Effects of collagen, chitosan and mixture on fibroblast responses and angiogenic activities in 2D and 3D in vitro models

Despite accumulating evidences have demonstrated the potential of collagen and chitosan on tissue repair, it remains unclear on their combination effects. Here, we examined the regenerative effects of single collagen, chitosan and their mixture on fibroblasts and endothelial cells at cellular levels. The results showed that fibroblast responses, as indicated by high proliferative rate, increased spheroid diameter and migrated area existing from spheroid edge, and decreased wound area, were significantly promoted by either collagen or chitosan stimulation. Similarly, both collagen and chitosan resulted in increased endothelial cell proliferation and migration with accelerated tube- like network formation and upregulated VEcadherin expression, although collagen strongly provided this effect. While the 1:1 mixture (100:100 mu g/ mL of chitosan to collagen) treatment caused a reduction in fibroblast viability, the lower ratio of chitosan (1:10 mixture; 10:100 mu g/mL) did not produce any impact on both fibroblast and endothelial cell viabilities. The 1:10 mixture also significantly enhanced the additional effects on fibroblast responses and angiogenic activities as shown by higher endothelial growth, proliferation and migration with accelerated capillary-like network formation than those treated with the single substance. Further investigation of signaling proteins found that collagen significantly increased expressions of p-Fak, p-Akt and Cdk5 whereas chitosan upregulated p-Fak and Cdk5 expressions. Comparing to the single treatments, p-Fak, p-Akt and Cdk5 were higher expressed in the 1:10 mixture. These observations indicate that proper collagen-chitosan mixture provides the combination effects on fibroblast responses and angiogenic activities when a high concentration of collagen is used, possibly through Fak/Akt and Cdk5 signaling pathways. Therefore, this study helps to define the clinical use of collagen and chitosan as promising biomaterials for tissue repair.