Crosslinking is a key step in the production of stable all-natural polymeric scaffolds for tissue engineering, as it slows the degradation and increases the mechanical properties of the material. In this study, we investigate the crosslinking parameters of a natural, anisotropic scaffold produced from gelatine, chitosan and cellulose through a modified freeze drying protocol, with the goal of maintaining the porous architecture of the scaffold while improving its degradation and mechanical strength. Genipin and EDC were selected as the two crosslinking alternatives, while crosslinker concentration and solvent system (ethanol-to-water ratio) were the optimized parameters. The degree of crosslinking was quantified through a 2,4,6-Trinitrobenzene Sulfonic Acid assay, and the scaffolds were further tested for hydrolytic and enzymatic degradation, swelling and mechanical properties. Scaffolds achieve ultimate tensile strength values of up to 4 MPa, in the relevant physiological range for tendon applications, and crosslinking degrees in the range of 70% - 90%. While scaffolds processed with both crosslinkers maintain the desired pore alignment, genipin was the most successful at delaying the degradation of the material, with 85% of the initial mass of the scaffold remaining after 21 days of immersion in PBS. The solvent system of the crosslinking solution was investigated, with varying ratios of ethanol to water, finding that adding water is necessary for optimal swelling, homogeneous crosslinking and low cytotoxicity of the scaffolds, highlighting the importance of this parameter in the genipin crosslinking process. Genipin crosslinked scaffolds were found to be capable of sustaining the attachment and proliferation of tendon derived stem cells up to 21 days in both 21% and 2% oxygen environments, yielding a strong stable scaffold suitable for supporting tendon regeneration in-vitro.
Diaz et al. (Fri,) studied this question.