Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan.

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.

Adhesion and proliferation of human dermal fibroblasts on collagen matrix.

The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon. Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy). By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous. By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel. By transmission electron microscopy, collagen fibrils showed the typical banding. Human dermal fibroblasts were seeded on the collagen matrix, previously equilibrated in growth medium. Fibroblast proliferation stopped in the second week and was always significantly lower than that of the same cell strain seeded on plastic and cultured in parallel. By light microscopy, after six days culture, cells formed a confluent multilayer on the surface of the gel. By scanning and transmission electron microscopy, fibroblasts appeared flat and adherent to the matrix. Contacts of cells among themselves and with the collagen fibrils were observed. Fibroblasts never moved into the collagen gel. In conclusion, human dermal fibroblasts can be grown in a three-dimensional matrix made by horse tendon that, on the other hand, seems to condition their proliferation rate.