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FGF-7 Expression Enhances the Performance of Bioengineered Skin
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doi:10.1016/j.ymthe.2004.04.013
FGF-7 Expression Enhances the Performance of Bioengineered Skin Gulsun Erdag,1 Daniel A. Medalie,1 Hinne Rakhorst,1 Gerald G. Krueger,2 and Jeffrey R. Morgan1,3,* 1
Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School and Shriners Hospital for Children, Boston, MA 02114, USA 2 Department of Dermatology, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA 3 Center for Biomedical Engineering and Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA
*To whom correspondence and reprint requests should be addressed at Brown University, G-B 393, Biomed Center, 171 Meeting Street, Providence, RI 02912, USA. Fax: (401) 863-1753. E-mail: Jeffrey_ [email protected].
Available online 4 June 2004
To improve the performance of bioengineered skin, we used a recombinant retrovirus encoding FGF-7 to modify diploid human keratinocytes genetically. Control or FGF-7-expressing keratinocytes were seeded onto acellular human dermis to form bioengineered skin. Gene-modified skin secreted significant levels of FGF-7 and formed a thicker and hyperproliferative epidermis with about four times the number of cells per square centimeter. Secretion of an endogenous trophic factor, VEGF, was increased f5-fold. Migration of FGF-7-expressing keratinocytes was stimulated as was the self-healing of bioengineered skin expressing FGF-7. When tested in a bacterial infection model, the antimicrobial properties of FGF-7-expressing skin were increased >500-fold against both gram-negative and gram-positive bacteria. After transplantation to full-thickness wounds on athymic mice, skin expressing FGF-7 was revascularized more rapidly. These results demonstrate that genetic modification can be used to enhance performance and that expression of FGF-7 augments several properties important to the wound-healing properties of bioengineered skin. Key Words: fibroblast growth factor, skin, wound healing, vascularization, infection, tissue engineering
INTRODUCTION Burns and chronic ulcers of the skin are significant medical problems in need of new treatments. Annually in the United States, there are more than 1 million burn injuries (Burn Incidence and Treatment in the U.S.A., 2000 Fact Sheet, American Burn Association, http://www.ameriburn.org), an estimated 800,000 diabetic ulcers leading to about 80,000 amputations (National Diabetes Fact Sheet 2002, Centers for Disease Control, http://www.cdc.gov/ health/diabetes.htm), and about 1 million venous ulcers [1], of which, only 40 – 50% are healed by standard therapies. Bioengineered skin substitutes are a potential treatment for the closure of skin defects and methods are well established for the culture of large numbers of human epidermal keratinocytes as well as dermal fibroblasts [2 – 4]. Cultured autologous keratinocytes have been used as living cell sheets for the treatment of burn wounds, and allogeneic keratinocytes and fibroblasts in a collagen matrix have been combined to form bilayered skin constructs for the treatment of chronic skin ulcers [5,6].
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We and others have been exploring the possibility of enhancing the function and thus the performance of bioengineered skin by genetic modification of the cells [7 – 9]. Unlike gene therapy for inherited diseases in which the gene to be delivered is usually known, it is unclear which gene will have a significant impact on the performance of tissue-engineered skin. Since normal bioengineered skin is thought to promote healing by the local synthesis of endogenous growth factors, one area of interest has been to use genetic modification to program the cells to produce higher levels of growth factors. Genemodified skin that secretes elevated levels of insulin-like growth factor-1, platelet-derived growth factor A chain and B chain, vascular endothelial growth factor (VEGF), hepatocyte growth factor, or epidermal growth factor (EGF) has been made [7 – 12]. These studies have demonstrated the feasibility of this approach and have shown that growth factor production can influence one facet of the performance of bioengineered skin. In this report, we show that unlike other growth factors tested, production of fibroblast growth factor-7
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(FGF-7) by genetically modified skin enhances several significant aspects of the performance of bioengineered skin. Compared to controls, gene-modified skin producing FGF-7 (i) has a thicker, more hyperproliferative epidermis that heals more rapidly after injury; (ii) has increased antimicrobial activity against gram-negative and gram-positive bacteria; (iii) secretes more endogenous trophic factors such as VEGF; and (iv) is more rapidly revascularized after transplantation. Bioengineered skin expressing FGF-7 appears to be activated in a way that may make it more effective at closing problematic wounds.
RESULTS Bioengineered Skin with Gene-Modified Keratinocytes Secretes FGF-7 and Higher Levels of Other Trophic Factors such as VEGF We modified diploid human keratinocytes genetically with a recombinant retrovirus encoding FGF-7 and mea-
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sured the levels of secreted FGF-7 by ELISA. FGF-7 was continuously secreted by confluent keratinocytes grown on tissue culture plastic and accumulated in the medium at a rate of approximately 0.6 ng/106 cells/24 h. In control unmodified cells, FGF-7 levels were undetectable (detection limit <15 pg/ml) (Fig. 1A). We seeded the same cells onto the papillary surface of acellular human dermis raised to the air/liquid interface for 7 days to form a stratified and differentiated epidermis. The rate of FGF-7 accumulation from this bioengineered skin was 3.24 ng/ cm2/24 h. FGF-7 levels in matched samples of control, unmodified keratinocytes were undetectable (Fig. 1B). Keratinocytes naturally synthesize and secrete numerous growth factors involved in autocrine and paracrine signaling. We measured the levels of VEGF secreted by our bioengineered skin. Skin formed with control unmodified keratinocytes secreted VEGF at a rate of 1.8 ng/ cm2/24 h, while skin formed with keratinocytes expressing FGF-7 secreted VEGF at a rate of 9.1 ng/cm2/24 h, an approximately fivefold increase per surface area ( P < 0.01)
FIG. 1. Gene-modified bioengineered skin secretes FGF-7 and increased levels of VEGF. A time course of FGF-7 secretion was determined by removing portions of the culture medium of confluent cultures of control or genetically modified keratinocytes grown (A) on tissue culture plastic or (B) as bioengineered skin. Levels of FGF-7 protein were determined by ELISA. (C) Culture media from bioengineered skin were also assayed for levels of VEGF by ELISA ( P < 0.01, FGF-7 vs control). (D) To determine cell number, bioengineered skin was treated with dispase, followed by trypsin and Hoechst staining to facilitate the counting of nucleated cells (*P < 0.001, FGF-7 vs control).
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(Fig. 1C). When normalized to cell number, rates of VEGF secretion by control and gene-modified skin were comparable because there were significantly more (f4-fold) keratinocytes per square centimeter ( P < 0.001) in the bioengineered skin expressing FGF-7 (Fig. 1D). The increased cell number was due to the fact that the epidermis formed by FGF-7-expressing cells is thicker and the cells were more densely packed. FGF-7 Expression Speeds Migration and Healing of Bioengineered Skin To evaluate the effect of FGF-7 on keratinocyte migration, we seeded control and FGF-7-expressing cells into microfabricated polydimethylsiloxane silicone elastomer (PDMS) stencils on plastic tissue culture dishes to form cell islands of precisely defined sizes. After cell attachment, we removed the stencils and measured island size over time. For control keratinocytes, island size increased from 4.5 to 6.2 mm2 in 24 h. For FGF-7-expressing keratinocytes, migration was significantly enhanced. Island size increased from 4.4 to 10.9 mm2 during the same 24-h period ( P < 0.01) (Fig. 2A). To determine if FGF-7 expression influenced healing of bioengineered skin, we used a biopsy punch to create 1.5mm-diameter wounds in control and FGF-7-expressing skin. We monitored wound closure by live cell staining (SYTO 13) and histology over 5 days. The rate of wound closure by skin expressing FGF-7 was significantly increased compared to that of control skin ( P < 0.001) (Figs. 2B, 2C, and 2D). Wounds were nearly closed by 3 days for skin expressing FGF-7, whereas wound closure required >5 days for control bioengineered skin. FGF-7 Expression Stimulates the Antimicrobial Response of Bioengineered Skin Previously, we have shown that bioengineered skin can mount an active antimicrobial response if stimulated with selected cytokines such as IL-1 and IL-6 [13]. To determine if FGF-7 expression altered the antimicrobial properties of bioengineered skin, we inoculated control and FGF-7-expressing skin with Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. After 24 h of incubation at 37jC, we made an extract of the skin and counted the number of viable bacteria (colonyforming units, CFU). Bacterial load of E. coli, S. aureus, or P. aeruginosa was significantly reduced in skin expressing FGF-7 compared to control skin ( P < 0.01) (Figs. 3A, 3B, and 3C). Control experiments with recombinant FGF-7 protein showed that the FGF-7 protein itself does not have a direct antimicrobial activity (data not shown). Most likely, FGF-7’s action is indirect by inducing keratinocyte expression of antimicrobial peptides and proteins. To determine if FGF-7 induced the expression of genes involved in the antimicrobial response, we isolated total RNA from control and FGF-7-expressing skin and used
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semiquantitative RT-PCR to measure mRNA levels of selected genes. Compared to control skin, levels of mRNA encoding the antimicrobial peptides human h-defensin-2 (HBD-2) and LL-37 were elevated in skin expressing FGF-7 as were mRNA levels encoding the antimicrobial proteins lysozyme, bactericidal/permeability-inducing protein (BPI), and phospholipase A2 (PLA2). Levels of mRNA encoding human h-defensin-1 (HBD-1) and antileukoprotease (ALP) were unchanged (Fig. 3D). Bioengineered Skin Expressing FGF-7 Forms a Hyperproliferative Epidermis That Promotes More Rapid Revascularization of the Acellular Dermis after Transplantation We seeded control or FGF-7-expressing keratinocytes onto acellular human dermis, grew them in vitro for 1 week, and then grafted them to full-thickness wounds on athymic mice. Seven days after transplantation, both control and gene-modified skin had formed a stratified and differentiated epidermis with well-defined basal, spinous, granular, and cornified layers; however, FGF-7expressing cells formed an epidermis that was significantly thicker with more cell layers from the basal to cornified layers (Figs. 4A and 4B). The thicker hyperplastic epidermis formed by FGF-7-expressing cells was also present at 6 weeks posttransplantation (Figs. 4C and 4D). To determine if the FGF-7-expressing epidermis was also hyperproliferative, we stained tissue sections for Ki67, a nuclear cell antigen associated with proliferation. At 1 and 6 weeks after transplantation, the FGF-7-expressing epidermis had significantly more Ki67+ cells than control grafts (Figs. 4E, 4F, 4G, and 4H). Unlike control grafts, in which Ki67+ cells were located predominately in the basal layer, abundant Ki67+ cells were present in the basal and the suprabasal layer, another indication of the hyperproliferative phenotype. Since bioengineered skin expressing FGF-7 also secretes increased levels of endogenous VEGF, we determined if revascularization of the acellular dermis was affected. We transplanted control and FGF-7-expressing bioengineered skins to full-thickness wounds on athymic mice. In this model, when the panniculus carnosus and its associated connective tissue are removed, revascularization of the acellular dermis of the graft occurs predominately from the edges, thus enabling the quantitation of the revascularization process. At 1 week after transplantation, there was little revascularization of control grafts, whereas more buds of blood vessels were present at the corners and edges of the grafts expressing FGF-7. By 2 weeks, revascularization of the FGF-7 grafts proceeded rapidly to the center of the graft, covering almost 80% of the graft compared to the slower revascularization of control grafts. At 3 weeks, all FGF-7 grafts were completely revascularized, whereas control grafts had significant central areas that had yet to be revascularized (Fig. 5A). The rate of revasculariza-
