Hydrosurgery as a safe and efficient debridement method in a clinical wound unit.

OBJECTIVE: Hydrosurgical debridement allows removal of non-viable tissue, preserving healthy tissues. This study was designed to analyse whether hydrosurgery, used in a clinical wounds unit, is an effective and safe method that may reduce debridement time. METHODS: Patients' wounds had the following characteristics: wounds with devitalised tissue needing rapid debridement, wounds with cavities, or non-healing wounds. Hydrosurgical debridement uses a pressurised stream of saline (0.9% sodium chloride) and a vacuum around this stream to remove the devitalised tissue of the wound, preserving healthy surrounding tissues. RESULTS: This prospective study comprised of 53 wounds from 39 patients. The wound aetiology included 39.7% arterial insufficiency, 22.6% pressure ulcers (PUs), 15.1% diabetic foot ulcers (DFUs), 9.4% venous leg ulcers (VLUs), and 13.2% from other aetiologies. The percentage of wounds according the size was the following: 32.1% (<10cm2), 43.4% (10-49cm2), 15.1% (50-99cm2), and 9.4% (≥100cm2). Superficial wounds were 43.4% of the total and 56.6% of wounds had cavities. Pain associated with the hydrosurgery was mild to moderate. There were no hydrosurgery-related adverse events. For effective debridement, the required sessions were as follows: one procedure (73.6%), two procedures (18.9%) and three procedures (7.5%). There was a statistical significant direct correlation (r=0.307) between the number of required sessions and wound size. All patients improved in a week (>80% of granulation tissue). CONCLUSION: We demonstrate that hydrosurgery is an effective and rapid debridement method that can be used safely in the outpatient setting.

Is population genetic structure of vascular plants shaped more by ecological or geographic factors? A study case on the Mediterranean endemic Centaurea filiformis (Asteraceae).

All known populations of the Sardinian endemic Centaurea filiformis Viv. (Asteraceae) were studied in order to understand the impact of both geographic and ecological factors on the genetic structuring of this species. Fourteen populations and 234 individuals were sampled. The demographic structure of the populations and the reproductive ecology were estimated in 28 plots. Population genetic analyses were based on SSR markers. Genetic structure was investigated by spatial Bayesian methods. Average densities of 0.51 individuals m-2 were detected, with a prevalence of adults. Ten species of pollinators were identified; C. filiformis ability to self-pollinate and myrmecochory were demonstrated experimentally. The populations displayed an average heterozygosity value of He  = 0.576 and high genetic differentiation (overall FST  = 0.218). Bayesian analysis suggests that five is the most probable number of gene pools of origin. A strong correlation between geographic distances and genetic distances among populations was highlighted. The demographic population structure of C. filiformis is dominated by adults, suggesting that it is a stable-regressive or senile species, investing more in local persistence than colonisation ability. Despite the scattered distribution, the populations studied do not present evidence of genetic erosion. The analysis of genetic differentiation reveals very high differentiation levels among populations, thus indicating that effective barriers exist against gene flow. A general conclusion is that population distribution results in a clear genetic structure for the populations studied, and that geography and not ecology is shaping the present distribution of this species.

Enhanced collagen synthesis and transcription by peak E, a contaminant of L-tryptophan preparations associated with the eosinophilia myalgia syndrome epidemic.

The pathogenesis of the eosinophilia myalgia syndrome (EMS) remains unclear. Several abnormal constituents have been found in the L-tryptophan lots responsible for the illness, particularly, 1,1-ethylidenebis[L-tryptophan], also called peak E or EBT, and 3-phenylamino-alanine or peak 5. However, the role of these contaminants in the pathogenesis of EMS and in the development of fibrosis is unknown. We now report that peak E, a dimer of L-tryptophan, is a potent stimulus for human dermal fibroblast DNA and collagen synthesis. Peak E (0.1-1.0 microM) increased DNA synthesis up to four-fold (P = 0.0001) in a dose-dependent manner (r = 0.987). When added to monolayer cultures for 2 to 24 h, peak E (0.5 to 100 microM) caused a progressive, more than threefold increase in alpha 1(I) procollagen mRNA levels and collagenous protein. No increase in procollagen mRNA levels was found after the addition of another major L-tryptophan contaminant, peak 5, or with L-tryptophan itself. Transient transfection with a 2.5-kb alpha 1(I) procollagen promoter-luciferase construct showed that peak E causes a twofold upregulation of promoter activity (P = 0.022). Contraction of collagen gels, consisting of human dermal fibroblasts incorporated into a type I collagen lattice, was enhanced two-fold by exposure to peak E (P = 0.001). We conclude that a major constituent of contaminated batches of L-tryptophan, peak E, is a potent stimulus for fibroblast activation and collagen synthesis. This stimulatory action of peak E may provide a direct mechanism for the development of fibrosis in EMS.

