Study On Construction Of Double-crosslinked Bioactive Glass Composite Hydrogel And Sequential Treatment Of Tumor Related Bone Defects

Bone tissue has a special microenvironment,which the tumors of other tissues are highly prone to metastasize to bone tissue to form bone tumors.At present,surgical resection supplemented with chemoradiotherapy is generally used for bone tumors in clinic,while complete removal of tumor cells is difficult with surgical treatment due to the aggressive growth of bone tumors.Meanwhile,large bone defects caused by tumor resection are often difficult to heal,which seriously affects the postoperative recovery and quality of life of patients.Therefore,combined with advanced tumor cell killing technology,it is of great significance to develop bone repair materials with high osteogenic activity,local killing of residual tumor cells and low side effects,so as to cure the irregular bone defect formed by bone tumor surgery.Micro nano bioactive glass（MNBG）is a kind of inorganic non-metallic bone regeneration materials with bone conductivity,bone induction and bone binding ability.In recent years,the preparation of scaffold materials based on MNBG has become a common means to construct bone tissue engineering implants.However,from the perspective of biomimetic,natural polymer hydrogel has the most similar structure to extracellular matrix.It is the best biocompatible material and has most flexible processing method in tissue engineering.Therefore,this study first combines MNBG with natural polymer hydrogel to prepare organic-inorganic composite hydrogel materials.The influence of MNBG addition on the physical and chemical properties of natural polymer hydrogels is studied in detail.The operation rules of MNBG ion release and mineralization behavior in hydrogels are expounded.The osteogenic induction effect and mechanism of MNBG doped composite hydrogels in critical size bone defects were explored in the form of membrane materials.Further,the MNBG was photothermal functionalized by elemental doping,and an injectable in-situ forming composite hydrogel was constructed.The effect of photothermal on killing tumor cells in vitro and in vivo was evaluated.The osteogenic activity after hydrogels in-situ molding in bone defects was studied,and its application value as tumor related bone defect repair material was clarified.The main research work and conclusions of this study are as follows:（1）Micro nano bioactive glass（MNBG）was prepared by sol-gel method and template method using DDA as template.Further,the double crosslinked gelatin/hyaluronic acid-MNBG composite hydrogel（GA/HA-BG）was prepared by Schiff base reaction and HRP/H₂O₂ enzyme cross-linking reaction.The addition of MNBG enhanced the crosslinking degree of natural polymer hydrogels,while enhancing the mechanical strength and reducing the swelling effect without affecting the adhesive properties to the bone tissue.MNBG and humoral fluid in hydrogels can carry out fast ion exchange,fully release Si O₄^4-,Ca²⁺and other bioactive ions,and only when MNBG content reaches 10 wt%or more,can the composite hydrogel surface stabilize the formation of hydroxyapatite（HA）mineralized layer in humoral environment.The formation of HA layer can slow down the degradation rate of hydrogel materials and make the composite hydrogel possess the ability of bone binding.（2）GA/HA-BG composite hydrogel was further made into biomimetic membrane material,and its bone induction effect in bone defect was evaluated and its mechanism was studied.The results showed that the addition of BG enhanced the adhesion,proliferation and migration of natural polymer hydrogel,and promoted the differentiation of bone marrow mesenchymal stem cells（BMSCs）into osteoblasts.In vitro vascular experiments showed that Si O₄^4-and Ca²⁺released by composite hydrogel could promote VEGF expression in human umbilical vein endothelial cells（HUVECs）by activating PI3K/Akt/HIF-1αpathway and promote HUVECs vascularization.In vivo experimental early results showed that the composite hydrogel membrane shielded the ingrowth of epithelial tissue into the bone defect area,induced multicentric osteogenesis in the defect area while promoted intraregional vascular regeneration;the later results indicate that the composite hydrogel degradation time matched the rate of bone regeneration and could continuously guide bone defect regeneration and accelerate neovascular maturation,which is of great significance for the repair of critical size bone defects that lacking blood supply.（3）According to the special repair needs of tumor related bone defects,BG was functionally modified by element doping.Copper doped nano bioactive glass microsphere（Cu BGM）with photothermal effect was prepared by doping Cu and controlling sintering temperature.The research shows that the photothermal effect of Cu BGM is mainly due to the Cu O crystal formed in the calcination process.The photothermal conversion is realized through the surface plasmon resonance effect（LSPR）of Cu O under near-infrared light.An injectable in-situ alginate/polyethylene glycol-Cu BGM composite hydrogel（SA/PEG-Cu BGM）was constructed by DA click chemical reaction and ion crosslinking reaction.The hydrogel can be rapidly heated and formed under near infrared light.The molding time can be controlled freely by the amount of Cu BGM doping and near infrared power.With the release of Cu²⁺,the composition of photothermal agent can be completely degraded in a short period.（4）The tumor cells killing effect and the ability of bone repair of SA/PEG-Cu BGM composite hydrogel were systematically evaluated.Both in vitro and in vivo experiments indicate that the photothermal effect of SA/PEG-Cu BGM composite hydrogel can effectively kill tumor cells and inhibit tumor growth.In vivo osteogenesis experiments,the composite hydrogel could be rapidly formed by NIR after injected into the femur defect,and the bone tissue was continuously induced to the defect area in subsequent treatment.The photothermal response injectable in-situ formed SA/PEG-Cu BGM composite hydrogel has excellent effect of filling in situ for the bone defect caused by tumor resection.It can be used for tumor cell cleaning and continuous induction of bone regeneration at the initial stage of implantation.