Doc Lab Report

3.1. Synthesis and characteristics of DAC Periodate oxidation specifically cleaves the vicinal glycols in polysaccharides to form their dialdehyde derivatives. Periodic oxidation results in complete range of aldehyde derivatives of DAC (oxidation levels between 0 and 100%) depending on the quantity of periodate employed. Each α-glycol group consumes one molecule of periodate and under given conditions, the rate of the reaction is dependent principally on the stereochemistry of the α-glycol group. DAC is precipitated out in heterogeneous medium of 3:1 t-butyl alcohol: water as a dispersion eventhough, the oxidation is carried out in aqueous medium. Table 1 lists the characteristics of DAC at different amounts of periodate equivalence. DAC

The crosslinking efficiency after treating with collagen solution at various oxidation levels of DAC is given in Table 2. It is observed that DAC results in crosslinking and an increase in oxidation level enhances the crosslinking efficiency by the formation of stable crosslinks with amino groups of collagen. DAC at 33% oxidation results in crosslinking efficiency of 25% as compared to 91% at 99% oxidation of DAC. The increase in crosslinking efficiency of DAC in stabilization of collagen is due to decrease in the amino groups of lysine of collagen that has covalently crosslinked with increasing aldehyde groups of DAC as compared to native collagen. Hence, in DAC-99 treated collagen it was observed that crosslinking efficiency increased considerably, which could have induced both inter-molecular and intra-molecular chain crosslinking (Figure 1). Higher oxidation level of DAC and pH, facilitate higher crosslinking due to the presence of a large number of aldehyde groups that crosslink with amino groups in collagen. The high crosslinking ability of DAC with collagen can be attributed to strong binding between the two, as DAC can have both covalent and non-covalent interactions with collagen. Hence, aldehydic functionality in DAC covalently crosslinks with amino groups of collagen and the hydroxyl groups can involve in hydrogen bonding interaction

The stability of the DAC treated collagen fibre against enzymatic degradation was studied by analyzing the rate of hydrolysis of collagen on treatment with bacterial collagenase. Bacterial collagenase preferentially cleaves X-Gly (X is most frequently a neutral amino acid) bond of the -Gly-Pro-X-Gly-Pro-X- sequence in the non polar regions of the collagen molecule. Bacterial collagenases from Clostridium histolyticum cleave collagen at multiple sites (French et al., 1992). Degradation of collagen (based on hydroxyproline released) for native GTAD and DAC (different oxidation levels) crosslinked collagen matrix by collagenase at 16 h was determined. Significant reduction in the degradation of collagen was observed for the fibres treated with DAC-99 compared to native RTT collagen matrix. DAC-99 treated collagen matrix exhibited 6% degradation of collagen as against 99% degradation in the case of native collagen at 16 h period of incubation. As seen from Table 2, the enzyme stability of the collagen matrix increases with increase in oxidation of DAC at pH 8. DAC interacts with collagen through covalent and hydrogen bonding. The stability of DAC treated collagen fibres against collagenase were brought about by protecting and masking the active sites in collagen by crosslinking (through interaction with DAC) recognized by collagenase (Golomb et