Graftskin, a human skin equivalent, is effective in the management of noninfected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial.

OBJECTIVE: We assessed in a randomized prospective trial the effectiveness of Graftskin, a living skin equivalent, in treating noninfected nonischemic chronic plantar diabetic foot ulcers. RESEARCH DESIGN AND METHODS: In 24 centers in the U.S., 208 patients were randomly assigned to ulcer treatment either with Graftskin (112 patients) or saline-moistened gauze (96 patients, control group). Standard state-of-the-art adjunctive therapy, which included extensive surgical debridement and adequate foot off-loading, was provided in both groups. Graftskin was applied at the beginning of the study and weekly thereafter for a maximum of 4 weeks (maximum of five applications) or earlier if complete healing occurred. The major outcome of complete wound healing was assessed by intention to treat at the 12-week follow-up visit. RESULTS: At the 12-week follow-up visit, 63 (56%) Graftskin-treated patients achieved complete wound healing compared with 36 (38%) in the control group (P = 0.0042). The Kaplan-Meier median time to complete closure was 65 days for Graftskin, significantly lower than the 90 days observed in the control group (P = 0.0026). The odds ratio for complete healing for a Graftskin-treated ulcer compared with a control-treated ulcer was 2.14 (95% CI 1.23-3.74). The rate of adverse reactions was similar between the two groups with the exception of osteomyelitis and lower-limb amputations, both of which were less frequent in the Graftskin group. CONCLUSIONS: Application of Graftskin for a maximum of 4 weeks results in a higher healing rate when compared with state-of-the-art currently available treatment and is not associated with any significant side effects. Graftskin may be a very useful adjunct for the management of diabetic foot ulcers that are resistant to the currently available standard of care.

The development of bioengineered skin.

Driven by the need for donor tissue for patients suffering from extensive burns, alternatives to autologous and cadaver-derived tissue have been under development for the past 20 years. Unilayered and bilayered models representing the skin's epidermal and/or dermal components have been developed using both cells and matrix materials. In addition to their use in patients with extensive burns, trials using these products on refractory and challenging patients with both acute and chronic wounds have led to the commercial availability of some of these products.

Transforming growth factor-beta regulates the adhesive interactions between mononuclear cells and microvascular endothelium.

Adhesion of leukocytes to the vascular endothelium is essential for the movements of cells from the bloodstream into inflammatory sites. In the present study, dermal microvascular endothelial cells (DMEC) isolated from normal porcine skin retained the capacity to adhere 51Cr-labeled porcine peripheral blood mononuclear cells (PBMC), nylon-wool-purified T cells, and isolated monocytes. Transforming growth factor-beta 1 (TGF-beta) decreased the capacities of DMEC to support the adhesion of these cells in a dose-dependent manner. Maximal inhibition was observed with a TGF-beta dose of 0.25 ng/ml and an incubation time of 6-12 h. TGF-beta did not affect the morphology of DMEC and had no adverse effect on the viability of the treated cells. The blocking effects of TGF-beta on PBMC adhesion to DMEC was neutralized by a polyclonal turkey anti-TGF-beta antiserum but not by control turkey serum. Although pretreatment of PBMC with TGF-beta decreased the capacity of these cells to adhere to normal DMEC monolayers, kinetic studies demonstrated that these effects required between 4 and 8 h incubation time. In addition, preincubation of DMEC with TGF-beta completely blocked their response to the stimulating effects of TNF-alpha, IL-1-beta, or both cytokines. Furthermore, TGF-beta also abrogated the enhanced adhesiveness of DMEC pretreated with TNF-alpha and IL-1-beta. These findings suggest that TGF-beta may play an important role in the down-regulation of inflammatory responses by decreasing vascular endothelial adhesiveness for mononuclear cells and monocytes.

