Up-regulation of prostaglandin EP4 receptor messenger RNA in fetal rabbit skin wound.

BACKGROUND: Scar formation and subglottic stenosis often cause health problems in surgical otolaryngology. However, fetal wounds demonstrate scarless healing. The underlying mechanism remains poorly understood. We isolated differentially expressed genes by comparison between nonwounded with wounded skin of fetal and adult rabbits. METHODS: Skin incisional wounds were made in fetal (21 to 23 days' gestation) and adult rabbits. Nonwounded and wounded skin were harvested 12 hours after surgery. Total RNA was extracted. By means of messenger RNA differential display, differentially expressed complementary DNA fragments were isolated, cloned, and sequenced. The expressed transcripts were verified by reverse RNA dot blot and semiquantitative reverse transcription and polymerase chain reaction. RESULTS: One complementary DNA tag that was induced in fetal skin wounds and repressed in adult skin wounds was isolated. The sequence of this complementary DNA (352 base pairs) encodes the messenger RNA for the E-prostanoid (EP) 4 receptor for prostaglandin E(2) (PGE(2)). The truly differential expression of the transcript was confirmed. In normal skin, the EP4 receptor messenger RNA levels were higher in adults than in fetuses. Twelve hours after wounding, the EP4 receptor transcript was remarkably induced in fetal skin wounds but repressed in adult skin wounds. CONCLUSIONS: Our study demonstrates the differential expression of the EP4 receptor messenger RNA in fetal and adult skin before and 12 hours after wounding. Our results suggest that prostaglandin E(2) is involved in the differential cellular responses and in the regulation of the intracellular signal transduction through its binding to EP4 receptor during fetal wound repair.

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

BACKGROUND/PURPOSE: Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS: Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. RESULTS: Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS: With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Treatment of chronic suppurative otitis media with topical tobramycin and dexamethasone.

OBJECTIVES: To investigate the safety and efficacy of a topical combination of tobramycin and dexamethasone in a primate model of chronic suppurative otitis media (CSOM) and to explore the contribution of the added topical steroid for the treatment of CSOM. DESIGN: Blinded, randomized, placebo-controlled trial. SUBJECTS: Sixty juvenile cynomolgus monkeys randomized into the following 6 treatment groups of 10 monkeys each: 0.3% tobramycin (group 1), combined 0.3% tobramycin-0.1% dexamethasone (group 2), combined 1.0% tobramycin-0.33% dexamethasone (group 3), 0.1% dexamethasone (group 4), vehicle (group 5), and phosphate-buffered saline solution (group 6). INTERVENTIONS: Chronic suppurative otitis media was established by inoculating the right ear with Pseudomonas aeruginosa. After 4 weeks of drainage, animals were treated according to the group assignment with 3 drops twice daily for 7 weeks. Hearing thresholds were monitored with repeated auditory brainstem response testing (ABR), and clinical response was monitored with repeated otoscopic examinations and cultures throughout the study. Cytocochleograms were evaluated for quantification of outer hair cell loss. RESULTS: Rapid resolution of otorrhea and eradication of P aeruginosa occurred in all groups receiving tobramycin. The inclusion of dexamethasone accelerated the resolution of otorrhea and negative yields of cultures compared with tobramycin alone. Otorrhea and positive culture findings persisted in the groups not treated with topical antibiotic. Results of ABRs at 4 and 8 weeks and cytocochleograms for outer cell hair loss were not affected by drug administration. Perilymph samples collected at the end of the study showed no detectable tobramycin. CONCLUSIONS: Combined tobramycin-dexamethasone ear drops were safe and effective in the monkey CSOM model. Dexamethasone enhanced the efficacy of tobramycin.

Transplantation of virally transduced cells into the dermis of immunocompetent and immunodeficient (SCID) mice to determine gene expression profile and differential donor cell survival.

