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Biomedical subjects

F A Auger

Publications and source records attributed to F A Auger.

At least 19 recordsLinked to original sources
PubMed2001

A human tissue-engineered vascular media: a new model for pharmacological studies of contractile responses.

Our method for producing tissue-engineered blood vessels based exclusively on the use of human cells, i.e., without artificial scaffolding, has previously been described (1). In this report, a tissue-engineered vascular media (TEVM) was specifically produced for pharmacological studies from cultured human vascular smooth muscle cells (VSMC). The VSMC displayed a differentiated phenotype as demonstrated by the re-expression of VSMC-specific markers and actual tissue contraction in response to physiological stimuli. Because of their physiological shape and mechanical strength, rings of human TEVM could be mounted on force transducers in organ baths to perform standard pharmacological experiments. Concentration-response curves to vasoconstrictor agonists (histamine, bradykinin, ATP, and UTP) were established, with or without selective antagonists, allowing pharmacological characterization of receptors (H1, B2, and P2Y1, and pyrimidinoceptors). Sustained agonist-induced contractions were associated with transient increases in cytosolic Ca2+ concentration, suggesting sensitization of the contractile machinery to Ca2+. ATP caused both Ca2+ entry and Ca2+ release from a ryanodine- and caffeine-sensitive store. Increased cyclic AMP or cyclic GMP levels caused relaxation. This human TEVM displays many of functional characters of the normal vessel from which the cells were originally isolated, including contractile/relaxation responses, cyclic nucleotide sensitivity, and Ca2+ handling mechanisms comparable to those of the normal vessel from which the cells were originally isolated. These results demonstrate the potential of this human model as a versatile new tool for pharmacological research.

Adenosine Triphosphate↗
PubMed2000

Role of wound healing myofibroblasts on re-epithelialization of human skin.

In human skin, large burned surfaces heal using two concomitant phenomena: re-epithelialization and dermal neoformation. Numerous studies report the role of interactions between keratinocytes and fibroblasts, but the relationship between wound healing myofibroblasts and keratinocytes is not clear, even though these two cell types coexist during healing. We investigated the influence of myofibroblasts on keratinocyte growth and differentiation using an in vitro skin model. A histological study was performed to determine the speed and quality of epithelialization. When the dermis was populated with fibroblasts, a continuous epidermis was formed in 7-10 days. In contrast, with wound healing myofibroblasts or without cell in dermis, the complete reepithelialization never occurred over the 10-day period studied. After 7 further days of epidermal differentiation, histology showed an epidermis more disorganized and expression of basement membrane constituents was reduced when wound healing myofibroblasts or no cells were added in the dermis instead of fibroblasts. These results suggest that wound healing myofibroblasts are not efficient to stimulate keratinocyte growth and differentiation. Treatment of fibroblasts with TGFbeta1 induced an increase of epidermal cell differentiation as seen when myofibroblasts were present. However, this cytokine did not change re-epithelialization rate and induced an increase of basement membrane matrix deposition in opposition to myofibroblasts. Thus, TGFbeta1 action is not sufficient to explain all the different keratinocyte reactions towards fibroblasts and wound healing myofibroblasts. Our conclusion is that myofibroblasts seem to have a limited role in the re-epithelialization process and might be more associated with the increased extracellular matrix secretion.

Basement Membrane↗
PubMed2000

Can we produce a human corneal equivalent by tissue engineering?

Tissue engineering is progressing rapidly. Bioengineered substitutes are already available for experimental applications and some clinical purposes such as skin replacement. This review focuses on the development of reconstructed human cornea in vitro by tissue engineering. Key elements to consider in the corneal reconstruction, such as the source for epithelial cells and keratocytes, are discussed and the various steps of production are presented. Since one application of this human model is to obtain a better understanding of corneal wound healing, the mechanisms of this phenomenon as well as the function played both by membrane-bound integrins and components from the extracellular matrix have also been addressed. The analysis of integrins by immunohistofluorescence labelling of our reconstructed human cornea revealed that beta(1), alpha(3), alpha(5), and alpha(6) integrin subunits were expressed but alpha(4) was not. Laminin, type VII collagen and fibronectin were also detected. Finally, the future challenges of corneal reconstruction by tissue engineering are discussed and the tremendous applications of such tissue produced in vitro for experimental as well as clinical purposes are considered.

Biomedical Engineering↗
PubMed2000

Multistep production of bioengineered skin substitutes: sequential modulation of culture conditions.

Many studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboidal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.