Lymphocyte adhesion to psoriatic dermal endothelium is mediated by a tissue-specific receptor/ligand interaction.

Dermal lymphocytic infiltrates are characteristic of psoriasis and may be involved in the pathogenesis of the disease. We have utilized an in vitro lymphocyte adherence assay to examine the mechanism(s) mediating lymphocyte migration into psoriatic skin. In this assay, we assessed the binding of lymphocytes overlaid onto frozen biopsy sections of normal and psoriatic skin. Lymphocytes isolated from human blood and rat thoracic duct bound specifically to dermal endothelia in psoriatic plaques but not to those of uninvolved skin from psoriatic patients or skin from normal individuals; analysis of the binding properties of B cells and T lymphocyte subsets revealed a preferential binding of CD4+ T cells compared with CD8+ T cells or B cells. This lymphocyte-endothelial interaction is an energy- and calcium-dependent process and involves surface protein and carbohydrate moieties, requirements similar to those found in lymphocyte interaction with post-capillary high endothelial venules (HEV) in lymphoid tissues. However, preincubation of lymphocytes with antibodies directed against surface molecules mediating adhesion to HEV of peripheral lymph node and gut-associated lymphoid tissue did not interfere with the capacity of lymphophocytes to bind to the skin. The results of this study support the hypothesis that emigration of lymphocytes from vasculature into psoriatic skin is promoted by the presence of specialized endothelia in psoriatic dermis capable of mediating specific lymphocyte-endothelial interactions.

Transforming growth factor-beta differentially regulates the adhesiveness of normal and psoriatic dermal microvascular endothelial cells for peripheral blood mononuclear cells.

Lymphocytes adhere to dermal microvascular endothelial cells (DMEC) as the first step in their migration from the bloodstream into diseased skin. Psoriasis is characterized by an intense T-lymphocytic infiltrate in the dermis, which may be a consequence of the abnormal regulation of endothelial adhesiveness by cytokines released locally. In the present study, we investigated the effects of tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1), IL-4, and transforming growth factor-beta 1 (TGF-beta) on the adhesiveness of DMEC isolated from psoriatic plaques or normal skin for human peripheral blood mononuclear cells (PBMC). The results showed that DMEC from both normal and psoriatic skin retain the capacity to adhere to 51Cr-labeled PBMC. Pretreatment of DMEC from normal skin with human recombinant IL-1 or TNF alone or in combination for 8 h significantly (p less than 0.01) enhanced their capacity to adhere to human PBMC. Similarly, treatment of normal DMEC with IL-4 also increased endothelial adhesiveness, although this cytokine required an incubation period of 24 h. In parallel studies, DMEC from psoriatic plaques were found to respond to the stimulatory effects of TNF, IL-1, and IL-4 in similar dose- and time-dependent manner. In contrast, although pretreatment of normal DMEC with TGF-beta (0.1 to 0.25 ng/ml) for 6 to 12 h significantly reduced (p less than 0.01) both the unstimulated and IL-1- and TNF-stimulated endothelial adhesiveness for normal PBMC, TGF-beta had no effect on the binding of unstimulated or cytokine-stimulated psoriatic DMEC to PBMC, even at concentrations as high as 2 ng/ml and incubation period of 36 h. These results suggest that cytokines stimulate the adhesiveness of DMEC through distinct pathways and provide evidence that TGF-beta may play an important regulatory role in the control of lymphocyte extravasation into normal skin. The altered responsiveness of psoriatic DMEC to TGF-beta may contribute to the intense dermal lymphocytic infiltrates in psoriasis.

Lymphocyte recognition of psoriatic endothelium: evidence for a tissue-specific receptor/ligand interaction.

