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Characterization of Epidermal Wound Healing in a Human Skin Organ Culture Model: Acceleration by Transplanted Keratinocytes1

1998; Elsevier BV; Volume: 111; Issue: 2 Linguagem: Inglês

10.1046/j.1523-1747.1998.00265.x

1523-1747

Ingrid Moll, Pia Houdek, Hannelore Schmidt, Roland Moll,

Skin and Cellular Biology Research

Few data are available on early regeneration of human epidermis in vivo. We have established a supravital skin organ culture model for epidermal wound healing by setting a central defect (3 mm diameter) in freshly excised skin specimens and culturing under air exposure. Re-epithelialization was followed for up to 7 d by histology and immunohistologic analysis of various markers for differentiation and proliferation. In 12 of 19 cases (63%; 5% fetal calf serum) or six of 21 cases (29%; 2% fetal calf serum), the wounds were re-epithelialized spontaneously after 7 d. After transplantation to the wounds of 1–2 × 106 dissociated allogenic cultured epidermal or about 1 × 106 autologous outer root sheath keratinocytes, 18 of 21 cases (86%; 5% fetal calf serum) or 17 of 21 cases (81%; 2% fetal calf serum) were healed within the same period. Histologically, early neoepithelium (3 d) was disordered after keratinocyte transplantation, whereas later (7 d) it had gained a more ordered stratification, exhibiting a thin discontinuous granular and a compact horny layer. At this stage, not only hyperproliferative (CK 6) but also, abundantly, maturation-associated cytokeratins (CK 1, CK 10) were detected immunohistochemically. Analyses of regenerated epidermis after transplantation of (i) keratinocytes labeled in vitro with BrdU and (ii) heterosexual keratinocytes by immunohistochemistry and fluorescence in situ hybridization for the Y chromosome, respectively, clearly showed that external keratinocytes are physically integrated into the regenerated epidermis and extendedly contribute to its formation. The data presented here demonstrate improvement and acceleration of epidermal re-epithelialization by transplantation of keratinocytes. Few data are available on early regeneration of human epidermis in vivo. We have established a supravital skin organ culture model for epidermal wound healing by setting a central defect (3 mm diameter) in freshly excised skin specimens and culturing under air exposure. Re-epithelialization was followed for up to 7 d by histology and immunohistologic analysis of various markers for differentiation and proliferation. In 12 of 19 cases (63%; 5% fetal calf serum) or six of 21 cases (29%; 2% fetal calf serum), the wounds were re-epithelialized spontaneously after 7 d. After transplantation to the wounds of 1–2 × 106 dissociated allogenic cultured epidermal or about 1 × 106 autologous outer root sheath keratinocytes, 18 of 21 cases (86%; 5% fetal calf serum) or 17 of 21 cases (81%; 2% fetal calf serum) were healed within the same period. Histologically, early neoepithelium (3 d) was disordered after keratinocyte transplantation, whereas later (7 d) it had gained a more ordered stratification, exhibiting a thin discontinuous granular and a compact horny layer. At this stage, not only hyperproliferative (CK 6) but also, abundantly, maturation-associated cytokeratins (CK 1, CK 10) were detected immunohistochemically. Analyses of regenerated epidermis after transplantation of (i) keratinocytes labeled in vitro with BrdU and (ii) heterosexual keratinocytes by immunohistochemistry and fluorescence in situ hybridization for the Y chromosome, respectively, clearly showed that external keratinocytes are physically integrated into the regenerated epidermis and extendedly contribute to its formation. The data presented here demonstrate improvement and acceleration of epidermal re-epithelialization by transplantation of keratinocytes. cytokeratin Whereas the majority of studies dealing with cutaneous wound healing have focused on dermal repair mechanisms and wound ground remodeling (for a general review, seeClark, 1996Clark R.A.F. Overview and general considerations of wound repair.in: Clark R.A.F. