Growth factors and extracellular matrix proteins during ... - Springer Link

Cell Tissue Res (2002) 307:79–89 DOI 10.1007/s004410100450

REGULAR ARTICLE

Elisa Tamariz-Domínguez Federico Castro-Muñozledo · Walid Kuri-Harcuch

Growth factors and extracellular matrix proteins during wound healing promoted with frozen cultured sheets of human epidermal keratinocytes Received: 15 January 2001 / Accepted: 20 July 2001 / Published online: 14 November 2001 © Springer-Verlag 2001

Abstract In a murine model of full-thickness wounds, healing is stimulated by the application of human frozen cultured epidermal sheets. With immunofluorescence techniques, we studied, during this process, the spatial and temporal pattern of expression of: transforming growth factor-α (TGF-α); transforming growth factor-β (TGF-β) isoforms 1, 2, and 3; platelet-derived growth factor (PDGF); and the extracellular matrix proteins fibronectin, collagen IV, and tenascin. The growth factors, with the exception of PDGF, were found to be located in the frozen cultured sheet of keratinocytes before and after its application to the wound, whereas collagen IV and tenascin were deposited in the connective tissue under the frozen cultures. None of these factors were detected in control wound beds. Monoclonal antibodies against collagen IV and tenascin showed that both were of murine origin. We propose that the frozen cultures of human keratinocytes promote faster reepithelialization through the release of growth factors such as TGF-α which directly enhance migration and proliferation of murine keratinocytes, and through the stimulation of murine subepithelial cells, by TGF-β, to secrete basement membrane proteins such as collagen IV, laminin, and tenascin, which provide a provisional substrate that improves migration of the murine epidermal cells. Keywords Cultured epidermis · Keratinocyte · Epithelialization · Wound healing · Mouse (NMRI) · Human

This work was supported in part by a CONTACyT grant # G28272-N. E. Tamariz-Domínguez · F. Castro-Muñozledo W. Kuri-Harcuch (✉) Department of Cell Biology, Centro de Investigación y Estudios Avanzados del IPN, Apdo. Postal 14–740, Mexico City 07000, Mexico e-mail: [email protected] Tel.: +52-5-7477000 ext. 5521, Fax: +52-5-7477081

Introduction Healing of full-thickness wounds requires reepithelialization from the margins of the wound through keratinocyte proliferation and migration over a provisional extracellular matrix converted to granulation tissue that is then remodeled to neodermis (Clark 1996). In attempts to achieve an accelerated rate of tissue repair, the exogenous addition of single growth factors such as plateletderived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and TGF-β to animal wounds usually stimulated granulation tissue formation or reepithelialization (Grotendorst et al. 1985; Lynch et al. 1987; Mustoe et al. 1987; Schultz et al. 1987; Ksander et al. 1989; Greenhalgh et al. 1990; Quaglino et al. 1990; Pierce et al. 1991a, 1991b; Benn et al. 1996); however, wound healing seems to depend on a combination of various types of growth factors (Lynch et al. 1987; Greenhalgh et al. 1990) and extracellular matrix proteins (Quaglino 1990; Pierce et al. 1991b; Juhasz et al. 1993; Gailit and Clark 1994). Keratinocytes secrete cytokines and extracellular matrix proteins that modulate cell proliferation, migration, and differentiation during skin wound healing. Among cytokines, TGF-α, TGF-β, and PDGF are involved in tissue repair after wounding (Rappolee et al. 1988; Ksander et al. 1989; Pierce et al. 1989, 1991b; Levine et al. 1993; Benn et al. 1996; Gold et al. 1997; Martin 1997). TGF-α is present early after wounding as it is secreted initially by platelets and later by macrophages and keratinocytes (Coffey et al. 1987; Schultz et al. 1991; Nanney and King 1996). TGF-α promotes keratinocyte migration and proliferation (Barrandon and Green 1987; Cha et al. 1996) and, by increasing the expression of α2integrin, it mediates keratinocyte migration on collagens I and IV (Chen et al. 1993). The application of TGF-α to second-degree burns in pigs accelerated epidermal regeneration (Schultz et al. 1987). TGF-β is chemotactic for monocytes, fibroblasts, and neutrophils. It is secreted at the wound site by many cell

