Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: Influence of crosslinking with diphenylphosphorylazide on matrix reorganization, growth, and biosynthetic and proteolytic activities

With the rapid development of tissue engineering and gene therapy, collagen -based biomaterials frequently are used as cell transplant devices. In this study we determined the behavior of mouse fibroblasts cultured for up to 6 weeks in control sponges treated by severe dehydration and used commercial ly as hemostatic agents and in two sponges (DPPA 2 and 3) crosslinked by di phenylphosphorylazide, a method developed in our laboratory. Growth capacit y, biosynthetic and proteolytic activities, and matrix reorganization were followed over time in cultures and compared with similar data for fibroblas ts in monolayer culture on plastic and in floating or attached collagen gel s. Control sponges with and without seeded mouse fibroblasts showed rapid p artial denaturation or contraction, weight loss, and severe calcification ( 13-18% Ca) after 6 weeks. In contrast, the crosslinked sponges showed only slightly decreased size and weight, and the calcification was inhibited (0. 2% Ca) in the presence of cells. Mouse fibroblasts seeded on the crosslinke d sponge surface at 50,000-200,000 cells/cm(2) progressively penetrated the matrix and proliferated to give the same constant cell density after 3 wee ks (around 600,000 cells/sponge). A specific, two- to threefold decrease in collagen synthesis was observed between 1 and 3 or 6 weeks, due mainly to a decrease in the fraction secreted into the medium (25-30% instead of 45-5 0%). No collagenase 3 activity was detected in the culture medium under any condition or time whereas 25% gelatinase A was found by gelatin zymography to be in an active form in cultures within sponges as compared with less t han 10% in monolayers and more than 50% in floating collagen gel. A small a mount of gelatinase B was observed after 1 week in sponge cultures and was completely absent thereafter. These results show that the biosynthetic and proteolytic behavior of mouse fibroblasts cultured in crosslinked collagen scaffolds is different from that in monolayers or in floating collagen gels and more similar to that previously described in attached collagen gels. ( C) 2000 John Wiley & Sons, Inc.