Preparation of [3H] Collagen for Studies of the Biologic Fate of Xenogenic Collagen Implants In Vivo

Reduction of a commercially available, pepsin-solubilized, bovine dermal collagen (Vitrogen 100) with sodium [3H] borohydride provided radiolabeled collagen preparations with specific activities ranging from 7.1-12.0 μCi/mg collagen. These specific activities were 2-3 times greater than those obtained by reduction of intact rat tail tendon collagen under similar conditions. The α, β and higher aggregate components of type I collagen were radiolabeled as well as the α component of a small amount of type III collagen present in the samples. Fractionation of cyanogen bromide peptides showed that α(I)CB7, α(I)CB8, and α2(I)CB3, 5 were the predominant peptides labeled by this procedure. Amino acid analysis indicated that the majority of the radioactivity was in reducible cross-links, precursors of these cross-links, and in hexosyllysine residues.Reconstitution experiment S comparing this radiolabeled collagen with nonlabeled collagen with nonlabeled collagen showed them to be indistinguishable. Bacterial collagenase digestion of this reconstituted fibrillar collagen in both in a lightly cross-linked (glutaradehyde 0.0075%) and noncross-liked form provided evidence that digestion of labeled and nonlabeled collagens proceeded at similar rates. Thus, labeling did not change the properties of the collagen. Cross-linking made the preparation refractory to proteolytic degradation.Injection of fibrillar collagen preparation, spiked with radiolabled collagen, into the guinea pig dermis followed by quantitation of the amount of radioactivity recovered from implant sites as a function of time, indicated that the lightly cross-lined samples also were more resistant to degradation sites as a function of time, indicated that the lightly cross-linked samples also were more resistant to degradation in vivo than the noncross-linked preparation. The half-life of noncross-linked collagen was about 25 days. These degradation rates were much faster than observed for similar, nonlabeled samples injected into the dermis of humans, presumably due to a higher metabolic activity in the guinea pig dermis