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types such as macrophages, platelets, fibroblasts, endothelial cells, smooth muscle cells, and keratinocytes (Roberts and Sporn 1996). TGF-β also stimulates production of collagen and fibronectin by fibroblasts (Ignotz and Massague 1986; Rossi et al. 1988), and it modulates integrin expression in keratinocytes (Hebda 1988; Zambruno et al. 1995). Exogenous application of TGF-β1 and -β2 to wounds increases granulation tissue formation and tensile strength (Mustoe et al. 1987; Ksander et al. 1989; Benn et al. 1996), which in turn could lead to stimulation of scarring (Shah et al. 1995). PDGF is chemotactic for fibroblasts, monocytes, and neutrophils, and it induces the secretion by fibroblasts of extracellular matrix proteins that promote granulation tissue formation (Grotendorst et al. 1985; Lynch et al. 1987; Greenhalgh et al. 1990). Overexpression of a transfected PDGF gene in sheets of cultured human keratinocytes transplanted onto athymic mice promoted thickening of connective tissue and an increased number of blood vessels (Eming et al. 1995). Extracellular matrix components have an important function during wound healing, from the formation of blood clot up to remodeling of the scar tissue (Gailit and Clark 1994). Fibronectin is found in the early provisional extracellular matrix and it is chemotactic for neutrophils, monocytes, fibroblasts, and endothelial cells, which contribute to granulation tissue formation (Grinnell 1984). Fibronectin promotes the attachment and spreading of keratinocytes in vitro, and fibronectin receptors are expressed in the keratinocytes obtained from wounded skin (Grinnell et al. 1987). Tenascin is secreted by both fibroblasts (Rettig et al. 1994) and keratinocytes (Latijnhouwers et al. 1997). In the fetus, tenascin is induced early during wound repair and seems to enhance keratinocyte migration and wound closure (Whitby and Ferguson 1991). Tenascin is expressed later in granulation tissue and beneath the migrating epidermal cells at the dermal-epidermal junction in adult wounds but not in fetal wounds (Mackie et al. 1988; Murakami et al. 1989; Fassler et al. 1996; Latijnhouwers et al. 1997). The application of cultured sheets of human allogeneic keratinocytes, both fresh and preserved by freezing, to partial-thickness wounds, skin donor sites, and venous and diabetic ulcers of the leg promotes faster reepithelialization and formation of granulation tissue (Madden et al. 1986; De Luca et al. 1989; Phillips et al. 1989; Bolívar-Flores et al. 1990; Beele et al. 1991; De Luca et al. 1992; Fratianne et al. 1993; Núñez-Gutiérrez et al. 1996; Rivas-Torres et al. 1996; Bolívar-Flores and KuriHarcuch 1999; Alvarez-Diaz et al. 2000). We have shown previously that frozen cultures applied to fullthickness wounds in immunocompetent mice stimulate granulation tissue formation and accelerate reepithelialization (Tamariz et al. 1999). The mechanism of action of the frozen cultures in promoting tissue repair remains unknown; it might require not only release of soluble growth factors but also the deposition of extracellular matrices (Tamariz et al. 1999).

By indirect immunostaining of PDGF, TGF-α, and the TGF-β isoforms, fibronectin, collagen IV, and tenascin, we show that murine full-thickness wounds treated with cultured keratinocytes are altered in their spatial and temporal pattern of deposition of growth factors and extracellular matrices. This combination of growth factors and extracellular matrices released or induced by the frozen cultured sheets may be responsible for the improvement of the wound healing process.