Epidermotropic lymphocytes are an essential cellular component of the skin-associated lymphoid tissues (SALT). Dermal lymphocytic infiltrates are also characteristics of inflammatory dermatoses, such as psoriasis, and may be involved in the pathogenesis of the disease, although the mechanisms by which lymphocytes are recruited into these sites are not known. We have used an in vitro lymphocyte adherence assay to test the hypothesis that specialized endothelial cells are present in inflamed skin, and are capable of supporting lymphocyte adherence and promoting lymphocyte emigration. In this assay, we assessed the binding of lymphocytes overlaid onto frozen sections of normal and psoriatic skin. Peripheral blood mononuclear cells (PBMC) from patients, from healthy volunteers, and from rat thoracic duct bound specifically to dermal endothelia in psoriatic plaques, in steroid-resistant areas of plaques, but not in uninvolved skin or skin from healthy individuals. Analysis of the binding properties of lymphocyte subsets revealed preferential adherence by CD4+ T cells as compared with CD8+ T cells and to B cells. Interestingly, PBMC from patients undergoing ultraviolet light therapy failed to adhere to autologous skin or to lesion-containing skin sections from untreated patients. Additional studies indicate that the lymphocyte-endothelial interaction is an energy- and calcium-dependent process and involves surface glycoprotein and carbohydrate moieties, requirements similar to those found in specific lymphocyte interactions with high endothelial venules in lymph nodes during the homing process. Pretreatment of lymphocytes with antibodies directed against homing receptors mediating migration into lymph nodes and into gut-associated lymphoid tissues, however, did not interfere with lymphocyte adherence to psoriatic endothelium. In contrast, anti-lymphocyte function associated antigen (LFA)-1 antibody partially inhibited lymphocyte binding to chronic plaques. We conclude that a tissue-specific receptor/ligand interaction independent of LFA-1 directs lymphocyte emigration from the dermal microvasculature into the psoriatic dermis and that LFA-1 plays only an accessory role in the adhesion process.

Transforming growth factor-beta receptor binding and function are decreased in psoriatic dermal endothelium.

T lymphocyte adhere to dermal microvascular endothelial cells (DMEC.) as the first step in their emigration from the blood vasculature into diseased skin. Earlier studies have shown that the adhesiveness of cultured DMEC. from normal skin for lymphocytes can be blocked by transforming growth factor-beta1 (TGF-beta1). In contrast, TGF-beta1 has no effect on the adhesive properties of DMEC from psoriatic plaques, and this response is attenuated by the addition of interleukin-4 (IL-4). In the present study, we show that both TGF-beta1 and TGF-beta2, and to a lesser extent TGF-beta3 isoforms block the ability of normal but not psoriatic DMEC to bind lymphocytes. Pretreatment with TGF-beta1 selectively inhibited the tumor necrosis factor-alpha(TNF-alpha)-stimulated expression of E-selecting on normal DMEC but had no psoriatic DMEC. Scatchard analysis revealed both low- and high-affinity receptors on normal DMEC. The baseline number of high-affinity TGF-beta receptors was significantly reduced on psoriatic DMEC, whereas IL-4 treatment of DMEC altered the binding affinity but not the number of receptors. The protein and mRNA transcripts of type I and type II TGF-beta receptor genes were detectable in psoriatic DMEC. A reduction in the autophosphorylation the TGF-beta type II receptor protein, a constitutively active serine/threonine kinase, however, was detected in psoriatic DMEC. These in vitro finding suggest that reduction of TGF-beta receptor expression and function may contribute to lymphocyte infiltration into psoriatic plaques in vivo by allowing dermal microvascular endothelium to escape form the negative regulation by TGF-beta.

Hypoxia upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts.

In this report, we have investigated the secretion and synthesis of transforming growth factor-beta 1 (TGF-beta 1) by human dermal fibroblast cultures in response to hypoxia (2% oxygen), and have compared it to standard oxygen culture conditions (15% oxygen at the cell surface). Sandwich enzyme-linked immunosorbent assay (SELISA) showed a selective and progressive increase in secretion of the TGF-beta 1 isoform in response to hypoxia, up to ninefold after cultures were exposed to low oxygen for 72 h; TGF-beta 2 peptide levels were not increased. We then investigated the transcriptional regulation of the TGF-beta 1 gene in response to low and standard oxygen tensions. In the first 24-48 h, TGF-beta 1 mRNA levels decreased steadily in both oxygen environments. This mRNA decline continued for up to 72 h in standard oxygen but not in cultures exposed to low oxygen tension. At 72 h, steady-state TGF-beta 1 mRNA levels were 8 times greater in low compared to standard oxygen, and this increase was reversible upon re-exposure of fibroblast cultures to standard oxygen tension for 24 h. Elevated TGF-beta 1 m-RNA levels in both low and standard oxygen declined steadily and with the same half-life after the addition of actinomycin D, suggesting that hypoxia increased TGF-beta 1 transcription rather than mRNA stability. We conclude that low oxygen tension upregulates the synthesis of TGF-beta 1 by human dermal fibroblasts, and leads to increased secretion of this peptide.