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FIG. 2. FGF-7 expression enhances cell migration and self-healing of bioengineered skin. To measure cell migration, control (unmodified) or FGF-7-modified keratinocytes were seeded into PDMS stencils. Stencils were removed after the cells attached and image analysis was used to quantify cell migration at 12 and 24 h. (A) Shown are photos of the PDMS stencil of squares with 2-mm sides, control cells, and FGF-7-expressing cells after 24 h of migration. The means F SEM of a total of 18 samples from two experiments are plotted (*P < 0.01 FGF-7 vs control). (B) To measure self-healing, 1.5-mm circular wounds were created in the center of bioengineered skin. The wounded skins were placed on a second layer of acellular dermis and cultured for additional 5 days. Samples were stained every 24 h with SYTO 13 and observed by fluorescence microscopy (original magnification 4). (C) Self-healing was quantified by measuring the remaining unclosed wound area of seven or eight skins for each time point in duplicate experiments (*P < 0.001 FGF-7 vs control). (D) After image analysis, samples were fixed in formalin and paraffin samples were stained with H&E (original magnification 10).
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FIG. 3. FGF-7 expression enhances the antibacterial activity of bioengineered skin. Bioengineered skins were inoculated with (A) 3.2 104 E. coli, (B) 4.6 104 S. aureus, or (C) 6.5 104 P. aeruginosa. The number of viable bacteria, in colony-forming units (CFU), was determined after 24 h. Results are expressed as means F SEM of triplicates from two separate experiments (*P < 0.01, FGF-7 vs control). (D) RNA was isolated from bioengineered skin and RT-PCR was used to determine mRNA levels of HBD-1, HBD-2, LL-37, ALP, lysozyme, BPI, and PLA2 in composite grafts of control and FGF-7 keratinocytes. Expression levels were normalized to G3PDH mRNA expression in control and FGF-7 skin. Data are consistent with results obtained in two separate experiments repeated three times (REL, relative expression level).
tion of FGF-7-expressing grafts was significantly higher than that of control grafts ( P < 0.01, FGF-7 vs control) (Fig. 5B).
DISCUSSION In the skin, FGF-7 is normally not synthesized by keratinocytes, rather it is a paracrine factor secreted by dermal fibroblasts whose expression is significantly upregulated by injury to the skin [14 – 16]. Keratinocytes express the receptor for FGF-7, and FGF-7’s ability to stimulate keratinocyte proliferation in vitro has been well characterized [17]. The importance of signaling via the FGF-7 receptor pathway in vivo has also been documented. Transgenic mice expressing a dominant-negative FGF-7 receptor demonstrated epidermal atrophy with dermal thickening and a delay in the reepithelialization of cutaneous wounds [16]. Conversely, the epidermis of mice expressing FGF-7 driven by a keratin 14 promoter was significantly thicker, was more proliferative than that of control
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animals, and had increased cell numbers as well as crowding of the basal layer [18]. In this study, we have genetically modified bioengineered skin to produce FGF-7 and produced a transgenic human epidermis expressing FGF-7. Like the studies with transgenic mice, the human epidermis was thicker with increased cell numbers. In a prior study, we also showed that the basal layer of FGF-7-expressing epidermis was crowded with increased cell density [19]. In contrast to the murine epidermis, proliferative cells of the FGF-7expressing epidermis were not confined exclusively to the basal layer, but were also present in the suprabasal layer of the hyperproliferative epidermis, perhaps indicating a species-specific difference in the epidermis. The increased thickness of the FGF-7-expressing epidermis is probably a function of both an increased rate of cell proliferation and a delay in cell differentiation as demonstrated in a prior study [18]. This study also demonstrated that despite the increased thickness and hyperproliferative status of the FGF-7-expressing epider-
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FIG. 4. Grafts of bioengineered skin expressing FGF-7 are hyperproliferative. Bioengineered skin of (A, C, E, G) control unmodified and (B, D, F, H) modified keratinocytes seeded onto acellular dermis was grafted to full-thickness excisional wounds on the back of athymic mice. (A, B, E, F) One week and (C, D, G, H) 6 weeks after transplantation, samples were taken and stained for hematoxylin and eosin (A, B, C, D) or immunostained for the proliferation marker, Ki67 (E, F, G, H).
mis, final differentiation and epidermal barrier formation were unaltered. FGF-7 is also well known for its ability to stimulate keratinocyte migration [20]. This activity was observed in our cells genetically modified to express FGF-7. Migration of FGF-7 cells was enhanced versus control when migration of the cells was assayed on tissue culture plastic. Cell migration plays a significant role in the processes of wound healing and reepithelialization. Bioengineered skin, which is a three-dimensional living tissue construct,
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has been shown to heal itself after injury [21,22]. In our study, the self-healing rate of the FGF-7 skin was accelerated compared to normal controls. Self-healing in this model is a function of both cell migration and cell proliferation, thus the increase in self-healing of FGF-7 skin may be due to increases in both of these processes. Also contributing to the increase in self-healing may be the fact that the FGF-7 epidermis is thicker with a greater cell density prior to the injury. These additional cells present at the start of injury may contribute to the
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FIG. 5. FGF-7 expression speeds revascularization. (A) Bioengineered skin with either control or FGF-7expressing keratinocytes was transplanted to fullthickness skin wounds on athymic mice and samples were harvested at 1, 2, and 3 weeks. The underside of each graft along with the surrounding mouse tissue was photographed (original magnification 25). (B) Image analysis was used to compute the total graft area and the area revascularized, which was plotted as a function of time (*P < 0.01 FGF-7 vs control).
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closing of the wound. Due to its thicker and hyperproliferative nature, the entire FGF epidermis resembles the thick hyperproliferative epidermis seen at the edges of wounds during the normal wound-healing process. One activity we observed in bioengineered skin expressing FGF-7 that has not been previously attributed to FGF-7 is the stimulation of the innate immune response. In our study, FGF-7 expression significantly enhanced the antimicrobial properties of bioengineered skin against both gram-negative and gram-positive bacteria. It is well documented that as part of a first line of immune defense, epidermal keratinocytes can synthesize antimicrobial proteins/peptides and that expression can be enhanced by inflammatory mediators such as IL-1 and IL-6 [13,23]. In a previous study, we showed that the antimicrobial properties of bioengineered skin can be enhanced by treatment with IL-1 or IL-6 [13]. The antimicrobial properties of the FGF-7 skin in the present study were greater than that achieved by IL-1 or IL-6 stimulation. It is unclear if FGF-7 stimulates the innate immune response directly via the FGFR signal transduction pathway or indirectly by upregulation of IL-1 or IL-6 production by keratinocytes. It is also unclear which of the antimicrobial peptides or proteins are responsible for the enhanced antibacterial state of FGF-7 skin. Regardless of the mechanism, it appears that in addition to its ability to stimulate events important for reepithelialization (proliferation and migration), FGF-7 also provides an important link between the wound healing and innate immune responses of the epidermis. Indirect actions of FGF-7 are most likely the reason the bioengineered skin expressing FGF-7 is more rapidly revascularized after transplantation. FGF-7 is well known as a growth factor whose activity is restricted to epithelial cells, so FGF-7 secreted by the bioengineered skin is probably not the factor that directly stimulates the growth of murine blood vessels into the acellular dermis. There is one report that FGF-7 stimulates the proliferation of microvascular endothelial cells [24]. Nevertheless, enhanced revascularization is more likely an indirect effect, due to the fact that bioengineered skin expressing FGF-7 also produces more VEGF. The FGF-7-expressing grafts have more cells per square centimeter and FGF-7 is known to stimulate keratinocyte expression of VEGF [25]. Bioengineered skin composed of allogeneic cells has been approved for the treatment of chronic ulcers of the skin [6]. After application to a wound, it is widely accepted that the allogeneic cells are not permanently engrafted, but rather are gradually replaced by surrounding cells of the host [26]. In fact, one mechanism by which bioengineered skin is thought to promote healing is via the local synthesis of growth factors that stimulate the edges of the wound to restart the healing process [22]. Regardless of the mechanism, it is clear that more often than not, multiple application of bioengineered skin is required to affect healing. This suggests that the perfor-
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mance and the healing properties of bioengineered skin may not yet be optimal. Our studies with bioengineered skin genetically modified to express FGF-7 suggest that in addition to its ability to secrete FGF-7, the skin has undergone several important and fundamental changes to its structure and function so that it now more closely resembles the activated epidermis present in normal healing wounds. These changes may improve the effectiveness of bioengineered skin in treating chronic ulcers of the skin. The thick hyperproliferative epidermis of the FGF-7 skin with enhanced self-healing properties may improve graft take, integration, and resistance to trauma. The elevated antimicrobial properties of FGF-7 skin may improve graft survival in contaminated wounds and help to combat bacteria colonizing chronic wounds. Last, the increased production of endogenous trophic factors such as VEGF by the thicker, more proliferative FGF-7 skin may enhance graft take by stimulating revascularization and may act in concert with the locally produced FGF-7 to stimulate the wound’s edges to restart the healing process.