Cell therapy and bioengineering hold great promise as therapeutic approaches using cells and cell-derived factors to treat various pathologic or trauma-induced states. One possible application is the transplantation of cells into wounded tissue to help regulate tissue repair. Cells engineered for optimal wound healing may help to minimize scarring following surgery or to enhance the rate of healing of chronic wounds. The purpose of the current study was to determine the effect of a viral insert, the LacZ-bearing, first generation adenovirus AdRGD, on the survival of dermally transplanted murine skin allogenic fibroblasts. The LacZ insert facilitated quantitation of both cell survival and gene expression and was used here to measure viable cell number. In addition to bearing the LacZ marker, the AdRGD vector is capable of carrying therapeutic gene inserts, so this study tested the feasibility of gene therapy for wound healing. Murine skeletal muscle PP6 (i.e., Pre-Plate 6) myogenic stem cells served as an alternate donor cell type. Cells were labeled with the LacZ-bearing AdRGD adenovirus vector and injected (50,000 cells/site) into the dorsal skin of adult normal, immunocompetent mice as well as in immunodeficient SCID mice. Skin biopsies were taken on days 0, 1, 2, 3, and 7 post-transplant, and assayed for LacZ expression. Soon after transplant (day 1), cell numbers underwent a transient decrease, but by day 2 post-transplant they were present in appreciable numbers. Between days 2-7 post-transplant, both allogenic fibroblasts and PP6 myogenic stem cells maintained survivability in similar numbers. Further, survival of transplanted cell types was similar in both normal, immunocompetent as well as SCID mice during this time period. There were no signs of acute inflammation or rejection in any of the samples. This study shows that AdRGD-transduced cells are not immunogenic in the mouse skin model and the cells show similar survival for the first 7 days post-transplantation independent of the cell type or immunocompetence of the host.

Transplanted fetal fibroblasts: survival and distribution over time in normal adult dermis compared with autogenic, allogenic, and xenogenic adult fibroblasts.

Cell therapy is a widely applicable therapeutic approach using cells and cell elements, frequently from fetal or young animals, for their beneficial effects. This study evaluated the host response to and tolerance of transplanted fetal skin fibroblasts. Cultured fibroblasts from adult rabbit skin (autogenic and allogenic), 21-day fetal rabbit skin (allogenic), and adult pig skin (xenogenic) were labeled with a fluorescent vital dye CM-DiI, injected intradermally into the dorsal skin of adult rabbits at multiple sites and then biopsied over an 8-week period. Each cell type showed a biphasic distribution curve with an early phase (0 to 28 days) and a late phase (28 to 56 days). In the early phase, cells showed a rise and fall in total cell density (reflecting an increase and then a decrease in total cell number), followed by a slow decrease in cell density with cells still detectable at 56 days. Fetal cells showed the highest survival at the end of the study. None of the groups showed clinical or histologic signs of acute inflammation or rejection. This study demonstrated that (1) transplanted fibroblasts are well tolerated by an immunologically competent host, (2) CM-DiI-labeled cells are detectable in vivo for at least 8 weeks, and (3) fetal fibroblasts have a distribution and survival profile that is distinct from that of adult fibroblasts.

Wound management of the airway mucosa: comparison with skin in a rabbit model.

In comparison to the extensive study of skin wound healing, there have been few reports investigating mucosal wound healing. Our primary objective was to compare the natural progression of wound healing in airway mucosa to skin in a rabbit model. Split-thickness skin wounds and subglottic mucosal wounds created by drill injury were compared on days 0, 1, 3, 5, 7, 14 and 21 after injury. Histologic examination was performed by a veterinary pathologist blinded to sample identity. Subglottic wounds showed a 'fibrinous clot' overlying the epithelium, analogous to the fibrin crust in skin wounds. Re-epithelialization started on day 5 in the subglottic epithelium and was complete by day 14; fibroplasia and fibrosis in the lamina propria were present on days 7-21. This wound healing profile paralleled the skin epidermis and dermis, respectively. The epithelial changes, however, were temporally extended in the airway. Our secondary objective was to determine the effects of treating airway mucosa with a bioresorbable membrane, modified sodium hyaluronate and carboxymethylcellulose (modified HA/CMC), placed over the subglottic wounds of four rabbits after drill injury. Subglottic wounds treated with modified HA/CMC showed a more mature epithelium and less fibrosis on day 21. In this pilot study, the application of a bioresorbable membrane improved mucosal wound healing at both the epithelial and lamina propria levels. Clearly, a larger study must be performed to confirm this interesting observation.

Upregulation of messenger RNA for inflammatory cytokines in middle ear mucosa in a rat model of acute otitis media.