Human dermal fibroblast clones derived from single cells are heterogeneous in the production of mRNAs for alpha 1(I) procollagen and transforming growth factor-beta 1.

Fibroblast clonal heterogeneity has been reported for growth and protein synthesis, but quantitative studies of synthetic phenotype at the pretranslational level have been limited because of difficulty in reliably growing large numbers of clonal cells. We have recently shown a unique stimulatory activity of low oxygen tension in the early phases of clonal growth, which can be used to establish clonal fibroblast cultures suitable for Northern analysis. Using this methodology, we have measured mRNA levels of alpha 1(I) procollagen and transforming growth factor-beta 1 (TGF-beta) both at baseline and after TGF-beta stimulation in a total of 43 clones derived from single cells and from seven different cell strains. We report a remarkable baseline heterogeneity, commonly four- to six-fold, in procollagen mRNA levels among clones and between clones and their parent cultures. Conversely, differences in baseline TGF-beta mRNA levels among clones were either not present or less than onefold. The clonal phenotypic expression of alpha 1(I) procollagen mRNA remained stable after eight additional cell passages. TGF-beta stimulation of itself (autoinduction) was highly variable among clones (range of increases 30% to 150%), and up-regulation of procollagen mRNA levels after TGF-beta stimulation was detected in only 15 (54%) of 28 clonal cultures (range of increases 30% to 353%). A notable lack of correlation was found between baseline mRNA levels of TGF-beta and alpha 1(I) procollagen in clonal cultures. In conclusion, fibroblast clonal populations are remarkably heterogeneous in their baseline procollagen mRNA levels and in their response to TGF-beta.

Transforming growth factor-beta: selective increase in glycosaminoglycan synthesis by cultures of fibroblasts from patients with progressive systemic sclerosis.

Transforming growth factor-beta (TGF-beta) has been found in all cells examined thus far, and has been shown to play an important role in inflammation and connective tissue formation. We now report that TGF-beta, alone or in combination with epidermal growth factor (EGF), led to a preferential increase in glycosaminoglycan synthesis by cultures of dermal fibroblasts from patients with progressive systemic sclerosis (PSS) when compared with normal fibroblasts (p less than 0.001). Transforming growth factor-beta increased collagen synthesis to the same extent in both PSS and normal fibroblasts, whereas EGF had no stimulatory activity on collagen synthesis. The addition of EGF to cultures incubated with TGF-beta led to a decrease in collagen synthesis compared with the effect seen with TGF-beta alone (p less than 0.02). These studies suggest that TGF-beta may play an important role in the accumulation of connective tissue seen in PSS and that the combined action of multiple growth factors may modulate the synthetic activity of human dermal fibroblasts.

TGF-beta3 stimulates and regulates collagen synthesis through TGF-beta1-dependent and independent mechanisms.

In contrast to the TGF-beta1 and beta2 isoforms, TGF-beta3 has shown the ability to downregulate scarring and fibrosis in vivo under certain experimental conditions. In this study, we determined the direct effects of TGF-beta3 on cultures of human dermal fibroblasts. TGF-beta3 (0.1 to 100 pg per ml) increased DNA synthesis up to 50% (p < 0.01, r = 0.970), collagen protein synthesis up to 200% (dose range of 0.1 to 5 ng per ml, p < 0.001, r = 0.990), and increased alpha1(I) procollagen mRNA levels (r = 0.999), with maximal effects (200% of control) observed by 24 h. Collagen lattice contraction was increased by more than 50% in response to TGF-beta3 (p < 0.001), and to a similar extent as the TGF-beta1 isoform. Stimulation of collagen synthesis and of alpha1(I) procollagen mRNA levels in response to TGF-beta3 was partially blocked by a TGF-beta1-specific anti-sense oligonucleotide but was still detectable (35% greater than baseline) when TGF-beta3 was added to dermal fibroblasts from TGF-beta1 knock-out mice. In contrast with these stimulatory effects, however, downregulation of alpha1(I) procollagen, alpha1(III) procollagen, and TGF-beta1 mRNA levels toward baseline occurred when TGF-beta3 (0.1 to 5 ng per ml) was added simultaneously and in combination with TGF-beta1. We conclude that stimulation of collagen synthesis by TGF-beta3 occurs through TGF-beta1-dependent and independent pathways. By downregulating the response to TGF-beta1 and by shifting from one pathway to the other, TGF-beta3 can dampen and provide fine-tuning to the overall TGF-beta's induced program of collagen deposition.

