Influence of different crosslinking treatments on the physical properties of collagen membranes

The physical properties of collagen-based biomaterials are profoundly influenced by the method and extent of crosslinking. In this study, the influence of various crosslinking treatments on the physical properties of reconstituted collagen membranes was assessed. Five crosslinking agents viz., GTA, DMS, DTBP, a combination of DMS and GTA and acyl azide method were used to stabilize collagen matrices. Crosslinking density, swelling ratio, thermo-mechanical properties, stress–strain characteristics and resistance to collagenase digestion were determined to evaluate the physical properties of crosslinked matrices. GTA treatment induced the maximum number of crosslinks (13) while DMS treatment induced the minimum (7). Of the two diimidoesters (DMS and DTBP), DTBP was a more effective crosslinking agent due to the presence of disulphide bonds in the DTBP crosslinks. Ts for DTBP and DMS crosslinked collagen were 80°C and 70°C, and their HIT values were 5.4 and 2.85 MN/m2, respectively. Low concentration of GTA (0.01%) increased the crosslinking density of an already crosslinked matrix (DMS treated matrix) from 7 to 12. Lowest fracture energy was observed for the acyl azide treated matrix (0.61 MJ/m3) while the highest was observed for the GTA treated matrix (1.97 MJ/m3). The tensile strength of GTA treated matrix was maximum (12.4 MPa) and that of acyl azide treated matrix was minimum (7.2 MPa). GTA, DTBP and acyl azide treated matrices were equally resistant to collagenase degradation with ≈6% solubilization after 5 h while the DMS treated was least stable with 52.4% solubilization after the same time period. The spatial orientation of amino acid side chain residues on collagen plays an important role in determining the crosslinking density and consequent physical properties of the collagen matrix.