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Biomedical subjects

M Ponec

Publications and source records attributed to M Ponec.

At least 163 records · Page 9Linked to original sources
PubMed1977

Plasma levels of triamcinolone acetonide as determined by radioimmunoassay after topical application.

In this paper a method for the radioimmunological determination of triamcinolone acetonide (TAAc) in plasma is described. The antiserum used in the radioimmunological determinations showed high specifity for TAAc; the minimal detectable amount was 0.2 ng. The course of the plasma concentration-time curve of TAAc after intravenous injection into rats was analysed. The concentration of TAAc in the blood of the patients after topical application of the drug is very low. No detectable amount of TAAc was found in the blood of out-patients. Hospitalized patients treated with TAAc under plastic occlusion showed plasma levels ranging from 0.8 to 3.4 ng/ml. This level remained constant throughout the treatment.

Animals↗
PubMed1976

Penetration of corticosteroids through the skin in relation to the vehicle.

Results of in vitro studies on the penetration of a corticosteroid hydrocortisone 17-butyrate (HCN) in relation to the vehicle are reported. When HCB is dissolved completely in the vehicle, penetration is enhanced, and only under this condition an increase in HCB concentration (in the range 0.05-0.2%) leads to an increase in the penetration. Evaluation of the efficiency of various vehicles (an oil/water cream, Plastibase or gels) in which HCB was dissolved in propylene glycol or ethanol, showed that Plastibase with propylene glycol was the most effective for both 0.1 and 0.2% HCB. The results of experiments in which HCB was applied in ethanolic solutions suggests that at least initially HCB and ethanol together penetrate through the epidermis.

Administration, Topical↗
PubMed1976

Dependence of corticosteroid penetration on the vehicle.

The penetration of corticosteroids through epidermal membranes in vitro proved to be better when the corticosteroid was dissolved in, or salicylic acid was added to, the vehicle. Hydrocortisone 17 butyrate (HCB) penetration from an ethanolic solution or a vehicle containing propylene glycol (PG) exceeds the penetration from an oil-in-water (O/W) cream or a petrolatum-polyethylene base (Plastibase), both without PG. The addition of PG led to higher penetration from a cream containing 0.2% HCB as compared with that from one containing 0.1% HCB. When the atmosphere on the donor side of the chamber was kept dry with a molecular sieve, the penetration from an O/W cream containing PG was enhanced, whereas that from an O/W cream without PG was substantially diminished.

Adrenal Cortex Hormones↗
PubMedDate not supplied

Incorporation of linoleic acid by cultured human keratinocytes.

Linoleic acid is required for the formation and maintenance of the epidermal barrier, but most of the current in vitro keratinocyte culture systems are linoleic acid-deficient. The aim of the present study was to examine the efficiency of linoleic acid uptake in human keratinocyte cultures grown under submerged and air-exposed conditions in serum-free medium. The water-insoluble linoleic acid was bound to carrier molecules (cyclodextrin or bovine serum albumin). Comparable results were obtained with home-made and commercially available linoleic acid complexes. In the submerged cultures, the increase of the linoleic acid medium concentration (ranging from 0 to 20 microg/ml) resulted in a gradual increase in the linoleic acid cellular content, which exceeded 1.4 times the value found in native epidermis when the highest concentration of linoleic acid was used. The addition of linoleic acid did not alter the profile of the other epidermal fatty acids, with the exception of oleic acid, which decreased in parallel with the increasing linoleic acid content. While the content of linoleic acid found in phospholipids was similar to that in native epidermis, a large excess of linoleic acid was detected in triglycerides, the synthesis of which was markedly increased in cultures grown submerged in medium containing higher concentrations of linoleic acid. Under air-exposed conditions, the dermal substrate used seemed to be the most limiting factor for efficient linoleic acid supplementation. A low linoleic acid cellular content was detected when an inert filter was used. De-epidermized dermis was found to be the most permeable substrate for linoleic acid complexes. The cellular linoleic acid content increased in a parallel with the increasing linoleic acid concentration (ranging from 4 to 30 microg/ml), but the overall amount incorporated was lower than that in submerged cultures. The content of linoleic acid in the phospholipid and ceramide fractions isolated from reconstructed epidermis grown under air-exposed conditions was close to that of native epidermis, but the triglycerides remained abnormally enriched in linoleic acid, indicating persistence of some anomalies in epidermal lipogenesis in vitro.