Materials and methods Human epidermal keratinocytes, strain HE-120, were obtained from the foreskin of a newborn and they were serially cultured as described by Rheinwald and Green (1975), with certain modifications (Bolívar-Flores et al. 1990). The 3T3 fibroblasts used as feeder layers (Rheinwald and Green 1975) were cultured and lethally treated with mitomycin C as described previously (BolívarFlores and Kuri-Harcuch 1999; Tamariz et al. 1999). Keratinocytes from the 13th culture passage of strain HE-120 were used for epidermal sheet production; the cultured sheets were backed with petrolatum-coated gauze (Bolívar-Flores et al. 1990) and were frozen at –70°C until further use (Tamariz et al. 1999). After thawing, the cultured sheets did not contain proliferative cells, but some metabolic activity was shown by the fluorescein diacetate staining (Tamariz et al. 1999). NMRI mice (30 g) were used for experiments according to the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85–23, revised 1985, updated 1996). Mice were anesthetized and two 1-cm2 full-thickness wounds down to muscle fascia were made on the back of each mouse. Wounds in each mouse were randomized; one wound was covered with a single application of a frozen and thawed sheet of cultured human epidermal cells, and the other one was covered with petrolatum-coated gauze as control (Tamariz et al. 1999). Opsite (Smith and Nephew Medical, UK) was applied on top of the petrolatum-coated gauze, wrapped with sterile gauze, and held in place with adhesive tape. Mice were caged with water and food ad libitum. At least six mice were anesthetized and killed for each indicated healing time; the wounded tissue and its surrounding normal skin were carefully removed, embedded with tissue freezing medium (Jung), and frozen at –70°C until cryosectioning. Immunostaining Cryosections (8 µm) in silanized glass slides were air-dried and then hydrated in phosphate-buffered saline (PBS) for 10 min. After blocking with 20% bovine or rabbit serum in PBS (v/v), slides were incubated with the primary antibodies for 1 h (see Table 1). They were washed with PBS and incubated again with the bovine or rabbit serum for 1 h and then incubated with the fluorochrome-labeled secondary antibody (see Table 1). For antiPDGF, secondary antibody was biotinylated and a third incubation with streptavidin-FITC was carried out. Slides were washed with PBS and mounted in Dako fluorescent mounting medium (Dako Corporation, Calif., USA). Staining with monoclonal antibodies was carried out with the Vector mouse on mouse immunodetection kit (Vector laboratories, Burlingame, Calif. USA) using 3′,3-diaminobenzidine as a peroxidase substrate (Vector Laboratories, Burlingame, Calif., USA) following the manufacturer's instructions.

81 Table 1 Antibodies and reagents used for immunostaining Antigen

Antibody specificity

Dilution

Source

Tenascin

Polyclonal (rabbit) against human tenascin Monoclonal against human tenascin Polyclonal (rabbit) against mouse fibronectin Polyclonal (rabbit) against mouse collagen IV Monoclonal against human collagen IV Polyclonal (goat) against rat, bovine and human TGF α Polyclonal (goat) against human PDGF-AB

1:50 1:50 1:75 1:50 1:40 1:25 1:50

Polyclonal (rabbit) against the carboxy terminus of the precursor form of human TGF-β1 Polyclonal (rabbit) against the carboxy terminus of the precursor form of human TGF-β2 Polyclonal (rabbit) against the carboxy terminus of the precursor form of human TGF-β3 Polyclonal (goat) Polyclonal (rabbit)

1:15

Polyclonal (rabbit)

1:400 5 µg/ml

Chemicon (Temecula Calif.) Chemicon (Temecula Calif.) Chemicon (Temecula, Calif.) Chemicon (Temecula, Calif.) Chemicon (Temecula, Calif.) Chemicon (Temecula, Calif.) Upstate Biotechnology (Lake Placid, N.Y.) Santa Cruz Biotechnology (Santa Cruz,Calif.) Santa Cruz Biotechnology (Santa Cruz,Calif.) Santa Cruz Biotechnology (Santa Cruz,Calif.) ICN Biomedicals (Costa Mesa, Calif.) Zymed (Carlton Court, San Francisco, Calif.) Dako (Carpinteria, Calif.) Boehringer Mannheim (USA)

Fibronectin Collagen IV TGF-α PDGF-AB TGF-β1 TGF-β2 TGF-β3 Anti-rabbit (FITC) Anti-goat (FITC) Anti-goat (Biotin) Streptavidin (FITC)

Results In a previous report, we showed the effects of frozen cultures of human keratinocytes on wound healing by quantitating the rate of reepithelialization of full-thickness skin wounds in immunocompetent NMR1 mice (Tamariz et al. 1999). The cultured sheets accelerated the rate of reepithelialization by about 76%, even though the cultures contained no keratinocytes capable of multiplication (Tamariz et al. 1999). In such experiments, the cultured human epidermal sheet is lost 4–6 days after its application onto the wound bed, and most wounds treated with frozen cultures completely healed after 10 days, whereas control wounds required 16 days. Treatment with the frozen cultures also promoted early formation of granulation tissue and laminin deposition over the surface of the wound bed. Although stimulation of wound healing and reepithelialization has been attributed to cytokine secretion by the cultured keratinocytes at the wound site, our previous results suggest that the accelerated reepithelialization and granulation tissue formation observed in the presence of the cultured sheets might be also due to relatively insoluble factors originating from the cultured cells, or to the stimulation of their expression in the host tissue. Therefore we studied, using immunochemical methods, the expression of growth factors and extracellular matrix proteins, whose formation was influenced by the application of frozen human cultured epidermal sheets to fullthickness wounds in mice. The cultured keratinocyte sheets, when thawed, were strongly stained by antibodies against TGF-α, TGF-β1, -β2, and -β3 (Fig. 1), but not by antibodies against PDGF, fibronectin, collagen IV, or tenascin. This shows that frozen cultures possessed the TGF class of factors, but not extracellular matrix proteins or PDGF.

