The effect of chemical modification on the enzymatic degradation of acellular matrix (ACM) processed biomaterials

In this study, the effects of specific chemical modifications of amino add sidechains on the in vitro degradation of 'native' collagen (obtained from acellular matrix (ACM) processed arteries) and 'purified' type I collagen (extracted from bovine Achilles tendon) was studied. Two monofunctional epoxides of different size and chemistry were used to modify lysine with or without methylglyoxal modification of arginine. Carboxyl groups of aspartic and glutamic acids were modified with glycine methyl ester. The reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used as a basis for comparison of the effects of crosslinking with chemical modification. EDC treatment was studied under two different pH conditions: (i) pH controlled at an optimal value of 5.5 and (ii) a simpler-but industrially significant--uncontrolled pH system. Biochemical, thermomechanical, tensile mechanical, shear stiffness and multi-enzyme (collagenase, cathepsin B, (acetyl)trypsin) in vitro enzyme analyses were used to determine the effects of each modification. Carboxyl capping had no effect on the structure of native ACM collagen as determined by thermo- or tensile mechanical testing. Increased collagen solubilization by enzymes, with the exception of cathepsin B, was observed after carboxyl capping. In contrast, lysine modification destabilized native ACM collagen with the larger, hydrophobic epoxide having the greater effect. In general, enzymatic solubilization of collagen was either unaltered or decreased after modification with the smaller, hydrophilic epoxide, whereas the larger, hydrophobic epoxide increased solubilization. Analysis of collagen fragments solubilized by trypsin and acetyltrypsin revealed that sites of cleavage were altered after lysine and arginine modification. Differences were also observed in the solubilization of (i) purified type I collagen and (ii) native ACM collagen by collagenase. In comparison, both EDC treatments were equally effective in stabilizing native ACM collagen against solubilization by enzymes in vitro. EDC crosslinking of ACM arteries significantly increased thermal denaturation temperatures, treatment with pH control having the greatest effect. Crosslinking without pH control, however, consumed more lysine residues and increased shear stiffness to a greater degree (21x compared to 14x with pH control). The observed differences are attributed to differences in the location or type of EDC crosslinks formed which differentially affected mechanical behaviour without affecting the increase in resistance to enzymatic degradation. In summary, chemical modification without crosslinking can prevent degradation. This effect however, is not as broad-based as that produced by EDC crosslinking. The effects of chemical modification depended on the modifying reagent, the amino acid(s) modified, and architecture of the substrate. The ability to modulate the enzyme degradation of tissue-derived materials as demonstrated in this study may, however, facilitate the design of novel engineering scaffolds for tissue regeneration or collagen based drug delivery systems.