Biomedical subjects

M Ponec

Publications and source records attributed to M Ponec.

At least 73 records · Page 4Linked to original sources

PubMed1994

Role of extracellular calcium in the regulation of 1,25-dihydroxyvitamin D3 formation in cultured human keratinocytes.

Cultured normal human keratinocytes (NHK) provide a useful experimental model for studies of processes occurring during terminal differentiation, since the extent of keratinocyte maturation can be manipulated experimentally by modulation of extracellular calcium concentration. When NHK are maintained in low calcium (0.06 mM) medium they proliferate but do not stratify. Raising the level of calcium to 1-2 mM results within a few hours in induction of keratinocyte differentiation. Results of the present study show that formation of 1,25-(OH)2D3 is higher in NHK grown at 0.06 mM than in NHK grown at 1.6 mM calcium concentration. After 2 h exposure of low calcium cultures to 1.6 mM calcium the 1,25-(OH)2D3 production starts to decrease. On the other hand, exposure of cells cultured in 1.6 mM calcium medium to 0.06 mM calcium concentration induced already within 4 h an increase in 1,25-(OH)2D3 formation which was not accompanied by a decrease in cornified envelope formation. Thereby, the present study demonstrated that calcium can regulate 1,25-(OH)2D3 formation independently of changes in keratinocyte differentiation.

Calcitriol↗

PubMed1994

Biocompatibility of a biodegradable matrix used as a skin substitute: an in vivo evaluation.

Synthetic biodegradable polymeric matrices, with a dense top layer and porous under-layer, made of a (poly)ether/(poly)ester (PEO:PBT) copolymer called Polyactive, and also of poly-L-lactide (PLLA), are under investigation as part of a cell-seeded skin substitute for third-degree, large-scale skin defects. The biocompatibility of subcutaneously implanted matrices representing large body surface areas, were studied at 2, 4, 13, 26, and 52 weeks in rats. To investigate local or systemic effects, the weight development of the complete animal and of their hearts, kidneys, lungs, livers, and spleens, as well as the macroscopic and histologic appearance of the implants and organs, were monitored. Early inflammatory response was associated with surgical implantation trauma. All matrices showed neovascular and fibrous tissue ingrowth into the porous underlayer within 2-4 weeks after implantation. Copolymeric and PLLA matrices increasingly fragmented and liquified. After 1 year, small polymeric fragments embedded in fibrous, vascularized tissue could be retrieved at the implantation site. No systemic effects of the implants on the organs or on the animal as a whole were observed. These results and earlier studies on (skin) cell substrate properties and physicochemical characteristics of the matrices indicate the suitability of the matrices as part of a cell-seeded skin substitute.

Animals↗

PubMed1994

Improvement of epidermal differentiation and barrier function in reconstructed human skin after grafting onto athymic nude mice.

To determine whether epidermis reconstructed in vitro at the air-liquid interface on de-epidermized dermis has the capacity to normalize the expression of differentiation-specific markers, its lipid composition and stratum corneum barrier properties, human skin equivalents were transplanted onto athymic nude mice and investigated at different stages ranging from 1 to 4 months after grafting. Indirect immunofluorescence with species- or non-species-specific antibodies revealed that as early as 1 month after transplantation keratinization, and involucrin, loricrin and transglutaminase patterns were normalized. Human melanocytes were observed in the basal layer of the pigmented graft. As revealed by high-performance thin-layer chromatography and transmission electron microscopy after ruthenium tetroxide fixation, the lipid profile and the intracellular lamellar organization were similar to those found in natural epidermis. Transepidermal water loss measurements and penetration studies showed that the barrier properties of the reconstructed epidermis after transplantation were comparable to those of normal human skin.

Animals↗

PubMed1994

Validation of an in vivo extraction method for human stratum corneum ceramides.