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 3-50Google Scholar), comparatively little attention has been paid to the question of epidermal regeneration. In partial-thickness epidermal wounds, the re-epithelialization arises from viable epidermal cells at the wound edges as well as (depending on the depth of the wound) from those parts of the epidermal appendices remaining in the wound bed itself (Eisen et al., 1955Eisen A.Z. Holyoke J.B. Lobitz W.C. Responses of the superficial portion of the human pilosebaceous apparatus to controlled injury.J Invest Dermatol. 1955; 15: 145-156Abstract Full Text PDF Scopus (51) Google Scholar). Re-epithelialization is defined as being the reconstruction of keratinocytes into a stratified epidermis that, after wound healing, provides a permanent cover and restores the function of the skin. During the processes involved in epidermal wound healing, keratinocytes undergo a series of behavioral changes (Paladini et al., 1996Paladini R.D. Takahashi K. Bravo N.S. Coulombe P.A. Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: Defining a potential role for keratin 16.J Cell Biol. 1996; 132: 381-397Crossref PubMed Scopus (330) Google Scholar), including cell migration, proliferation, and differentiation of keratinocytes at the wound margin. Migration has been shown to be the initial event, which includes the movement of suprabasal cells over basal cells (Marks and Nishikawa, 1973Marks S. Nishikawa T. Active epidermal movement in human skin in vitro.Br J Dermatol. 1973; 88: 245-248Crossref PubMed Scopus (29) Google Scholar;Garlick and Taichman, 1994Garlick J.A. Taichman L.D. Fate of human keratinocytes during reepithelialization in an organotypic culture model.Lab Invest. 1994; 6: 916-924Google Scholar), this event is followed by a mitotic burst (Viziam et al., 1964Viziam C.B. Maltotsy A.G. Mescon H. Epithelialization of small wounds.J Invest Dermatol. 1964; 43: 499-507Abstract Full Text PDF PubMed Scopus (60) Google Scholar;Garlick and Taichman, 1994Garlick J.A. Taichman L.D. Fate of human keratinocytes during reepithelialization in an organotypic culture model.Lab Invest. 1994; 6: 916-924Google Scholar). Differentiation of the newly formed epidermis begins a short distance behind the migrating tip before re-epithelialization has been completed (Odland and Ross, 1968Odland G. Ross R. Human wound repair.J Cell Biol. 1968; 39: 135-151Crossref PubMed Scopus (321) Google Scholar;Garlick and Taichman, 1994Garlick J.A. Taichman L.D. Fate of human keratinocytes during reepithelialization in an organotypic culture model.Lab Invest. 1994; 6: 916-924Google Scholar). All processes of epidermal wound healing are under the influence of chemical attractants, such as various growth factors, cytokines, and extracellular matrix proteins, which are secreted locally by inflammatory cells or by fibroblasts, endothelial cells, or keratinocytes themselves (Eisinger et al., 1988Eisinger M. Sadan S. Silver I.A. Flick R.B. Growth regulation of skin cells by epidermal cell-derived factors: implications for wound healing.Proc Natl Acad Sci USA. 1988; 85: 1937-1941Crossref PubMed Scopus (87) Google Scholar;Moulin, 1995Moulin V. Growth factors in skin wound healing.Eur J Cell Biol. 1995; 68: 1-7Abstract Full Text PDF PubMed Google Scholar;Martin, 1997Martin P. Wound healing-Aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3749) Google Scholar). The chronology of the various events of re-epithelialization and epidermal differentiation in vivo has mainly been studied using various animal models (Viziam et al., 1964Viziam C.B. Maltotsy A.G. Mescon H. Epithelialization of small wounds.J Invest Dermatol. 1964; 43: 499-507Abstract Full Text PDF PubMed Scopus (60) Google Scholar;Mansbridge and Knapp, 1987Mansbridge J.N. Knapp A.M. Changes in keratinocyte maturation during wound healing.J Invest Dermatol. 1987; 89: 253-263Abstract Full Text PDF PubMed Google Scholar); however, owing to the different structure of animal skin (Regauer and Compton, 1990Regauer S. Compton C.C. Cultured keratinocyte sheets enhance spontaneous re-epithelialization in a dermal explant model of partial-thickness wound healing.J Invest Dermatol. 