MATERIALS AND METHODS Keratinocyte culture, genetic modification, and preparation of bioengineered skin. Swiss mouse 3T3-J2 and virus-producing cells were passed in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% bovine calf serum (HyClone, Logan, UT, USA) and penicillin – streptomycin (100 IU/ ml – 100 Ag/ml; Boehringer Mannheim Co., Indianapolis, IN, USA) and incubated in a humidified 10% CO2 atmosphere at 37jC. Normal human keratinocytes derived from neonatal foreskins were isolated and cultured following the method described by Rheinwald and Green [27]. Keratinocytes were cocultivated with 3T3-J2 mouse fibroblasts (originally provided by H. Green, Harvard Medical School, Boston, MA, USA) pretreated with 15 Ag/ml mitomycin C (Boehringer Mannheim Co.). Keratinocyte culture medium (KCM) was a 3:1 mixture of DMEM and Ham’s F12 medium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) (JRH Bioscience, Lenexa, KS, USA), adenine 1.8 10 4 M (Sigma Chemical Co., St. Louis, MO, USA), cholera toxin 10 10 M (Vibrio cholerae, Type Inaba 569 B; Calbiochem, La Jolla, CA, USA), hydrocortisone 0.4 Ag/ml (Calbiochem), insulin 5 Ag/ml (Novo Nordisk, Princeton, NJ, USA), transferrin 5 Ag/ml (Boehringer Mannheim Co.), triiodo-Lthyronine 2 10 9 M (Sigma), and penicillin – streptomycin 100 IU/ ml – 100 Ag/ml and was changed every 3 – 4 days. Beginning with the first medium change, EGF (Collaborative Biomedical Products, Bedford, MA, USA) was added at 10 ng/ml. Cells were subcultured by first removing the feeder layer cells with a brief ethylenediaminetetraacetic acid (EDTA) wash (5 mM in phosphate-buffered saline PBS) and then treating the keratinocytes with trypsin – EDTA. A recombinant retrovirus encoding FGF-7 was prepared by inserting a cDNA encoding human FGF-7 (kindly provided by Dr. William LaRochelle of the National Cancer Institute) into the NcoI and BamHI site of the MFG retroviral vector [28]. To generate a virus-producing cell line, MFG-FGF-7 plasmid DNA was transfected into the c-CRIP packaging cell line and a high-titer clone isolated as described [29]. Keratinocytes were genetically modified as previously described [29]. Briefly, preconfluent cultures were dissociated and the cells were passed onto mitomycin Ctreated virus-producing cells. Six days after cocultivation, modified cells were dissociated and parallel cultures were prepared for protein analysis,
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migration assay, antibacterial assay, and the preparation of bioengineered skin. Unmodified control cells were cultured on 3T3-J2 cells in parallel. Bioengineered skin was prepared as previously described [13]. Briefly, acellular dermis was prepared by exposing human cadaver skin (obtained from Shriners Hospital Skin Bank) to three rapid freeze – thaw cycles in liquid nitrogen to devitalize the cells and incubating at 37jC for 1 week in sterile PBS with 100 Ag/ml gentamicin, 10 Ag/ml ciprofloxacin, 2.5 Ag/ ml amphotericin B, and 100 IU/ml – 100 Ag/ml penicillin – streptomycin. After 1 week, the epidermis was separated from the dermis and the dermis was maintained in antibiotic cocktail at 4jC for an additional 4 weeks. To prepare bioengineered skin, the acellular dermis was cut into 1-cm2 pieces and each piece was placed on a 35-mm tissue culture dish, papillary side up. Media used were slight modifications of those developed by Ponec et al. [30]. Control or FGF-7-modified keratinocytes were seeded onto each piece of dermis (5 105 cells/piece) in a seeding medium composed of DMEM/F12 (3/1), FBS 1%, cholera toxin 10 10 M, hydrocortisone 200 ng/ml, insulin 5 Ag/ml, ascorbic acid 50 Ag/ml (Sigma), and penicillin – streptomycin 100 IU/ml – 100 Ag/ml. The next day, culture medium was changed to priming medium, which was the same as seeding medium but supplemented with bovine serum albumin (BSA) 24 AM (Sigma), fatty acid cocktail (oleic acid 25 AM, linoleic acid 15 AM, arachidonic acid 7 AM, palmitic acid 25 AM) (Sigma), L-carnitine 10 AM (Sigma), and L-serine 1 mM (Sigma). Bioengineered skin was maintained in this medium submerged for an additional 2 days. At day 4, the skins were placed on a stainless steel mesh and were raised to the air – liquid interface for 7 days. The air – liquid interface medium was composed of serum-free priming medium supplemented with 1 ng/ml EGF. The medium was changed every 2 days. Measurement of FGF-7 and VEGF secretion. To assay FGF-7 production, modified cells or control cells were grown to confluence in 10-cm2 dishes using a mitomycin C-treated fibroblast feeder layer and serum containing KCM. The cells were washed twice with DMEM and then 30 ml of fresh KCM without EGF was added to the plates. Aliquots were removed over a 4-day period and kept frozen prior to analysis. For bioengineered skin, control or FGF-7 skin was cultured for 7 days at the air – liquid interface. Subsequently 8-mm biopsy punches were cut and placed in 24well plates on stainless steel meshes. One milliliter of air – liquid interface medium was added to each well. At 12, 24, and 48 h, medium was collected from the skins, and the volume of the collected medium was measured and subsequently frozen at 20jC until further analysis. After all samples were collected, ELISAs were performed for FGF-7 and VEGF (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Quantification of nucleated cells on bioengineered skin. To assess the number of nucleated cells per surface area of skins, keratinocytes in the epidermis of 8-mm punch biopsies from bioengineered skin at the 7th day of air – liquid interface were counted. The epidermis was removed by treating with 2.5 mg/ml dispase in DMEM, at 37jC for 4 h. Then the epidermal sheets were incubated overnight in 0.25% trypsin/EDTA at 4jC. As a nuclear stain, Hoechst 33342 (Molecular Probes, Eugene, OR, USA) was added to the trypsin. The next day, the trypsin/EDTA was warmed up to 37jC for 20 min and neutralized using 10% FBS in DMEM. A single-cell suspension was made from epidermis by gentle pipetting. The cell suspension was centrifuged and the cell pellet was resuspended in 1 ml of culture medium. Nucleated cells were counted using a hemacytometer and a Nikon Eclipse microscope, set for a combination of phase microscopy and fluorescence. The remnants of the epidermis and dermis were also checked for cells. No nucleated cells were observed, ensuring adequate trypsinization of the epidermis. Cell migration and self-healing assays. Photolithography of silicon wafers spin coated with SU-8 polymer (MicroChem Corp., Newton, MA, USA) was used to create the inverse pattern needed to create stencils of squares with 2-mm sides [31]. To make the stencils, a solution of liquid