Animals↗
PubMed1999

Physical characterization of the stratum corneum of an in vitro human skin equivalent produced by tissue engineering and its comparison with normal human skin by ATR-FTIR spectroscopy and thermal analysis (DSC).

An in vitro human skin equivalent may be obtained by culturing human keratinocytes on a collagen gel containing fibroblasts. The anchored skin equivalent cultured at the air-liquid interface closely resembles human skin and is acceptable for in vitro percutaneous absorption. However, it is still more permeable than human skin. Since intercellular lipids have been recognized to play an important role in skin permeability, infrared spectroscopy and differential scanning calorimetry were performed on the stratum corneum of bovine or human skin equivalents grown at different days of air-liquid culture. The symmetric and asymmetric CH(2) stretching vibrations suggested that for all days observed, the intercellular lipids were less organized than those in human skin, irrespective of whether bovine or human collagen was used. Different culture conditions were also tested and the medium without serum and no epidermal growth factor at the air-liquid culture showed results significantly more comparable to human skin. Actually, the thermal behavior of in vitro stratum corneum showed transitions at lower temperatures than human skin. The transition around 80 degrees C, in the form of a lipid-protein complex, was absent. These results showed that the structural arrangement of intercellular lipids and their thermodynamic properties hold a crucial role in the barrier function of the stratum corneum.

Animals↗
PubMed1999

Characterization of a new tissue-engineered human skin equivalent with hair.

We designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.

Adult↗
PubMed1999

A novel approach for studying angiogenesis: a human skin equivalent with a capillary-like network.

Angiogenesis results from an ordered set of events that can be modulated in vivo by a variety of angiogenesis-enhancing or inhibiting agents. We review in vitro angiogenesis models and the agents that enhance or inhibit angiogenesis. We also discuss a new in vitro angiogenesis model created within a skin equivalent. Briefly, endothelial cells were combined with the cutaneous cells of a standard skin equivalent and cultured in a chitosan cross-linked collagen-glycosaminoglycan scaffold of this endothelialized skin. This model enables the formation of capillary-like structures in a coculture environment containing newly synthesized extracellular matrix by fibroblasts and keratinocytes. Several morphological characteristics associated with the microvasculature in vivo were observed in the endothelialized skin equivalent such as histotypic organization of tubular structures, basement membrane deposition, and intercellular junction formation.

Animals↗
PubMed1998

Modulated response to cytokines of human wound healing myofibroblasts compared to dermal fibroblasts.

Myofibroblasts play an important role in normal wound healing. They are present transiently during tissue repair. Their differentiation from fibroblasts and their role in granulation tissues are most likely to be modulated by cytokines. As these cells are derived from normal fibroblasts, their responses to cytokines are assumed to be similar. Until now, however, the difficulties in obtaining and maintaining normal human wound healing myofibroblasts in vitro have hampered comparison. The present study was designed to determine the effect of TGF-beta 1 and IFN-gamma, two cytokines known to modulate fibroblast morphology, on wound healing myofibroblasts and to compare it to fibroblasts. Morphological and phenotypic changes were followed by light and electron microscopy (stress fibers) and immunofluorescence cytochemistry (alpha-SM actin). Functional parameters such as the capacity to synthesize collagen and collagen gel contraction were studied. Both cytokines induced a strong modification of growth rate and phenotypic and morphological parameters in fibroblasts whereas collagen synthesis was slightly changed. Furthermore, TGF-beta 1 increased contractile capacity of fibroblasts whereas IFN-gamma greatly decreased it. In myofibroblasts, TGF-beta 1 and IFN-gamma did not induce any variation of morphology or growth rate. Interestingly, a strong modulation of functional parameters was observed: collagen synthesis was highly modified and, as for fibroblasts, the contractile capacity was altered. However, inhibition of contraction by IFN-gamma was irreversible in myofibroblasts but not in fibroblasts. These results suggest that fibroblastic cells show modulated responses to cytokines according to their stage of differentiation during wound healing.

Adult↗
PubMed1998

Tissue-engineered human skin substitutes developed from collagen-populated hydrated gels: clinical and fundamental applications.

The field of tissue engineering has opened several avenues in biomedical sciences, through ongoing progress. Skin substitutes are currently optimised for clinical as well as fundamental applications. The paper reviews the development of collagen-populated hydrated gels for their eventual use as a therapeutic option for the treatment of burn patients or chronic wounds: tools for pharmacological and toxicological studies, and cutaneous models for in vitro studies. These skin substitutes are produced by culturing keratinocytes on a matured dermal equivalent composed of fibroblasts included in a collagen gel. New biotechnological approaches have been developed to prevent contraction (anchoring devices) and promote epithelial cell differentiation. The impact of dermo-epidermal interactions on the differentiation and organisation of bio-engineered skin tissues has been demonstrated with human skin cells. Human skin substitutes have been adapted for percutaneous absorption studies and toxicity assessment. The evolution of these human skin substitutes has been monitored in vivo in preclinical studies showing promising results. These substitutes could also serve as in vitro models for better understanding of the immunological response and healing mechanism in human skin. Thus, such human skin substitutes present various advantages and are leading to the development of other bio-engineered tissues, such as blood vessels, ligaments and bronchi.

