Study On Fabrication And Properties Of Injectable Bioactive Alginate/calcium Silicate Composite Hydrogels

The injectable materials have recently drawn great attention for tissue engineering and regeneration because of their unique fluid-to-solid transforming property. Because of their structural similarity to the extracellular matrix(ECM) of many tissues, injectable hydrogels hold a great promise for drug delivery, cell encapsulation, tissue engineering and regenerative medicine applications. Our previous studies have shown that calcium silicate(CS) and akermanite(Aker) possesses good bioactivity and can stimulate osteogenic differentiation in vivo. In this dissertation, we prepare in situ-forming injectable alginate/bioceramics composite hydrogels in the presence of D-gluconic acid δ-lactone(GDL) and amino acid(aspartic acid(Asp) or glutamic acid(Glu)). The physicochemical properties of the composite hydrogels, including gelling time, compressive strength, swelling ratios, in vitro biomineralization and so on, are characterized to obtain a better formula for the composite hydrogels. In addition, we explore the cell compatibility, osteogenesis and angiogenesis of the composite hydorgels in vitro and in vivo.Firstly, the SA/CS/GDL composite hydrogels are prepared by crosslinking SA with Ca2+ slowly released from CS itself in the presence of GDL. The gelling time, compressive strength and swelling ratio can be controlled and regulated with different amount of CS and GDL. The FTIR and SEM show that the SA/CS/GDL composite hydrogels have a stimulatory effect on the formation of hydroxyapatite. In addition, the SA/CS/GDL composite hydrogel can not only maintain the viability and proliferation of rat bone mesenchymal stem cells(rtBMSCs), but also stimulate rtBMSCs to produce alkaline phosphatase, the an early marker of osteoblastic differentiation, and promote angiogenesis of human umbilical vein endothelial cells(HUVECs).Secondly, based on the SA/CS/GDL composite hydrogel system, the injectable SA/Aker/GDL composite hydrogels are prepared by dual-ion crosslinking SA with Ca2+ and Mg2+ slowly released from Aker in the presence of GDL. The gel forming ability and injectability of the SA/Aker/GDL composite hydrogel can be well controlled with the combined action of Ca2+ and Mg2+. In vitro results indicate that the SA/Aker/GDL composite hydrogel is not only able to maintain the viability and proliferation of human bone marrow mesenchymal stem cells(hBMSCs) and human umbilical vein endothelial cells(HUVECs) but also shows stimulatory effects on osteogenic differentiation of hBMSCs and vascularization of HUVECs. In addition, in vivo results indicate that the SA/Aker/GDL composite hydrogel possesses osteoinductivity when it is injected subcutaneously in nude mice, which is the first time demonstration of osteoinductivity of silicate based biomaterials. Furthermore, when hBMSCs are encapsulated within the hydrogels, the composite hydrogel further stimulates the osteogenic differentiation and mineralization of hBMSCs and the ingrowth of blood vessels into hydrogels, indicating the stimulatory effects of the material on the interactions of seeded cells with the recruited host cells.Finally, to further improve the injectability and safety of the SA/Aker/GDL composite system, the injectable SA/Aker/Asp and SA/Aker/Glu composite hydrogels are developed by dual-ion crosslinking SA with Ca2+ and Mg2+ slowly released from Aker in the presence of Asp and Glu. Compared to the SA/Aker/GDL composite system, the physico-chemical properties of the SA/Aker/amino acid composite hydrogels, including gelling time, compressive strength and swelling ratio can be well tuned by changing the amount of amino acid. In addition, in vitro results show that the SA/Aker/amino acid hydrogels can not only maintain the viability and proliferation of hBMSCs and HUVECs but also promote osteogenic differentiation of hBMSCs and vascularization of HUVECs.Considering the research results above, three composite hydrogel systems all possess properties of tunable gelling time, good bioactivity, cytocompatibility and the effects of osteogenic and angiogenic differentiation. SA/Aker system with amino acid has a better gel forming ability and injectability compared to the other two systems. Three composite hydrogel systems possess great potential for bone regeneration and tissue engineering applications.