Design of an artificial skin. II. Control of chemical composition

Detailed methodology is described for the reproducible preparation of collagen--glycosaminoglycan (GAG) membranes with known chemical composition. These membranes have been used to cover satisfactorily large experimental full-thickness skin wounds in guinea pigs over the past few years. Such membranes have effectively protected these wounds from infection and fluid loss for over 25 days without rejection and without requiring change or other invasive manipulation. When appropriately designed for the purpose, the membranes have also strongly retarded wound contraction and have become replaced by newly synthesized, stable connective tissue. In our work, purified, fully native collagen from two mammalian sources is precipitated from acid dispersion by addition of chondroitin 6-sulfate. The relative amount of GAG in the coprecipitate varies with the amount of GAG added and with the pH. Since coprecipitated GAG is generally eluted from collagen fibers by physiological fluids, control of the chemical composition of membranes is arrived at by crosslinking the collagen--GAG ionic complex with glutaraldehyde, or, alternately, by use of high-temperature vacuum dehydration. Appropriate use of the crosslinking treatment allows separate study of changes in membrane composition due to elution of GAG by extracellular fluid in animal studies from changes in composition due to enzymatic degradation of the grafted or implanted membrane in these studies. Exhaustive in vitro elution studies extending up to 20 days showed that these crosslinking treatments insolubilize in an apparently permanent manner a fraction of the ionically complexed GAG, although it could not be directly confirmed that glutaraldehyde treatment covalently crosslinks GAG to collagen. By contrast, the available evidence suggests strongly that high-temperature vacuum dehydration leads to formation of chemical bonds between collagen and GAG. Procedures are described for control of insolubilized and "free" GAG in these membranes as well as for control of the molecular weight between crosslinks (Mc). The insolubilized GAG can be controlled in the range 0.5--10 wt. % while "free" GAG can be independently controlled up to at least 25 wt. %; Mc can be controlled in the range 2500--40,000. Studies by infrared spectroscopy have shown that treatment of collagen--GAG membranes by glutaraldehyde or under high-temperature vacuum does not alter the configuration of the collagen triple helix in the membranes. Neither do these treatments modify the native banding pattern of collagen as viewed by electron microscopy. Collagen--GAG membranes appear to be useful as chemically well-characterized, solid macromolecular probes of biomaterial--tissue interactions.