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PDMS (Sylgard 184; Dow Corning Corp., Midland, MI, USA) was poured over the master chip and polymerized by incubating at 65jC for 2 h. The PDMS stencils, with a pattern of squares, were carefully separated from the silicon chip, sterilized in 100% isopropanol, and then treated with 1% BSA to minimize cell adherence. Stencils were placed on plastic tissue culture dishes and seeded with control or FGF-7-modified keratinocytes (1.5 million/ml) in serum-free keratinocyte growth medium (KGM) (Clonetics, San Diego, CA, USA) supplemented with 0.5 Ag/ml hydrocortisone, 0.4% bovine pituitary extract, 0.1 ng/ml EGF, and 50 Ag/ml – 50 ng/ml gentamicin – amphotericin B (Clonetics). After the cells attached, the stencils were removed, and the plate was washed with KGM to remove any dead cells. Fresh KGM was added and at 0, 12, and 48 h after the removal of stencils, phase contrast images of cell islands (n = 9) were obtained and quantified using the image analysis software (Metamorph, Universal Imaging, Downingtown, PA, USA). To quantify the rate of self-healing of bioengineered skin, a small wound was created in the center of the skin with a 1.5-mm skin biopsy punch (Acuderm, Inc., Ft. Lauderdale, FL, USA) at day 5 of culture at the air – liquid interface. Wounded samples were placed on a second layer of acellular dermis and cultured for an additional 5 days. Samples were sacrificed every 24 h and stained with fluorescent nucleic acid dye SYTO 13 (Molecular Probes), which labels the live cells green. Images were obtained by a Nikon Eclipse E800 microscope equipped with a Spot 32 camera and the software Metamorph. Wound healing was analyzed by outlining and measuring the unclosed wound area. Seven to eight bioengineered skins were sacrificed for each time point, and the experiment was repeated twice. Samples were fixed in 10% formalin and processed for histology as described below. Assay of antibacterial properties. The bacteria infection model for bioengineered skin has been previously described [13]. E. coli (ATCC 43827), P. aeruginosa (ATCC 27853), and S. aureus (ATCC 29213) were maintained on Luria Berlani agar, Nutrient agar (Sigma), and Tripticase soy agar, respectively. Organisms from a single colony were inoculated into 10 ml of related broth and cultured overnight on a platform shaker at 300 rpm at 37jC. Standard curves for each organism were created by plating various dilutions on agar plates, counting colonies, and measuring the optical densities of these dilutions at 650 nm. Bacterial concentrations were calculated by using the relationships C = (6.82 108 cfu/ml) A650 for E. coli, C = (6.26 108 cfu/ml) A650 for S. aureus, and C = (4.28 108 cfu/ml) A650 for P. aeruginosa, where C is the bacterial concentration (CFU/ml) and A650 is the absorbance at 650 nm. To test the antimicrobial properties of bioengineered skin, the surface of the skin was exposed for 5 s to a 2-mm-diameter metal probe held at 140jC to destroy the epidermal barrier. Into this well-like area, 3.2 104 E. coli, 6.5 104 P. aeruginosa, or 4.6 104 S. aureus in 2 Al of PBS were inoculated. Antibiotics were excluded from the medium for the antibacterial assays. The skins were then maintained in an incubator at 37jC and 10% CO2 for 24 h. At the end of the incubation, the skins were removed from the steel meshes, placed in plastic tubes containing 0.5 ml of sterile PBS, and homogenized with a tissue homogenizer (Tissue Tearor; Fisher Scientific, Pittsburgh, PA, USA) for 2 min. Serial dilutions of the homogenates were made and 100 Al of each dilution was plated onto appropriate agar plates. After 24 h of incubation at 37jC, the number of CFU was determined. To measure mRNA levels of antimicrobial peptides/proteins, total RNA was extracted from control and FGF-7 skin using the Clontech nucleospin kit (Clontech, Palo Alto, CA, USA), according to the manufacturer’s instructions. cDNA synthesis and PCR amplification were made with a GeneAmp RT-PCR kit (Perkin – Elmer, Branchburg, NJ, USA). The primers for HBD-1, HBD-2, LL-37, ALP, lysozyme, BPI, and PLA2 are based on published sequences as described elsewhere [13]. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (Clontech) was used as a control amplimer set. RT-PCR was performed for each mRNA using a Perkin – Elmer DNA Thermal Cycler 480. Analysis of PCRs was done in 1.5% agarose gels and visualized by ethidium bromide staining. The results were quantified by scanning using a Fluor-S Imager coupled to Multi-Analyst/PC Software (Bio-Rad Laboratories, Hercules, CA, USA). Levels of mRNA were calculat-
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ed relative to G3PDH mRNA levels and the value of peptide/G3PDH expression in control was set as 1. Transplantation of bioengineered skin. Four- to eight-week-old female Swiss nu/nu mice (Massachusetts General Hospital, Boston, MA, USA) were anesthetized using 2,2,2-tribromoethanol (1.6 g/ml, 30 Al per gram of mouse) (Aldrich Chemical Company, Inc., Milwaukee, WI, USA). The grafting protocol was approved by the Subcommittee on Animal Care, Committee on Research, at the Massachusetts General Hospital. A square graft bed was created by excising approximately 1 cm2 of full-thickness skin from the back of the mouse. Panniculus carnosus and all the connective tissue under the skin were removed so that migration of blood vessels revascularizing the graft occurred only through the edges of the graft, not from the underlying tissues. Bioengineered skin of control or FGF-7-modified keratinocytes was placed into the bed and held in place by 6-O nylon sutures at the corners. Grafts were covered with Vaseline gauze, Tegaderm transparent dressing, a Band-Aid, and adhesive bandages. Grafts at 1, 2, 3, and 6 weeks were harvested along with some surrounding mouse skin (n = 25 – 27 grafts per time point). The specimens were placed epidermal side down, and digital images of the underside of the grafts showing the graft borders and the blood vessels were obtained using a camera coupled to a dissecting microscope with 25 magnification. Images were analyzed, and vascularized and nonvascularized areas of the grafts were quantified using the software Metamorph. Afterward, specimens were bisected, and half was embedded in Tissue-Tek OCT compound (Sakura Finetek, Inc., Torrance, CA, USA) and snap frozen for immunostaining. The other half was fixed in 10% formalin and embedded in paraffin for routine histology. Paraffin-embedded samples were sectioned (6 Am) and stained with hematoxylin and eosin (H&E). For immunostaining, frozen sections (6 Am) were fixed in acetone for 5 min and air-dried. To detect the presence of Ki67, sections were washed with PBS for 5 min and then incubated with blocking solution (3% BSA, 1% normal goat serum, 0.02% Na azide in PBS) for 1 h at 37jC. Sections were incubated with the IgG fraction of rabbit anti-human Ki67 (Pharmingen, Torrey Pines, CA, USA) for 1 h at 37jC. Slides were washed 3 15 min in PBS and incubated with fluorescein-conjugated affinity-purified goat anti-rabbit IgG antibody (1:100) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. The slides were coverslipped using 1% npropyl gallate mounting solution (Sigma). All antibodies were diluted in blocking solution. Control sections were not exposed to primary antibody. Neonatal foreskins served as tissue controls. Statistical analysis. Student’s t test was used to compare cell numbers in control versus FGF-7 epidermis and to compare bacterial counts in control versus FGF-7 skin. Data for VEGF ELISA, keratinocyte migration on plastic, self-healing, and vascularization measurements were tested for significance using Tukey’s test for pairwise comparisons.
ACKNOWLEDGMENTS This work was supported in part by the NIH (HD-28528) and the Shriners Hospitals for Children. RECEIVED FOR PUBLICATION DECEMBER 22, 2003; ACCEPTED APRIL 18, 2004.
REFERENCES 1. Margolis, D. J., Bilker, W., Santanna, J., and Baumgarten, M. (2002). Venous leg ulcer: incidence prevalence in the elderly. J. Am. Acad. Dermatol. 46: 381 – 386. 2. Green, H., Kehinde, O., and Thomas, J. (1979). Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Natl. Acad. Sci. USA 76: 5665 – 5668. 3. Bell, E., Ehrlich, H. P., Buttle, D. J., and Nakatsuji, T. (1981). Living tissue formed in vitro and accepted as skin-equivalent tissue of full-thickness. Science 211: 1052 – 1054. 4. Boyce, S. T. (1996). Cultured skin substitutes: a review. Tissue Eng. 2: 255 – 266.