1990; 95: 341-346Abstract Full Text PDF PubMed Google Scholar) and the complex nature of the wounded microenvironment, it is difficult to obtain, for human skin, acceptably precise data concerning the proliferative, migratory, and differentiation character of keratinocytes of the wound margin and of migrating cell sheets during the various sequential stages of re-epithelialization in vivo (see alsoOliver et al., 1991Oliver A.M. Kaawach W. Weiler Mithoff E. Watt A. Abramovich D.R. Rayner C.R. The differentiation and proliferation of newly formed epidermis on wounds treated with cultured epithelial allografts.Br J Dermatol. 1991; 125: 147-154Crossref PubMed Scopus (31) Google Scholar). To circumvent these difficulties of studying human epidermal wound healing we have developed a novel skin organ culture model, which involves the removal of the total epidermis from the center of the specimen, so that the resulting defects are healed via keratinocyte outgrowth from its margins. We have previously used the basic (without wounding) organ culture model for studies dealing with the development of dermatitis and sunburn cells after ultraviolet irradiation. 1Bohnert E, Moll I, Jung EG: Formation and quantitation of SBCs in UV-irradiated, short-term primary, and organotypic keratinocyte cultures. Arch Dermatol Res 285:70, 1993 (abstr.)1Bohnert E, Moll I, Jung EG: Formation and quantitation of SBCs in UV-irradiated, short-term primary, and organotypic keratinocyte cultures. Arch Dermatol Res 285:70, 1993 (abstr.)Hintner et al., 1980Hintner H. Fritsch P.O. Foidart J.-M. Stingl G. Schuler G. Katz S.L. Expression of basement membrane zone antigens at the dermoepidermal junction in organ cultures of human skin.J Invest Dermatol. 1980; 74: 200-204Abstract Full Text PDF PubMed Scopus (71) Google Scholar have also used related organ culture models for studying the expression of basement membrane zone antigens. The viability and efficiency of such models have further been demonstrated by similar skin organ cultures (also called supravital skin models) established byPope et al., 1995Pope M. Betjes M.G.H. Hirmand H. Hoffman L. Steinman R.M. Both dendritic cells and memory T lymphocytes emigrate from organ cultures of human skin and form distinctive dendritic-T-cell conjugates.J Invest Dermatol. 1995; 104: 11-17Abstract Full Text PDF PubMed Scopus (93) Google Scholar,Lukas et al., 1996Lukas M. Stössel H. Hefel L. et al.Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model.J Invest Dermatol. 1996; 106: 1293-1299Abstract Full Text PDF PubMed Google Scholar, andRomani, 1996Romani N. Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model.J Invest Dermatol. 1996; 106: 1293-1299Crossref PubMed Scopus (105) Google Scholar to study complex aspects of cutaneous dendritic cells. From these various applications of skin organ cultures, it is clear that they allow the investigation of complex processes in human skin biology without the intrusion of systemic influences. In this study, a supravital skin organ culture model was, by central wounding, applied to epidermal wound healing in order to study the process of re-epithelialization in the absence of circulating factors, immunologic reactions involving lymph nodes, and general inflammatory reactions. This human skin organ culture made it further possible to study the early effects as well as the fate of transplanted cultured epidermal keratinocytes and freshly isolated human outer root sheath (ORS) keratinocytes; these latter ones are especially viable and proliferative in culture (Yang et al., 1993Yang J.S. Lavker R.M. Sun T.T. Upper human hair follicles contain a subpopulation of keratinocytes with superior in vitro proliferative potential.J Invest Dermatol. 1993; 101: 652-659Abstract Full Text PDF PubMed Google Scholar;Moll et al., 1995Moll I. Schönfeld M. Jung E.G. Applikation von Keratinozyten in der Therapie von Ulcera crurum.Hautarzt. 1995; 46: 548-552Crossref PubMed Scopus (18) Google Scholar[BC1]) after their topical application to wounds and thus are already being introduced into the therapy of chronic wounds (Moll et al., 1995Moll I. Schönfeld M. Jung E.G. Applikation von Keratinozyten in der Therapie von Ulcera crurum.Hautarzt. 1995; 46: 548-552Crossref PubMed Scopus (18) Google Scholar) and the closure of fresh excisions (Böhm et al., 1992Böhm K. Gerhardt H.J. Kaschke O. Winter H. Böhm F. Sönnichsen N. Neumann S. Wundverschluss mit Zellsuspension im Haut- und Schleimhautbereich.Dermatol Monatsschr. 