1:20 1:15 1:500 1:250

Immunolocalization of growth factors and extracellular matrix proteins PDGF and fibronectin The thawed sheet of cultured human keratinocytes did not show PDGF or fibronectin staining (Fig. 1). In contrast, immunoreactive PDGF was present in the newly formed murine epithelium of both control and treated wounds. PDGF expression was also evaluated in the open wound bed at various times after wounding. Staining in control and treated wounds was similarly faint at the 1st day, then it became strong at 2 days and 3 days, and began to fade after 4 days, until it was undetectable at day 10 (not shown). Also, at all days studied, the polyclonal antibody against fibronectin showed similar strong staining in both control and treated wound beds (not shown). Transforming growth factor-α The frozen cultured sheet showed strong staining for TGF-α (Fig. 1). The open control wounds did not show TGF-α staining at any day (Fig. 2, 1C, 2C, 4C, 6C); the sheet of human keratinocytes remained strongly stainable during the 1st day and 2nd day, and became fainter at the 4th day and 6th day. (Fig. 2, 1T, 2T, 4T, 6T). At all times analyzed, up to 10 days, when the reepithelialization of treated wounds was complete, an intense immunoreactivity against TGF-α was seen in the advancing murine epithelium of treated and control wounds (not shown). Western blot analysis with the same antibody in extracts from the keratinocyte sheet demonstrated the presence of the TGF-α, suggesting that immunodetection of this growth factor seemed to be specific (not shown).

82 Fig. 1 Immunostaining of frozen cultured human epidermal sheets. Thawed human epidermal cultures were labeled with antibodies (see Table 1) against fibronectin (Fn), collagen IV (Col-IV), tenascin (TN), and platelet-derived growth factor (PDGF); these antibodies did not show staining of the cultured sheet. Transforming growth factor-α (TGF-α) was stained in all keratinocyte layers. TGF-β1 showed strong staining in the keratinocyte suprabasal layers; TGF-β2 showed weak and diffuse staining of all keratinocyte layers; TGF-β3 showed strong labeling of the keratinocyte basal layer. Dotted line and arrows indicate the basal surface of the cultured sheet. Bar 10 µm

Fig. 2 Immunostaining of TGF-α in the open wound bed. Control wounds do not show staining against TFGα on any day (1C, 2C, 4C, 6C); however, there is an intense staining of the keratinocyte cultured sheet on the wound bed at 1 day and 2 days (1T, 2T), becoming fainter at 4 days and 6 days (4T, 6T). Arrowheads frozen cultured epidermal sheet. Dotted lines indicate the surface of the wound bed (1C, 2C, 4C, 6C) (LCT loose connective tissue). Bar 10 µm

Transforming growth factor-β We tested for each of the isoforms TGF-β1, -β2, and-β3. These experiments were carried out with specific antibodies raised against a peptide fragment of the carboxy terminus of the corresponding precursor isoform of human origin (Table 1). The frozen cultured sheet showed strong staining for each of the isoforms; the presence of TGF-β isoforms in the cultured sheets was also confirmed by Western blot analysis (not shown). TGF-β1

and -β2 were detected in all epidermal layers of the frozen cultures, whereas TGF-β3 was seen in the basal layer (Fig. 1). TGF-β1, -β2, and -β3 were also evaluated in the open wound bed at various times after wounding; in control wounds none of the isoforms were detected at any time during healing (Fig. 3, 1C–6C). However, in treated wounds, each isoform showed an intense staining in the grafted cultured sheet up to 2 days for TGF-β2, and up to 4 days for TGF-β1 and -β3 (Fig. 3, 1T–6T). In all three

