A model for the study of wounds in normal elderly adults and patients with peripheral vascular disease or diabetes mellitus.

The purpose of the study was to test the hypothesis that significant delays in cutaneous wound healing could be demonstrated using standard wounds and high quality histological methods in patients with severe peripheral vascular disease (PVD) and/or diabetes mellitus (DM) compared to healthy elderly controls. Additionally, we proposed that standard wounds on the arms of elderly controls would heal more rapidly than comparable wounds on the legs. In order to test these hypotheses we developed and characterized a partial thickness wound model which could be used safely in human subjects. The study population consisted of 25 elderly normal volunteers, 17 patients with PVD, and 24 patients with DM. Standard wounds were created using a Simplate II bleeding-time device. A total of 309 wounds ranging in age from 1 to 25 days were determined to be suitable for analysis. A global index of wound maturity was developed based on selected epidermal and dermal events of repair which could be scored histologically. The superficial component (within 0.1 mm of the epidermis) and deep components of dermal wounds were analyzed separately. Simultaneously created arm and leg wounds were studied in 15 of the elderly controls. Transcutaneous partial pressure of oxygen (TcPO2) measurements were used to estimate the severity of cutaneous ischemia. Data analysis revealed that the most striking differences observed were in dermal events of repair. Control wounds were more mature than dermal wounds from patients with PVD (P < 0.05). A significant reduction in the number of neutrophils and macrophages (P < 0.05) was demonstrated in 7-day-old wounds of patients with PVD compared to controls. Patients with DM showed a similar trend of reduced wound maturity but it did not reach statistical significance. Wounds created in skin with TcPO2 > 20 were more mature than wounds with TcPO2 < or = 20 (P < 0.05) and arm wounds were more mature than leg wounds (P < 0.01). The most significant difference noted in this wound model was that the superficial compartment of dermal wounds was significantly more mature than the deep compartment (P < 0.001). Good agreement was observed between two independent scorers of wound histology and no complications were noted in either patients or controls when using this human wound model. We conclude that the model described allows evaluation of both epidermal and dermal events of repair with relative safety even in patients with PVD and DM.(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation of tissue constituents with the acoustic properties of skin and wound.

The purpose of this study was to compare measurements of ultrasound properties of skin and wound tissue with measurements of material properties such as total collagen concentration, acetic acid soluble collagen concentration, water concentration, and morphologic properties. Using a scanning laser acoustic microscope (SLAM), both ultrasonic speed and attenuation coefficient values were obtained for control skin (2-3 cm from the wound), for skin immediately adjacent to wounds (within 0.3 mm), as well as for wound tissue itself. The attenuation coefficient and speed measurements were lowest for wound tissue followed by adjacent skin and then control skin. As the wounds healed there appeared to be an increase in both speed and attenuation coefficient although the wound age at which these increases started and the length of time for which they continued varied from one dog to the next. The precision of duplicate sample measurement of wave speed was +/- 1.7% for control skin, whereas that for attenuation coefficient it was +/- 16%. Both ultrasonic speed and attenuation coefficient were directly correlated with tissue collagen concentration and inversely correlated with tissue water concentration (p less than 0.001). Attenuation coefficient correlated best (r = 0.73) with acetic acid soluble collagen concentration which reflects the changes in collagen taking place during the repair process. These attenuation measurements made at 100 MHz using the SLAM were compared for control skin and wound samples with measurements made at 10-40 MHz using backscatter acoustic techniques (BAT). The tissue samples analyzed by each ultrasound technique were from adjacent locations on the animals.

Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction.

Wound contraction can substantially reduce the amount of new tissue needed to reestablish organ integrity after tissue loss. Fibroblasts, rich in F-actin bundles, generate the force of wound contraction. Fibronectin-containing microfibrils link fibroblasts to each other and to collagen bundles and thereby provide transduction cables across the wound for contraction. The temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction have not been determined. To establish these relationships, we used a cutaneous gaping wound model in outbred Yorkshire pigs. Granulation tissue filled approximately 80% of the wound space by day 5 after injury while wound contraction was first apparent at day 10. Neither actin bundles nor fibronectin receptors were observed in 5-d wound fibroblasts. Although fibronectin fibrils were assembled on the surfaces of 5-d fibroblasts, few fibrils coursed between cells. Day-7 fibroblasts stained strongly for nonmuscle-type F-actin bundles consistent with a contractile fibroblast phenotype. These cells expressed fibronectin receptors, were embedded in a fibronectin matrix that appeared to connect fibroblasts to the matrix and to each other, and were coaligned across the wound. Transmission EM confirmed the presence of microfilament bundles, cell-cell and cell-matrix linkages at day 7. Fibroblast coalignment, matrix interconnections, and actin bundles became more pronounced at days 10 and 14 coinciding with tissue contraction. These findings demonstrate that granulation tissue formation, F-actin bundle and fibronectin receptor expression in wound fibroblasts, and fibroblast-matrix linkage precede wound contraction.

