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.

Dichotomy in response to indomethacin in UV-C and UV-B induced ultraviolet light inflammation.

In subjects irradiated with both UV-C and UV-B ultraviolet light (UVL), 10 mug of intradermal indomethacin decreased the redness in all 13 of the UV-B irradiated areas but in only 2 of 13 of the UV-C irradiated areas. Higher doses of intradermal indomethacin (50 mug and 100 mug) decreased the redness produced by UV-C irradiation in 6 subjects. It is suggested that the failure of 10 mug of indomethacin to decrease the redness of the UV-C induced inflammation, while decreasing the redness in the UV-B induced inflammation, is consistent with the possibility that prostaglandins participate in UV-B but not UV-C induced inflammation.

Prostaglandin and DNA synthesis in human skin: possible relationship to ultraviolet light effects.

The effect of prostaglandin E2 (PGE2) on DNA synthesis in human skin was evaluated. PGE2 (1 mug) was infected intradermally into normal buttock skin of 15 volunteers followed by tritiated thymidine for autoradiographic quantitation of DNA synthesizing cells. Controls of normal saline, histamine (50 mug), and lower doses of PGE2 were also injected into 8 of the volunteers. Forty-eight hours after injection of 1 mug and 0.1 mug PGE2 there was a 264% and 62% increase, respectively, in the number of DNA synthesizing epidermal cells/high-power field as compared to saline controls. These differences were statistically significant (p smaller than 0.01). Histamine (50 mug) produced a statistically significant 36% higher labeling index compared to its saline controls (p smaller than 0.05). Many types of skin injury, including ultraviolet light (UVL) irradiation, produce an increase in the number of DNA synthesizing cells about 48 hr after the stimulus. Our findings suggest that PGE, a putative mediator of UVL-induced inflammation, may be one of the chemical mediators for the UVL-induced increase in DNA synthesizing cells. Histamine may also contribute to the increase in DNA synthesizing cells following UVL-induced inflammation.

"Inert" vehicles do affect wound healing.

The effect of a single daily application of U.S.P. petrolatum, an oil-in-water vanishing cream or a lotion on the rate of epidermal wound healing was determined in domestic white pigs. The superficial wounds were made with a dermatome and were not infected. In these studies, applications of U.S.P. white petrolatum retarded the rate of epidermal healing by 17% compared to untreated control wounds. Applications of an oil-in-water vanishing cream increased the rate of epidermal healing by 24% and a lotion increased the rate 15% compared to untreated control wounds.

New methods for assessing epidermal wound healing: the effects of triamcinolone acetonide and polyethelene film occlusion.

Epidermal healing of superficial, excised wounds in domestic white pigs was evaluated visually and histologically after separation of the epidermis and dermis. The visual determination of epidermal healing correlated well with the histologic studies of surface re-epithelialization. Wounds healed 40% faster when occluded with polyethelene film. Topical triamcinolone acetonide treatment delayed healing (62% slower than control).

Ultraviolet radiation-induced inflammation and leukocytes.

To evaluate the role of leukocytes in inflammation induced by ultraviolet radiation, the response of guinea pigs make leukopenic with cyclophosphamide was compared with the response of nonleukopenic animals. The response of leukopenic animals to UVB (295 nm) was significantly altered. Leukopenic animals and saline-treated (i.e., nonleukopenic) animals responded similarly to (1) UVC (250 nm), (2) UVA (340 nm) given after 8-methoxypsoralen pretreatment (PUVA), (3) intradermal histamine and (4) a topically applied prostaglandin analogue. These results suggest that leukocytes are important in UVB-induced inflammation but not in the inflammation induced by UVC or PUVA.

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.

Effect of basement membrane entactin on epidermal cell attachment and growth.

A preparation of laminin and entactin (Matrix), extracted from the basement membrane of the murine cell line M1536-B3 was used to evaluate the effect of entactin on epidermal cell attachment and growth in culture. Cell growth on Matrix was significantly higher than on laminin or plastic, in the presence or absence of serum. In attachment assays, the attachment of cells to laminin (137% of control) or to Matrix (158% of control) was significantly higher when compared with plastic (p less than 0.01), and the attachment to Matrix was higher than to laminin (p less than 0.05). Varying the amount of laminin or Matrix used as a substratum showed each enhanced cell attachment to the same maximum value (approximately 50% attached cells or 160% of control), but maximal attachment was achieved with lower amounts of Matrix (15 micrograms Matrix vs 15-30 micrograms laminin, p less than 0.05). Inhibition studies with anti-entactin and anti-laminin antibodies were used to assay the specific contribution of entactin to cell attachment to Matrix. Pretreatment of the substratum with increasing amounts of anti-entactin antibody decreased cell attachment to Matrix in a concentration-dependent manner, with cell attachment to Matrix eventually falling to the same level as that obtained with laminin. There was no effect with anti-entactin on cell attachment to laminin or to plastic controls, and nonspecific rabbit IgG had no effect on any group. Similar experiments were performed using 2 different concentrations of anti-laminin antibody. At a low concentration, anti-laminin antibody decreased attachment to laminin (to a level equivalent to the plastic control). At a higher concentration anti-laminin decreased attachment to Matrix, but to a level that was still greater than the plastic control. The anti-laminin antibody had no effect on attachment to the plastic control and nonspecific rat IgG in equivalent amounts had no effect on attachment to any of the substrata. These results indicate that Matrix, containing laminin and entactin, enhanced cell attachment above the level seen with laminin alone, and that this effect was probably due to the presence of entactin in the Matrix.