A rat model for acute otitis media has been established and was used to delineate the temporal expression of messenger RNA for key inflammatory cytokines. Inoculation with live Streptococcus pneumoniae induced a rapid expression of tumor necrosis factor alpha (within 6 hours) followed by upregulation of message for interleukin (IL)-6 (peak at 12 to 24 hours, remaining elevated through 120 hours) and IL-10 (peak at 24 hours). Inducible nitric oxide synthase message was also selectively increased following live bacterial inoculation (peak at 12 to 24 hours). Although there was a detectable inflammatory response to killed bacteria, it was minimal, was of short duration, and preceded the peak for live bacteria; only expression of IL-6 was significantly increased in this group (peak at 12 hours, remaining elevated through 72 hours). We interpret this to be due to an inflammatory response to bacterial products (such as lipopolysaccharide) in the heat-killed bacterial inoculum. The phosphate-buffered saline (PBS)-inoculated group exhibited a transient increase of IL-6 message, which indicates that this cytokine is a sensitive marker of the acute response to trauma. Otherwise, PBS invoked only a slight reaction in the mucosa with respect to the other inflammatory mediators being measured.

Fetal airway wound repair: a new frontier.

PURPOSE: Fetal dermal repair is regenerative and scarless until middle to late gestation, when there is a transition to fibrotic repair. Fetal skeletal muscle and tendon undergo repair with fibrosis similar to the process in adults. This study addresses whether fetal mucosal healing is regenerative and scarless. METHODS: Anesthetized pregnant rabbits underwent laparotomy and controlled hysterotomy at 21 to 23 days' gestation (term is 31 days). A midline thyrotomy was made, followed by cricoidotomy and circumferential cauterization of the subglottic mucosa. A similar insult was applied to weanlings. The data were collected in 2 groups. One group was followed to term and killed at 4 weeks. A second group was killed after 6 days (30 days' gestation). The weanlings were killed at similar points. The larynges were harvested and processed for histological and morphometric analysis. RESULTS: Three litters were followed to term. Of these, 1 was not recovered; in the other two, 7 of 8 manipulated fetuses were found and 3 of 8 were viable. The fourth litter was harvested after 6 days; all 4 injured fetuses were recovered and viable. All animals in the fetal injury groups healed with complete regeneration of the airway mucosa. In contrast, weanlings injured post partum had mucosal inflammation, necrosis, and ulceration; squamous metaplasia and basal cell hyperplasia were also found. There were fibrosis, granulation tissue, and inflammation in the lamina propria; chondritis, cartilaginous necrosis, chondrolysis, and perichondritis were also found. CONCLUSIONS: Fetal airway mucosal healing is regenerative and, thus, scarless. This study provides further support for the thesis that skin and mucosa respond to injury similarly in both the developmental and postpartum stages, and that subglottic stenosis is reasonably thought of as the "hyperplastic scar" of the airway. These results have potential therapeutic applications for mucosal wound management.

Acquired subglottic stenosis--depth and not extent of the insult is key.

In contrast to skin, mucosal wound healing has not been extensively studied. Subglottic stenosis (SGS) is an excellent model for such investigation. The main objective of this pilot study was to develop a chronic model of SGS in a small animal (i.e. rabbit). In so doing, a serendipitous observation was made that the development of SGS is directly related to depth of the injury and is independent of circumferential extent. Animals with deep injury (i.e. deep to the lamina propria, reaching the perichondrium), independent of age and circumferential extent, experienced respiratory obstruction resulting from edema and granulation tissue formation and died or had to be sacrificed in the acute period. This was in contrast to no risk of mortality in the more superficially injured group. Histology was used to characterize this model of SGS. In the mucosal epithelium, or mucosa, changes of inflammation, squamous metaplasia, basal cell hyperplasia, necrosis and ulceration were only seen acutely and total regeneration of the epithelium was achieved by the end of the study period. In contrast, changes within the lamina propria, including chronic inflammatory cellular infiltrates and fibroplasia, were lasting and resulted in fibrotic repair, not regeneration. These findings are quite similar to the healing events in skin and suggest that SGS is the mucosal equivalent of a 'keloid' or, perhaps more appropriately, a 'hypertrophic scar.' Likewise, cartilage degeneration and deformation were persistent markers of the chronic phase of healing. Like the lamina propria, the response to injury was reparative. Therefore, injury to the connective tissue is a critical component of development of SGS.

Nitric oxide inhibits the synthesis of type-II collagen without altering Col2A1 mRNA abundance: prolyl hydroxylase as a possible target.

