Evaluation of the immunomodulatory potential of chitosan-grafted-poly(e-caprolactone) copolymers and their use for bone tissue regeneration

摘要

Introduction: The use of biodegradable biomaterials as scaffolds in bone tissue engineering is a useful tool to conduct tissue development. At the same time biomaterials have an impact on the host immune response. The induced immune response is essential since it can facilitate the healing process. It is therefore important to predict or promote the proper immune response after implantation. The aim of the present study was to prepare chitosan-grafted-poly(ε-caprolactone) copolymers (CS-g-PCL) with PCL contents of 20 wt% and 50 wt% and evaluate (ⅰ) their immunomodulatory potential by analyzing the differentiation of primary bone marrow derived macrophages (BMDM) cultured on copolymer films and (ⅱ) the pre-osteoblastic cell response on films. Methods: The CS-g-PCL copolymers were prepared by chemical grafting of PCL-COOH onto the chitosan (CS) backbone [2] and characterized by FTIR and 1H NMR spectroscopy. We employed primary bone marrow derived macrophages (BMDM) for the immunological study, and MC3T3-E1 pre-osteoblastic cells for the investigation of the osteogenic response. We performed resazurin-based assays for the quantification of living cells and their proliferation, scanning electron microscopy and laser fluorescence confocal microscopy for the visualization of cell morphology after immunocytochemical staining, flow cytometry analysis for immunophenotypic characterization, semi-quantitative RT-PCR analysis for gene expression. Results and Discussion: Films of the CS-g-PCL copolymers were used as substrates for the culture of BMDM. The results showed that cells adhere well, survive and proliferate on the CS-g-PCL copolymer surfaces. Moreover, the CS-g-PCL(50) surface seems to favor the differentiation of MO into M2 without affecting the polarization into an M1 phenotype, while the CS-g-PCL(20) surface weakens the polarization into M1 without affecting the polarization into an M2 phenotype. Regarding the osteogenic behavior we observe a flattened cell morphology of MC3T3-E1 cells on CS-g-PCL similar to the tissue culture treated polystyrene control. In addition, CS-g-PCL copolymers allow for cell differentiation as observed through characteristic markers for early and late stages of bone morphogenesis, such as collagen type Ⅰproduction, alkaline phosphatase activity, and calcium biomineralization. Conclusion: We have successfully synthesized novel CS-g-PCL copolymers and prepared thin films on glass substrates. In vitro experiments of BMDM onto CS-g-PCL films have shown a strong cell attachment and good cell proliferation after 7 days in cell culture. The CS-g-PCL copolymer with the higher PCL content exhibited anti-inflammatory action on macrophages which was attributed to their higher CS content. We demonstrate an enhanced osteogenic response of pre-osteoblastic cells on CS-g-PCL copolymers and their potential as scaffolding material in bone tissue engineering.