1992; 178: 22-26Google Scholar). This model also allowed the sequential appearance of keratinocyte differentiation markers to be analyzed. Skin samples of human trunk skin (23 females, 17 males; age range, 15–63 y) were obtained during the routine removal of epidermal cysts and various tumors; the samples used were localized at least 2 cm from any lesion. All cell and organ culture experiments were performed in the Cell Culture Laboratory of the Department of Dermatology, Mannheim Medical School (Mannheim, Germany). All skin samples (n = 101) were used immediately after excision. Each sample was trimmed into a piece with a diameter of 6 mm and a punch biopsy (diameter 3 mm) including the epidermis and the upper dermis was removed from its center. Each piece was placed dermis down on gauze on a culture disk (Falcon; diameter 1 cm) filled with Dulbecco's modified Eagle's medium (supplemented with hydrocortisone, 2% or 5% fetal calf serum, penicillin, and streptomycine) in such a way that the medium was only in contact with the underside of the sample so that the epidermis remained constantly exposed to the air. These cultures were incubated at 37°C with 10% CO2 for 7 d, the medium being changed every other day. In some cases, allogenic keratinocytes cultured from epidermis (n = 29) or freshly isolated autologous ORS keratinocytes (n = 26) were transplanted to the wound site at day 1 (see below). At days 2–7, the cultures were snap-frozen in isopentane pre-cooled in liquid nitrogen and stored at –80°C until use or were fixed in 4% buffered formaldehyde and embedded in paraffin. To obtain human keratinocyte cultures, skin samples were trypsinized (0.25% in phosphate-buffered saline, PBS) overnight at 4°C, and the single-cell suspensions were placed in keratinocyte basic medium supplemented with epidermal growth factor, bovine pitituary extract, insulin, hydrocortisone, and glutamine. On reaching 70% confluence, the cultures were passaged (for details, seeLimat and Noser, 1986Limat A. Noser F.K. Serial cultivation of single keratinocytes from the outer root sheath of human scalp hair follicles.J Invest Dermatol. 1986; 87: 485-488Abstract Full Text PDF PubMed Scopus (110) Google Scholar;Moll et al., 1995Moll I. Schönfeld M. Jung E.G. Applikation von Keratinozyten in der Therapie von Ulcera crurum.Hautarzt. 1995; 46: 548-552Crossref PubMed Scopus (18) Google Scholar). Prior to transplantation to the skin organ culture model, the cultured keratinocytes were trypsinized using 0.1% trypsin and 0.02% ethylenediamine tetraacetic acid in PBS for 5 min at 37°C. The cells (about 200,000) were collected in a small volume (≈10 μl) of cell culture medium and applied, using a fine pipet, to the central wound area of the specimen. To evaluate the localization, integration, migration, or replication of the transplanted allogenic keratinocytes, they were labeled with bromodeoxyuridine (BrdU) for 48 h (Gratzner, 1982Gratzner H.G. Monoclonal antibody to 5-bromo- and 5-iododeoxyridine: a new reagent for detection of DNA replication.Science. 1982; 218: 474-478Crossref PubMed Scopus (2361) Google Scholar) prior to transplantation. ORS keratinocytes (see below) were similarly labeled for some experiments. ORS keratinocytes were obtained from freshly plucked anagen hair follicles using 0.1% trypsin and 0.02% ethylenediamine tetraacetic acid in PBS for 1 h at 37°C and then via mechanical separation using pipets. A total of ≈106–2 × 106 autologous keratinocytes suspended in ≈10 μl cell culture medium were applied, using fine pipets, to each wound in the skin organ culture 1 d after its establishment (day 1; see below). From each frozen or paraffin-embedded skin organ culture specimen, serial sections (frozen sections: 5–7 μm thick; paraffin sections: 3–4 μm thick) through its central area were cut. Hematoxylin and eosin staining was performed for morphologic analyses. Healing was considered to have occurred when no part of the central area lacked an epithelium, even though this might be only one to three cell layers thick in some cases. All samples that failed to show this criterion were classified as being unhealed. The remaining sections were used for immunohistochemical staining. To evaluate the state of differentiation and proliferation, acetone-fixed (10 min at –20°C) frozen sections or deparaffinized paraffin sections were stained immunohistochemically using various primary antibodies (listed in Table 1).Table IPrimary antibodies used for immunohistochemistryAntibodySpecificityFrozen (F)/Paraffin (P) sectionsSourceMoAb K 8.60CK 1, 10, 11FProgen Biotechnik, Heidelberg, GermanyMoAb DE-K10CK 10paApplication of 0.001% trypsin subsequent to microwave oven heating (see Materials and Methods).BioGenex/Diaplus, Neu-Isenburg, GermanyMoAb Ks 2.342.7.1CK 2eF, PProgen BiotechnikMoAb AE 14CK 5FKindly provided by Dr. T.