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5. Gallico, G. G., 3rd, O’Connor, N. E., Compton, C. C., Kehinde, O., and Green, H. (1984). Permanent coverage of large burn wounds with autologous cultured human epithelium. N. Engl. J. Med. 311: 448 – 451. 6. Falanga, V., et al. (1998). Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group. Arch. Dermatol. 134: 293 – 300. 7. Eming, S. A., Snow, R. G., Yarmush, M. L., and Morgan, J. R. (1996). Targeted expression of insulin-like growth factor to human keratinocytes: modification of the autocrine control of keratinocyte proliferation. J. Invest. Dermatol. 107: 113 – 120. 8. Eming, S. A., Medalie, D. A., Tompkins, R. G., Yarmush, M. L., and Morgan, J. R. (1998). Genetically modified human keratinocytes overexpressing PDGF-A enhance the performance of a composite skin graft. Hum. Gene Ther. 9: 529 – 539. 9. Supp, D. M., Supp, A. P., Bell, S. M., and Boyce, S. T. (2000). Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor. J. Invest. Dermatol. 114: 5 – 13. 10. Eming, S. A., Yarmush, M. L., Krueger, G. G., and Morgan, J. R. (1999). Regulation of the spatial organization of mesenchymal connective tissue: effects of cell-associated versus released isoforms of platelet-derived growth factor. Am. J. Pathol. 154: 281 – 289. 11. Hamoen, K. E., and Morgan, J. R. (2002). Transient hyperproliferation of a transgenic human epidermis expressing hepatocyte growth factor. Cell Transplant. 11: 385 – 395. 12. Andree, C., et al. (1994). In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair. Proc. Natl. Acad. Sci. USA 91: 12188 – 12192. 13. Erdag, G., and Morgan, J. R. (2002). Interleukin-1alpha and interleukin-6 enhance the antibacterial properties of cultured composite keratinocyte grafts. Ann. Surg. 235: 113 – 124. 14. Aaronson, S. A., et al. (1991). Keratinocyte growth factor: a fibroblast growth factor family member with unusual target cell specificity. Ann. N. Y. Acad. Sci. 638: 62 – 77. 15. Werner, S., Peters, K. G., Longaker, M. T., Fuller-Pace, F., Banda, M. J., and Williams, L. T. (1992). Large induction of keratinocyte growth factor expression in the dermis during wound healing. Proc. Natl. Acad. Sci. USA 89: 6896 – 6900. 16. Werner, S., et al. (1994). The function of KGF in morphogenesis of epithelium and reepithelialization of wounds. Science 266: 819 – 822. 17. Miki, T., Fleming, T. P., Bottaro, D. P., Rubin, J. S., Ron, D., and Aaronson, S. A. (1991). Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science 251: 72 – 75. 18. Guo, L., Yu, Q. C., and Fuchs, E. (1993). Targeting expression of keratinocyte growth factor to keratinocytes elicits striking changes in epithelial differentiation in transgenic mice. EMBO J. 12: 973 – 986. 19. Andreadis, S. T., Hamoen, K. E., Yarmush, M. L., and Morgan, J. R. (2001). Keratinocyte growth factor induces hyperproliferation and delays differentiation in a skin equivalent model system. FASEB J. 15: 898 – 906. 20. Tsuboi, R., Sato, C., Kurita, Y., Rubin, J. S., Ron, D., and Aaronson, S. A. (1993). Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes. J. Invest. Dermatol. 101: 49 – 53. 21. Garlick, J. A., and Taichman, L. B. (1994). Effect of TGF-b1 on re-epithelialization of human keratinocytes in vitro: an organotypic model. J. Invest. Dermatol. 1: 554 – 559. 22. Falanga, V., et al. (2002). Wounding of bioengineered skin: cellular and molecular aspects after injury. J. Invest. Dermatol. 119: 653 – 660. 23. Gallo, R. L., and Huttner, K. M. (1998). Anti-microbial peptides: an emerging concept in cutaneous biology. J. Invest. Dermatol. 111: 739 – 743. 24. Gillis, P., et al. (1999). Keratinocyte growth factor induces angiogenesis and protects endothelial barrier function. J. Cell Sci. 112: 2049 – 2057. 25. Frank, S., Hubner, G., Breier, G., Longaker, M. T., Greenhalgh, D. G., and Werner, S. (1995). Regulation of vascular endothelial growth factor expression in cultured keratinocytes. J. Biol. Chem. 270: 12607 – 12613. 26. Gielen, V., Faure, M., Maudit, G., and Thivolet, J. (1987). Progressive replacement of human cultured epithelial allografts by recipient cells as evidenced by HLA class I antigens expression. Dermatologica 175: 166 – 170. 27. Rheinwald, J. G., and Green, H. (1975). Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331 – 343. 28. Ohashi, T., et al. (1992). Efficient transfer and sustained high expression of the human glucocerebrosidase gene in mice and their functional macrophages following transplantation of bone marrow transduced by a retroviral vector. Proc. Natl. Acad. Sci. USA 89: 11332 – 11336. 29. Morgan, J. R., Barrandon, Y., Green, H., and Mulligan, R. C. (1987). Expression of an exogenous growth hormone gene by transplantable human epidermal cells. Science 237: 1476 – 1479. 30. Ponec, M., et al. (1997). The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C. J. Invest. Dermatol. 109: 348 – 355. 31. Folch, A., and Toner, M. (1998). Cellular micropatterns on biocompatible materials. Biotech. Prog. 14: 388 – 392.
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FGF-7 Expression Enhances the Performance of Bioengineered Skin Gulsun Erdag,1 Daniel A. Medalie,1 Hinne Rakhorst,1 Gerald G. Krueger,2 and Jeffrey R. Morgan1,3,* 1
Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School and Shriners Hospital for Children, Boston, MA 02114, USA 2 Department of Dermatology, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA 3 Center for Biomedical Engineering and Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA
*To whom correspondence and reprint requests should be addressed at Brown University, G-B 393, Biomed Center, 171 Meeting Street, Providence, RI 02912, USA. Fax: (401) 863-1753. E-mail: Jeffrey_ [email protected].
Available online 4 June 2004
To improve the performance of bioengineered skin, we used a recombinant retrovirus encoding FGF-7 to modify diploid human keratinocytes genetically. Control or FGF-7-expressing keratinocytes were seeded onto acellular human dermis to form bioengineered skin. Gene-modified skin secreted significant levels of FGF-7 and formed a thicker and hyperproliferative epidermis with about four times the number of cells per square centimeter. Secretion of an endogenous trophic factor, VEGF, was increased f5-fold. Migration of FGF-7-expressing keratinocytes was stimulated as was the self-healing of bioengineered skin expressing FGF-7. When tested in a bacterial infection model, the antimicrobial properties of FGF-7-expressing skin were increased >500-fold against both gram-negative and gram-positive bacteria. After transplantation to full-thickness wounds on athymic mice, skin expressing FGF-7 was revascularized more rapidly. These results demonstrate that genetic modification can be used to enhance performance and that expression of FGF-7 augments several properties important to the wound-healing properties of bioengineered skin. Key Words: fibroblast growth factor, skin, wound healing, vascularization, infection, tissue engineering
INTRODUCTION Burns and chronic ulcers of the skin are significant medical problems in need of new treatments. Annually in the United States, there are more than 1 million burn injuries (Burn Incidence and Treatment in the U.S.A., 2000 Fact Sheet, American Burn Association, http://www.ameriburn.org), an estimated 800,000 diabetic ulcers leading to about 80,000 amputations (National Diabetes Fact Sheet 2002, Centers for Disease Control, http://www.cdc.gov/ health/diabetes.htm), and about 1 million venous ulcers [1], of which, only 40 – 50% are healed by standard therapies. Bioengineered skin substitutes are a potential treatment for the closure of skin defects and methods are well established for the culture of large numbers of human epidermal keratinocytes as well as dermal fibroblasts [2 – 4]. Cultured autologous keratinocytes have been used as living cell sheets for the treatment of burn wounds, and allogeneic keratinocytes and fibroblasts in a collagen matrix have been combined to form bilayered skin constructs for the treatment of chronic skin ulcers [5,6].
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We and others have been exploring the possibility of enhancing the function and thus the performance of bioengineered skin by genetic modification of the cells [7 – 9]. Unlike gene therapy for inherited diseases in which the gene to be delivered is usually known, it is unclear which gene will have a significant impact on the performance of tissue-engineered skin. Since normal bioengineered skin is thought to promote healing by the local synthesis of endogenous growth factors, one area of interest has been to use genetic modification to program the cells to produce higher levels of growth factors. Genemodified skin that secretes elevated levels of insulin-like growth factor-1, platelet-derived growth factor A chain and B chain, vascular endothelial growth factor (VEGF), hepatocyte growth factor, or epidermal growth factor (EGF) has been made [7 – 12]. These studies have demonstrated the feasibility of this approach and have shown that growth factor production can influence one facet of the performance of bioengineered skin. In this report, we show that unlike other growth factors tested, production of fibroblast growth factor-7
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(FGF-7) by genetically modified skin enhances several significant aspects of the performance of bioengineered skin. Compared to controls, gene-modified skin producing FGF-7 (i) has a thicker, more hyperproliferative epidermis that heals more rapidly after injury; (ii) has increased antimicrobial activity against gram-negative and gram-positive bacteria; (iii) secretes more endogenous trophic factors such as VEGF; and (iv) is more rapidly revascularized after transplantation. Bioengineered skin expressing FGF-7 appears to be activated in a way that may make it more effective at closing problematic wounds.
RESULTS Bioengineered Skin with Gene-Modified Keratinocytes Secretes FGF-7 and Higher Levels of Other Trophic Factors such as VEGF We modified diploid human keratinocytes genetically with a recombinant retrovirus encoding FGF-7 and mea-
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sured the levels of secreted FGF-7 by ELISA. FGF-7 was continuously secreted by confluent keratinocytes grown on tissue culture plastic and accumulated in the medium at a rate of approximately 0.6 ng/106 cells/24 h. In control unmodified cells, FGF-7 levels were undetectable (detection limit <15 pg/ml) (Fig. 1A). We seeded the same cells onto the papillary surface of acellular human dermis raised to the air/liquid interface for 7 days to form a stratified and differentiated epidermis. The rate of FGF-7 accumulation from this bioengineered skin was 3.24 ng/ cm2/24 h. FGF-7 levels in matched samples of control, unmodified keratinocytes were undetectable (Fig. 1B). Keratinocytes naturally synthesize and secrete numerous growth factors involved in autocrine and paracrine signaling. We measured the levels of VEGF secreted by our bioengineered skin. Skin formed with control unmodified keratinocytes secreted VEGF at a rate of 1.8 ng/ cm2/24 h, while skin formed with keratinocytes expressing FGF-7 secreted VEGF at a rate of 9.1 ng/cm2/24 h, an approximately fivefold increase per surface area ( P < 0.01)
FIG. 1. Gene-modified bioengineered skin secretes FGF-7 and increased levels of VEGF. A time course of FGF-7 secretion was determined by removing portions of the culture medium of confluent cultures of control or genetically modified keratinocytes grown (A) on tissue culture plastic or (B) as bioengineered skin. Levels of FGF-7 protein were determined by ELISA. (C) Culture media from bioengineered skin were also assayed for levels of VEGF by ELISA ( P < 0.01, FGF-7 vs control). (D) To determine cell number, bioengineered skin was treated with dispase, followed by trypsin and Hoechst staining to facilitate the counting of nucleated cells (*P < 0.001, FGF-7 vs control).