Recruitment of mononuclear cells by endothelial cell binding into wounded skin is a selective, time-dependent process with defined molecular interactions.

Wound healing involves a complex series of interactions between cells in the dermis and epidermis, and important relationships exist between keratinocytes and resident dermal cells. Monocytes and lymphocytes secrete cytokines that are capable of stimulating dermal repair and influencing keratinocyte and fibroblast migration and proliferation, although the mechanism by which mononuclear cells are recruited into the wound is unknown. We have tested the hypothesis that in wounded skin specialized endothelial cells are induced to mediate peripheral blood mononuclear cell (PBMC) emigration from the vasculature into the dermis. For this purpose, partial-thickness wounds made with a keratome on the backs of domestic pigs were excised 0 to 9, 12, 15, and 21 d after wounding. The biopsies were then tested for the capacity to adhere selectively to PBMC. The results indicated that PBMC overlaid onto sections of wounds from day 4 to 15 adhered selectively to dermal endothelium, with two distinct peaks of adherence observed on day 7 and day 12. In contrast, PBMC did not adhere to the tissue sections when overlaid onto frozen sections of normal skin or 0-, 1-, 2-, 3-, and 21-d-old wounded skin. Additional studies on the binding properties of PBMC subsets revealed that monocytes adhered maximally at day 7, whereas T cells adhered optimally at day 12 post-wounding. Furthermore, the adhesion process was energy and magnesium dependent but not calcium dependent and involved surface protein and carbohydrate moieties on PBMC surface. Pre-treatment of PBMC with monoclonal antibodies against the LFA-1 adhesive receptors inhibited the binding by greater than 80%, suggesting that LFA-1 adhesive receptors play an important role in the binding process. These studies provide evidence that the recruitment of monocytes and lymphocytes into wounds is an active, dynamic, and regulated process mediated at least in part by specific adhesive interactions between mononuclear leukocytes and dermal endothelial cells.

Adherence of human helper/memory T-cell subsets to psoriatic dermal endothelium.

Dermal lymphocytic infiltrates are characteristic features of psoriasis and may be involved in the pathogenesis of the disease. We have previously shown that specialized endothelial cells are present in the dermis of psoriatic skin and are capable of supporting lymphocyte adherence and promoting lymphocyte extravasation. In this study, we investigated the dermal endothelial binding properties of human lymphocyte subsets and the role of LFA-1 molecules in the adhesion process. It was found that both human T cells and B cells adhered to frozen sections of psoriatic plaques, but with different cell dose-response relationships. In addition, quantitative assessment of lymphocyte adhesion demonstrated that human CD4+T cells and the CDw29+ (helper-inducer) subset adhered preferentially to the papillary dermis as compared with CD8+ T cells or the CD45R+ (suppressor-inducer) subset. Moreover, human lymphocytes adhered to untreated psoriatic plaques, but not to uninvolved skin or to steroid-treated, clinically and histologically resolved lesions. Preincubation of T lymphocytes with saturating amounts of monoclonal antibody 60.3 against the surface membrane CD18 glycoprotein complex inhibited partially their capacity to bind to untreated psoriatic plaques. These observations suggest that the emigration of human CD4+ T cell and the CDw29+ helper-inducer subset is promoted by selective adherence to psoriatic dermal endothelia, with LFA-1 molecules playing an accessory role in the binding process.

Stimulation of collagen synthesis by the anabolic steroid stanozolol.