A topical acetone/diethylether (A/E) lipid extraction method was evaluated for its suitability for use in the study of stratum corneum lipids in various skin disorders. Its efficiency was compared in vitro with topical chloroform/methanol (C/M) extraction and with the classical 'integral' C/M extraction (submerged tissue) of stratum corneum or whole epidermis. To estimate the depth of lipid removal by A/E extraction, light microscopic and freeze-fracture electron microscopic studies were carried out on A/E and C/M topically treated skin samples. The in vivo experiments consisted of topical A/E extraction and of classical C/M extraction of scrapings of the stratum corneum. Transepidermal water loss (TEWL) was measured before and after topical A/E extraction and after every scraping procedure, and correlated with TEWL values found after stripping of the stratum corneum. The total amount of lipid found with both topical extraction procedures was lower than that found with the integral extraction of the stratum corneum. Light microscopy showed that topical C/M extraction induced cell damage in the living epidermal cell layers. Great interindividual variation in overall lipid composition was shown in the in vitro experiments irrespective of the extraction protocol used. However, the ceramide (CER) profiles in a single skin sample from the same subject were similar irrespective of the protocol used, and a uniformity in the CER profiles was found in skin samples from different subjects. Similar results were obtained with in vivo topical A/E extractions: marked interindividual variation was seen in overall lipid composition, but not in the CER profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

The involvement of protein kinase C in proliferation and differentiation of human keratinocytes--an investigation using inhibitors of protein kinase C.

Protein kinase C, the major cellular receptor for tumour-promoting phorbol esters, has been suggested as playing a key role in the regulation of proliferation and differentiation of epidermal cells. In the present study, we investigated the effects of various well-characterized inhibitors of protein kinase C on proliferation and differentiation of SV 40-transformed and normal human keratinocytes. The drugs were found to inhibit cell proliferation in a dose-dependent manner, displaying similar effects in both cell types and reflecting their potencies in inhibiting purified protein kinase C. In contrast, keratinocyte differentiation induced by treatment with a calcium ionophore or spontaneously, i.e. by exposure of cells grown in the presence of low calcium concentration (0.06 mM) to normal calcium concentration (1.6 mM), was not inhibited by the compounds tested. The potent protein kinase C inhibitor, staurosporine, was found even to enhance cell differentiation. Therefore, the present study provides evidence that the classical protein kinase C pathway plays a critical role in the regulation of keratinocyte proliferation rather than in calcium-induced differentiation.

Alkaloids↗

PubMed1994

Degradative behaviour of polymeric matrices in (sub)dermal and muscle tissue of the rat: a quantitative study.

Bilayered matrices, made of synthetic degradable polymers, are developed for use as a dermal regeneration template in large surface area full-thickness skin defects. The porous underlayer is designed to allow ingrowth of dermal components and the dense top layer will serve as a substrate for keratinocytes. Considering the importance of the degradation kinetics of tissue regeneration templates, quantification of matrix degradation up to 1 yr post-implantation, and histological and ultrastructural examination of the implants was performed. In this study a moderate foreign body reaction was observed at both the intramuscular and subcutaneous implantation sites, including the presence of mono- and multinucleated phagocytes. Poly(L-lactic acid) underlayers tended to elicit a stronger cellular infiltrate than co-polymeric ones. In the course of the implantation periods this inflammatory response subsided and neovascular ingrowth and the formation of fibrous tissue in the pores was observed. Matrix degradation was demonstrated, starting with the fragmentation of the constituent polymers into increasingly smaller particles. During the implantation period, fragments showed progressive surface erosion. Poly(L-lactic acid) fragments had a more rounded off appearance as compared to co-polymeric ones. Implant surface area had decreased to less than 20%, 1 yr post-implantation. At both implantation sites and with all matrices, polymer particles were observed inside phagocytic cells. Degradation kinetics were similar with the different matrices. Implants fragmented more rapidly at the subcutaneous implantation site as compared to the intramuscular one. Although the data suggest biomaterial degradation, remnants of matrices could still be retrieved 1 yr post-implantation.

Animals↗

PubMed1994

Improved barrier structure formation in air-exposed human keratinocyte culture systems.

The epidermis (including stratum corneum) of human keratinocytes cultured at the air-liquid interface attached to an appropriate substrate shows a morphology closely mimicking that of its in vivo counterpart. In spite of the histologic similarities, the barrier function seems to be impaired. The aim of the present study was to characterize development and structure of the epidermal permeability barrier in two human skin recombinants using electron microscopy (including ruthenium tetroxide-post fixation technique) and analysis of lipid composition. The epidermis was reconstructed by growing human keratinocytes either on de-epidermized dermis or on a bovine collagen-containing matrix with active fibroblasts (Living Skin Equivalent). Ultrastructurally both culture systems showed a) an abnormal lamellar body delivery system, b) disturbance of transformation into lamellar lipid bilayers, c) an impaired structural organization and distribution of the epidermal lipids in the intercellular spaces. In either of the systems used, prolongation of the culture period did not induce any significant improvement in the stratum corneum lipid organization. Whereas the Living Skin Equivalent showed only sparse lamellar bodies, the number of lamellar bodies in the human keratinocyte culture on de-epidermized dermis grown in regular medium seemed to be comparable to native skin. Contrary to the Living Skin Equivalent, the keratinocyte culture on de-epidermized dermis contained a higher number of intracorneocytic lipid droplets correlating with a higher triglyceride content in the lipid analyses. By reconstructing the keratinocyte culture on de-epidermized dermis with the same medium as used for the Living Skin Equivalent, both lipid composition (lower triglyceride, higher ceramide contents) and structural organization were improved, and regular lamellar lipid bilayers comparable to those of native skin appeared.

