Property peculiarities of the atelocollagen–hyaluronan conjugates crosslinked with a short chain di-oxirane compound

• The 1.3 nm length 1,4-butanediol-diglycidyl ether (BDDGE) was used as a crosslinker.
• Crosslinking atelocollagen and hyaluronan with BDDGE revealed unexpected effects.
• Increasing BDDGE amounts induces two thresholds in rheological behavior.
• In excess crosslinker induces a quasi-denaturation side-effect over atelocollagen.
• Optimal ratios: 1:5 aK:NaHyal w/w DM, and 1:2.5 to 1:5 M α-NH2:BDDGE.

Minimal amounts of a short-chain bifunctional crosslinker of about 1.3 nm length, the 1,4-butanediol-diglycidyl ether (BDDGE), were used to generate atelocollagen–hyaluronan conjugates in hydrogel state. Two a priori constraints were considered in recipe/procedure developing: (i) working in nondenaturing conditions, and (ii) ensuring a low cytotoxicity of the final product. Both atelocollagen (aK) and hyaluronan (NaHyal) were accurately purified to reduce their molecular-weight dispersity, in order to ensure the reproducibility of hydrogels characteristics. 1:5 aK:NaHyal weight ratios and 1:2.5 to 1:5 α-NH2:BDDGE molar ratios were found to be the most favorable recipe prescriptions that allow the obtaining of rheo-mechanically stable hydrogels, able to be manipulated during cell culturing protocols. Experiments revealed two unexpected effects due to the crosslinking reactions mediated by a short-chain molecule: (i) the occurrence of two thresholds in the rheological behavior of the hydrogels, related with the amount of added crosslinker, and (ii) a quasi-denaturation side-effect induced over the protein component by large or in excess amounts of crosslinker.

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