There is evidence that anabolic steroids, which are derived from testosterone and have markedly less androgenic activity, promote tissue growth and enhance tissue repair; however, the mechanisms involved in their anabolic activities remain unclear. In this report, we measured the effect of the anabolic steroid stanozolol on cell replication and collagen synthesis in cultures of adult human dermal fibroblasts. Stanozolol (0.625-5 microg per ml) had no effect on fibroblast replication and cell viability (p = 0.764) but enhanced collagen synthesis (p < 0.01) in a dose-dependent manner (r = 0.907). Stanozolol also increased (by 2-fold) the mRNA levels of alpha1 (I) and alpha1 (III) procollagen and, to a similar extent, upregulated transforming growth factor-beta1 (TGF-beta1) mRNA and peptide levels (p < 0.001). There was no stimulation of collagen synthesis by testosterone. The stimulatory effects of stanozolol on collagen synthesis were blocked by a TGF-beta1 anti-sense oligonucleotide, by antibodies to TGF-beta, and in dermal fibroblast cultures derived from TGF-beta1 knockout mice. We conclude that collagen synthesis is increased by the anabolic steroid stanozolol and that, for the most part, this effect is due to TGF-beta1. These findings point to a novel mechanism of action of anabolic steroids.

Proliferation and collagen synthesis by human dermal fibroblasts cultured from different body sites.

Distal areas in systemic sclerosis (scleroderma), such as the dorsal skin of the hand are more frequently involved and more indurated than proximal areas. On the contrary, the observation often made after surgical excision or trauma is that distal body areas heal more slowly than proximal areas. A possible explanation may be that dermal fibroblasts from distal body parts are more capable, when stimulated, to synthesize greater or lesser amounts of collagen and proliferate at different rates than dermal fibroblasts from more proximal skin. In this study, cultures of dermal fibroblasts from three different body sites (arm, forearm, and hand) of healthy volunteers were investigated for their proliferative activity and collagen synthesis after stimulation in 3% or 10% fetal bovine serum. No significant differences were observed in cell proliferation or in the relative or absolute collagen synthesis by fibroblasts cultured from the hand, forearm or upper arm. We conclude that other in vivo factors are responsible for the observed differences in fibrosis and healing at different body sites. Moreover, if clonal expansion of different fibroblast phenotypes occurs in these physiologic or disease states, it must be of a magnitude that overcomes the fundamental proliferative and biosynthetic baseline.

Retrovirally mediated gene transfer in a skin equivalent model of chronic wounds.

Given that treatment for chronic wounds is unsatisfactory, it is likely that gene therapy may be tested as a therapeutic modality in this difficult clinical problem. Actively proliferating cells in wounds are also a good target for retroviral transduction, an increasingly useful method for gene therapy. However, it is unclear how gene therapy may best be used in chronic wounds, and experimental models are urgently needed to study and manipulate gene transfer in the context of chronic wounds. In this report, partial- and full-thickness wounds were made in vitro in a human living skin equivalent (LSE) consisting of fully differentiated keratinocytes layered over a collagen matrix seeded with fibroblasts. To mimic a chronic wound situation, we used tissue culture conditions which, as in a chronic wound, allowed fibroblast but not keratinocyte proliferation or migration. The wounded LSE was then placed over a transduced cell line (PA317) which produced a replication defective retrovirus containing as a histological marker the bacterial beta galactosidase gene. Using this close and direct exposure to the virus-producing cell line, distinct staining for beta-galactosidase was observed in partial-thickness wounds, and was limited to fibroblasts away from the upper site of injury and immediately overlying the retrovirus-producing cell monolayer. Expression of beta-galactosidase was uniformly present at the wound edges and along the base of the entire partial thickness wound. These studies demonstrate that, in in vivo conditions mimicking a chronic wound, an intimate apposition of the injured LSE with the virus-producing cell line is needed for gene transfer. Using this in vitro model system, gene transfer protocols may be optimized prior to beginning in vivo studies in chronic wounds.

Growth inhibition of primary keratinocytes following transduction with a novel TGFbeta-1 containing retrovirus.

Growth and migration of keratinocytes are known to be affected by the addition of exogenous cytokines, such as TGFbeta-1, to culture media. We have developed a retroviral vector, LNTbeta-1, that confers constitutive expression of human TGFbeta-1 to transduced cells. Keratinocytes were exposed to retroviral particles generated in serum-free media, and infected cells were selected for with Geneticin. Transduced keratinocytes remained in culture as single cells instead of a normally grouped growth pattern. While these transduced keratinocytes survived in culture for several weeks, they did not proliferate and seemed arrested in their growth. Keratinocytes transduced with retrovirus not containing the TGFbeta-1 gene appeared normal in their growth pattern. These findings indicate that high-level endogenous expression of TGFbeta-1 in keratinocytes can at least inhibit, and possibly arrest, growth.