The hamster flank organ model: is it relevant to man?

The critical role that androgens play in the etiology of acne has led to a search for topically active antiandrogens and the frequent use of the flank organ of the golden Syrian hamster as an animal model. 17-alpha-propyltestosterone (17-PT) has been identified as having potent antiandrogenic activity in the hamster model, and this report describes its clinical evaluation. Two double-blind placebo controlled studies comparing 4% 17-PT in 80% alcohol versus vehicle alone were conducted. One study examined 17-PT sebosuppressive activity in 20 subjects. The second study examined its efficacy in 44 subjects having mild to moderate acne. A third study measured in vitro percutaneous absorption of 17-PT through hamster flank and monkey skin, and human face skin in-vivo, using radioactive drug. 17-PT was found to be ineffective in reducing either the sebum excretion rate or the number of inflammatory acne lesions. Failure of 17-PT to show clinical activity was not a result of poor percutaneous absorption. Total absorption in man was 7.7% of the dose and only 1.0% in the hamster. The sebaceous gland of hamster flank organ is apparently more sensitive to antiandrogens than the human sebaceous gland.

A method for localizing the early products of nonenzymatic glycosylation in fixed tissue.

The process of nonenzymatic glycosylation (NEG) may play a significant role in the development of chronic complications of diabetes. Early products of NEG can be measured by various biochemical methods. A method has been developed to localize these early products of glycosylation in vivo in fixed tissue sections of normal and diabetic skin using monoclonal antibodies specific for glucitollysine, which is formed when the early products of NEG are chemically reduced in vitro. Carnoy's-fixed, paraffin-embedded skin samples from six diabetic and 13 nondiabetic subjects were sectioned, mounted on glass slides, and reduced for one h in 100 mM NaBH4. Immunolocalization was by the avidin--biotin immunoperoxidase method. Diabetic skin consistently stained more intensely for glucitollysine than nondiabetic skin. Staining around vessels, in particular, and of the collagenous matrix in general, was markedly enhanced in diabetic skin compared with nondiabetic skin. Antigens present in both the epidermis and the eccrine structures reacted with the antibody in both diabetic and nondiabetic skin but with greater intensity in the diabetic skin. This study has shown that it is possible to localize the early products of NEG in tissue sections using monoclonal antibodies. The findings correlate with biochemical data that show increased NEG in diabetics compared with nondiabetics. This technique should prove valuable for further investigations of the role of NEG in the pathogenesis of diabetes.

An assessment of human epidermal repair in elderly normal subjects using immunohistochemical methods.

The purpose of this study was to document a timetable for selected events of epidermal repair in standard partial thickness incised wounds on the legs of normal elderly human subjects. A Simplate-II bleeding-time device was used for producing the wounds, and immunohistochemical techniques were employed for evaluation of the wounds. Antibodies to filaggrin and Ulex europeus I demonstrated little or no staining on migrating epithelium, but staining was apparent whenever epidermal closure had occurred. Bullous pemphigoid antigen was present in the basement membrane zone at all time points examined, including beneath migrating epithelium, whereas antibodies to laminin and type IV collagen were found only at the most lateral aspects of 2-, 3-, and 5-day wounds. Staining progressed centrally by day 7 and was present as a complete linear band beneath most 14-day wounds. The Simplate-II device provides a standard, easy to use, commercially available, sterile, relatively safe method of producing wounds for systematic studies in humans.

Measurement uncertainty assessment of the scanning laser acoustic microscope and application to canine skin and wound.

Accuracy and precision were experimentally evaluated for the scanning laser acoustic microscope (SLAM) by measurements on homogeneous liquids of known ultrasonic properties. Using aqueous solutions of bovine serum albumin, the attenuation coefficient accuracy and precision are +/-12% and +/-15%, respectively. Using Dow Corning 710, a silicone oil, the speed accuracy and precision are +/-2.9% and +/-0.4%, respectively. Precision was assessed using duplicate samples of canine skin and wound tissue. The estimated precisions in the measurement of the attenuation coefficient and speed were +/-16% and +/-1.7%, respectively.

Ultrasonic assessment of skin and wounds with the scanning laser acoustic microscope.

