Many substances are used in the production of biomaterials: metals (titanium), ceramics (alumina), synthetic polymers (polyurethanes, silicones, polyglycolic acid (PGA), polylactic acid (PLA), copolymers of lactic and glycolic acids (PLGA), polyanhydrides, polyorthoesters) and natural polymers (chitosan, glycosaminoglycans, collagen). With the rapid development in tissue engineering, these different biomaterials have been used as three-dimensional scaffolds and cell transplant devices. The principal biochemical and biological characteristics of the collagen-based biomaterials are presented, including their interactions with cells (fibroblasts), distinct from those of synthetic polymers, and their potential use in gene therapy through the formation of neo-organs or organoids.

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 commercially as hemostatic agents and in two sponges (DPPA 2 and 3) crosslinked by diphenylphosphorylazide, a method developed in our laboratory. Growth capacity, biosynthetic and proteolytic activities, and matrix reorganization were followed over time in cultures and compared with similar data for fibroblasts in monolayer culture on plastic and in floating or attached collagen gels. Control sponges with and without seeded mouse fibroblasts showed rapid partial 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 crosslinked 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 weeks (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 45-50%). 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 than 10% in monolayers and more than 50% in floating collagen gel. A small amount 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.

Biodegradable scaffolds, along with cells, are important components of most tissue-engineered constructs. In the study, there is a comparison of the behaviour of human fibroblasts cultured for up to six weeks in four different collagen-based three-dimensional matrices, in the form of sponges composed of pure native type I collagen (control), of collagen-GAG-chitosan (CGC) and of collagen cross-linked by two concentrations of diphenylphosphorylazide (DPPA-2 and DPPA-3). Variations in size and weight of the sponges, as well as fibroblast growth and migration, and total protein and collagen synthesis, are determined with time in culture. Owing to their low thermal stability, the partial denaturation and dissolution of the control sponges after incubation at 37 degrees C lead to considerable contraction and low cell proliferation. CGC sponges, stabilised by ionic interactions between the different components, show, after six weeks, limited contraction (20%) and weight increase (10% when seeded) and high cell growth (threefold increase). Similar results are obtained with weakly, cross-linked (DPPA-2) collagen sponges. Highly cross-linked (DPPA-3) sponges do not contract, whereas weight gain and cell proliferation are no different from those found with CGC and DPPA-2 sponges. Similar levels of total protein and collagen synthesis are shown for fibroblasts seeded in different matrices, with a slight general decrease (twofold) after three weeks, a much lower value than that observed with fibroblasts in culture within a contracted collagen gel (sixfold). Furthermore, the fraction of neo-synthesised collagen deposited in the sponges after six weeks represents more than 60% of the total, compared with only 10% obtained with fibroblasts in monolayer culture or 30% within a collagen gel. These results indicate that the matrices, particularly the CGC and DPPA-2 sponges, provide excellent supports for fibroblast growth and the formation of dermal and skin equivalents.