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(Fig. 1C). When normalized to cell number, rates of VEGF secretion by control and gene-modified skin were comparable because there were significantly more (f4-fold) keratinocytes per square centimeter ( P < 0.001) in the bioengineered skin expressing FGF-7 (Fig. 1D). The increased cell number was due to the fact that the epidermis formed by FGF-7-expressing cells is thicker and the cells were more densely packed. FGF-7 Expression Speeds Migration and Healing of Bioengineered Skin To evaluate the effect of FGF-7 on keratinocyte migration, we seeded control and FGF-7-expressing cells into microfabricated polydimethylsiloxane silicone elastomer (PDMS) stencils on plastic tissue culture dishes to form cell islands of precisely defined sizes. After cell attachment, we removed the stencils and measured island size over time. For control keratinocytes, island size increased from 4.5 to 6.2 mm2 in 24 h. For FGF-7-expressing keratinocytes, migration was significantly enhanced. Island size increased from 4.4 to 10.9 mm2 during the same 24-h period ( P < 0.01) (Fig. 2A). To determine if FGF-7 expression influenced healing of bioengineered skin, we used a biopsy punch to create 1.5mm-diameter wounds in control and FGF-7-expressing skin. We monitored wound closure by live cell staining (SYTO 13) and histology over 5 days. The rate of wound closure by skin expressing FGF-7 was significantly increased compared to that of control skin ( P < 0.001) (Figs. 2B, 2C, and 2D). Wounds were nearly closed by 3 days for skin expressing FGF-7, whereas wound closure required >5 days for control bioengineered skin. FGF-7 Expression Stimulates the Antimicrobial Response of Bioengineered Skin Previously, we have shown that bioengineered skin can mount an active antimicrobial response if stimulated with selected cytokines such as IL-1 and IL-6 [13]. To determine if FGF-7 expression altered the antimicrobial properties of bioengineered skin, we inoculated control and FGF-7-expressing skin with Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. After 24 h of incubation at 37jC, we made an extract of the skin and counted the number of viable bacteria (colonyforming units, CFU). Bacterial load of E. coli, S. aureus, or P. aeruginosa was significantly reduced in skin expressing FGF-7 compared to control skin ( P < 0.01) (Figs. 3A, 3B, and 3C). Control experiments with recombinant FGF-7 protein showed that the FGF-7 protein itself does not have a direct antimicrobial activity (data not shown). Most likely, FGF-7’s action is indirect by inducing keratinocyte expression of antimicrobial peptides and proteins. To determine if FGF-7 induced the expression of genes involved in the antimicrobial response, we isolated total RNA from control and FGF-7-expressing skin and used
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semiquantitative RT-PCR to measure mRNA levels of selected genes. Compared to control skin, levels of mRNA encoding the antimicrobial peptides human h-defensin-2 (HBD-2) and LL-37 were elevated in skin expressing FGF-7 as were mRNA levels encoding the antimicrobial proteins lysozyme, bactericidal/permeability-inducing protein (BPI), and phospholipase A2 (PLA2). Levels of mRNA encoding human h-defensin-1 (HBD-1) and antileukoprotease (ALP) were unchanged (Fig. 3D). Bioengineered Skin Expressing FGF-7 Forms a Hyperproliferative Epidermis That Promotes More Rapid Revascularization of the Acellular Dermis after Transplantation We seeded control or FGF-7-expressing keratinocytes onto acellular human dermis, grew them in vitro for 1 week, and then grafted them to full-thickness wounds on athymic mice. Seven days after transplantation, both control and gene-modified skin had formed a stratified and differentiated epidermis with well-defined basal, spinous, granular, and cornified layers; however, FGF-7expressing cells formed an epidermis that was significantly thicker with more cell layers from the basal to cornified layers (Figs. 4A and 4B). The thicker hyperplastic epidermis formed by FGF-7-expressing cells was also present at 6 weeks posttransplantation (Figs. 4C and 4D). To determine if the FGF-7-expressing epidermis was also hyperproliferative, we stained tissue sections for Ki67, a nuclear cell antigen associated with proliferation. At 1 and 6 weeks after transplantation, the FGF-7-expressing epidermis had significantly more Ki67+ cells than control grafts (Figs. 4E, 4F, 4G, and 4H). Unlike control grafts, in which Ki67+ cells were located predominately in the basal layer, abundant Ki67+ cells were present in the basal and the suprabasal layer, another indication of the hyperproliferative phenotype. Since bioengineered skin expressing FGF-7 also secretes increased levels of endogenous VEGF, we determined if revascularization of the acellular dermis was affected. We transplanted control and FGF-7-expressing bioengineered skins to full-thickness wounds on athymic mice. In this model, when the panniculus carnosus and its associated connective tissue are removed, revascularization of the acellular dermis of the graft occurs predominately from the edges, thus enabling the quantitation of the revascularization process. At 1 week after transplantation, there was little revascularization of control grafts, whereas more buds of blood vessels were present at the corners and edges of the grafts expressing FGF-7. By 2 weeks, revascularization of the FGF-7 grafts proceeded rapidly to the center of the graft, covering almost 80% of the graft compared to the slower revascularization of control grafts. At 3 weeks, all FGF-7 grafts were completely revascularized, whereas control grafts had significant central areas that had yet to be revascularized (Fig. 5A). The rate of revasculariza-
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FIG. 2. FGF-7 expression enhances cell migration and self-healing of bioengineered skin. To measure cell migration, control (unmodified) or FGF-7-modified keratinocytes were seeded into PDMS stencils. Stencils were removed after the cells attached and image analysis was used to quantify cell migration at 12 and 24 h. (A) Shown are photos of the PDMS stencil of squares with 2-mm sides, control cells, and FGF-7-expressing cells after 24 h of migration. The means F SEM of a total of 18 samples from two experiments are plotted (*P < 0.01 FGF-7 vs control). (B) To measure self-healing, 1.5-mm circular wounds were created in the center of bioengineered skin. The wounded skins were placed on a second layer of acellular dermis and cultured for additional 5 days. Samples were stained every 24 h with SYTO 13 and observed by fluorescence microscopy (original magnification 4). (C) Self-healing was quantified by measuring the remaining unclosed wound area of seven or eight skins for each time point in duplicate experiments (*P < 0.001 FGF-7 vs control). (D) After image analysis, samples were fixed in formalin and paraffin samples were stained with H&E (original magnification 10).
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FIG. 3. FGF-7 expression enhances the antibacterial activity of bioengineered skin. Bioengineered skins were inoculated with (A) 3.2 104 E. coli, (B) 4.6 104 S. aureus, or (C) 6.5 104 P. aeruginosa. The number of viable bacteria, in colony-forming units (CFU), was determined after 24 h. Results are expressed as means F SEM of triplicates from two separate experiments (*P < 0.01, FGF-7 vs control). (D) RNA was isolated from bioengineered skin and RT-PCR was used to determine mRNA levels of HBD-1, HBD-2, LL-37, ALP, lysozyme, BPI, and PLA2 in composite grafts of control and FGF-7 keratinocytes. Expression levels were normalized to G3PDH mRNA expression in control and FGF-7 skin. Data are consistent with results obtained in two separate experiments repeated three times (REL, relative expression level).
tion of FGF-7-expressing grafts was significantly higher than that of control grafts ( P < 0.01, FGF-7 vs control) (Fig. 5B).
DISCUSSION In the skin, FGF-7 is normally not synthesized by keratinocytes, rather it is a paracrine factor secreted by dermal fibroblasts whose expression is significantly upregulated by injury to the skin [14 – 16]. Keratinocytes express the receptor for FGF-7, and FGF-7’s ability to stimulate keratinocyte proliferation in vitro has been well characterized [17]. The importance of signaling via the FGF-7 receptor pathway in vivo has also been documented. Transgenic mice expressing a dominant-negative FGF-7 receptor demonstrated epidermal atrophy with dermal thickening and a delay in the reepithelialization of cutaneous wounds [16]. Conversely, the epidermis of mice expressing FGF-7 driven by a keratin 14 promoter was significantly thicker, was more proliferative than that of control
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animals, and had increased cell numbers as well as crowding of the basal layer [18]. In this study, we have genetically modified bioengineered skin to produce FGF-7 and produced a transgenic human epidermis expressing FGF-7. Like the studies with transgenic mice, the human epidermis was thicker with increased cell numbers. In a prior study, we also showed that the basal layer of FGF-7-expressing epidermis was crowded with increased cell density [19]. In contrast to the murine epidermis, proliferative cells of the FGF-7expressing epidermis were not confined exclusively to the basal layer, but were also present in the suprabasal layer of the hyperproliferative epidermis, perhaps indicating a species-specific difference in the epidermis. The increased thickness of the FGF-7-expressing epidermis is probably a function of both an increased rate of cell proliferation and a delay in cell differentiation as demonstrated in a prior study [18]. This study also demonstrated that despite the increased thickness and hyperproliferative status of the FGF-7-expressing epider-
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FIG. 4. Grafts of bioengineered skin expressing FGF-7 are hyperproliferative. Bioengineered skin of (A, C, E, G) control unmodified and (B, D, F, H) modified keratinocytes seeded onto acellular dermis was grafted to full-thickness excisional wounds on the back of athymic mice. (A, B, E, F) One week and (C, D, G, H) 6 weeks after transplantation, samples were taken and stained for hematoxylin and eosin (A, B, C, D) or immunostained for the proliferation marker, Ki67 (E, F, G, H).
mis, final differentiation and epidermal barrier formation were unaltered. FGF-7 is also well known for its ability to stimulate keratinocyte migration [20]. This activity was observed in our cells genetically modified to express FGF-7. Migration of FGF-7 cells was enhanced versus control when migration of the cells was assayed on tissue culture plastic. Cell migration plays a significant role in the processes of wound healing and reepithelialization. Bioengineered skin, which is a three-dimensional living tissue construct,
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has been shown to heal itself after injury [21,22]. In our study, the self-healing rate of the FGF-7 skin was accelerated compared to normal controls. Self-healing in this model is a function of both cell migration and cell proliferation, thus the increase in self-healing of FGF-7 skin may be due to increases in both of these processes. Also contributing to the increase in self-healing may be the fact that the FGF-7 epidermis is thicker with a greater cell density prior to the injury. These additional cells present at the start of injury may contribute to the
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FIG. 5. FGF-7 expression speeds revascularization. (A) Bioengineered skin with either control or FGF-7expressing keratinocytes was transplanted to fullthickness skin wounds on athymic mice and samples were harvested at 1, 2, and 3 weeks. The underside of each graft along with the surrounding mouse tissue was photographed (original magnification 25). (B) Image analysis was used to compute the total graft area and the area revascularized, which was plotted as a function of time (*P < 0.01 FGF-7 vs control).