The aim of the present study was to test the hypothesis that ultrasonic propagation properties in skin and wound tissue would correlate with material properties such as collagen content, water content, and tensile strength of those tissues. Both ultrasonic speed and ultrasonic attenuation coefficient were directly correlated with tissue collagen content, [r = 0.80 and r = 0.56, respectively (p less than 0.001)]. In addition, ultrasonic speed and attenuation coefficient were inversely correlated with tissue water content, [r = -0.57 and r = -0.73, respectively (p less than 0.001)]. Tensile strength also correlated very significantly with ultrasonic speed (r = 0.90, p less than 0.001), and significantly with attenuation coefficient (r = 0.58, p less than 0.001). The results demonstrate the feasibility of using ultrasound for noninvasively determining the material properties of biologic tissues including healing cutaneous wounds.

Erythema gyratum repens unassociated with internal malignancy.

A case report of erythema gyratum repens occurring in a 68-year-old man is presented. Evaluation and follow-up for development of malignancy over a 39-month period failed to reveal evidence of malignancy. The patient died of an unrelated cause. Autopsy did not demonstrate any evidence of malignancy.

Congenital abnormalities of the skin: future directions.

The inherited abnormalities of the skin are easily recognizable and readily accessible. They involve structures of different embryonic origins, are amenable to study in in vitro systems, are available for study in laboratory and domestic animal models, and are eminently suited to the type of interdisciplinary research presented at this workshop. Biochemists, geneticists, and clinicians in various disciplines all profit by this exchange of information. Ultimately, the gain is for the patient in the treatment and prevention of inherited dermatologic disorders.

Regional development of the human epidermis in the first trimester embryo and the second trimester fetus (ages related to the timing of amniocentesis and fetal biopsy).

The epidermis was sampled from multiple body sites of whole human embryos and fetuses of 45- and 65-days, 16- and 21-weeks estimated gestation age to determine whether a regionally dependent, variable rate of interfollicular epidermal development exists. Surface characteristics and thickness of the epidermis were evaluated by scanning electron and light microscopy, respectively. It was concluded that all epidermal development proceeded simultaneously during the first trimester with the exception of the foot which was more advanced in both thickness and state of differentiation. During the second trimester the epidermis of both the head and the foot showed more precocious development, particularly in relation to the special sense organs and to the plantar surface. The interfollicular epidermis of the majority of the body, however, was approximately equivalent in the state of development. These data have potential relevance to prenatal diagnosis of inherited skin disease from amniocentesis and/or fetal biopsy specimens; the present survey of the total epidermal surface will allow one to predict the types of skin-derived cells that should be present in the amniotic fluid at a given age, and to evaluate a fetal biopsy from one region and be confident that it is an accurate index of fetal skin development, age and status in general.

Age-related changes in the mechanical properties of human skin.

The mechanical properties of skin have been studied both in vivo and in vitro by a variety of test methods. These properties are well matched to the function of the skin, and they depend on the geometry of the collagen and elastin networks of the dermis. The time dependence of these properties is thought to be related to the "ground substance" components of the dermis. Age-related changes in the mechanical properties are a function of the degradation of the elastin network and of some as yet undefined changes in the viscoelastic properties of the "ground substance."

A collagen and elastic network in the wing of the bat.

Bundles of collagen fibrils, elastic fibres and fibroblasts are organized into a network that lies in the plane of a large portion of the bat wing. By ultrastructural (TEM and SEM) and biochemical analyses it was found that individual bundles of the net are similar to elastic ligaments. Although elastic fibres predominate, they are integrated and aligned in parallel with small bundles of collagen. A reticulum of fibroblasts, joined by focal junctions, forms a cellular framework throughout each bundle. Because of the unique features of the fibre bundles of the bat's wing, in particular their accessibility, and the parallel alignment of the collagen fibrils and elastic fibres in each easily isolatable fibre bundle, they should prove a most valuable model for connective tissue studies, particularly for the study of collagen-elastin interactions.

Intraendothelial tubular aggregates in experimental wounds.

Intraendothelial tubular aggregates were found in small dermal blood vessels in three healing superficial wounds in patients receiving systemic corticosteroids. Similar inclusions were also demonstrated in two wounds in healthy subjects, but were not found in normal (unwounded) skin of either healthy subjects or steroid-treated patients. Tubular aggregates of similar structure have previously been described as occurring in 'connective tissue' or 'auto-immune' diseases, including lupus erythematosus and dermatomyositis, both in the skin and in internal organs. Although it has been suggested that such aggregates are of viral origin, their morphogenesis and significance remain undertermined. Our findings do not support a viral cause, but suggest that these structures are probably a product of regenerating endothelium.