Collagenase in wound healing: effect of wound age and type.

Collagenase is believed to be important for cell migration and collagen remodeling during tissue repair and regeneration. We have investigated collagenase concentrations in different types of surgically inflicted wounds in pigs. Collagenase was extracted from tissue homogenates of wounds by heating to 60 degrees C for 6 min in 0.1 M CaCl2. The molecular weight of latent collagenase was about 52 kDa. Activated collagenase produced the characteristic 3/4 fragment of collagen. Collagenase was assayed by the use of radiolabeled telopeptide-free collagen. To detect maximal collagenase activity, extracts were reduced and alkylated to destroy inhibitors, then activated with aminophenylmercuric acetate. Sutured incisions showed peak collagenase content on postoperative day 1 and thereafter steadily declining concentrations. Granulation tissue from non-sutured large defect full-thickness wounds showed high collagenase content on postoperative day 5 and then a sharp decline to day 7 followed by a slowly declining curve to postoperative day 21. Partial-thickness wounds exhibited a different time course, with collagenase increasing to peak concentrations on postoperative days 3-5; however, a large proportion of the detected collagenase was due to the adherent scab. By day 7 collagenase concentrations approached the low concentrations of normal skin when epithelialization was complete and the scab rejected. In general, collagenase shows an early maximum and then declines with postoperative time, with the sharpest decline occurring when epithelialization is complete.

The healing of superficial skin wounds is stimulated by external electrical current.

We studied the effects of direct electric current supplied by an energized silver-coated electrode on dermal and epidermal wound healing. Keratome-induced wounds (0.3 mm deep) on the skin of young domestic pigs were treated with either an energized (50-300 microA) electrode (DC), an unenergized electrode (placebo), or left untreated. Wounds were excised on days 1-7 after wounding and the epidermis was separated from the dermis. The epidermal sheet was evaluated for reepithelialization and the dermis was assayed for collagen biosynthetic capacity. Dermal collagen production among treatments did not differ markedly on days 1-4 after wounding. However, a highly significant increase (p less than 0.001) in the collagen synthetic capacity was observed on days 5, 6, and 7 in wound treated with DC. There was no significant difference in collagen synthesis among treatments when collagen production was corrected for DNA content. The rate of wound epithelialization was also significantly accelerated (p less than 0.05) in DC-treated wounds. These results suggest that the proliferative and/or migratory capacity of epithelial and connective tissue cells involved in repair and regeneration can be affected by an electrical field.

Detection of basement membrane zone antigens during epidermal wound healing in pigs.

Bullous pemphigoid (BP) antigen, laminin, and type IV collagen, 3 distinct antigens of basement membrane, were studied by indirect immunofluorescence in the epidermal-dermal junction of re-epithelializing wounds. Partial thickness wounds were made with a dermatome in the skin of white Yorkshire pigs. After 2 or 3 days, the wound site and the surrounding normal skin were excised and cryostat sections were studied using BP sera as well as whole antisera and affinity purified antibodies to laminin and type IV collagen. Laminin and type IV collagen were detected in the basement membrane zone of normal epidermis and at the re-epithelializing epidermal-dermal junction for a variable distance into the healing wound but both were absent from the more distal migrating epidermis. In contrast, BP antigen extended from the basement membrane zone of normal skin throughout the entire epidermal-dermal junction of dermis. These results suggest that in the re-epithelization of superficial wounds laminin and type IV collagen are not present in the initial epidermal-dermal interaction of the migrating epithelium but that BP antigen may be important in this early interaction.

The effect of a semiocclusive dressing on the microbial population in superficial wounds.

Superficial wounds in a Yorkshire pig were treated with a semiocclusive polyurethane film dressing (PUD) or left open. The number and types of microflora present in the wounds each day was determined by a scrub technique, selective media, and the spiral plating system. In wounds covered with the PUD, the number of organisms increased, and there were more gram-negative pathogens. We concluded that microorganisms in wound beds multiply and survive better beneath a semiocclusive dressing than with air exposure.

Occlusive dressings. Does dressing type influence the growth of common bacterial pathogens?

We studied the effect of different occlusive dressings and of air exposure on the growth of four pathogenic bacteria in wounds. Partial-thickness wounds on domestic pigs were inoculated with Staphylococcus aureus, Clostridium perfringens, Bacteroides fragilis, or Pseudomonas aeruginosa. Each wound was covered with three dressings (DuoDERM, Opsite, or Vigilon), or left exposed to air. Groups of wounds were sampled at 24, 48, and 72 hours. Staphylococcus aureus reached high levels beneath all of the dressings and in the air-exposed wounds. The numbers of C perfringens and B fragilis were greatly reduced in the air-exposed wounds and slightly reduced in the Opsite-covered wounds. The numbers of P aeruginosa were greatest in the Opsite- and Vigilon-covered wounds. The results indicate that occlusive dressings are not indicated in wounds that clinically appear to be grossly contaminated or that may contain anaerobic organisms.