83 Fig. 3 Immunostaining of TGF-β1, TGF-β2, and TGF-β3 in the wound. TGF-β1 shows a strong fluorescent signal in the frozen culture on day 1 and day 3 after its application to the wound (1T, 3T), becoming fainter at day 4 (4T) and almost disappearing at day 6 (6T). TGF-β2 staining was weak in the frozen cultures at 1 day and 2 days (1T, 2T), almost disappearing at 4 days (4T), and becoming undetectable at 6 days (6T). TGF-β3 stained in the basal cells of the frozen cultures at 1 day, 2 days, 4 days, and 6 days (1T, 2T, 4T, 6T) In all cases, control wounds did not show labeling for any of the TGF-β isoforms (1C–6C). Dotted lines indicate the surface of the wound (LCT loose connective tissue). Bar 10 µm

cases, at 6 days of treatment only remnant fragments of the grafted cultured sheet showed some staining for each of the isoforms (Fig. 3, 1T–6T). Immunoreactivity for the three antibodies was similar in the newly formed murine epithelium of both control

and treated wounds; immunostaining was seen in all epidermal layers. In both wounds, the epithelium was strongly immunostained during the first 3–4 days after wounding, then staining became fainter and slightly detectable at day 8, with the exception for TGF-β2, which

84 Fig. 4 Immunostaining of collagen IV in the wound. Treated wounds showed collagen deposition under the frozen culture in wounds as early as 1 day after application of the frozen sheet (1T), and the collagen staining continued to be strong for at least 4 days (4T); the antibody also stained collagen IV at the basement membrane of blood vessels (arrows) found in granulation tissue (6T, inset). Arrowheads frozen cultured epidermal sheet. In contrast, the antibody did not show any staining of control wounds at 1–6 days of wound healing (1C, 2C, 4C, 6C). Dotted lines indicate the surface of the wound bed (1C, 2C, 4C, 6C) (LCT loose connective tissue). Bar 15 µm

Fig. 5 Immunostaining of tenascin in the wound. With polyclonal antibody, tenascin was strongly stained in the wound under the frozen cultures at 2 days and 3 days (2T, 3T), but staining was absent on the 1st day after treatment (1T) and diffuse in the mesenchyme at 6 days (6T). Arrowheads frozen cultured epidermal sheet. In control wounds, staining of the mesenchyme was absent during day 1 and day 2 (1C, 2C) and diffuse and weaker than in treated wounds at later times (3C, 6C). Dotted lines indicate the surface of the wound bed (1C, 2C) (LCT loose connective tissue). Bar 10 µm

showed strong staining even at 8 days (not shown). The antibody against TGF-β3 also showed similar staining of the newly formed murine epithelium in both control and treated wounds; however, staining was weaker and more diffuse than with the antibodies against TGF-β1 and -β2 (not shown) Collagen IV Open control wounds at all days analyzed showed poor staining with the antibody against collagen IV (Fig. 4, 1C–6C). However, treated wounds demonstrated a strong and well-defined stained band under the cultured sheet beginning 1 day after its application and continu-

ing for 4 days; thereafter it became less intense and poorly defined (Fig. 4, 1T–6T). A monoclonal antibody against human collagen IV did not show any staining under the cultured epithelium at any day analyzed (Fig. 6). This shows that the collagen IV accumulated in the treated wounds is not of human origin but is murine. On the other hand, the antibody against collagen IV showed similar labeling under the newly formed epithelium in both control and treated wounds. After 2 days, collagen IV staining was more diffuse near the outgrowth tip, but it was stronger and well defined under the epithelium that had already migrated (not shown). A similar pattern was observed in control wounds but at corresponding later days than in the treated wounds.

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Fig. 6 Immunostaining of collagen IV (Col) with a monoclonal antibody against human collagen IV, which did not cross-react against mouse collagen (Mo), showed an intense staining of the connective tissue under the normal human skin (Hm). 2d 2-day treated wound is not stained with the monoclonal antibody under the frozen cultured epidermal sheet (arrowhead). Bar 12 µm. TN Immunostaining of tenascin with a monoclonal antibody against human tenascin shows an intense staining under the human skin (Hm), but did not stain the mouse tissue (Mo). 3d 3-day treated wounds were not stained with the monoclonal antibody under the frozen cultured epidermal sheet (arrowhead). Bar 12 µm

