerated skin structure as well as sparse melanocytes in the dermis (as shown by the arrow). These results were repeated in 4 of 4 animals in 2 independent experiments. We believe that the amount of dermal melanocytes was not sufficient to produce the blue skin color as seen in the grafts illustrated in FIGS. 5A-C and FIGS. 6A-C.

This example was repeated, but the fresh dermal cells were replaced with the fresh dermal cells of Example 2 which were passaged 3 times at a 1.5 split in Chang's media in accordance with the procedure of Example 3. FIG 7C illustrates that the graft of the combination containing passaged dermal cells possessed decreased hair follicles without a homogeneous blue skin color. This result was repeated in 3 of 3 animals in 1 experiment.

In this Example, we have found that fresh dermal cells appeared to inhibit the capacity of NIH3T3 cells to induce CBN-like moφhology. Furthermore, the results of this Example support the belief that this inhibitory capacity of fresh dermal cells may reside in their non-FP cells component in view of the fact that removal of the hair-inductive property (presumably by removal of FP cells during passage) did not prevent the passaged fresh dermal cells from inhibiting CBN-like changes.

Example 10: Inhibition of Tyrosinase Induction bv Fresh Dermal Cell Conditioned Medium

About 60,000 fresh dermal cells of Example 2 were evenly distributed into about 6 dishes (150 mm diameter) with 30 ml media A at approximately 20% confluence and cultured for 7 days at 37'C. At time of media harvest, the cells were 100% confluent. After collecting and combining the conditioned media (CM) from each dish, the resulting mixture was filtered through a 0.45 μM mesh filter and stored at -70"C. CM were similarly prepared from fresh dermal cells of both Black Swiss pups, as well as those from ts TFP cells.

Cloudman S91 melanoma cells were grown in media according to the standard conditions and processes set forth in Ortow et al., 94 J Invest Dermatol, 461-464 (1990)(^*Orlow^*), then stimulated to produce eumelanin by addition of cholera toxin in a volumetric ratio of melanoma cells/media:toxin of about 1000:1 such that the final concentration of cholera toxin in the mixture was about 1x10^"10M.

Each CM was assayed for the ability to inhibit cholera toxin-induced pigmentation by adding each CM, in various amounts as evidenced in FIG. 8, to the dish of melanoma cell/media simultaneously with the addition of the cholera toxin thereto. The resulting mixtures were placed into a Forma Scientific Steri-cult 200 incubator and maintained under the conditions set forth in Orlow for 3 days.

After the resulting cell mixture was removed from the incubator, the media was aspirated away from the S91 melanoma cells. The remaining S91 melanoma cells were then fixed in 1 mL of a formalin/0.2% glutaraldehyde solution. This fixative solution was removed and replaced with 3 ml of PBS. To this PBS solution was added an amount of a L-Dopa pigmentation substrate was added thereto such that the final concentration of substrate in the well was 0.01%, based on the total well volume.

The resulting mixture was subjected to image analysis in a Optimax V HR image analyzer which quantified the amount of pigmentation in a defined area (μM²).

Since dermal cells of a Swiss Webster albino mouse produce the agouti protein, a natural inhibitor of pigmentation produced by FP cells, we further generated CM from the dermal cells of Black Swiss mice, a fresh dermal source that does not produce agouti, via a similar process. To eliminate non-specific inhibitory effects, another CM derived from ts TFP cells was similarly prepared.

FIG 8 shows that maximal pigmentation of the melanoma cells in the presence of control media (i.e. unconditioned media consisting of 10%FCS/EMEM and identified by ^*10%FCS/EMEM" in FIG. 8) was approximately 4000 μM². CM from Swiss Webster Albino fresh dermal cells (identified as "agouti" in FIG. 8), added to the media in an amount of, for example, 0.5 ml, resulted in approximately 80% inhibition of pigmentation relative to the control. Similarly, CM from Black Swiss fresh dermal cells (identified as "non-agouti^* in FIG. 8) added to the media in an amount of, for example, 0.5 ml, resulted in approximately 95% inhibition of pigmentation. CM from tsTFP cells (identified as "1.61 ts TFP" in FIG. 8), when similarly added to the media did not inhibit pigmentation. In Example 10, we have found that fresh dermal cells appeared to secrete a factor that inhibited pigmentation in vitro. Furthermore, this factor appeared to be unrelated to the agouti protein, and was specific to fresh dermal cells. These results further supported the in vivo data from Example 9 which appeared to indicate that fresh dermal cells contained a pigmentation inhibitor.

Claims

We claim:

1. A method for regenerating skin at a cutaneous wound or bum site in an animal comprising: applying to a wound or a bum a composition comprising hair bud cells and dermal cells in an amount effective to result in skin tissue which has the appearance and histological structure similar to normal skin.