Tissue engineering and the development of Apligraf, a human skin equivalent.

In recent years, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This review details the pioneering work of numerous investigators that led to the following precursors of tissue-engineered skin replacement: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular collagen matrices, and cellular matrices. It also discusses the rationale for the development of the newer products and describes the technical advances leading to the development of Apligraf, a tissue-engineered human skin product.

Patient referrals to a dermatologist. The referring physician's perspective.

BACKGROUND AND DESIGN: As changes in the health care system aim to reduce the cost of medical care, physicians need to understand and be able to justify their reasons for referring patients. To learn the basis for making referrals, we asked 116 referring physicians why they made referrals to one university-based physician. We also assessed the referring physicians' satisfaction. RESULTS: A total of 112 of the 116 physicians responded in reference to 114 patients. The responses fit into five categories. Fifty of 114 patient were referred for therapy; 16 patients, for a diagnosis; and 16, for a combination of diagnosis and therapy. Other reasons in descending order of frequency were (1) a nondermatologist referring the patient to a dermatologist, (2) self-referral, ie, the patient was not referred by the physician, and (3) the patient's personality was difficult. The referring physicians were satisfied in 94 instances. CONCLUSIONS: The most frequent reason for referral was for therapy. This observation may be generally true for many physicians, since it is consistent with results of two other studies, in different fields, that we were able to find. Eighty-two percent of referrals were satisfactory to the referring physicians. Most of the physicians who were unsatisfied with the referral process stated they had not received a follow-up letter from the consulted physician.

High-dose methylprednisolone in the treatment of bullous pemphigoid.

Eight hospitalized patients with active bullous pemphigoid were treated with high-dose intravenous methylprednisolone pulse therapy followed by moderate maintenance doses of oral prednisone. Blistering decreased within 24 hours of the first pulse dose in seven of the eight patients. When blistering recurred, it was less severe than it had been before pulse therapy was given. Our earlier experience and data from the literature show that most patients with bullous pemphigoid will respond to oral corticosteroids and/or immunosuppressive treatment. Pulse therapy has a role in the treatment of bullous pemphigoid, and a mechanism of action is proposed.

Protein C and protein S plasma levels in patients with lipodermatosclerosis and venous ulceration.

Lipodermatosclerosis of the lower extremity, with or without ulceration, is a common manifestation of severe venous disease and the result of sustained venous hypertension. The latter is generally a sequela of deep vein thrombosis. Factors that enhance clot formation or impair fibrinolysis contribute to the pathogenesis of venous disease. It is already established that faulty fibrinolysis may play a pathogenic role in patients with venous disease. We examined the possibility that patients with venous disease have abnormally low plasma levels of proteins C and S, two proteins whose deficiencies have been reported to cause an increased frequency of thromboembolic disease. Using immunologic and functional assays for plasma proteins C and S, we found that 4 (21%) of 19 patients with lipodermatosclerosis and leg ulcers had abnormally low levels of protein C or protein S. One of 7 patients with lipodermatosclerosis without ulceration had a profoundly depressed level of protein C and a history of cerebral stroke at a young age. Plasma levels of protein C were normal in five patients with arterial insufficiency severe enough to cause leg ulceration. We conclude that abnormally low plasma levels of proteins C and S may be found in patients with lipodermatosclerosis and venous ulceration. As with the abnormally low fibrinolytic activity in these patients, our findings indicate a possible propensity for increased thrombotic disease.

Electrical stimulation and wound healing.

Living tissues possess direct current surface electropotentials that regulate, at least in part, the healing process. Following tissue damage, a current of injury is generated that is thought to trigger biological repair. In addition, exogenous electrical stimuli have been shown to enhance the healing of wounds in both human subjects and animal models. Intractable ulcers have demonstrated accelerated healing and skin wounds have resurfaced faster and with better tensile properties following exposure to electrical currents. This article examines the bioelectric properties of living systems and reviews the existing literature on electrical stimulation and wound healing.

Human essential fatty acid deficiency: treatment by topical application of linoleic acid.

An essential fatty acid (EFA) deficiency developed in a 19-year-old man who was being maintained on a long-term regimen of fat-free, intravenous hyperalimentation fluids. The EFA deficiency was reversed after 21 days by daily, topical application of linoleic acid to the patient's skin. The ratio of eicosatrienoic acid (20:3, n-9) to eicosatetraenoic acid (20:4, n-6) decreased to normal levels in the skin and serum with clinical improvement of the EFA deficiency syndrome. The cutaneous manifestations (scalp dermatitis, alopecia, and depigmentation of hair) were reversed with continued, topical application of safflower oil, which contains 60% to 70% linoleic acid.