Adult↗

PubMed1994

Fumaric acid derivatives evoke a transient increase in intracellular free calcium concentration and inhibit the proliferation of human keratinocytes.

Systemic administration of fumaric acid (FA) derivatives was originally an empirical antipsoriatic treatment, which showed promising clinical results. In the present study, FURA-2-loaded suspensions of cultured normal keratinocytes and SV40-transformed keratinocytes (SVK-14 cells) were used to study the effects of FA derivatives on the intracellular free calcium concentration ([Ca2+]i). Monomethylfumarate (MMF), dimethylfumarate (DMF) and monoethylfumarate (MEF) induced a rapid, transient [Ca2+]i increase in both cell types. This immediate increase reached maximal values of 396 nmol/l 10s after addition of MMF, and fell to basal values within 90-120 s (173 nmol/l for normal keratinocytes and 68 nmol/l for transformed keratinocytes). This increase was not affected by the prior addition of EGTA, indicating that FA derivatives released Ca2+ mainly from intracellular stores into the cytoplasm. Subsequently, dose-dependent inhibitory effects of FA derivatives on keratinocyte proliferation were demonstrated. The results of these experiments revealed that DMF was the most potent, MMF and MEF intermediate, and FA and malonic acid the least potent growth inhibitors. These antiproliferative effects of FA derivatives might be linked to the observed, transient [Ca2+]i elevations.

Anticarcinogenic Agents↗

PubMed1994

Immunohistochemical studies on regeneration in cultured epidermal autografts used to treat full-thickness burn wounds.

The normal pattern of healing following culture grafting is not well-established. The present study describes the immunohistochemical expression of differentiation markers during various stages of wound healing after resurfacing full-thickness burn wounds with cultured epidermal autografts. For this purpose, biopsy specimens were obtained from six patients 6 days to 4.5 years after transplantation. A panel of monoclonal antibodies was used against various differentiation-specific protein markers, including cytokeratins, involucrin, transglutaminase and filaggrin. Findings from early and late biopsies were compared with site-matched normal skin and cultured epidermal sheets. The persistence of cytokeratins 6 and 16, and to a lesser extent, the abnormal distribution of involucrin up to 4.5 years after resurfacing burns with cultured autografts was observed. These findings confirm previous studies--that the newly formed epidermis after culture grafting remains in a hyperproliferative state for a long time, which may be caused by the absence of a modulating dermal factor. Our results suggest that keratinocyte maturation following culture autografting does not return to normal for at least 4.5 years after healing of full-thickness burn wounds.

Adolescent↗

PubMed1994

Lipid composition and barrier function of human skin after grafting onto athymic nude mice.

The barrier properties of human epidermis grafted for 1-3 months onto nude mice are compared with normal human skin. Beside penetration studies with tritiated water and measurements of transepidermal water loss (TEWL), we analyzed the epidermal lipids by high-performance thin layer chromatography and evaluated the ultrastructure of the intercorneocyte lipid arrangement by freeze fracture electron microscopy (FFEM). The permeability of human skin for tritiated water and the TEWL exhibit no significant changes after grafting onto nude mice. FFEM analysis showed that grafted epidermis has the same morphological pattern as normal human epidermis. Regular desmosomes and lamellar lipid structures are present. Grafting did not qualitatively affect the lipid composition of human epidermis. Ceramides which contribute largely to the barrier function, have the same distribution profile.

Animals↗

PubMed1994

Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface.

