Effects of Extracellular Matrices on Human Keratinocyte Adhesion and Growth and on its Secretion and Deposition of Fibronectin in Culture

Abstract

Extracellular matrices (ECMs) play an important role as components of basement membrane of normal human skin and in migrating epidermal cells in wound healing. We investigated the effects of various ECMs on human keratinocyte adhesion and growth as well as on its secretion and deposition of fibronectin (FN) in vitro using a serum-free, low-calcium culture system. Since cell adhesion is the first step of cell growth, we performed cell adhesion assay for 14 h. Human keratinocytes adhered best on FN and less well on types I/III collagen, type IV collagen, and heparan sulfate proteoglycan (HSPG) as compared with bovine serum albumin (BSA) (control) or laminin (La). Cell growth assayed for 7-8 days on the dishes coated with various extracellular matrices revealed significantly increased keratinocyte growth on FN and on types I/III collagen in comparison with that on type IV collagen, HSPG, BSA (control), or La. Morphology of keratinocytes and of their colonies on FN and types I/III collagen was strikingly different from that of the control; the colonies were not so compact as in the control, but rather loose and larger; each keratinocyte was spread out more on these substrata. These morphologic features seemed to correlate with the increased keratinocyte growth on these extracellular matrices. Both immunofluorescence study for FN with keratinocytes in 8-day culture on various extracellular matrices and enzyme-linked immunosorbent assay for FN measurement on substratum or in conditioned medium with keratinocytes in 5-day culture demonstrated that extracellular matrices modulated the secretion and deposition of FN by human keratinocytes in culture; the keratinocyte growth correlated with the amount of FN detected on substratum but not with that in medium. Based on the results of the present investigation, we think that the growth of human keratinocytes depends on the amount of FN on substratum.