-T. Sun, New York, University, New York, U.S.A.MoAb KA 12CK 6F, PaApplication of 0.001% trypsin subsequent to microwave oven heating (see Materials and Methods).Progen BiotechnikMoAb Ks 8.12CKs 13, 15, 16bBecause CK 13 is generally absent in epidermis and the reactivity of this antibody with CK 15 is only minor, the results were taken as indicating the presence of CK 16.FBio-Makor, Rehovot, IsraelMoAb E3CK 17F, PaApplication of 0.001% trypsin subsequent to microwave oven heating (see Materials and Methods).Progen BiotechnikMoAb V9VimentinFBoehringer Mannheim, Mannheim, Germany; DakoMoAb VIM-3B4VimentinpaApplication of 0.001% trypsin subsequent to microwave oven heating (see Materials and Methods).Progen Biotechnik; DakoMoAb MIB-1Ki 67F, PDianova, Hamburg, GermanyMoAb BMC 9318BrdUFBoehringer Mannheima Application of 0.001% trypsin subsequent to microwave oven heating (see Materials and Methods).b Because CK 13 is generally absent in epidermis and the reactivity of this antibody with CK 15 is only minor, the results were taken as indicating the presence of CK 16. Open table in a new tab For frozen sections indirect immunoperoxidase staining was performed using peroxidase-coupled goat antibodies against mouse or rabbit immunoglobulins (Dako, Hamburg, Germany) as secondary antibodies, with 3,3′-diaminobenzidine (DAB) and H2O2 being employed for the staining reactions (for details, seeFranke and Moll, 1987Franke W.W. Moll R. Cytoskeletal components of lymphoid organs I. Synthesis of cytokeratin 8 and 18 and desmin in subpopulations of extrafollicular reticulum cells of human lymph nodes, tonsils, and spleen.Differentiation. 1987; 36: 145-163Crossref PubMed Scopus (191) Google Scholar). For paraffin sections (seeDemirkesen et al., 1995Demirkesen C. Hoede N. Moll R. Epithelial markers and differentiation in adnexal neoplasms of the skin: an immunohistochemical study including individual cytokeratins.J Cutan Pathol. 1995; 22: 518-535Crossref PubMed Scopus (100) Google Scholar), the avidin-biotin-peroxidase complex (ABC) method (ABC Elite Kit; Vector, Burlingame, CA) was applied. After deparaffination and rehydration of the sections, endogenous peroxidase activity was blocked using 1% H2O2 in methanol for 30 min. Prior to the application of the primary antibody, an antigen retrieval step consisting of microwave oven heating (3–5 times for 5 min, 600 W) in 10 mM sodium citrate buffer (pH 6.0) was employed. For some antibodies (Table 1), this was followed by a very mild trypsinization step (0.001% trypsin in 50 mM Tris-HCl, pH 8.0, containing 0.001% CaCl2 for 15 min at 37°C). Generally, blocking was performed using 10% horse serum in PBS for 15 min. Primary antibodies were applied for 1 h at 37°C using a Shandon Sequenza apparatus (Shandon-Life Sciences, Frankfurt/Main, Germany). As secondary antibodies, biotinylated anti-mouse IgG antibodies (1:100; Vector) were applied for 30 min at room temperature. Subsequently, the ABC Elite reagent (Vector) was applied for 30 min at room temperature. The staining reaction was developed using 3,3′-diaminobenzidine and H2O2. For mild counterstaining, Mayer's hematoxylin solution was used. Negative controls were performed either by applying PBS instead of a primary antibody or by using an irrelevant primary antibody reacting with human tissues other than human skin. These controls always yielded the expected negative results. For the preparation of paraffin sections for fluorescence in situ hybridization, a modification of the method described byNeubauer et al., 1994Neubauer S. Liehr T. Tulusan H.A. Gebhart E. Interphase cytogenetics by FISH on archival paraffin material and cultured cells of human ovarian tumors.Int J Oncol. 