Search PubMed⌕ Search

Biomedical subjects

F A Auger

Publications and source records attributed to F A Auger.

At least 55 records · Page 3Linked to original sources
PubMed1995

Early basement membrane formation following the grafting of cultured epidermal sheets detached with thermolysin or Dispase.

The basement membrane zone is important for graft adhesion and stability. The aim of the present study was to visualize the regeneration of the basement membrane and determine the sequential appearance of its constituents in the early postgrafting period of cultured human epidermal sheets. A keratinocyte single cell suspension, devoid of dermal fibroblast contamination, was obtained from human skin by a two-step tissue digestion method with thermolysin and trypsin. After culturing, epidermal sheets were generated, detached enzymatically by incubating with thermolysin (for 20-30 min) or Dispase (for 45-60 min), and deposited on a muscular graft bed of athymic mice. Immunohistochemistry and ultrastructural analyses were performed on biopsies harvested 2, 4 and 21 days postgrafting. Bullous pemphigoid antigens and laminin were detected at the dermo-epidermal junction, showing an almost continuous line 2 days postgrafting. Type IV collagen was generally absent at this time, but it was detected 4 days postgrafting. Type VII collagen was labelled as a discontinuous line of increasing intensity from 2 to 21 days postgrafting. Ultrastructural analysis revealed hemidesmosomes and a discontinuous lamina densa 2 days postgrafting, and a complete basement membrane with a continuous lamina densa, hemidesmosomes and anchoring fibrils 21 days postgrafting. The sequence of appearance of major basement membrane components was similar for cultured sheets detached with thermolysin or Dispase. However, it differed from that of other wound healing models. Results are discussed in terms of the variable keratinocyte migration requirement between various wound healing models.

Animals↗
PubMed1995

A method for the evaluation of tensile properties of skin equivalents.

In vitro production of anchored skin equivalent is a new therapeutical option for burn patients. A skin equivalent is a combined culture of dermal and epidermal layers. The dermal layer provides important mechanical properties, such as tensile resistance and nonlinear elasticity, to the skin equivalent during its development. Prior to any in vivo human transplantation, the tensile properties of cutaneous equivalents have to be evaluated as a function of its structural components, in view of establishing the culture conditions leading to the best mechanical resistance and stretchability characteristics. However, the handling and clamping of skin equivalents are frequent causes of tearing and lack of repeatability in the measuring of tensile properties. A new indentation method involving a specially designed culture dish has been developed to minimize the risk of damage. Using this new culture dish, cutaneous equivalents were installed on an indentation apparatus. The central loading of a spherical tip was transmitted to the central area of a circular anchored cutaneous equivalent and was recorded with tip position. The tests were achieved at a constant low deflection rate of the tip. This new and accurate method gave repeatability in three central load-deflection characteristics of anchored dermal equivalent: the high-modulus (0.15 g mm-1), the central load of rupture (1.49 g), the rupture deflection (0.470 mm). This indentation test is expected to be an efficient tool in the evaluation of various skin equivalent models tensile properties.

Biological Dressings↗
PubMed1995

Study of the tensile properties of living skin equivalents.

The living skin equivalent is one of the more advanced clinical applications in the field of tissue engineering. It is a promising therapeutic option for burn victims and a strong potential for manifold in vitro experiments. However, researchers have encountered major drawbacks in the reconstruction of the dermal layer. Peripheral anchorage of the dermal equivalent component has been a valuable solution to many of these problems. In this work, we have carried out the mechanical analysis of skin equivalent models, based on this dermal anchoring technique, with a study of their biaxial tensile properties. Differences between models were related to the origin of collagen, either bovine or human, and on the culture techniques: immersion or at the air-liquid interface. The study was accomplished in vitro using 25.4-mm-diameter disk-shaped specimens with an indentation test. In appropriate wet condition, the specimens were punctured with a spherical tip at a quasi-static rate. We measured the load applied against the tip vs deflection up to the breaking point. Our results show that skin equivalents presented a typical exponential load-deflection relationship. All skin equivalents presented large extensibility up to 1.41 expressed in a ratio of deflection vs specimen's radius. The maximum tensile strength (0.871-1.169 Newton) and energy calculations (3.75-6.432 N.mm) was offered by living skin equivalent, made with human types I and III collagens, cultured at the air-liquid interface. In these conditions, our results suggest the tensile properties of living skin equivalents were enhanced due to the development of well stratified stratum corneum.

