Development of bioactive humanized collagen I-PCL composites with enhanced mechanical properties for tissue engineering applications

The hierarchical assembly of collagen critically affected its biological activity, with type I collagen (COL1) being especially important for tissue organization and cell adhesion. This study aimed to identify COL1 fragments that could be biosynthesized by Pichia pastoris while retaining functional bioactivity. We fragmented COL1 and selected variants based on higher thermal stability (T_m), net charge, and predicted bioactivity. Three of the ten variants were successfully secreted by P. pastoris and formed triple-helix at 4 °C. For practical applications, we focused on two variants that maintained their triple-helical conformation above 15 °C, and further confirmed their higher-order structures using microscopy and rheological analysis. The variant COL109 demonstrated enhanced promotion of osteoblast differentiation but exhibited weak mechanical strength when preparing hydrogels. To address this, we enhanced its structural integrity by fusing it with an adhesion tag of (GPP)₁₀ motif. The resulting construct of COL109(GPP)₁₀ showed improved mechanical strength. Furthermore, we combined COL109(GPP)₁₀ with polycaprolactone (PCL), showing that higher PCL concentrations increased mechanical strength but reduced bioactivity and water retention, while a 1:1 ratio of COL109(GPP)₁₀ to PCL achieved an optimal balance. By promoting wound healing and cell differentiation, the composite demonstrates strong potential for use in tissue engineering.