1994; 4: 317-321PubMed Google Scholar was used. Sections from paraffin-embedded tissue were mounted on poly L-lysine-coated slides, air-dried overnight at 60°C, deparaffinized in xylene, rinsed in ethanol (100%), air-dried, and baked at 80°C for 1 h. Digestion with 0.02% proteinase K was carried out at 4°C for 10 min, followed by proteinase K treatment for another 30 min at 37°C. The slides were dehydrated and dried for 30 min at 80°C. In situ hybridization was carried out according to standard protocols (Verma and Babu, 1994Verma R.S. Babu A. Human Chromosomes: Principles and Techniques. 2nd edn. McGraw-Hill, New York1994: 280-287Google Scholar), using a specific probe for the Y centromere (classical satellite probe from ONCOR, Heidelberg, Germany) labeled with biotin that was detected by fluorescein-avidin (ONCOR). Nuclear counterstaining was achieved with 4′,6-diamidino-2-phenylindole-2HCl (Serva, Heidelberg, Germany). Sections were mounted in glycerol-PBS containing the anti-fading agent p-phenyldiamine (Sigma, Deisenhofen, Germany). In this study, we established and evaluated a human skin organ culture model for epidermal wound healing and we further investigated the effects of transplanted autologous freshly isolated ORS keratinocytes and cultured allogenic keratinocytes for epidermal regeneration in this model. Forty cases of skin organ cultures with central wounds were maintained for 7 d (see Materials and Methods), and the samples were then immediately either frozen or fixed in formalin and embedded in paraffin, before being cut into serial sections, so that the center could be examined histologically to see whether complete re-epithelialization had occurred. The results are presented in Table 2. Complete healing was found in six of 21 cases (DMEM with 2% fetal calf serum) and in 12 of 19 cases (DMEM with 5% fetal calf serum). In another six specimens maintained for only 2 d, no healing was observed histologically.Table IIKeratinocytes improved re-epithelialization in the skin organ culture modelControlsFresh ORSCultured epidermalkeratinocyteskeratinocytesDay 75% fetal calf serum/DMEM12aNumber of healed wound specimens./19bTotal number of specimens. (63%)cPercentage of wound specimens that healed.8/10 (80%) 10/11 (91%)2% fetal calf serum/DMEM6/21 (29%)7/9 (78%) 10/12 (83%)Day 35% fetal calf serum/DMEM0/6 (0%)4/7 (57%) 5/6 (83%)a Number of healed wound specimens.b Total number of specimens.c Percentage of wound specimens that healed. Open table in a new tab When the wound was totally re-epithelialized, a two- to four-layered epithelium in the center and an even more stratified epithelium towards the wound margins were found to have developed. Most of the cells of the upper layers were very flat, with spindle-shaped nuclei oriented parallel to the skin surface, whereas the basal cells were more or less cuboid in shape (Figure 1a). A thin discontinuous stratum granulosum layer was only inconsistently developed and was more frequently absent. The uppermost layer was formed by a relatively thin parakeratotic stratum corneum layer (see also below, Figure 2a); however, the regenerated epidermis was devoid of ridges (Figure 1a).Figure 2Markers of hyperproliferation and terminal differentiation are detectable in spontaneously regenerated epidermis. Immunoperoxidase staining of central parts of the spontaneously regenerated epidermis of skin organ cultures (day 7) using antibodies against different CK (frozen sections). All keratinocytes are homogeneously stained by antibody KA12 against the "hyperproliferative" CK 6 (a), whereas the stainings using antibody E3 (against CK 17;b) and antibody K 8.60 (against the terminal differentiation markers CK 1 and 10;c) are heterogeneous and most prominent among suprabasal keratinocytes. Scale bars, 50 μm.View Large Image Figure ViewerDownload (PPT) In general, the wound margins were more or less acanthotic, with the upper prickle and granular cells being enlarged, exhibiting vacuolization, and obviously undergoing pyknosis and cell necrosis. In contrast, the lower prickle cells and their nuclei were spindle-shaped with their long axis oriented parallel to the skin surface, although basal cells