Air↗
PubMedDate not supplied

Epidermal lipid metabolism of cultured skin substitutes during healing of full-thickness wounds in athymic mice.

Cultured epidermal keratinocytes provide an abundant supply of biologic material for wound treatment. Because restoration of barrier function is a definitive criterion for efficacy of wound closure and depends on the lipids present in the epidermis, we analyzed lipid composition of the epidermis in cultured skin substitutes in vitro and after grafting to athymic mice. The cultured skin substitutes were prepared from human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates. After 14 days of incubation, cultured skin substitutes were grafted orthotopically onto full-thickness wounds in athymic mice. Samples for lipid analysis were collected after 14 and 34 days of in vitro incubation, and 3 weeks and 4 months after grafting. Both in vitro samples show disproportions in epidermal lipid profile as compared with the native human epidermis, i.e., a low amount of phospholipids (indicating imbalance in proliferation and differentiation); a large excess of triglycerides (storage lipids); and low levels of free fatty acids, gluco-sphingolipids, cholesterol sulfate, and ceramides-suggesting abnormal composition of stratum corneum barrier lipids. Fatty acid analysis of cultured skin substitutes in vitro revealed insufficient uptake of linoleic acid, which resulted in increased synthesis of and substitution with monounsaturated fatty acids, mainly oleic acid. These abnormalities were partially corrected by 3 weeks after grafting; and 4 months after grafting, all epidermal lipids, with some minor exceptions, were synthesized in proportions very similar to human epidermis. Results of this study show that grafting of cultured skin substitutes to a physiologic host permits the recovery of lipid in proportion to that required for barrier formation in normal human epidermis.

Journal Article↗
PubMedDate not supplied

Dermal regeneration in full-thickness wounds in Yucatan miniature pigs using a biodegradable copolymer.

The aim of this study was to assess the performance of a biodegradable dermal substrate in deep dermal skin defects. The substrate consisted of a synthetic biodegradable matrix called Polyactive, which is an elastomeric poly (ether)/ poly (ester) block copolymer. This matrix was manufactured either as a porous substrate, with gradually changing pore size (BISKIN-M), or as a bilayer consisting of a porous underlayer with a fully dense surface layer (BISKIN). Cell-free matrices and matrices seeded with autologous or allogeneic porcine fibroblasts were applied to full-thickness skin wounds in Yucatan miniature pigs. Biopsies were taken at different time intervals up to 24-months post-transplantation. Although all BISKIN substrates showed little or no adherence to the wound bed, the adherence of the BISKIN-M substrates to the underlying wound was achieved within minutes after application. Therefore, only BISKIN-M Polyactive substrates were further evaluated. Wound contraction was inhibited by both cell-free and fibroblast-populated substrates. All substrates showed extensive neovascular and fibrous tissue ingrowth within 2-weeks post-transplantation. Furthermore, during this time period, matrix degradation was observed, starting with the fragmentation of the polymers into particles, which were phagocytized by macrophages. These processes occurred actively up to 3 months and ceased thereafter. Cell-free substrates degraded faster, and also, the collagen deposition was lower as compared with cell-seeded substrates. The tissue surrounding the remnants of the Polyactive substrates after 24-months post-transplantation consisted of a mature connective tissue. The newly formed collagen had the same distribution pattern as observed in normal native dermis. We conclude therefore that treatment of full-thickness skin defects with fibroblast-populated BISKIN-M Polyactive substrates leads to satisfactory dermal regeneration.

Absorbable Implants↗
PubMedDate not supplied

A modified culture system for epidermal cells for grafting purposes: an in vitro and in vivo study.

