Tuneable microfibrillar collagen structures within dense chitosan hydrogels

Chitosan-type I collagen hydrogels are paradigms of polysaccharide-protein assemblies with applications as biomaterials. However, preparing physical hydrogels combining them at comparable, high concentrations (>20 mg mL^-1) within interpenetrated networks remains challenging. Here, we could combine chitosan and collagen solutions at 25 mg mL^-1 to prepare two different types of concentrated hydrogels. When neutralized under ammonia vapours, mixed solutions form composite hydrogels, where collagen fibers exhibiting an unusual, branched morphology occupy a chitosan network porosity. In contrast, neutralization by immersion in liquid ammonia yielded hybrid networks where collagen microfibrils were associated with chitosan nanoaggregates. Structural variations impacted the mechanical behaviour and biological properties, assessed by 2D cultures of fibroblasts, of these hydrogels. Differences in gelation kinetics between the two biomacromolecules in the two processes appeared as a key factor driving the mixed network structuration. This work discloses a new route to obtain dense hydrogels from binary biopolymer systems and offers additional insights into the underlying gelation process.