Tenascin The open control wounds did not show tenascin staining at days 1 and 2 after wounding, but, at the 3rd day, staining although sparse was seen in the mesenchymal tissue (Fig. 5, 1C, 2C, 3C); after 6 days it was more homogeneously distributed throughout this mesenchyme (Fig. 5, 6C). The antibody did not show staining of tenascin in the thawed cultures (Fig. 1) or under the grafted sheet during the 1st day after its application on the open wound (Fig. 5, 1T). However, at 2 days and 3 days, tenascin was intensely stained under the cultured sheet in the loose mesenchymal tissue beneath (Fig. 5, 2T, 3T). After day 4 and up to day 8, tenascin was more diffusely distributed in the mesenchymal tissue underneath the cultured sheet and in its few remaining fragments, since it was partially lost by day 6 (Fig. 5, 6T; see Tamariz et al. 1999). At all times, tenascin staining was less abundant in control than in the treated wounds (Fig. 5). Monoclonal antibody against human tenascin did not show staining under the cultured sheet at any day analyzed (Fig. 6), indicating that tenascin found in the treated wounds is of murine origin. The antibody against tenascin showed similar labeling at the dermal-epidermal junction of the newly formed murine epithelium in both control and treated wounds at all days studied; tenascin was observed at the basement membrane zone of the advancing basal keratinocytes (not shown)

Discussion Wound healing depends on a combination of growth factors (Lynch et al. 1987; Greenhalgh et al. 1990) and extracellular matrix proteins (Quaglino 1990; Pierce et al. 1991b; Juhasz et al. 1993; Gailit and Clark 1994). Reepithelialization and epidermal differentiation is regulated by the supporting mesenchyme at the wound bed, since migration and a properly organized epithelium depend on its interaction with the adequate mesenchymal substratum (Dodson 1967). In this work, we have applied immunochemical techniques to study the early events related to growth factors and extracellular matrix proteins, whose formation was influenced by the application of frozen human cultured epidermal sheets to full-thickness wounds in mice. Growth factors TGF-α is present early during wound healing and seems to have a relevant role particularly in reepithelialization (Schultz et al. 1991; Nanney and King 1996). In addition, TGF-α has a more potent effect in promoting in vitro keratinocyte migration than epidermal growth factor (EGF; Barrandon and Green 1987; Cha et al. 1996). TGF-α also stimulates the formation of tubular-like structures by human omental microvascular endothelial cells. Since tubulogenesis is blocked with antibodies against TGF-α, it has been suggested that this cytokine might stimulate skin angiogenesis (Ono et al. 1992). The application of TGF-α to skin wounds in vivo also accelerates epidermal regeneration (Schultz et al. 1987, 1991). Our results also show that improved healing of treated wounds is associated with the presence of TGFα in the cultured sheets. This result has led us to suppose that the accelerated reepithelialization that we observed in wounds treated with the frozen cultures (Tamariz et al. 1999) could be due to growth factors such as TGF-α. This stimulatory effect could be due to a direct stimulation of keratinocyte migration by the TGF-α released from the frozen cultures. Also, TGF-α, in addition to other angiogenic factors, could be promoting an earlier formation of blood vessels in the new

