Synthesis Of RGD-Grafted Composite Hydrogel And Cell Differentiation Study Under Low-Intensity Pulsed Ultrasound Stimulation

Bone tissue engineering provides a potential approach for the production of three-dimensional implants. However, there are still some challenges associated with three-dimensional bone regeneration including creating the regenerated tissue that has similar structural and mechanical properties to the natural bone and achieving successful integration of scaffolds with the host bone. Among them, the level of formation of new bone and vascularization is considered as a critical factor for the clinical application of bone tissue engineering. Hydrogels have good cell compatibility and similar structures to the extracellular matrix (ECM) so that they can be used as tissue engineering scaffolds. However, hydrophilic surfaces of hydrogels are not beneficial for the adsorption of protein and not conducive to the adhesion and growth of cells and in turn to the formation of new bone and vascularization. An approach to overcome it is to use RGD to modify hydrophilic surfaces. Schiff base reaction between amine groups and aldehyde groups offers one possibility for the modification of RGD of hydrophilic surfaces of hydrogels whereas polysaccharides can provide aldehyde groups for Schiff base reaction by oxidization of linear polysaccharides and their source is very rich. In this study, an approach using a RGD-grafted oxidized sodium alginate/N-succinyl chitosan (RGD-OSA/NSC) hydrogel as a scaffold and low-intensity pulsed ultrasound (LIPUS) as mechanical stimulation was proposed to achieve a high level of formation of new bone and vascularization. To evaluate the ability of blood vessels and osteoblasts performance under the LIPUS effect of composite hydrogel material, the effect of hydrogels on endothelial and osteogenic differentiation of hMSCs in vitro was investigated. The results showed that RGD-OSA/NSC composite hydrogels presented good biological properties in attachment, proliferation and differentiation of cells. The MTT cell viability assay showed that the total number of cells increased more significantly in the LIPUS-stimulated groups with RGD than that in the control ones; the similar results were obtained for ALP activity/staining and mineralized nodule formation assay of osteogenic induction and immunohistochemical test of endothelial induction. Compare with LIPUS-RGD-OSA/NCS experimental group and the Control-RGD-OSA/NCS experimental group and LIPUS-OSA/NCS experimental group. The positive synergistic effect of LIPUS and RGD on the enhancement of proliferation and differentiation of hMSCs was observed. These findings suggest the hybrid use of RGD modification and LIPUS might provide one approach to achieve a high level of formation of new bone and vascularization in bone tissue engineering scaffold implants.