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P Contard

Publications and source records attributed to P Contard.

5 recordsLinked to original sources
PubMed1993

Collagen fibrillogenesis in a three-dimensional fibroblast cell culture system.

The purpose of this study was to follow collagen fibril formation in a newly developed three dimensional cell culture system. Human neonatal foreskin fibroblasts were grown on a nylon mesh in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. Fibrillogenesis was initiated by the addition of 50 micrograms/ml ascorbate to confluent cultures. Sample meshes were processed for electron microscopy or immuno-electron microscopy. Fibrils approximately 20-30 nm in diameter, with 67 nm periodicity, were first detected five days after the addition of ascorbate. As cultures progressed, cells organized into parallel layers between which collagen fibers continued to form and increase in diameter. By day 50, fiber diameter ranged from 30 to 80 nm and large bundles were seen. No collagen fibril formation occurred in control cultures to which no ascorbate was added. However, large amounts of microfibrils were observed. Antibodies against the aminopropeptide of type I procollagen were found to bind to fibrils with diameters less than 34 nm while antibodies against the aminopropeptide of type III collagen bound primarily to fibers which ranged from 35-54 nm in diameter. We believe that this system, which morphologically resembles a normal dermis, will serve as an excellent model for the study of collagen fibrillogenesis.

Cells, Cultured↗
PubMed1993

Culturing keratinocytes and fibroblasts in a three-dimensional mesh results in epidermal differentiation and formation of a basal lamina-anchoring zone.

The purpose of this study was to characterize an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 3 and 5 weeks and then processed for electron microscopy. Keratinocytes showed reconstruction of an epidermis consisting of a basal layer with hemidesmosomes, a stratified epithelium with tonofilaments and desmosomes, a granular layer with keratinosomes and keratohyaline granules, and a transitional stratum corneum. Anchoring filaments, lamina densa, anchoring fibrils, bundles of elastin-associated microfibrils (diameters 10 nm) and fine collagen fibrils were formed. Collagen fibrils near the epidermis were much thinner than those in the lower levels. The present study shows that the dermal model containing metabolically active fibroblasts in their natural environment will support epidermal morphogenesis and differentiation including the formation of a basal lamina and anchoring zone.

Basement Membrane↗
PubMed1993

Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

Cell Adhesion Molecules, Neuronal↗
PubMed1991

Elastin-associated microfibrils (10 nm) in a three-dimensional fibroblast culture.

The purpose of this study is to present a three-dimensional dermal fibroblast model. Skin fibroblasts cultured in this system deposit large amounts of collagen and microfibrils. Fibroblasts were seeded onto a nylon filtration mesh and incubated in the presence or absence of ascorbic acid. Collagen fibril formation was found in the presence of ascorbic acid whereas microfibril formation was seen independent of ascorbic acid supplementation. Immunoelectron microscopy revealed that microfibrils were labeled with fibrillin at 67 nm periodicity. Isolated microfibrils studied by rotary shadowing had a beaded appearance consisting of beads linked to each other by a filamentous structure. The spaces between the beads ranged from 10.00-33.33 nm, suggesting that these microfibrils may have an extension-contraction mechanism. Furthermore, the size and spacing of the beads were similar to that seen in microfibrils from tissues (measured after rotary shadowing). Fibroblasts cultured in a three-dimensional mesh represent an effective in vitro model with which to study microfibril formation.

Cells, Cultured↗