Unraveling the Critical Role of Oxidized Carboxymethyl Cellulose’s Molecular Weight in the Multiscale Cross-Linking of Collagen Matrix

Although periodate-oxidized polysaccharides are effective cross-linkers for collagen matrix (CM), the multiscale structural changes of CM induced during cross-linking remain unclear. The mass transfer and cross-linking behaviors of oxidized carboxymethyl cellulose (OCMC) with different molecular weights within CM were investigated. Medium Mw OCMC-5 (9.53 × 10³ g/mol) showed optimal penetration and cross-linking efficiency, superior to higher or lower Mw OCMCs. OCMC-5 cross-linked CM exhibited enhanced stability, as evidenced by a smaller fibril D-period (65.62 nm), larger fibril diameter (142.13 nm), and higher porosity (70.72%). Real-time in situ SAXS analysis elucidated the microscale cross-linking mechanism. This process involved an intermolecular cross-linking and formation of carbinolamine intermediates between OCMC and collagen during basification, resulting in fibril compression. Subsequent heating promoted the intermediates dehydration into stable Schiff base cross-links, ultimately improving CM stability and porosity. These findings provide a theoretical basis for understanding cross-linking mechanisms and optimizing collagen-based biomaterials.