Adolescent↗
PubMed1994

Human wound healing fibroblasts have greater contractile properties than dermal fibroblasts.

Contractile and phenotypic properties of human fibroblasts from healing wounds were compared to those of dermal fibroblasts using in vitro models. Wound fibroblasts were recovered from implants, made of a polyvinyl alcohol sponge threaded into a perforated silicone tube, 12 days after their subcutaneous implantation in human volunteers. Dermal fibroblasts were isolated from the skin of healthy subjects. Two morphologically different fibroblast populations were observed in cells cultured from implants. In order to characterize these fibroblast populations, intracellular alpha-actin expression was studied by immunofluorescence labeling of cells cultured in monolayer. This protein was detected in less than 1% of the dermal fibroblasts. By contrast, 30 to 40% of wound fibroblasts were labeled and contained fiber networks of alpha-actin. These results confirm the presence of myofibroblasts in human wound healing tissues. The contractile property of fibroblasts and myofibroblasts was evaluated using a three-dimensional cell culture model (fibroblast populated collagen gels). Cells were incorporated in a collagen matrix and cultured for 14 days. The surface area of collagen gels was measured every day. Our results show that wound fibroblasts strongly contract collagen gels during the first 24 hr (surface area at 24 hr = 20-55% of initial surface area) in comparison to dermal fibroblasts (surface area at 24 hr = 70-75% of initial surface area). This superior level of contraction was observed until the fifth day of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Visual analogue thermometer: a valid and useful instrument for measuring pain in burned patients.

This study assessed the psychometric qualities of a new pain rating instrument--the visual analogue thermometer (VAT)--which was developed to measure pain in burned patients. The validity and utility of the VAT was assessed and compared with a conventional numeric (NUM) and adjective pain scale (ADJ) with a group of 103 burned patients and 51 nurses. Analyses of the results support the concurrent and construct validity of the VAT as a pain measure. Furthermore, the VAT gave more sensitive and precise pain measures than the ADJ and/or NUM scales. No major difference between the three scales emerged in the patients' preference. The same was true for the nurses' evaluation except for those who had more clinical experience with the VAT and who tended to prefer this scale for its accuracy and ease of utilization. The VAT appears to be a valid, sensitive and clinically useful tool to measure pain in burned patients. A systematic pain assessment procedure which can be easily implemented in burn care facilities is presented.

Adult↗
PubMed1993

Role and innocuity of Tisseel, a tissue glue, in the grafting process and in vivo evolution of human cultured epidermis.

Cultured epidermal sheets are currently used for burn wound treatment but reported results on graft take are variable. This study was designed to evaluate the role and influence of Tisseel, a fibrin glue, in the take of cultured human epidermal sheets in an athymic mouse model. On days 4, 10 and 21 post-grafting, histology, electron microscopy and immunofluorescence staining confirmed the presence of a human epithelium and the development of a basement membrane. Tisseel was detectable on day 4 only, but overall treated and untreated grafts were similar. The use of Tisseel enhanced the mechanical stability of these fragile grafts, increased the percentage of graft take, and its innocuity on the in vivo evolution of cultured epidermal sheets was demonstrated. For these reasons, we think that Tisseel may be advantageous in a clinical setting.

Animals↗
PubMed1993

Improvement of human keratinocyte isolation and culture using thermolysin.

We propose a modification of the conventional keratinocyte isolation method which has shown a significant improvement in the purity, colony forming efficiency (c.f.e.) and growth capacity of the isolated epidermal cell population. This method utilized thermolysin since it selectively digests the dermo-epidermal junction. Following separation from the dermis, the epidermis was digested with trypsin to obtain a single cell suspension. Compared with the conventional procedure, this isolation method was shorter and resulted in (i) cells displaying a higher colony forming efficiency, (ii) cells reaching confluence 1-3 days earlier, (iii) cells not contaminated by fibroblasts, (iv) a cell population containing all the basal layer keratinocytes. These cells were suitable for the establishment of primary cultures and could be subcultured. Such cell populations should be advantageous in studies of epithelial-mesenchymal interactions in which keratinocyte populations, free of fibroblasts, are desirable. In the treatment of extensively burned patients using cultured epidermal sheets, the main problem remains the time required for their production. Thus, the absence of fibroblast overgrowth of the keratinocyte cultures and the significantly reduced time to obtain confluent cultures and epidermal sheets with our method have very important implications for the treatment of large burn wounds.

