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Biomedical subjects

L Germain

Publications and source records attributed to L Germain.

At least 37 records · Page 2Linked to original sources
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

Antiangiogenic effects of the oral administration of liquid cartilage extract in humans.

BACKGROUND: The antiangiogenic properties of shark cartilage extracts have been demonstrated in animal models but there are no data in human subjects. MATERIALS AND METHODS: A placebo or one of two doses of a liquid shark cartilage extract was orally administered daily, from Day 1 to Day 23 of the study protocol, to 29 healthy male volunteers randomized into three groups. On Day 12, a polyvinyl alcohol sponge threaded in a perforated silicone tubing was inserted subcutaneously on the anterior side of the arm and removed on Day 23. Evaluation of endothelial cell density, with factor VIII immunostaining, an indirect measurement of angiogenesis, was performed on histological sections of the implant using a semiquantitative numerical scale ranging from 1 (low density) to 5 (high density). The hydroxyproline content of the sponges was measured by HPLC. RESULTS: The mean endothelial cell density was significantly lower in groups that had received the liquid cartilage extract: grades 2.24 +/- 0.10, 2.47 +/- 0.10, and 3.15 +/- 0.11 for 7 and 21 ml liquid cartilage extract and placebo, respectively (P < 0.01 for both comparisons). No grade 1 was observed in the placebo group, whereas 9 treated subjects received a grade 1. Hydroxyproline content of the sponges did not differ between groups and there was no significant correlation between hydroxyproline content and endothelial cell density in the sponges. CONCLUSIONS: These results demonstrate that the liquid cartilage extract contains an antiangiogenic component bioavailable in humans by oral administration. This is the first report of an inhibition of wound angiogenesis in healthy men.

Administration, Oral↗
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

The type I keratin 19 possesses distinct and context-dependent assembly properties.

Keratins (K), the cytoplasmic intermediate filament (IF) proteins of epithelial cells, are encoded by a multigene family and expressed in a tissue- and differentiation-specific manner. In human skin, keratinocytes of the basal layer of epidermis and the outer root sheath of hair follicles express K5 and K14 as their main keratins. A small subpopulation of basal cells exhibiting stem-cell like characteristics express, in addition, K19. At 40 kDa, this keratin is the smallest IF protein due to an exceptionally short carboxyl-terminal domain. We examined the assembly properties of K19 and contrasted them to K14 in vitro and in vivo. Relative to K5-K14, we find that K5-K19 form less stable tetramers that polymerize into shorter and narrower IFs in vitro. When transiently co-expressed in cultured baby hamster kidney cells, the K5 and K19 combination fails to form a filamentous array, whereas the K5-K14 and K8-K19 ones readily do so. Transient expression of K19 in the epithelial cell lines T51B-Ni and A431 results in its integration into the endogenous keratin network with minimal if any perturbation. Collectively, these results indicate that K19 possesses assembly properties that are distinct from those of K14 and suggest that it may impart unique properties to the basal cells expressing it in skin epithelia.

Animals↗
PubMed1998

TGF-beta receptor expression on human keratinocytes: a 150 kDa GPI-anchored TGF-beta1 binding protein forms a heteromeric complex with type I and type II receptors.

Keratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-beta has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-beta receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-beta receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-beta1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-beta isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-beta receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-beta1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-beta receptors on keratinocytes, supporting the heterotetrameric model of TGF-beta signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-beta by markedly downregulating all four TGF-beta binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-beta1 binding protein forms a heteromeric complex with the TGF-beta signalling receptors suggests that this GPI-anchored protein may modify TGF-beta signalling in human keratinocytes.

Adult↗
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↗
PubMed1997

From newborn to adult: phenotypic and functional properties of skin equivalent and human skin as a function of donor age.

The skin's most important function is to act as a barrier against fluid loss, microorganism infections, and percutaneous absorption. To fulfill this role, keratinocytes proliferate and differentiate to produce a protective layer: the stratum corneum. Because stem cells are responsible for the production of differentiated progeny and stem cells (K19-expressing cells) are less abundant in skin from older donors, the purpose of this study was to establish whether histological and functional properties of differentiating skin is influenced by donor age. The in vitro model developed for the evaluation of skin properties (Michel et al., 1995) was used to produce skin equivalents from newborn, child, and adult keratinocytes. Throughout maturation, skin equivalents were compared with corresponding skin biopsies for keratin, filaggrin, and transglutaminase expression. Percutaneous absorptions of hydrocortisone also were measured and correlated with lipid content. After 1 wk of immersed culture, the epidermal layer of newborn skin equivalents was thicker than child and adult epidermis. As expected, a greater proportion of cutaneous stem cells was present in newborn compared with children and adult skin equivalents. No age-related difference was observed for differentiation markers. When skin equivalents were cultured at the air-liquid interface, cell differentiation and stratum corneum formation were induced, and the age-related variation in the thickness of the epidermal layer disappeared. Percutaneous absorption through these matured skin equivalents did not vary with age. Their lipid density and profile were similar. Accordingly, skin biopsies exhibited comparable percutaneous absorption profiles independently of donor age. In conclusion, although newborn skin equivalents, or skin biopsies, contained more stem cells than child and adult counterparts, no age-related histological difference was observed in the differentiated tissues. Moreover, the functional barrier property of skins and matured skin equivalents did not vary with age. Therefore, both newborn and adult keratinocytes produce useful in vitro models to study epidermal differentiation processes involved in both normal and pathological states.

Absorption↗
PubMed1997

Use of in vitro reconstructed skin To cover skin flap donor site.

BACKGROUND: The skin flap technique is widely used in reconstructive surgery for the coverage of deep burns of the face, neck, and joints. Facial deformities and joint contractures are avoided by transplanting vascularized full-thickness skin on wounds. The major drawback of this technique is the injury inflicted upon the donor site, which corresponds to a third degree burn. The usual technique to cover the flap donor site is the transplantation of split-thickness autografts. In the case of patients with deep and extensive burns, the harvesting of good quality autografts is often difficult because of multiple scars. In order to avoid additional trauma to the patient by split-thickness skin harvesting, we have experimented the use of a new model of in vitro reconstructed skin graft for flap donor site coverage in a mouse model. MATERIALS AND METHODS: The reconstructed skin was grafted on the back of nude mice at the skin flap donor site, while flap was used to cover a wound generated on joint of the posterior leg. RESULTS: A 100% graft take was achieved (16 mice were used) and a limited contraction of the reconstructed skin was observed 30 days posttransplantation (78% of the initial surface area of the graft remained). Histological analysis of the graft demonstrated healing of a well differentiated epidermis laying on a dense dermis. CONCLUSIONS: Since this technique would prevent additional trauma to the patient while achieving a good healing of the wound, it may be a useful approach in the coverage of skin flap donor site in humans.

Animals↗