A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitro by culturing human or porcine epidermal keratinocytes on polyester filter substrate at the air-liquid interface. In addition, after 2 weeks of culture, hemidesmosome-like structures were formed along the basal area of the plasma membrane of the basal cells at the cell-filter interface. When grafted onto full-thickness skin wounds in pigs, the take of cell sheets detached from the filter with dispase was significantly higher (about 70%) in comparison to mechanically detached keratinocytes (about 15%). With dispase-treated keratinocytes alone, basement membrane formation took place within 7 days postgrafting as judged from the presence of a lamina lucida and positive staining for type IV collagen. Also, numerous hemidesmosomes and anchoring fibrils were observed at the basal cell-"neodermis" interface. The fully differentiated epidermis, generated by culturing keratinocytes at the air-liquid interface and detached from the substrate by dispase-treatment, is less fragile and easier to handle than epidermal autografts obtained by conventional culturing methods. Detachment by a short dispase-treatment appeared in our hands the only method for successful and complete epithelial regeneration in full-thickness wounds.

Animals↗
PubMedDate not supplied

Epidermal growth factor and keratinocyte growth factor differentially regulate epidermal migration, growth, and differentiation.

Various growth factors such as epidermal growth factor and keratinocyte growth factor have been reported to promote wound closure and epidermal regeneration. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of epidermal growth factor and keratinocyte growth factor on keratinocyte proliferation, migration and differentiation. Our results show that epidermal growth factor supplemented cultures share many of the features which are observed during regeneration of wounded epidermis: a thickening of the entire epidermis, an enhanced rate of proliferation and migration, and an increase in keratin 6, keratin 16, skin-derived antileukoproteinase, involucrin and transglutaminase 1 expression. The increase in transglutaminase 1 protein is accompanied by an increase in the amount of active transglutaminase 1 enzyme. Surprisingly no increase in keratin 17 is observed. Prolonging the culture period for more than two weeks results in rapid senescence and aging of the cultures. In contrast, keratinocyte growth factor supplemented cultures have a tissue architecture that is similar to healthy native epidermis and remains unchanged for at least 4 weeks of air-exposure. The rate of proliferation and the expression of keratins 6, 16 and 17, skin-derived antileukoproteinase, involucrin and transglutaminase 1 is similar to that found in healthy epidermis and furthermore keratinocyte migration does not occur. When the culture medium is supplemented with a combination of keratinocyte growth factor and a low concentration of epidermal growth factor, skin-derived antileukoproteinase, involucrin and keratins 6, 16 and 17 expression is similar to that found in cultures supplemented with keratinocyte growth factor alone and in healthy epidermis. Only high transglutaminase 1 expression remains similar to that observed in cultures supplemented with epidermal growth factor alone. Our results show that the regulation of keratinocyte growth, migration and differentiation depends on the availability of these growth factors. Epidermal growth factor may play a dominant early role in wound healing by stimulating keratinocyte proliferation and migration while keratinocyte growth factor may play a role later in the repair process by stabilizing epidermal turnover and barrier function.

Cell Division↗
PubMedDate not supplied

In vivo human skin barrier modulation by topical application of fatty acids.

The in vivo effects of fatty acids on skin barrier function were assessed by measuring: (i) transepidermal water loss (TEWL), (ii) diffusion lag times for hexyl nicotinate (HN), and (iii) irritant skin response using laser Doppler velocimetry (LDV) in combination with visual scoring. Two classes of fatty acids have been investigated: straight-chain saturated fatty acids (SFA), having 6-12 carbon atoms, and unsaturated fatty acids (UFA): oleic, linoleic, alpha-linolenic and arachidonic acids. It has been reported that these acids can enhance the permeation of various compounds across the skin. After topical and occlusive application as a solution in propylene glycol (PG) for 3 h on the volar arm of human subjects, SFA only caused a slight irritation and increase in TEWL. The diffusion lag times of HN were reduced by the application SFA to the same extent as and not more than by the application of the pure solvent PG. In contrast, the application of UFA caused a significant increase in TEWL and LDV (irritation) responses. The TEWL values after oleic acid application were higher than those observed for the other three acids, while the irritation potential of arachidonic acid was the highest among UFA. As with SFA, sites treated with UFA did not show significantly different lag times of HN diffusion from PC-treated sites. The data suggest that the degree of irritation and the degree of barrier modulation for fatty acids are not necessarily correlated.