The addition of human recombinant interleukin-1beta (IL-1beta) to cultures of lapine articular chondrocytes provoked the synthesis of large amounts of NO and reduced the production of type-II collagen. NG-Monomethyl-l-arginine (L-NMA), an inhibitor of NO synthase, strongly suppressed the production of NO and partially relieved the inhibition of collagen synthesis in response to IL-1beta. The NO donor S-nitrosoacetylpenicillamine (SNAP), on the other hand, inhibited collagen production. IL-1 lowered the abundance of Col2A1 mRNA in an NO-independent manner. Collectively, these data indicate that IL-1 suppresses collagen synthesis at two levels: a pretranslational level which is NO-independent, and a translational or post-translational level which is NO-mediated. These effects are presumably specific as L-NMA and SNAP had no effect on total protein synthesis or on the distribution of newly synthesized proteins between the cellular and extracellular compartments. Prolyl hydroxylase is an important enzyme in the post-translational processing of collagen, and its regulation and cofactor requirements suggest possible sensitivity to NO. Extracts of cells treated with IL-1 or SNAP had lower prolyl hydroxylase activity, and L-NMA was partially able to reverse the effects of IL-1. These data suggest that prolyl hydroxylase might indeed be a target for NO. Because underhydroxylated collagen monomers fail to anneal into stable triple helices, they are degraded intracellularly. Inhibition of prolyl hydroxylase by NO might thus account for the suppressive effect of this radical on collagen synthesis.

Removal of necrotic tissue with an ananain-based enzyme-debriding preparation.

An enzymatic debriding preparation was formulated with purified enzyme derived from a crude pineapple stem extract. The primary component of this preparation was the sulfhydryl protease ananain which represented >/=85% of the proteolytic activity. The remaining proteolytic activity in the preparation was contributed by a co-purifying homologous cysteine protease comosain. Taken together these two proteases provided a protein purity of greater than 95% as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This ananain-based enzyme preparation exhibited both gelatinolytic and fibrinolytic activity in vitro. Ananain-based enzyme preparation was formulated in a hydrophilic cream vehicle at concentrations ranging from 115 to 260 U/gm vehicle. Ananain-based enzyme preparation formulated in this fashion is referred to as Vianain debriding agent. Vianain was applied to partial-thickness cutaneous burn wounds produced in the skin of domestic pigs. A maximum of two 4-hour applications of Vianain provided complete debridement of eschar from the partial-thickness burn wounds as judged by light and electron microscopic analyses of biopsy specimens harvested before and after debridement. Wounds debrided with Vianain exhibited more rapid reepithelialization as compared with wounds that were not debrided. Wounds on pigs that were hyperimmunized to ananain-based enzyme preparation before burning and debridement with Vianain exhibited a similar enhancement in reepithelialization as compared with wounds treated with vehicle alone. The capacity of Vianain to debride necrotic tissue was also evaluated in a guinea pig ischemic ulcer model. Full-thickness ischemic lesions were created on the back of guinea pigs. Vianain was applied to the hardened necrotic tissue for 6 hours per day for up to a maximum of 5 days. Complete debridement of these wounds was accomplished within 4 to 5 days. Treatment of ischemic cutaneous ulcerations in this animal model with two commercially available enzyme-debriding agents provided little or no debridement of the necrotic tissue. In vitro, Vianain treatment of surgically debrided human tissue samples, obtained from patients with burn injury or cutaneous ulcers, showed that the protease preparation was effective in rapidly digesting these necrotic tissues.

Effects of sex hormones on protein and collagen content of the temporomandibular joint disc of the rat.