We present a study on modification of culture conditions in serially cultured human bronchial epithelial cells (HBEC), necessary to achieve bronchial epithelial cells similar to the native epithelium. Cells were obtained from bronchial biopsies and serially cultured using a previously described method (In Vitro Cell. Dev. Biol. 1993; 29A:379-387). At the air-liquid interface, the second and the subsequent passages of HBEC cultures were grown 7 to 31 days, in medium containing fetal calf serum, using de-epidermized dermis or collagen discs as substratum. Scanning and transmission electron microscopy revealed ciliogenesis after 7 days and maturation of the cilia up to 31 days, irrespective of whether de-epidermized dermis or collagen membrane was used. The transmission electron microscopy of the developing cilia showed fibrogranular masses, procentrioles, basal bodies, and in the mature cilia a normal ultrastructure of the axoneme, the nine doublets, the central pair, radial spokes, and dynein arms in the ciliary shaft. In contrast, the submerged cultures showed no signs of ciliogenesis in the same time course. Results of experiments, in which cell seeding density, the substrate used, and the manner of nutrient supplementation were modulated, revealed that the air-exposure of the cultured HBEC is a necessary requirement for the ciliogenesis. The development pathway of ciliated cells in air-exposed HBEC cultures was similar to the differentiation and maturation pattern in human fetal tracheal cells. The in vitro model of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy offers an attractive model for future studies on the function of human bronchial epithelial cells under normal and pathologic conditions.

Bronchi↗

PubMed1994

SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes.

Skin-derived antileukoproteinase (SKALP), otherwise known as elafin, is a recently discovered epidermal proteinase inhibitor with specificity for polymorphonuclear leukocyte (PMN)-derived elastase and proteinase-3; in addition to the proteinase-inhibiting domain, SKALP contains several transglutaminase substrate motifs. SKALP is virtually absent in normal human epidermis but is found in a number of inflammatory skin diseases, including psoriasis. Here we report the induction and processing of SKALP in vivo and in vitro. SKALP expression in vivo could be demonstrated following injury in normal human epidermis, using histology, western blotting, northern blotting and a functional assay. In vitro, SKALP expression was studied in conventional submerged keratinocyte culture systems and in keratinocytes cultured in an air-liquid interface model. Induction of SKALP activity in epidermis could be measured as early as 16 hours after skin injury; immunohistological examination showed that SKALP expression was confined to the outer layers of the stratum spinosum and the stratum granulosum. Northern blot analysis revealed a 0.8 kb transcript, both in vivo (psoriatic skin, injured skin) and in vitro (cultured keratinocytes). Western blot analysis showed that the major SKALP form in vivo was a low molecular mass fragment, containing the antiproteinase domain. In all cultures that were positive for SKALP, larger (8-10 kDa) forms of SKALP, containing the N-terminal transglutaminase substrate motifs in addition to the antiproteinase domain, were found. SKALP expression in cultured cells was found to be dependent on the system used. In a submerged culture system, SKALP could be induced by fetal calf serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Air↗

PubMed1993

Co-culture of human melanocytes and keratinocytes in a skin equivalent model: effect of ultraviolet radiation.

Melanocytes grown in pure monolayer culture lack the three-dimensional organization and many of the cellular interactions that exist in vivo. This can be partially overcome by growing melanocytes together with other epidermal cells in skin equivalent models. In this study skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in various ratios onto de-epidermized dermis. They were cultured in DMEM/Ham's F12 (3:1) for 3 days and then lifted to the air-liquid interface and maintained for 11 days. Histological examination revealed a structure that closely resembled human interfollicular epidermis. Melanocytes, identified by their dendritic appearance, positive dopa reaction and positive staining with a melanocyte-specific antibody (MEL5), were located in the basal layer. Melanin was seen both in melanocytes and in neighbouring keratinocytes. Whilst the skin equivalent became more pigmented following UV irradiation (total UVB 4760 J/m2 over 3 days), the quantity and distribution of melanin at the light microscopic level appeared to be unchanged. However, the number and dendricity of melanocytes increased, as did their staining with dopa and MEL5. These results indicate that melanocytes are functional and capable of responding to UV irradiation.

Cell Count↗

PubMed1993

Serial culturing of human bronchial epithelial cells derived from biopsies.

In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.

3T3 Cells↗

PubMed1993

Cell-seeding and in vitro biocompatibility evaluation of polymeric matrices of PEO/PBT copolymers and PLLA.