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granulation tissue exposed to the cultured sheets (Tamariz et al. 1999). The exogenous application of TGF-β to guinea pig, rat, or rabbit wounds (Mustoe et al. 1987; Ksander et al. 1989; Pierce et al. 1991b), or transfection of the TGF-β1 gene into rat skin (Benn et al. 1996) stimulates granulation tissue formation, collagen fibrillogenesis, collagen bundle formation, and increased wound strength. This effect is probably related to stimulation of fibronectin and collagen synthesis in fibroblasts (Ignotz and Massague 1986; Rossi et al. 1988), to downregulation of collagenase, and to stimulation of synthesis of tissue inhibitor metalloproteinases (Edwards et al. 1987; Overall et al. 1989). It has been suggested that, both in vivo and in vitro, TGF-β modulates epidermal keratinocyte integrins to a migratory phenotype (Zambruno et al. 1995) and stimulates keratinocyte migration (Zambruno et al. 1995; Roberts and Sporn 1996; Nickoloff et al. 1988; Chen et al. 1992). Before their application on the wound bed, the frozen human cultured keratinocytes showed strong immunostaining for TGF-β1, -β2, and -β3. These TGF-β isoforms remained in the cultured sheets for up to 6 days after their application to the wound bed. Consistent with previous reports showing that epithelial cells constitute the main source of TGF-βs in wounds (Levine et al. 1993), our control wounds did not have any detectable staining for these isoforms on their surface. Therefore, the TGF-β isoforms present on the treated wounds were released from the cultured sheets (Figs. 1, 3), contributing toward accelerating the formation of granulation tissue, as we have observed in the culture treated wounds (Tamariz et al. 1999). Although TGF-β stimulates fibronectin secretion by keratinocytes (Nickoloff et al. 1988) and upregulates fibronectin receptors (Takeshima and Grinnell 1985), we did not observe any difference in fibronectin expression between control wounds and those treated with the frozen cultures. Apparently the frozen cultures do not promote reepithelialization through stimulation of fibronectin secretion. It has been demonstrated that TGF-β1 stimulates the upregulation of laminin-5 (LN-5) synthesis (Korang et al. 1995) and the modulation of its processing mediated by metalloproteinases and tissue plasminogen activator (tPA; Decline and Rousselle 2001; Goldfinger et al. 1998, 1999; Gianelli et al. 1997). Also, TGF-β1 induces a migratory phenotype in tracheal epithelial cells (Boland et al. 1996) and epidermal keratinocytes (Decline and Rousselle 2001). On the other hand, addition of TGF-β1 to human keratinocyte cultures stimulates their migration through the deposition of nonprocessed LN-5 (Decline and Rousselle 2001). In contrast, the processed form of LN-5 predominates in mature epithelia (Decline and Rousselle 2001) and promotes cell adhesion and hemidesmosome formation (Goldfinger et al. 1999). Since we previously showed that the cultured epidermal sheets promote an earlier deposition of laminin into the wound bed (Tamariz et al. 1999), it is possible that the TGF-β isoforms released by the cultured

sheets into the wound might also stimulate reepithelialization through changes in synthesis, processing, and deposition of LN-5 by the newly formed epithelium. Exogenous addition of TGF-β1 and TGF-β2 in rat and pig skin wounds induces scar formation and accumulation of fibronectin and collagens I and III, as compared to TGF-β3, which had an antiscarring effect (Quaglino et al. 1990; Shah et al. 1994, 1995; Clark et al. 1997). In our full-thickness wounds in mice, we did not see a clear fibrotic response or overaccumulation of fibronectin in the wounds treated with the cultures. The cultured keratinocytes release TGF-β1 and -β2, but also significant staining for TGF-β3 was detected (Fig. 1). It is possible that TGF-β3 in the cultured sheet might overcome the scarring response to TGF-β1 and -β2. It is known that the exogenous addition of TGF-β3 or of neutralizing antibodies against TGF-β1 and -β2 significantly diminished the scarring effect of TGF-β1 and -β2, improving rearrangement of neodermis (Shah et al. 1994, 1995). It is well known that PDGF stimulates secretion of TGF-β by monocytes and fibroblasts (Pierce et al. 1989, 1991a), and TGF-β seems to enhance the expression of collagen types I and III in fibroblasts and granulation tissue (Ignotz and Massague 1986; Rossi et al. 1988; Quaglino et al. 1990). Our results showed no significant differences between treated and control wounds in the expression of PDGF. It seems that PDGF does not contribute significantly to accelerate healing of the wounds treated with the cultured sheets. Cultured keratinocytes have low concentrations of PDGF, about 2.1 ng/107cells per 24 h,and they do not show any stimulation of granulation tissue formation when they are applied on murine skin flaps (Eming et al. 1995). However, keratinocytes transfected with the PDGF gene, secreting about 300fold more PDGF (744 ng/107cells per 24 h) than nontransfected cells, stimulate granulation tissue formation under the skin flap (Eming et al. 1995). In addition, exogenous application of high concentrations of PDGF (in the range of 50 ng to 10 µg/wound) stimulate granulation tissue formation but not reepithelialization (Grotendorst et al. 1985; Lynch et al. 1987; Greenhalgh et al. 1990). Since we are using the same techniques for keratinocyte cultivation as Eming et al. (1995), PDGF in our cultured sheets should be at similar low levels of the nontransfected keratinocytes. Therefore, we can suggest that PDGF does not have a significant role in granulation tissue formation and reepithelialization of the wounds treated with frozen cultures. We suggest that in our model the induction of murine TGF-β by PDGF in the culture-treated wounds does not seem to be a determinant step, since the frozen cultures significantly express TGF-β. Extracellular matrix proteins We examined the appearance of some components of the dermoepidermal junction, mainly fibronectin, collagen IV, and tenascin that are closely associated with the base-