Cell Culture Techniques↗
PubMed1998

A completely biological tissue-engineered human blood vessel.

Mechanically challenged tissue-engineered organs, such as blood vessels, traditionally relied on synthetic or modified biological materials for structural support. In this report, we present a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) cultured with ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This TEBV featured a well-defined, three-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, SMC reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated LDL, produced PGI2, and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength over 2000 mmHg. This is the first completely biological TEBV to display a burst strength comparable to that of human vessels. Short-term grafting experiment in a canine model demonstrated good handling and suturability characteristics. Taken together, these results suggest that this novel technique can produce completely biological vessels fulfilling the fundamental requirements for grafting: high burst strength, positive surgical handling, and a functional endothelium.

Animals↗
PubMed1998

In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent.

For patients with extensive burns, wound coverage with an autologous in vitro reconstructed skin made of both dermis and epidermis should be the best alternative to split-thickness graft. Unfortunately, various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. Our purpose was to develop a vascular-like network inside tissue-engineered skin in order to improve graft vascularization. To reach this aim, we fabricated a collagen biopolymer in which three human cell types keratinocytes, dermal fibroblasts, and umbilical vein endothelial cells were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary-like structures in a highly differentiated extracellular matrix. Immunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the microvasculature in vivo (von Willebrand factor, Weibel-Palade bodies, basement membrane material, and intercellular junctions). We have developed the first endothelialized human tissue-engineered skin in which a network of capillary-like tubes is formed. The transplantation of this ESE on human should accelerate graft revascularization by inosculation of its preexisting capillary-like network with the patient's own blood vessels, as it is observed with autografts. In addition, the ESE turns out to be a promising in vitro angiogenesis model.

Basement Membrane↗
PubMed1998

Influence of endothelial cells on structure, biochemistry and functionality of epidermis reconstructed on synthetic porous membrane.

The model of keratinocytes cultured on a synthetic porous membrane at the air-liquid interface leads to the formation of a pluristratified and cornified epidermis with histological and biochemical characteristics near those observed in vivo. In the present study, we evaluated the effect of proliferative endothelial cells on epidermalization. Keratinocytes were grown in three culture conditions: in defined medium (DM; control), in medium previously conditioned by proliferative endothelial cells (CM) and in medium with proliferative endothelial cells (pEC). The structures of reconstructed epidermis were analyzed by electron microscopy, their biochemistry by DNA, protein and cytokine analyses and finally their functionality was evaluated by estradiol and water absorption testing. Ultrastructural analysis showed a well-developed and cornified epidermis for each culture condition. In addition, living epidermis was thinner in the presence of endothelial cells, revealing faster epidermal differentiation. DNA and protein analyses were in accordance with these results. Secreted soluble factors varied according to culture conditions. At 37 degreesC, the permeability of reconstructed epidermis in DM, in CM or with pEC was 5- to 10-fold higher than that of native human epidermis with both tracers. Laminin coating of the inserts led to similar absorption results except for the DM where the barrier function to estradiol was decreased 2-fold. At 32 degreesC, reconstructed and native epidermis were, respectively, 1.5- and 2-fold less permeable to estradiol compared to 37 degreesC. In conclusion, this model is adequate for fundamental and pharmacological studies since it allows the study of interactions between two cell types without their direct contact as well as percutaneous absorption tests directly performed in the modified culture chamber.

Cell Differentiation↗
PubMed1998

Expression of heat shock proteins in mouse skin during wound healing.

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.

Animals↗
PubMed1997

Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro.

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of alpha-smooth muscle (alpha-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, alpha-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor beta (TGFbeta) in FBS and PCS was determined and the effect of neutralizing anti-TGFbeta antibody was evaluated. It was determined that FBS contained more TGFbeta than PCS, but that essentially all the TGFbeta was latent in both sera. However, results obtained with anti-TGFbeta antibody show that active TGFbeta is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFbeta or an enzyme that is able to activate latent serum TGFbeta. Alternatively, sera may contain two different forms of latent TGFbeta, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds.

Animals↗