Chondroitin-6-sulphate incorporated into collagen gels for the growth of human keratinocytes: the effect of cross-linking agents and diamines

This study demonstrates the effect of the glycosaminoglycans, hyaluronic acid and chondroitin-6-sulphate (Ch6SO4), diamines and a carbodiimide cross-linking agent on the growth of human epidermal cells on collagen gels. Ch6SO4 incorporated into collagen gels stimulated cell growth rate, but the effect was found to be inconsistent. We found that approximately 50% of the incorporated Ch6SO4 in the gels leached out into the growth medium after the first 3 d in culture, and this is thought to lead to the inconsistent cell growth response. In order to minimize the elution of Ch6SO4 from the gels and thereby maximize its effect on the growth of the keratinocytes, 1–100 μg ml−1 Ch6SO4 was added in the medium. The results showed that Ch6SO4 at these concentrations in the medium did not stimulate the cell growth on either plain collagen gels or gels containing 20% Ch6SO4. As an alternative strategy, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and diamines (putrescine or diaminohexane) were used to immobilize Ch6SO4 onto the collagen gels and to cross-link the gels. The cross-linking process partially prevented the elution of Ch6SO4 from the gels. Interestingly, only putrescine, not diaminohexane, promoted the growth of keratinocytes on the cross-linked plain collagen gels. We proposed to develop an artificial skin substitute containing putrescine as a growth factor for the human epidermal cells.