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ment membrane and that should have important functions in keratinocyte migration. Several reports have shown that keratinocytes and dermal fibroblasts synthesize basement membrane components (Briggaman et al. 1971; Alitalo et al. 1982; Regauer et al. 1990; Marinkovich et al. 1992). The pattern of immunostaining we observed during wound repair showed that there are some important differences in control and treated wounds in the expression of extracellular matrix proteins. It has been reported that collagen IV promotes the transient anchorage and migration of keratinocytes at the leading edge of the wound (Kim et al. 1994; Legrand et al. 1999). When interaction of the migrating cells with collagen IV is interrupted with antibodies against this collagen type, migration is blocked and only cell spreading seems to take place (Legrand et al. 1999). In addition, it has been reported that keratinocytes show faster migration rates onto collagen IV substrata than on any other basement membrane components such as laminin (Woodley et al. 1988). Our studies showed deposition of collagen IV in the wound surface under the cultured sheet but not at the wound surface of control wounds. Based on the previously reported role of collagen IV in keratinocyte migration, our results suggest that deposition of collagen IV in the wound bed of the treated wounds might also improve the migration of the murine keratinocytes at the leading edge. In our studies, the pattern of tenascin expression in control wounds was similar to previous reports (Mackie et al. 1988), but in wounds treated with the frozen cultures, tenascin expression appeared earlier and more strongly than in control wounds. This is consistent with the previously observed acceleration of epithelialization (Tamariz et al. 1999). It has also been shown that an early expression of tenascin during fetal wound healing correlates with an early reepithelialization (Whitby and Ferguson 1991), probably because tenascin reduces the strength of cell adhesion that is normally seen on fibronectin substratum (Lotz et al. 1989), and inhibits integrin-mediated attachment to fibronectin, laminin, and GRGDS peptide as demonstrated in chick fibroblasts (Chiquet-Ehrismann et al. 1988). Our results suggest that the early expression of tenascin in the wounds treated with the frozen cultures might stimulate migration of the keratinocytes from the wound edge. It has been reported that tenascin expression could be induced in fibroblasts by TGF-β, PDGF-BB, basic FGF (bFGF), tumor necrosis factor-α (TNFα), or interleukin-1β (IL-1β; Tucker et al. 1993; Rettig et al. 1994), and in keratinocytes after stimulation with inflammatory cytokines such as TNFα, interferon-γ (IFN-γ), and IL-4 (Latijnhouwers et al. 1997, 1998). But in our study, monoclonal antibody against human tenascin did not show any accumulation under the cultured sheet, indicating that the accelerated formation of tenascin, originated from the murine fibroblasts, was possibly induced by TGF-β released by the frozen cultures. Since the antibody against fibronectin showed the same pattern and intensity of staining in both control and

treated wound beds as well as underneath the newly forming epithelium, we conclude that fibronectin deposition, although important for tissue repair in general, does not seem to have a significant role in the promotion of healing by the frozen human cultures. Therefore, in relation to extracellular matrix deposition, the only changes associated with enhancement of murine keratinocyte migration that we have identified so far are in collagen IV, tenascin, and laminin. The frozen human keratinocyte cultures do not have cells with the ability to grow (Tamariz et al. 1999). We suggest that the frozen cultures promote tissue repair, at least in part, through the release of, among other components, TGF-α and TGF-βs. TGF-α would stimulate keratinocyte migration, and TGF-βs would increase the deposition of collagen IV, laminin, and tenascin promoting reepithelialization through the formation of an early substrate containing these basement membrane proteins. Acknowledgements We are grateful to Dr. Howard Green for his critical reading of the manuscript. We thank the technical assistants of the Unit of Epidermal Technology at the Department of Cell Biology, Alicia Beltrán-Langarica, María Teresa Robledo, Margarita Valades, and Norma Barragán for their work in culturing the human epidermal sheets. We also thank Ms. María Elena Rojano for her secretarial assistance and Mr. Alberto Rodríguez for his technical assistance in preparing all materials.

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