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closing of the wound. Due to its thicker and hyperproliferative nature, the entire FGF epidermis resembles the thick hyperproliferative epidermis seen at the edges of wounds during the normal wound-healing process. One activity we observed in bioengineered skin expressing FGF-7 that has not been previously attributed to FGF-7 is the stimulation of the innate immune response. In our study, FGF-7 expression significantly enhanced the antimicrobial properties of bioengineered skin against both gram-negative and gram-positive bacteria. It is well documented that as part of a first line of immune defense, epidermal keratinocytes can synthesize antimicrobial proteins/peptides and that expression can be enhanced by inflammatory mediators such as IL-1 and IL-6 [13,23]. In a previous study, we showed that the antimicrobial properties of bioengineered skin can be enhanced by treatment with IL-1 or IL-6 [13]. The antimicrobial properties of the FGF-7 skin in the present study were greater than that achieved by IL-1 or IL-6 stimulation. It is unclear if FGF-7 stimulates the innate immune response directly via the FGFR signal transduction pathway or indirectly by upregulation of IL-1 or IL-6 production by keratinocytes. It is also unclear which of the antimicrobial peptides or proteins are responsible for the enhanced antibacterial state of FGF-7 skin. Regardless of the mechanism, it appears that in addition to its ability to stimulate events important for reepithelialization (proliferation and migration), FGF-7 also provides an important link between the wound healing and innate immune responses of the epidermis. Indirect actions of FGF-7 are most likely the reason the bioengineered skin expressing FGF-7 is more rapidly revascularized after transplantation. FGF-7 is well known as a growth factor whose activity is restricted to epithelial cells, so FGF-7 secreted by the bioengineered skin is probably not the factor that directly stimulates the growth of murine blood vessels into the acellular dermis. There is one report that FGF-7 stimulates the proliferation of microvascular endothelial cells [24]. Nevertheless, enhanced revascularization is more likely an indirect effect, due to the fact that bioengineered skin expressing FGF-7 also produces more VEGF. The FGF-7-expressing grafts have more cells per square centimeter and FGF-7 is known to stimulate keratinocyte expression of VEGF [25]. Bioengineered skin composed of allogeneic cells has been approved for the treatment of chronic ulcers of the skin [6]. After application to a wound, it is widely accepted that the allogeneic cells are not permanently engrafted, but rather are gradually replaced by surrounding cells of the host [26]. In fact, one mechanism by which bioengineered skin is thought to promote healing is via the local synthesis of growth factors that stimulate the edges of the wound to restart the healing process [22]. Regardless of the mechanism, it is clear that more often than not, multiple application of bioengineered skin is required to affect healing. This suggests that the perfor-
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mance and the healing properties of bioengineered skin may not yet be optimal. Our studies with bioengineered skin genetically modified to express FGF-7 suggest that in addition to its ability to secrete FGF-7, the skin has undergone several important and fundamental changes to its structure and function so that it now more closely resembles the activated epidermis present in normal healing wounds. These changes may improve the effectiveness of bioengineered skin in treating chronic ulcers of the skin. The thick hyperproliferative epidermis of the FGF-7 skin with enhanced self-healing properties may improve graft take, integration, and resistance to trauma. The elevated antimicrobial properties of FGF-7 skin may improve graft survival in contaminated wounds and help to combat bacteria colonizing chronic wounds. Last, the increased production of endogenous trophic factors such as VEGF by the thicker, more proliferative FGF-7 skin may enhance graft take by stimulating revascularization and may act in concert with the locally produced FGF-7 to stimulate the wound’s edges to restart the healing process.
MATERIALS AND METHODS Keratinocyte culture, genetic modification, and preparation of bioengineered skin. Swiss mouse 3T3-J2 and virus-producing cells were passed in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% bovine calf serum (HyClone, Logan, UT, USA) and penicillin – streptomycin (100 IU/ ml – 100 Ag/ml; Boehringer Mannheim Co., Indianapolis, IN, USA) and incubated in a humidified 10% CO2 atmosphere at 37jC. Normal human keratinocytes derived from neonatal foreskins were isolated and cultured following the method described by Rheinwald and Green [27]. Keratinocytes were cocultivated with 3T3-J2 mouse fibroblasts (originally provided by H. Green, Harvard Medical School, Boston, MA, USA) pretreated with 15 Ag/ml mitomycin C (Boehringer Mannheim Co.). Keratinocyte culture medium (KCM) was a 3:1 mixture of DMEM and Ham’s F12 medium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) (JRH Bioscience, Lenexa, KS, USA), adenine 1.8 10 4 M (Sigma Chemical Co., St. Louis, MO, USA), cholera toxin 10 10 M (Vibrio cholerae, Type Inaba 569 B; Calbiochem, La Jolla, CA, USA), hydrocortisone 0.4 Ag/ml (Calbiochem), insulin 5 Ag/ml (Novo Nordisk, Princeton, NJ, USA), transferrin 5 Ag/ml (Boehringer Mannheim Co.), triiodo-Lthyronine 2 10 9 M (Sigma), and penicillin – streptomycin 100 IU/ ml – 100 Ag/ml and was changed every 3 – 4 days. Beginning with the first medium change, EGF (Collaborative Biomedical Products, Bedford, MA, USA) was added at 10 ng/ml. Cells were subcultured by first removing the feeder layer cells with a brief ethylenediaminetetraacetic acid (EDTA) wash (5 mM in phosphate-buffered saline PBS) and then treating the keratinocytes with trypsin – EDTA. A recombinant retrovirus encoding FGF-7 was prepared by inserting a cDNA encoding human FGF-7 (kindly provided by Dr. William LaRochelle of the National Cancer Institute) into the NcoI and BamHI site of the MFG retroviral vector [28]. To generate a virus-producing cell line, MFG-FGF-7 plasmid DNA was transfected into the c-CRIP packaging cell line and a high-titer clone isolated as described [29]. Keratinocytes were genetically modified as previously described [29]. Briefly, preconfluent cultures were dissociated and the cells were passed onto mitomycin Ctreated virus-producing cells. Six days after cocultivation, modified cells were dissociated and parallel cultures were prepared for protein analysis,
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migration assay, antibacterial assay, and the preparation of bioengineered skin. Unmodified control cells were cultured on 3T3-J2 cells in parallel. Bioengineered skin was prepared as previously described [13]. Briefly, acellular dermis was prepared by exposing human cadaver skin (obtained from Shriners Hospital Skin Bank) to three rapid freeze – thaw cycles in liquid nitrogen to devitalize the cells and incubating at 37jC for 1 week in sterile PBS with 100 Ag/ml gentamicin, 10 Ag/ml ciprofloxacin, 2.5 Ag/ ml amphotericin B, and 100 IU/ml – 100 Ag/ml penicillin – streptomycin. After 1 week, the epidermis was separated from the dermis and the dermis was maintained in antibiotic cocktail at 4jC for an additional 4 weeks. To prepare bioengineered skin, the acellular dermis was cut into 1-cm2 pieces and each piece was placed on a 35-mm tissue culture dish, papillary side up. Media used were slight modifications of those developed by Ponec et al. [30]. Control or FGF-7-modified keratinocytes were seeded onto each piece of dermis (5 105 cells/piece) in a seeding medium composed of DMEM/F12 (3/1), FBS 1%, cholera toxin 10 10 M, hydrocortisone 200 ng/ml, insulin 5 Ag/ml, ascorbic acid 50 Ag/ml (Sigma), and penicillin – streptomycin 100 IU/ml – 100 Ag/ml. The next day, culture medium was changed to priming medium, which was the same as seeding medium but supplemented with bovine serum albumin (BSA) 24 AM (Sigma), fatty acid cocktail (oleic acid 25 AM, linoleic acid 15 AM, arachidonic acid 7 AM, palmitic acid 25 AM) (Sigma), L-carnitine 10 AM (Sigma), and L-serine 1 mM (Sigma). Bioengineered skin was maintained in this medium submerged for an additional 2 days. At day 4, the skins were placed on a stainless steel mesh and were raised to the air – liquid interface for 7 days. The air – liquid interface medium was composed of serum-free priming medium supplemented with 1 ng/ml EGF. The medium was changed every 2 days. Measurement of FGF-7 and VEGF secretion. To assay FGF-7 production, modified cells or control cells were grown to confluence in 10-cm2 dishes using a mitomycin C-treated fibroblast feeder layer and serum containing KCM. The cells were washed twice with DMEM and then 30 ml of fresh KCM without EGF was added to the plates. Aliquots were removed over a 4-day period and kept frozen prior to analysis. For bioengineered skin, control or FGF-7 skin was cultured for 7 days at the air – liquid interface. Subsequently 8-mm biopsy punches were cut and placed in 24well plates on stainless steel meshes. One milliliter of air – liquid interface medium was added to each well. At 12, 24, and 48 h, medium was collected from the skins, and the volume of the collected medium was measured and subsequently frozen at 20jC