PURPOSE: The effect of sex hormones on the protein and collagen content of the temporomandibular joint (TMJ) disc of adult male and female rats. MATERIALS AND METHODS: One hundred forty-four Wistar rats were assigned to 14 groups of 12 each. Two groups, one female and one male, served as a control and received no treatment, and two other groups (one female and one male) received a sham gonadectomy and placebo hormone. The remaining 10 groups (five males and five females) received either orchiectomy or ovariectomy, followed by administration of estrogen, progesterone, combined estrogen and progesterone, or testosterone. The total protein and collagen content of the TMJ disc were determined using the calorimetric hydroxyproline method. RESULTS: The collagen content of TMJ discs of control males was statistically greater than the collagen content of the control female rats. This difference disappeared after ovariectomy of females and orchiectomy of males. Also, there was a general trend for a decrease in collagen and protein content to be produced by estrogen, progesterone, and by estrogen combined with progesterone in castrated male and female rats, and by orchiectomy of male rats. There was also a trend toward an increase in collagen and protein content after ovariectomy in female rats and administration of testosterone to castrated male and female rats. However, the only statistically significant effect of the drugs tested was that of estrogen combined with progesterone in ovariectomized female rats (a lowering effect on the total protein) and of estrogen alone in orchiectomized male rats (a lowering effect on the collagen content). CONCLUSION: Steroid sex hormones have an effect on the collagen and protein content of the TMJ disc of the rat as indicated by the difference in the values between control males and females and by the disappearance of this difference on castration of both male and female animals. This was also manifested by the significant effect of estradiol on collagen content of castrated males, by the effect of estrogen combined with progesterone on the protein content of castrated females.

Expression of interleukin 2 receptors on human carcinoma cell lines and tumor growth inhibition by interleukin 2.

We have previously shown that human squamous cell carcinomas (SCC) express the interleukin 2 receptor (IL2R)-alpha and -beta chains, and that the ligand, IL2, directly inhibits growth of the tumor in vitro and in vivo in the tumor xenograft-nude mice model. We now show that the alpha and beta chains of IL2R are expressed on a variety of human carcinoma cell lines and on normal human keratinocytes in early-stage cultures. While all carcinoma cells in a population expressed IL2R-alpha and -beta proteins, in keratinocytes obtained from different normal donors, variable proportions of cells were positive, as measured by flow cytometry. The carcinoma lines and 2/5 keratinocyte lines studied were also found to contain transcripts for the IL2R-gamma chain detectable by combined reverse transcription-PCR (RT-PCR) and hybridization with the specific cDNA probe. Incubation of the gastric (HR) or renal cell carcinoma (RCC) cell lines, but not of other IL2R+ carcinoma cell lines or normal keratinocytes, in the presence of IL2 resulted in dose-dependent inhibition of tumor cell growth. Monoclonal antibodies (MAbs) specific for IL2R-beta chain completely reversed this growth inhibitory effect of IL2. The ligand, IL2, also down-regulated surface expression of its own receptor and of intercellular adhesion molecule-I (ICAM-I) or class I major histocompatibility complex (MHC) antigens on IL2R+ tumor cells. All carcinoma cells studied incubated in the presence of IL2 exhibited significantly increased sensitivity to growth-inhibitory effects of other cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta. IL2 inhibited growth of the HR cells by arresting a significant proportion of tumor cells in the G0/G1 phase of the cell cycle. Thus, IL2 can have direct effects on IL2R+ carcinoma cells, leading to changes in growth or to increases in sensitivity of tumor cells to cytostatic activities of other cytokines.

Mast cell and myofibroblast in wound healing.

Growing evidence in the literature indicates that mast cells are integrally involved in the process of dermal wound repair. They are resident cells of the normal dermis and have several cytokines stored in their granules that are stimulatory to fibroblasts. They also contain serine proteases that may be involved in remodeling of the extracellular matrix during healing. Mast cells are found in increased numbers in acute wounds and in certain chronic fibrotic diseases. Their influence on fibroblast growth and collagen production may be an important element in fibrosis. The effects of mast cell mediators on dermal fibroblasts are currently being explored by our laboratory and others. Myofibroblasts are implicated in the phenomenon of wound contraction. These phenotypically altered fibroblasts express some features of smooth muscle cells, notably actin filaments, and are abundant in granulation tissue. It has been proposed that they are responsible for wound contraction and possibly certain types of contracture. However, this hypothesis has been challenged by studies demonstrating the presence of myofibroblasts in wounds that do not contract, or the process of contraction in vitro in the absence of myofibroblasts. At this time the issue remains open to debate and further research.

Direct evidence for spatial and temporal regulation of transforming growth factor beta 1 expression during cutaneous wound healing.