Administration, Cutaneous↗
PubMedDate not supplied

Analysis of epidermal lipids of the healthy human skin: factors affecting the design of a control population.

The intervariability of studies on the lipids of human epidermis and stratum corneum is high because of the different origin of the skin samples and the variety of extraction methods used. In the present work, a high-performance thin-layer chromatographic technique has been used to study the parameters age, sex, and anatomical site for their effects on the lipid profiles recovered from healthy epidermal skin biopsy specimens. It was found that sex-related differences were seen at the level of the total ceramide concentration. Observed decreases in lipid concentration, due to ageing, depended on the anatomical site. Therefore, these variables should be controlled in a reproducible and standardized way in order to be able to study the direct relationship between skin condition and barrier lipid composition. Only when this relation is established, results of topical treatment can be scientifically evaluated.

Adult↗
PubMedDate not supplied

Culture and characterization of rat middle-ear epithelium.

This study was performed to design a method for the culture of rat middle-ear epithelium and to apply the method to investigate the characteristics of this epithelium. Culture of explants of middle-ear epithelium in the presence of the epidermal growth factor was successful, whereas serial cultivation required 3T3 feeder cells in addition to the epidermal growth factor. Cultured middle-ear epithelium was studied by phase-contrast microscopy, transmission and scanning electron microscopy, and combined light and scanning electron microscopy (LM/SEM). These techniques showed similarity between the cultured and the natural middle-ear epithelium. Explants and outgrowths showed both flat polygonal and ciliated epithelial cells. In serial cultivation, however, only the first of these cell types was observed. Frequently, a single primary cilium was found on the cell surface. Transmission electron microscopy showed cross-linked envelopes whose formation was promoted by ionophore X537A. Cytokeratin was demonstrated by immunoblotting, immunofluorescence, and immunoperoxidase methods, using an anti-cytokeratin monoclonal antibody. The model described here permits study of the differentiation of middle-ear epithelium in vitro and may be of future value for the study of chronic middle-ear diseases.

Animals↗
PubMedDate not supplied

Characterization and comparison of reconstructed skin models: morphological and immunohistochemical evaluation.

Reconstructed human skin equivalents are currently being investigated as in vitro models for the prediction of human skin toxicity and irritation responses. Three different industrial reconstructed skin models (EpiDerm, Episkin and SkinEthic) and one in-house equivalent were characterized and compared using light microscopy, immunohistochemistry and reduction of (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT). Their inter- and intra-batch variation was evaluated. Histological examination showed a completely stratified epithelium in all skin models, which closely resembled normal human epidermis. Low intra-batch variation in tissue architecture was observed in all skin models, but moderate to considerable inter-batch variation was noticed. Evaluation of the expression and localization of a number of differentiation-specific protein markers revealed that all skin models showed an aberrant expression of keratin 6, skin-derived antileukoproteinase, small proline rich proteins, involucrin and transglutaminase. Although variation within batches was low, in particular keratin 6, involucrin and skin-derived antileukoproteinase expression demonstrated some inter-batch variation. Reduction of MTT in vehicle-treated cultures showed high similarities between skin models, but marked differences were observed when 1.0% sodium lauryl sulfate was applied topically for 3 or 16 h. Most pronounced effects were noticed in SkinEthic cultures. Intra-batch variations were low and moderate variations were observed between batches. All skin models tested reproduced many of the characteristics of normal human epidermis and therefore provide a morphologically relevant in vitro means to assess skin irritation and other skin-related studies.

Biomarkers↗