until further analysis. After all samples were collected, ELISAs were performed for FGF-7 and VEGF (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Quantification of nucleated cells on bioengineered skin. To assess the number of nucleated cells per surface area of skins, keratinocytes in the epidermis of 8-mm punch biopsies from bioengineered skin at the 7th day of air – liquid interface were counted. The epidermis was removed by treating with 2.5 mg/ml dispase in DMEM, at 37jC for 4 h. Then the epidermal sheets were incubated overnight in 0.25% trypsin/EDTA at 4jC. As a nuclear stain, Hoechst 33342 (Molecular Probes, Eugene, OR, USA) was added to the trypsin. The next day, the trypsin/EDTA was warmed up to 37jC for 20 min and neutralized using 10% FBS in DMEM. A single-cell suspension was made from epidermis by gentle pipetting. The cell suspension was centrifuged and the cell pellet was resuspended in 1 ml of culture medium. Nucleated cells were counted using a hemacytometer and a Nikon Eclipse microscope, set for a combination of phase microscopy and fluorescence. The remnants of the epidermis and dermis were also checked for cells. No nucleated cells were observed, ensuring adequate trypsinization of the epidermis. Cell migration and self-healing assays. Photolithography of silicon wafers spin coated with SU-8 polymer (MicroChem Corp., Newton, MA, USA) was used to create the inverse pattern needed to create stencils of squares with 2-mm sides [31]. To make the stencils, a solution of liquid
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PDMS (Sylgard 184; Dow Corning Corp., Midland, MI, USA) was poured over the master chip and polymerized by incubating at 65jC for 2 h. The PDMS stencils, with a pattern of squares, were carefully separated from the silicon chip, sterilized in 100% isopropanol, and then treated with 1% BSA to minimize cell adherence. Stencils were placed on plastic tissue culture dishes and seeded with control or FGF-7-modified keratinocytes (1.5 million/ml) in serum-free keratinocyte growth medium (KGM) (Clonetics, San Diego, CA, USA) supplemented with 0.5 Ag/ml hydrocortisone, 0.4% bovine pituitary extract, 0.1 ng/ml EGF, and 50 Ag/ml – 50 ng/ml gentamicin – amphotericin B (Clonetics). After the cells attached, the stencils were removed, and the plate was washed with KGM to remove any dead cells. Fresh KGM was added and at 0, 12, and 48 h after the removal of stencils, phase contrast images of cell islands (n = 9) were obtained and quantified using the image analysis software (Metamorph, Universal Imaging, Downingtown, PA, USA). To quantify the rate of self-healing of bioengineered skin, a small wound was created in the center of the skin with a 1.5-mm skin biopsy punch (Acuderm, Inc., Ft. Lauderdale, FL, USA) at day 5 of culture at the air – liquid interface. Wounded samples were placed on a second layer of acellular dermis and cultured for an additional 5 days. Samples were sacrificed every 24 h and stained with fluorescent nucleic acid dye SYTO 13 (Molecular Probes), which labels the live cells green. Images were obtained by a Nikon Eclipse E800 microscope equipped with a Spot 32 camera and the software Metamorph. Wound healing was analyzed by outlining and measuring the unclosed wound area. Seven to eight bioengineered skins were sacrificed for each time point, and the experiment was repeated twice. Samples were fixed in 10% formalin and processed for histology as described below. Assay of antibacterial properties. The bacteria infection model for bioengineered skin has been previously described [13]. E. coli (ATCC 43827), P. aeruginosa (ATCC 27853), and S. aureus (ATCC 29213) were maintained on Luria Berlani agar, Nutrient agar (Sigma), and Tripticase soy agar, respectively. Organisms from a single colony were inoculated into 10 ml of related broth and cultured overnight on a platform shaker at 300 rpm at 37jC. Standard curves for each organism were created by plating various dilutions on agar plates, counting colonies, and measuring the optical densities of these dilutions at 650 nm. Bacterial concentrations were calculated by using the relationships C = (6.82 108 cfu/ml) A650 for E. coli, C = (6.26 108 cfu/ml) A650 for S. aureus, and C = (4.28 108 cfu/ml) A650 for P. aeruginosa, where C is the bacterial concentration (CFU/ml) and A650 is the absorbance at 650 nm. To test the antimicrobial properties of bioengineered skin, the surface of the skin was exposed for 5 s to a 2-mm-diameter metal probe held at 140jC to destroy the epidermal barrier. Into this well-like area, 3.2 104 E. coli, 6.5 104 P. aeruginosa, or 4.6 104 S. aureus in 2 Al of PBS were inoculated. Antibiotics were excluded from the medium for the antibacterial assays. The skins were then maintained in an incubator at 37jC and 10% CO2 for 24 h. At the end of the incubation, the skins were removed from the steel meshes, placed in plastic tubes containing 0.5 ml of sterile PBS, and homogenized with a tissue homogenizer (Tissue Tearor; Fisher Scientific, Pittsburgh, PA, USA) for 2 min. Serial dilutions of the homogenates were made and 100 Al of each dilution was plated onto appropriate agar plates. After 24 h of incubation at 37jC, the number of CFU was determined. To measure mRNA levels of antimicrobial peptides/proteins, total RNA was extracted from control and FGF-7 skin using the Clontech nucleospin kit (Clontech, Palo Alto, CA, USA), according to the manufacturer’s instructions. cDNA synthesis and PCR amplification were made with a GeneAmp RT-PCR kit (Perkin – Elmer, Branchburg, NJ, USA). The primers for HBD-1, HBD-2, LL-37, ALP, lysozyme, BPI, and PLA2 are based on published sequences as described elsewhere [13]. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (Clontech) was used as a control amplimer set. RT-PCR was performed for each mRNA using a Perkin – Elmer DNA Thermal Cycler 480. Analysis of PCRs was done in 1.5% agarose gels and visualized by ethidium bromide staining. The results were quantified by scanning using a Fluor-S Imager coupled to Multi-Analyst/PC Software (Bio-Rad Laboratories, Hercules, CA, USA). Levels of mRNA were calculat-
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ed relative to G3PDH mRNA levels and the value of peptide/G3PDH expression in control was set as 1. Transplantation of bioengineered skin. Four- to eight-week-old female Swiss nu/nu mice (Massachusetts General Hospital, Boston, MA, USA) were anesthetized using 2,2,2-tribromoethanol (1.6 g/ml, 30 Al per gram of mouse) (Aldrich Chemical Company, Inc., Milwaukee, WI, USA). The grafting protocol was approved by the Subcommittee on Animal Care, Committee on Research, at the Massachusetts General Hospital. A square graft bed was created by excising approximately 1 cm2 of full-thickness skin from the back of the mouse. Panniculus carnosus and all the connective tissue under the skin were removed so that migration of blood vessels revascularizing the graft occurred only through the edges of the graft, not from the underlying tissues. Bioengineered skin of control or FGF-7-modified keratinocytes was placed into the bed and held in place by 6-O nylon sutures at the corners. Grafts were covered with Vaseline gauze, Tegaderm transparent dressing, a Band-Aid, and adhesive bandages. Grafts at 1, 2, 3, and 6 weeks were harvested along with some surrounding mouse skin (n = 25 – 27 grafts per time point). The specimens were placed epidermal side down, and digital images of the underside of the grafts showing the graft borders and the blood vessels were obtained using a camera coupled to a dissecting microscope with 25 magnification. Images were analyzed, and vascularized and nonvascularized areas of the grafts were quantified using the software Metamorph. Afterward, specimens were bisected, and half was embedded in Tissue-Tek OCT compound (Sakura Finetek, Inc., Torrance, CA, USA) and snap frozen for immunostaining. The other half was fixed in 10% formalin and embedded in paraffin for routine histology. Paraffin-embedded samples were sectioned (6 Am) and stained with hematoxylin and eosin (H&E). For immunostaining, frozen sections (6 Am) were fixed in acetone for 5 min and air-dried. To detect the presence of Ki67, sections were washed with PBS for 5 min and then incubated with blocking solution (3% BSA, 1% normal goat serum, 0.02% Na azide in PBS) for 1 h at 37jC. Sections were incubated with the IgG fraction of rabbit anti-human Ki67 (Pharmingen, Torrey Pines, CA, USA) for 1 h at 37jC. Slides were washed 3 15 min in PBS and incubated with fluorescein-conjugated affinity-purified goat anti-rabbit IgG antibody (1:100) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. The slides were coverslipped using 1% npropyl gallate mounting solution (Sigma). All antibodies were diluted in blocking solution. Control sections were not exposed to primary antibody. Neonatal foreskins served as tissue controls. Statistical analysis. Student’s t test was used to compare cell numbers in control versus FGF-7 epidermis and to compare bacterial counts in control versus FGF-7 skin. Data for VEGF ELISA, keratinocyte migration on plastic, self-healing, and vascularization measurements were tested for significance using Tukey’s test for pairwise comparisons.
ACKNOWLEDGMENTS This work was supported in part by the NIH (HD-28528) and the Shriners Hospitals for Children. RECEIVED FOR PUBLICATION DECEMBER 22, 2003; ACCEPTED APRIL 18, 2004.
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