A bilayered matrix has been evaluated in vitro as a carrier for autografts of cultured epidermal keratinocytes and dermal fibroblasts, to be used as a skin substitute in deep dermal skin defects. A poly-L-lactide (PLLA) and an elastomeric and biodegradable poly(ethyleneoxide)-poly(butyleneterephthalate)(PEO-PBT++ +)copolymer, called Polyactive, were chosen as the constituents of the matrix. The substrate properties of the bilayers for human and rat epidermal keratinocytes and dermal fibroblasts were assessed. Keratinocytes attached and expanded into confluent sheets on both the routine cell culture plastic (TCPS) and the experimental substrates. Morphology of the cells cultured on the biomaterials was found to be comparable with the morphology of those grown on TCPS. In contrast to dense films, porous PEO:PBT copolymer and PLLA appeared poor substrates for fibroblasts. Long-term (in vivo) degradation of the biomaterials was mimicked in vitro to screen the biomaterials for any release of toxic substances. Culturing keratinocytes and fibroblasts in media based on the artificially aged biomaterials did not result in any negative effects on proliferative activity or morphological appearance of the cells.

Animals↗

PubMed1993

Randomized trial comparing cryopreserved cultured epidermal allografts with hydrocolloid dressings in healing chronic venous ulcers.

BACKGROUND: Cultured epidermal allografts have been successfully used to treat a variety of wounds. Their postulated mechanism of action is through release of cytokines that stimulate epithelialization. On the basis of previous experience we expected ulcers treated with cryopreserved cultured allografts (CCAs) to be healed by 6 weeks. Hydrocolloid dressings (HCDs) have also been reported to be effective in the treatment of venous ulcers. OBJECTIVE: Our purpose was to compare the effectiveness of CCAs with HCDs in healing chronic venous ulcers. METHODS: Forty-three patients with 47 ulcers were enrolled in a randomized controlled trial. Ulcers not healed by 6 weeks were changed to the other treatment. RESULTS: No difference in the number of healed ulcers between the two groups was observed at 6 weeks. Healing rate, percent reduction of initial ulcer size, and radial progression toward wound closure were significantly greater for CCAs than for HCDs. Pain relief was not significantly different. CONCLUSION: CCAs achieve more rapid healing and greater reduction in ulcer size than HCDs.

Adult↗

PubMed1993

A new biodegradable matrix as part of a cell seeded skin substitute for the treatment of deep skin defects: a physico-chemical characterisation.

A bilayered matrix, as an integral part of a cell seeded skin substitute for deep dermal skin injury, is described. The skin substitute has been given a dense top layer, to function as a substrate for keratinocyte culture, and a porous under layer for wound adhesion and to serve as a template for dermal regeneration. This porous under layer may be seeded with dermal fibroblasts for improvement of neodermis formation. The elastomeric hydrogel Polyactive¿, a biodegradable polyether/polyester block copolymer, has been selected as the top layer component and Polyactive or Poly-L-lactide (PLLA) as the constituents of the under layer; this to ensure that the matrix is degradable and fulfils certain mechanical requirements. Polyactive top layers were found to be highly permeable to water vapour (+/-30 g/m -2 h -1 kPa) -1 and to molecules up to 150 kD (human IgG). Tensile properties of the bilayered matrices were to a considerable extent dependent on top layer and under layer composition and thickness. Elasticity moduli were in the range of those previously reported for human skin. Varying the weight ratios of the soft/hard segments of the Polyactives and/or substituting PLLA for Polyactive as the under layer component, allows the modulation of the matrix to specific application site dependent demands.

Biodegradation, Environmental↗

PubMed1993

Cytotoxic effects of topical antimicrobial and antiseptic agents on human keratinocytes in vitro.

The cytotoxicity of commonly used antimicrobial and antiseptic agents to cultured human keratinocytes was investigated, combining a morphologic assay with a quantitative neutral red (NR) spectrophotometric assay. Main outcome criteria of response were the initial cytotoxicity (NR 90) and midpoint cytotoxicity (NR 50) and the highest tolerated dose (HTD), a concentration causing the first observed morphologic alterations. These values were compared with commonly administered clinical doses or calculated doses. Thirty-five agents were evaluated; five agents were also subjected to an assay for cell morphologic alterations, and HTD and percentage of cells taking up trypan blue and annihilation dose were assessed. These studies show that in clinical concentrations many, but not all, agents exert profound cytotoxic effects. The results suggest that under certain conditions cultured epithelial grafts may be exposed to clinical concentrations of neomycin, clindamycin, framycetin, erythromycin, gentamicin, and 0.1% solutions of povidone-iodine. Tetrachlorodecaoxygen anion complex (TCDO) should be further evaluated as a likely effective cleansing agent before culture grafting.

Anti-Bacterial Agents↗