Adolescent↗
PubMed1993

In vitro construction of a human blood vessel from cultured vascular cells: a morphologic study.

PURPOSE: The purpose of this study was to create a tubular vascular model exclusively made of human cells and collagen. METHODS: The blood vessel equivalent was constructed with the three following human cell types: vascular smooth muscle cells, endothelial cells, and fibroblasts. A tissuelike structure was obtained from the contraction of a tubular collagen gel (human origin) by vascular smooth muscle cells, which created a media-like structure. An adventitia-like tissue was added around the media-like structure by embedding fibroblasts into a collagen gel. An endothelium was established within the tubular structure after intraluminal cell seeding. RESULTS: Cell orientation and gel contraction were followed up over time. Vascular smooth muscle cells developed a complex tridimensional network and were oriented in a circular fashion around the tube's axis. In contrast, fibroblasts were randomly oriented. A viable, homogeneous, and well-characterized endothelium was observed. These endothelial cells showed a slightly elongated structure and were oriented parallel to this vascular equivalent axis. CONCLUSION: An in vitro tridimensional vascular model that exhibits some phenotypic characteristics of in vivo vascular cells could be useful in the study of events that lead to atherosclerotic plaque formations.

Cells, Cultured↗
PubMed1993

Cultured epithelium allografts: Langerhans cell and Thy-1+ dendritic epidermal cell depletion effects on allograft rejection.

The effects of in vitro dendritic cell (DC) depletion on the survival of epidermal sheet allografts were studied in a murine model. Newborn (1-3 days old) mouse skin was used. Langerhans cell (LC) and Thy-1+ dendritic epidermal cell (Thy-1+ DEC) depletion was achieved using: (1) a prolonged culture period (7 days), or (2) the anti-IA and anti-Thy-1.2 mAbs followed by complement treatment. DC (LC and Thy-1+ DEC) depletion was assessed on sheets and cultured cell suspensions by an indirect immunofluorescence procedure. They showed that, after 7 days of culture or after the antibody-complement treatment, epidermal cultures were depleted of LC and Thy-1+ DEC. Cultured sheets were grafted onto the muscle of H-2-incompatible recipients. The control experiments were: (1) full epidermis and DC undepleted sheet allografts, and (2) DC depleted and undepleted sheet isografts. The full epidermis was totally rejected after 9 days. However, no rejection sign was ever seen in any of the isografts. The in vitro produced and allografted epithelia did not show any necrotic sign until the 11th day postgrafting. However, on days 12-13, the DC depleted allograft's color changed from pink to brown. On days 14-16, degradation of the allografts resulted in a complete denudation of the underlying muscle. Immunohistological analysis of the allografts revealed the presence of a monocyte and lymphocyte infiltration starting from the 11th day postgrafting, with the presence of polymorphonuclear leukocytes on day 14. These results suggest that LC and Thy-1+ DEC depletions were not sufficient to prevent allograft rejection.

Animals↗
PubMed1992

Optimization of murine keratinocyte culture for the production of graftable epidermal sheets.

The aim of the present study was to optimize murine epidermal cell cultures in order to obtain graftable sheets. Newborn (1-3 days old) Balb/c mice skin were used to optimize culture media and plating cell concentration, then epidermal sheet production, and grafting. Epidermal cells were plated at various concentrations in different culture media containing low (0.1 mM) or high (greater than 1 mM) Ca2+ levels. After a 3 day culture at the 10(4) cells/cm2 plating cell concentration, the percentage of differentiated cells was more than 80% in the high Ca2+ culture medium and less than 50% with bulky cells in the low Ca2+ culture medium. Under these conditions confluence was not obtained. At the 10(5) cells/cm2 seeding inoculum, the percentage of confluence increased to 95-100% during the first 72 h of culture in both high and low Ca2+ culture media. Three-day-old culture showed stratified multilayer epidermal sheets in the high calcium medium, and monolayer epidermal sheets were present in the low calcium medium after seeding keratinocytes in fibronectin precoated flasks. Seven days after plating, post confluent cultures were composed of a high percentage of differentiated cells (90%) with an increase in shedding cells in the medium. Considering the above morphological observations, sheets obtained with 10(5) cells/cm2 in MCDB-153 (A), DME-HAM (B) or GMEM (C) media after 3 days in culture were grafted. Twenty days after grafting, histological analysis of biopsies showed an epidermal structure and organization comparable to normal murine epidermis without hair follicles. Epidermal transplants showed a complete basement membrane, hemidesmosomes, and tonofilament bundles. Sheets obtained after seven day culture in all media showed lower coverage of the wound bed. These studies point out the importance of the plating cell and Ca2+ concentrations, and the culture time for murine keratinocyte confluence and differentiation to obtain graftable epidermal sheets.