exhibited no apparent alteration (Figure 1b). The epithelium peripheral to the wound margins was mostly vital and normally stratified (Figure 1c). In some cases, the upper prickle cells and granular cells also had a vacuolized appearance. At day 7, the state of differentiation of the spontaneously regenerated epidermis was characterized in completely healed wounds by immunohistochemical analysis of cytokeratins. In all basal and some suprabasal cells, cytokeratins (CK) 5 and 14 – typical basal-cell-type CK – were found to be expressed (not shown). In addition, CK 6 and 16, which are markers of hyperproliferation, were widely expressed throughout the epithelium (Figure 2a), whereas CK 17 showed a more heterogeneous expression (Figure 2b). Among the terminal differentiation markers, CK 1 and 10 were detectable in some cases in a subpopulation of the spindle-shaped suprabasal keratinocytes in an irregular, mosaic-like pattern (Figure 2c); in contrast, filaggrin was barely detectable (not shown). Interestingly, vimentin was detected in some basal cells of the regenerated epidermis (not shown). We further studied cell proliferation by applying a Ki 67 antibody in these skin organ cultures. A high number of basal cells distributed equally in the regenerated epidermis were found to be Ki 67 positive at day 7, whereas suprabasal cells were negative (Figure 3). Another area of investigation concerned the epidermal wound margins of spontaneously and completely regenerated wounds, whose spindle-shaped cells in the lower suprabasal layers may well have been migrating keratinocytes. Also within the original wound margins of re-epithelialized wounds nearly 100% of the basal and some parabasal keratinocytes were positive for Ki 67 (Figure 4). In the original epidermis peripheral to the wound margins, Ki 67 positive cells were rare, but did still occur. In margins of wounds without healing after 7 d, spindle-shaped keratinocytes appeared fewer in number, but Ki 67 positive proliferative keratinocytes were present in similar numbers. Thus, it is clear that low proliferation was not the main reason for nonhealing. One day after the establishment of skin organ cultures, keratinocytes freshly isolated from autologous ORS were transplanted to the wound, a procedure analogous to that which we have established for chronic leg ulcers (Moll et al., 1995Moll I. Schönfeld M. Jung E.G. Applikation von Keratinozyten in der Therapie von Ulcera crurum.Hautarzt. 1995; 46: 548-552Crossref PubMed Scopus (18) Google Scholar). At day 7, the specimens were harvested and checked histologically (for details, see below) for re-epithelialization. In 80% of the cultures, total re-epithelialization was observed. Surprisingly, even only 2 d after the application of these keratinocytes, 57% of cultures (5% fetal calf serum in DMEM) were completely re-epithelialized. For comparison, we studied the effects of transplanting cultured allogenic epidermal keratinocytes (see Materials and Methods), for which the healing effects in chronic leg ulcers were found to be similar in extent (Leigh et al., 1987Leigh I.M. Purkis P.E. Navsaia H.A. Phillips T.J. Treatment of chronic venous ulcers with sheets of cultured allogenic keratinocytes.Br J Dermatol. 1987; 117: 591-597Crossref PubMed Scopus (203) Google Scholar;Beele et al., 1991Beele H. Naeyaert J.M. Goeteyn M. De Mil M. Kint A. Repeated cultured epidermal allografts in the treatment of chronic leg ulcers of various origins.Dermatologica. 1991; 181: 31-35Crossref Scopus (59) Google Scholar;Schönfeld et al., 1993Schönfeld M. Moll I. Maier K. Jung E.G. Keratinozyten aus der Zellkultur zur Therapie von Hautdefekten.Hautarzt. 1993; 44: 281-289PubMed Google Scholar). These results are summarized in Table 2. Because transplantations of freshly isolated autologous ORS or allogenic cultured epidermis keratinocytes improved the incidence of complete wound healing after 7 d (see above), it seemed of interest to examine the regenerating epidermis at various time points after the application of such keratinocytes, starting as early as day 3 (i.e., 48 h after keratinocyte application); evidently, such investigations are not possible with patients in vivo, illustrating the value of our in vitro model. Interestingl