The expression of transforming growth factor (TGF beta 1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti-CC and anti-LC) following cutaneous injury. The anti-LC antibody binds intracellular TGF beta 1 constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti-CC antibody does not react with the form of TGF beta 1 present in normal skin as previously shown. TGF beta 1 may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGF beta 1 protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness procine skin. Anti-CC reactive TGF beta 1 in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGF beta 1 protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGF beta 1 activity. One day post-injury anti-CC reactive TGF beta 1 is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post-injury anti-CC reactive TGF beta 1 is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGF beta 1 from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGF beta 1 activity. Anti-CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti-LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti-LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti-CC reactive TGF beta 1 disappears while constitutive anti-LC reactive TGF beta 1 persists. Thus, changes in the structure or conformation of TGF beta 1, its localization, and perhaps its activity vary in a spatial and temporal manner following cutaneous injury and correlate with physiological changes during wound healing.

Clinical trials with purified platelet releasate.

Clinical trials of PDWHF are ongoing and the final results are not yet available; however early lessons learned have allowed us to modify these trials. It is anticipated that the patient accrual will be completed within the next six months, and the last patient will complete the trial within the next year. At that time, we hope to have new insight into the role of PDWHF in the treatment of lower extremity ulcers. At this time, we are encouraged by the early improvement seen in patients entering the PDWHF versus saline trial. Preliminary results suggest that PDWHF improves the healing of diabetic ulcers of the lower extremity.

Basic fibroblast growth factor stimulation of epidermal wound healing in pigs.

Basic fibroblast growth factor (bFGF) has recently been shown to be a mitogen for keratinocytes. This observation has now been extended in a porcine model of epidermal wound healing. A single application of recombinant human bFGF given at the time of injury to healthy animals accelerated the rate of epithelialization by 20%; multiple applications gave no greater effect than the single application. Histologic analysis of biopsies of these partial-thickness wounds taken during bFGF-mediated healing supported the assessment of an enhanced rate of epithelialization and an earlier onset of dermal healing. Because no histologic abnormalities were observed, bFGF induced an acceleration of what appears to be the normal healing process.

Topical tretinoin and epithelial wound healing.

The effects of topical tretinoin on epithelial wound healing were studied using a porcine model. Eight animals were treated with 0.05% tretinoin cream daily for ten days prior to partial-thickness skin wounding. Daily tretinoin treatment was continued after wounding on two of eight animals. Treatment with topical tretinoin before wounding accelerated epithelial wound healing in partial-thickness wounds. Continued tretinoin treatment on the wounds themselves retarded reepithelialization. Biopsy specimens of wounds with continued tretinoin treatment revealed persistent inflammation and fibroplasia in the dermis. The healing epithelium itself displayed areas of spongiosis and intracytoplasmic pale eosinophilic periodic acid-Schiff-positive, diastase-resistant globules. These globules did not stain with alcian blue. These epidermal alterations may be a direct effect of tretinoin or may be secondary to the underlying inflammation. While the dermal inflammation associated with continued tretinoin treatment appears to retard reepithelialization, epithelial cell alterations may also play a role.

Stimulatory effects of transforming growth factor-beta and epidermal growth factor on epidermal cell outgrowth from porcine skin explant cultures.

Transforming growth factor-beta (TGF-beta) is known to stimulate dermal wound healing events (fibroplasia and fibrosis). In this study, the effect of TGF-beta on epidermal wound healing (re-epithelialization) was examined. Epidermal cell outgrowth from partial-thickness porcine skin explants was used as an in vitro model for epithelialization. All cultures were grown in medium with 1% fetal bovine serum, which was sufficient for explant viability but low enough to permit measurement of modulation by added factors. Because TGF-beta is known to act in concert with other growth factors, it was evaluated alone and in the presence of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The results indicate that TGF-beta produced earlier initiation of outgrowth, by 1-2 d compared with control cultures, and increased the rate of outgrowth during the migratory phase of culture (Days 1-3). Compared to controls, EGF alone produced a greater percentage of growing explants and an increased rate of outgrowth during the mitotic phase (Days 4-7). TGF-beta (1 or 10 ng/ml) and EGF (5 ng/ml) had an additive rather than a synergistic effect on outgrowth. PDGF-treated explants did not show enhanced growth when PDGF (2.5 units/ml) was added alone or together with TGF-beta and EGF. The ability of TGF-beta to produce earlier initiation of outgrowth was not due to an effect on mitosis, because TGF-beta did not increase the incorporation of [3H]thymidine into keratinocytes in the growing epidermal sheets. Rather, it is likely that TGF-beta facilitated keratinocyte migration, possibly by unmasking a receptor on the epidermal cell surface. These results suggest that TGF-beta may play a role in early epidermal wound healing.