2. The method of claim 1 further comprising allowing the composition to be infiltrated by blood vessels and mesenchymal cells from healthy tissue in the wound site of the human or animal.

3. The method of claim 1 wherein the composition is present in a relative amount of from about 2.5 μl to about 475 μl for a wound site or burn site having a surface area of from about 1 cm² to about 5 cm².

4. The method of claim 1 wherein the dermal cells were passaged during their preparation.

5. The method of claim 1 wherein said composition contains a hair bud:deπmal cell numeric ratio of about 1 :1000 to about 1 :20,000.

6. The method of claim 1 wherein said dermal cells are comprised of at least one of the following cells: follicular papilla fibroblasts, non-follicular papilla fibroblasts, endothelial cells, myocytes, adipocytes, and mixtures thereof.

7. A method for regenerating skin at a cutaneous wound or bum site in an animal comprising: applying to a wound or a bum a composition comprising hair bud cells and follicular papilla cells in an amount effective to result in skin tissue which has the appearance and histological structure similar to normal skin.

8. The method of claim 7 further comprising allowing the composition to be infiltrated by blood vessels and mesenchymal cells from healthy tissue in the wound site of the human or animal.

9. The method of claim 7 wherein the composition is present in a relative amount of from about 2.5 μl to about 475 μl for a wound site or bum site having a surface area of from about 1 cm² to about 5 cm².

10. The method of claim 7 wherein said composition contains a hair bud:follicular papilla cell numeric ratio of about 1 :1000 to about 1:20,000.

11. A method for regenerating skin at a cutaneous wound or burn site in an animal comprising: applying to a wound or a bum a composition comprising hair bud cells, dermal cells, and fibroblast cells selected from the group consisting of follicular papilla cells, non-follicular papilla cells, and mixtures thereof in an amount effective to result in skin tissue which has the appearance and histological structure similar to normal skin.

12. The method of claim 11 wherein the fibroblast cells are follicular papilla cells.

13. The method of claim 11 further comprising allowing the composition to be infiltrated by blood vessels and mesenchymal cells from healthy tissue in the wound site of the human or animal.

14. The method of claim 11 wherein the composition is present in a relative amount of from about 2.5 μl to about 475 μl for a wound site or bum site having a surface area of from about 1 cm² to about 5 cm².

15. The method of claim 11 wherein said composition contains a hair bud:dermal cell:fιbroblast cell numeric ratio of about 1 :1000:1000 to about 1 :20,000:20,000.

16. The method of claim 11 wherein the dermal cells were passaged during their preparation.

17. A method of preventing dermal melanocytosis during skin regeneration comprising: applying on a area in need of skin regeneration a composition comprising hair bud cells, fresh dermal cells, and fibroblast celts selected from the group consisting of follicular papilla cells, non-follicular papilla cells, and mixtures thereof.

18. The method of claim 17 wherein the fibroblast cells are follicular papilla.

19. The method of claim 1 further comprising allowing the composition to be infiltrated by blood vessels and mesenchymal cells from healthy tissue in the wound site of the human or animal.

20. The method of claim 17 wherein the composition is present in a relative amount of from about 2.5 μl to about 475 μl for a wound site or bum site having a surface area of from about 1 cm² to about 5 cm².

21. The method of claim 17 wherein said composition contains a hair bud:dermal cell:fιbroblast cell numeric ratio of about 1 :1000:1000 to about 1 :20,000:20,000.

22. The method of claim 17 wherein the dermal cells were passaged during their preparation.

23. A method for treating cutaneous wound or bum sites in an animal comprising: topically administering a formulation comprised of: hair bud cells, dermal cells, and fibroblast ceils selected from the group consisting of follicular papilla cells, non-follicular papilla cells, and mixtures thereof.

24. The method of claim 23 wherein the fibroblast cells are follicular papillae.

25. The method of claim 23 further comprising allowing the composition to be infiltrated by blood vessels and mesenchymal cells^* from healthy tissue in the wound site of the human or animal.

26. The method of claim 23 wherein the composition is present in a relative amount of from about 2.5 μl to about 475 μl for a wound site or bum site having a surface area of from about 1 cm² to about 5 cm².

27. The method of claim 23 wherein said composition contains a hair bud:dermal celhfibroblast cell numeric ratio of about 1 :1000:1000 to about 1 :20,000:20,000.

28. The method of claim 23 wherein the dermal cells were passaged during their preparation.

29. A composition for the topical treatment of wound or bum sites in animal skin comprising: a) hair bud cells; b) dermal cells; and c) fibroblast cells selected from the group consisting of non-follicular papilla cells, follicular papilla cells, and mixtures thereof.

30. The composition of claim 29 wherein the fibroblast cells are follicular papilla.

31. The composition of claim 29 wherein said composition contains a hair bud:dermal cel fibroblast cell numeric ratio of about 1:1000:1000 to about 1 :20,000:20,000.