In Vivo Immune Responses of Cross-Linked Electrospun Tilapia Collagen Membrane

Abstract

Collagen has been used extensively in tissue engineering applications. However, the source of collagen has been primarily bovine and porcine. In view of the potential risk of zoonotic diseases and religious constraints associated with bovine and porcine collagen, fish collagen was examined as an alternative. The aim of this study is to use tilapia fish collagen to develop a cross-linked electrospun membrane to be used as a barrier membrane in guided bone regeneration. As there is limited data available on the cytotoxicity and immunogenicity of cross-linked tilapia collagen, in vitro and in vivo tests were performed to evaluate this in comparison to the commercially available Bio-Gide® membrane. In this study, collagen was extracted and purified from tilapia skin and electrospun into a nanofibrous membrane. The resultant membrane was cross-linked to obtain a cross-linked electrospun tilapia collagen (CETC) membrane, which was characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), degradation studies, cytotoxicity studies, and cell proliferation studies. The membranes were also implanted subcutaneously in rats and the host immune responses were examined. The DSC data showed that cross-linking increased the denaturation temperature of tilapia collagen to 58.3°C ± 1.4°C. The in vitro tests showed that CETC exhibited no cytotoxicity toward murine fibroblast L929 cells, and culture of murine preosteoblast MC3T3-E1 cells demonstrated better proliferation on CETC as compared to Bio-Gide. When implanted in rats, CETC caused a higher production of interleukin IL-6 at early time points as compared to Bio-Gide, but there was no long-term inflammatory responses after the acute inflammation phase. This finding was supported with histology data, which clearly illustrated that CETC has a decreased inflammatory response comparable to the benchmark control group. In all, this study demonstrated the viability for the use of CETC as a tissue engineering scaffold and provides an insight on the in vivo immune responses toward xenogenic collagen scaffolds.