Collagen was purified from bovine Achilles tendon and crosslinked with dimethyl suberimidate (DMS) and glutaraldehyde (GTA). Under optimal conditions, the shrinkage temperature (Ts) was raised to 74 degrees C for collagen crosslinked with DMS and to 80 degrees C for those crosslinked with GTA. Crosslinking density measurements were done on the hydrothermally denatured collagen by the method based on the Flory-Rehner equation. GTA treatment was found to introduce more number of crosslinks than DMS. The maximum tension attained during heating (after shrinkage has occurred) was greater for GTA-treated collagen than for DMS and control. The control collagen membranes broke during heating (at 73 degrees C), while for the crosslinked membranes the tension kept on increasing up to 100 degrees C. The crosslinking density correlated well with the data determined from the in vitro and in vivo degradation studies. Uncrosslinked and DMS crosslinked collagen membranes were more susceptible to degradation by enzymes in vitro, while GTA-treated collagen was highly resistant to degradation. The biocompatibility of the collagen membranes was studied by subcutaneous implantation in rats. Uncrosslinked collagen membranes degraded within 14 days with the formation of granulation tissue. DMS crosslinked membranes degraded within 21 days and the area was replaced by numerous fibroblasts and newly formed collagen. No calcification was observed. For GTA-treated membranes, necrosis was observed after 7 days implantation and by 14 days the membrane had started to calcify.
Crosslinking agents are used for improving the physical properties and durability of collagenous implants, glutaraldehyde (GTA) being the most widely used. Many of these reagents, including GTA, are known to be cytotoxic and to induce calcification. Hence, it is desirable to develop new crosslinking methods for collagen, so that biocompatibility and physical properties are improved. In the present study, dimethyl 3,3' -dithiobispropionimidate (DTBP) has been tried as a novel crosslinking reagent for collagen. Collagen purified from rat tail tendon has been crosslinked with DTBP and GTA. An increase of 22 degrees C in shrinkage temperature is observed for collagen treated with DTBP under optimal conditions. Crosslinking density determination shows that DTBP induces 10 crosslinks per mole, compared to 13 by GTA. While the tensile strength of GTA-treated samples is greater than those treated with DTBP, the latter shows more extensibility. In vitro degradation studies show that both GTA- and DTBP-treated samples are resistant to degradation by collagenase. The biocompatibility of crosslinked collagen samples studied by subcutaneous implantation in rats show that while both GTA- and DTBP-treated collagen do not degrade for up to 4 weeks, ultrastructural and histological studies indicate that DTBP collagen is far more biocompatible than GTA-treated matrices.
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