Animals↗
PubMed1992

Peripheral anchorage of dermal equivalents.

Human fibroblasts can induce collagen gel contraction with different kinetics depending on the number of cells and on the collagen concentration within this lattice, which has been considered as a dermal equivalent. Skin equivalent is a combined culture of dermo-epidermal layers which may be of therapeutic value in the treatment of burn patients. However, the current production of the dermal equivalent component gives results that present many drawbacks for their eventual clinical use as a first step in obtaining a skin equivalent. These include: (i) final surfaces which are very small; less than 20% of the initial size (ii) excessive thickness which may hamper successful graft take (iii) fibroblasts that do not have an arrangement comparable with normal dermal tissue. We propose, as a solution to these problems, the utilization of a 5-mm-wide fibre-glass filter ring peripherally attached to the surface of the Petri dishes to prevent inordinate contraction while the fibroblasts reorganize the collagen gel. Using this technique the initial surface was preserved and the dermal equivalent contracted only in thickness. Histological analysis of these anchored equivalents confirmed an alignment of fibroblasts and collagen fibres resembling normal dermal tissue. We consider this method useful in the development of dermo-epidermal sheets for clinical purposes.

Bioprosthesis↗
PubMed1992

Influence of dermal equivalent maturation on the development of a cultured skin equivalent.

Histologic and immunofluorescence methods were used to analyse the presence of fibronectin, chondroitin-4-sulphate and chondroitin-6-sulphate, type III and IV collagens, laminin, and keratins to assess the maturation level of cultured dermal and skin equivalents. In a first phase, fibroblasts in monolayer culture were compared with dermal equivalents in which fibroblasts are embedded in a type I collagen gel. Different fluorescent patterns were observed depending on the culture system used. A sequential appearance of macromolecules was noticed in dermal equivalents. Fibronectin was first detected after 4 days of culture, whereas chondroitin-4-sulphate and chondroitin-6-sulphate and type III collagen were present after 7 days. In contrast, all three macromolecules were detected at 24 h of culture in fibroblastic monolayer cultures. In a second phase, the quality of our skin equivalents was evaluated according to the seeding time of epidermal cells upon dermal equivalents (1, 4, or 7 days). A satisfactory stratification was obtained when keratinocytes were seeded after 4 and 7 days of dermal equivalent culture. Laminin and fibronectin were detected at the dermo-epidermal junction, but type IV collagen was absent. Various keratins, as detected by the AE1, AE2, and AE3 antibodies, were present in the epidermal layer. Following keratinocyte confluence, a change in the organization pattern of type III collagen in the dermal fraction of the skin equivalent was also noticed. Our comparative results show that seeding of epidermal cells on a more mature dermal equivalent leads to improved differentiation status of the epidermal layer.

Cell Division↗
PubMed1992

[Murine surgical model for the study of cultured grafts].

The objective of the study was to establish an animal model for in vivo studies of cultured cutaneous equivalents. The model on athymic mice that we already described (López-Valle C.A. et al., Plast Reconstr Surg, 1992, 89, 139-143) satisfied the criteria of immobilization of the recipient site and physical stability of the graft, but still allowing complete movement freedom of the animal used. Nevertheless, this technique encountered two long term weaknesses; a) a significant grafted surface reduction caused by the wound contraction and b) the absence of a physical barrier between human and murin keratinocytes. We propose some modifications to this technique to correct both problems. Moreover, the implantation of polypropylene, instead of glass pellets, to generate granulation tissue on the recipient bed when needed, constitutes an easier method for both the surgeon and the animal. Finally, in order to minimize wound care, some modifications were made to the rodent cages. Immunohistological analyses of the biopsies, 21 days post-grafting, revealed a continuous basal membrane. This animal model allows in vivo studies on the behavior, cicatrization and immunology of human cultured skin equivalents.

Animals↗