The Role And Mechanism Of TEOS/GEL Hydrogel And GelMA-HAP-SN Nanocomposite Hydrogel In Bone Defect Repair

Chapter 1:The role and mechanism of engineered multifunctional TEOS/GEL hydrogels with osteogenic capacity in the repair of critical-size segmental bone defectsBackground:Treating segmental bone defects of critical size is a formidable clinical challenge.Preparing bone graft substitutes with significant osteoinductive properties is a feasible strategy for treating critical size bone defects.Methods:1.TEOS/GEL hydrogel preparation:A biocompatible hydrogel was designed by dynamic supramolecular assembly of polyvinyl alcohol(PVA),sodium tetraborate(Na2B4O7),and tetraethyl orthosilicate(TEOS).2.Characterization and analysis of TEOS/GEL hydrogels:The properties of supramolecular hydrogels were evaluated by rheological analysis,swelling ratio,degradation experiments and scanning electron microscopy(SEM).3.TEOS/GEL hydrogel has no impact on the activity and osteogenicity of bone marrow mesenchymal stem cells(BMSCs):the proliferation activity was detected by CCK-8,the calcification was detected by alizarin red staining;Q-PCR detected the expression of the osteogenic genes.4.Analysis of bone regeneration ability of bone mesenchymal stem cell-embedded TEOS/GEL hydrogel on rabbit segmental bone defects:Construction of rabbit segmental bone defects model,bone mesenchymal stem cell-embedded TEOS/GEL hydrogel transplantation,micro-CT analysis and histomorphometric staining(H&E staining).Results:1.Characterization and analysis of TEOS/GEL hydrogels:The lyophilized hydrogels have interconnected porous structures;with the increase of PVA and Na2B4O7 contents,the storage and loss moduli of the hydrogels increase correspondingly,while the concentration of TEOS/GEL has a significant effect on the storage modulus and loss modulus of the hydrogel have a little significant effect;the TEOS/GEL hydrogel is cut into two halves,and the two separate TEOS/GEL hydrogels can be reassembled into a whole within 30 min;The TEOS/GEL hydrogel reached swelling equilibrium in about 20min,and its maximum swelling ratio of the hydrogel is about 7%;the TEOS/GEL hydrogel could be completely degraded within 36 days.2.Compared with the control hydrogel group and Gel group,TEOS/GEL hydrogel had no impact on the activity of bone marrow mesenchymal stem cells(BMSCs),and could promote the deposition of mineralized nodules in bone marrow mesenchymal stem cells(BMSCs)and osteogenesis-related genes(Runx-2,Col-1,OCN and OPN).3.Compared with the control hydrogel group and the Gel group,the bone mesenchymal stem cell-embedded TEOS/GEL hydrogel significantly increased the bone volume/total volume(BV/TV)valueand induced the formation of bone bridges and marrow in the area of the bone defects and cavity recanalization.Conclusion:1.TEOS/GEL hydrogel has self-healing,low swelling rate,degradability and interconnected porous structure.2.TEOS/GEL hydrogel has good biocompatibility,promotes the ossification of bone marrow mesenchymal stem cells,and acts by activating the RUNX2 osteogenic signalling pathway.3.TEOS/GEL hydrogel-encapsulated with bone marrow mesenchymal stem cells promotes bone regeneration in segmental bone defects in rabbits.Chapter 2:The role and mechanism of GelMA-HAP-SN nanocomposite hydrogel encapsulating bone mesenchymal stem cells in the bone regeneration of critical-size calvarial defects in ratsObjective:The use of autologous bone grafts for treating bone defects is limited by insufficient donor site supply and morbidity.The development of biomimetic bone graft materials as an alternative approach has attracted unanimous attention.However,hurdles remain in designing osteomimetic materials that combine the biomimetic properties of bone extracellular matrix,osteoblasts,and osteoinductive components with a rapid and convenient strategy.Methods:1.Preparation of GelMA-HAP-SN nanocomposite hydrogel:In this study,bone-mimicking gelatin loaded with bone marrow mesenchymal stem cells(BMSCs),nano-hydroxyapatite(HAP)and nano-silicate(SN)was designed and prepared.-Methacrylate-based hydrogel(GelMA-HAP-SN)system.2.Characterization and analysis of GelMA-HAP-SN nanocomposite hydrogel:The morphology of nano-HAP and SN was observed by transmission electron microscope,and the internal microstructure of freeze-dried hydrogel sample was analysed by field emission scanning electron microscopy(FE-SEM);The viscosity of GelMA-HAP-SN nanocomposite hydrogel by rheological measurement;Fourier transform infrared spectroscopy(FTIR)and X-ray diffraction analysis of GelMA-HAP-SN nanocomposite hydrogel for chemical functional groups and crystal structure;Instaton 5542 mechanical tester analysis the mechanical structure;immersion analysis in PBS for degradation and swelling.3.Analysis of the effect of GelMA-HAP-SN nanocomposite hydrogel on bone marrow mesenchymal stem cells(BMSCs):Calcein AM and immunofluorescence staining were used to detect the viability,proliferation and migration.Alizarin red staining,Q-PCR,immunofluorescence and western blotting were used to observe the ossification of BMSCs.4.GelMA-HAP-SN nanocomposite hydrogel-encapsulated in bone mesenchymal stem cells promotes bone regeneration in the critical-size calvarial bone defect in rats:Construction of rat calvarial defects model,GelMA-HAP-SN nanocomposite hydrogel transplantation,Micro-CT analysis and histomorphometric staining(H&E staining,Goldner-Masson staining).Result:1.The GelMA-HAP-SN nanocomposite hydrogel is an interconnected porous network microstructure with injectability;SN enhances the shear recovery ability of the prepolymer;there is no chemical bond between the nanoparticles and the GelMA backbone;HAP and SN did not affect the swelling rate and degradation curves of GelMA-HAP-SN nanocomposite hydrogels.2.The GelMA-HAP-SN nanocomposite hydrogel constructed with 100 μg/mL HAP and 2%SN did not affect the viability of bone marrow mesenchymal stem cells(BMSCs)and promoted the proliferation of BMSCs significantly compared with those of the GelMA,GelMA-SN and GelMA-HAP groups.3.The expression of bone marrow mesenchymal stem cell osteogenic genes(RUNX2,ALP,OCN and OPN)and matrix mineralization in the GelMA-HAP-SN nanocomposite hydrogel group were significantly increased compared with those in the GelMA,GelMA-SN and GelMA-HAP groups.4.In the critical-size calvarial defects model in rats,the bone mineral density(BMD),new bone volume(BV)and their relationship with tissue volume in the GelMA-HAP-SN nanocomposite hydrogel repair group embedded with bone mesenchymal stem cells The ratio(BV/TV)was significantly increased compared with GelMA,GelMA-SN and GelMA-HAP groups.Conclusion:1.GelMA-HAP-SN nanocomposite hydrogel has a good characterization of ideal bone tissue repair engineering materials.2.GelMA-HAP-SN nanocomposite hydrogel promotes the ossification of bone mesenchymal stem cells by activating the osteogenic RUNX2 signalling pathway.3.GelMA-HAP-SN nanocomposite hydrogels embedded in bone mesenchymal stem cells promote bone regeneration in the critical-size calvarial bone defect in rats.