Investigation into cell growth on collagen/chondroitin-6-sulphate gels: the effect of crosslinking agents and diamines

Abstract

Artificial skin substitutes based on cultured autologous keratinocytes need to have sufficient strength and ease of handling to be utilized successfully by surgeons in the clinic. This may be achieved by crosslinking the collagen substratum on which the cells are cultured, which in this case is a collagen gel. Increased strength must be attained without detrimental effect on cell growth. The influence of potential crosslinking agents including the glycosaminoglycan, chondroitin-6-sulphate (Ch6SO4), the water soluble carbodiimide crosslinking agents 1-ethyl-3-(3-diaminopropyl) carbodiimide (EDAC), and 1,1-carbonyldiimidazole (CDI), and the polyamines putrescine, spermine and diaminohexane, on cell growth rate has been investigated. Incorporation of 20% Ch6SO4 into collagen gels caused an approximately 16% increase in keratinocyte growth, but had no significant effect on that of dermal fibroblasts. Pre-formed collagen gels (+/- Ch6SO4) were treated with the carbodiimides. This crosslinking treatment markedly inhibited fibroblast growth (EDAC 45% inhibition, CDI 70%), without affecting that of keratinocytes. Pre-formed collagen gels (+/-Ch6SO4 and carbodiimide) were treated with 0.1 M, 0.5 M or 1.0 M polyamine. Spermine inhibited the growth rate of both cell types at all concentrations tested, whereas putrescine and diaminohexane had little effect. The mechanical strength of these crosslinked gels is currently being assessed to determine the optimum composition in terms of cell growth and biocompatibility, and strength.