Construction of poly(octamethylene citrate)-based regenerative microenvironment to facilitate tenogenic and osteogenic differentiation of bone lineage cell

Abstract

In the past decades, the investigation of musculoskeletal tissue has witnessed a revolution in tissue engineering, cell biology, and molecular biology. Among them, tissue engineering demonstrated a promising future as a discipline combining material science and cell biology. Since its invention in 2014, a click chemistry modified poly(octamethylene citrate) (POC) has exhibited strong potential for use in orthopaedics tissue engineering. However, the applications of this novel biomaterial still need to be further investigated. Hence, this study aims to investigate the feasibilities of applying a click chemistry modified POC to construct an artificial tendon tissue microenvironment (TME) and fabricate an osteo-inductive biomaterial for the purpose of extending the applications of this novel biodegradable biomaterial. In the first study, the optimal cross-linking condition of POC was investigated by attempting nine groups of experimental parameters. A series of features were characterized, including thermophysical properties, hydrophilicity, acid release, dry/wet mechanical properties, and degradability. Next, the optimized cross-linking parameter of the click chemistry modified POC was adopted to construct a POC artificial tendon TME with Type I collagen coating. The tendon TME thus established was further examined in vitro to observe its tenogenic effects. The morphological behavior, cell attachment, cell proliferation, and tenogenic differentiation of bone marrow stem cells (BMSCs) on this POC artificial tendon TME were evaluated. Results suggested that POC artificial tendon TME efficiently induced the tenogenic differentiation of BMSCs and demonstrated excellent mechanical stability and suitable degradation rate. To promote the hydrophilicity and increase the stability of the biochemical component, oxygen plasma treatment and C2 peptide conjugation were adopted to modify the surface of POC artificial tendon TME. Characterizations illustrated that oxygen plasma treatment significantly enhanced the material hydrophilicity and promoted cell adhesion without affecting the mechanical property. After the chemical conjugation of the C2 peptide, the resulting POC-C2 artificial tendon TME was used in the in vitro study. Results suggested that C2 peptide is feasible to substitute Type I collagen and effectively induce tenogenic differentiation of BMSC. Finally, POC was applied to investigate the feasibility of fabricating an osteo-inductive biomaterial through the integration of oyster shell protein and POC. First, shell proteins from two species of oysters (Crassostrea Hong Kong genesis and Angulata) were extracted and purified. Then, these proteins were cultured with MC3T3-E1 (pre-osteoblast) to compare their osteogenic effect. In the next experiment, Hong Kong oyster shell proteins and Angulata shell proteins were covalently conjugated to the POC. The osteogenic effect of these osteo-inductive biomaterials was compared afterwards, and results suggested that shell proteins from both species of oysters demonstrated osteogenic effect, confirming that it was feasible to fabricate an osteo-inductive biomaterial through the integration of oyster shell proteins and POC. However, Hong Kong oyster shell protein exhibited superior osteogenic effect to Angulata shell protein. In terms of osteogenic effect, POC conjugated with Hong Kong oyster shell proteins demonstrated more potential for future clinical application.