Nanohydroxyapatite And Nanosilicate-Composite Injectable Biomimetic Hydrogel For Bone Regeneration

Part 1 Preparation and characterization of Nanohydroxyapatite and Nanosilicate-composite GelMA hydrogel and its physicochemical optimizationObjectiveTo prepare the GelMA-HAP-SN nanocomposite hydrogel by simply mixing the GelMA,HAP,and SN,and to characterize the physicochenmical features of the GelMA-HAP-SN nanocomposite hydrogel.MethodsGelMA macromolecular precursor was synthesized using type-A gelatin reacted wtih MA.By simply blending GelMA precursor,HAP and SN in PBS,then crosslinking under UV light irradiation,the GelMA-HAP-SN nanocomposite hydrogel samples were obtained.The microscopic morphology of the GelMA-HAP-SN nanocomposite hydrogel samples was observed by an electron microscopy.The viscoelastic properties and the Young’s modulus of the nanocomposite hydrogel samples were measured using a rheometer and a universal biomechanical testing machine,respectively.The swelling ratio of the nanocomposite hydrogel samples was evaluated by the mass differences.The degradation behavior of the nanocomposite hydrogels was analyzed by in vitro degradation experiments.The chemical groups of the nanocomposite hydrogel samples and the relative crystallinity were analyzed using FT-IR and XRD,respectively.ResultsThe morphology of GelMA under the electron microscope showed a honeycomb-like porous network with smooth inner walls,and the uniformly loaded HAP roughed the inner wall.The nanoparticles of HAP and SN were rhombus-shaped and disc-shaped in nanoscale,respectively.The introduction of HAP and SN did not significantly change the chemical structure,degradation behavior and swelling ratio of GelMA,but narrowed the pore size.In addition,the introduction of SN reduced the UV irradiation time required for GelMA crosslinking.The Young’s modulus of GelMA-HAP-SN nanocomposite hydrogel was positively correlated with the concentration of the introduced nanoparticles,and was at the optimal range for the osteogenic differentiation of MSC when 100 μg/ml of HAP and 2%(w/v)of SN were jointly mixxed.The shear thinning properties of the GelMA-HAP-SN nanocomposite hydrogel precursor increase with the elevation of introduced SN concentration.ConclusionThe GelMA-HAP-SN nanocomposite hydrogel possesses ideal injectable and rheological properties,with its microstructure suitable for MSC attachment and growth,and the biomimetic microenvironment for osteogenic differentiation.Part 2 Biocompatibility and in vitro osteogenesis assessment of Nanohydroxyapatite and Nanosilicate-composite GelMA hydrogelObjectiveTo prepare the MSC encapsulated GelMA-HAP-SN nanocomposite hydrogel samples for further in vitro osteogenic culturing,verifying the biocompatibility and in vitro osteogenesis,and to determine the optimal concerntrations of the HAP and SN.MethodsTo fabricate MSC encapsulated GelMA-HAP-SN nanocomposite hydrogel samples,primary MSC from rat femoral bone marrow were extracted,isolated and cultured in vitro,and then were seeded into the GelMA-HAP-SN nanocomposite hydrogel.Osteogenic differentiation of encapsulated MSC was induced in vitro using osteogenic induction medium.Cytotoxicity of different concentrations of HAP or SN nanoparticles was determined by CCK-8 assay.Cell viability and spreading activity of encapsulated MSC in the hydrogel samples were assessed using Live/Dead assay and DAPI/Phalloidin staining.Quantitative analysis of matrix mineralization of the samples was conducted via Alizarin Red staining.The expression of osteogenic proteins and genes of the encapsulated MSC in the samples were analyzed by immunofluorescence staining and qRT-PCR.ResultsMSC encapsulated in GelMA-HAP-SN nanocomposite hydrogel samples demonstrated favorable cell viability and spreading motion.The results of CCK-8 assay indicated the minimal cytotoxicity of the introduced nanoparticles were at 100μg/ml of HAP and 2%(w/v)of SN.The results of Alizarin Red staining suggested the GelMA-HAP-SN nanocomposite hydrogel possessed the most significant matrix mineralization,and the encapsulated MSC demonstrated high expression levels of four types of osteogenic genes and the corresponding proteins.ConclusionThe GelMA-HAP-SN nanocomposite hydrogel demonstrated the favorable biocompatibility,remarkable matrix mineralization and superior in vitro osteogenesis.The optimal concentrations of HAP and SN were 100 μg/ml and 2%(w/v),respectively.Part 3 In vivo osteogenesis assessment of Nanohydroxyapatite and Nanosilicatecomposite GelMA hydrogel in the rat critical cranial defectObjectiveTo evaluate the in vivo osteogenesis of the MSC encapsulated GelMA-HAP-SN nanocomposite hydrogel samples with optimal nanoparticle concentrations.MethodsThe adult male SD rats were randomly selected to establish an 8mm circular cranial defect pattern.The MSC encapsulated GelMA-HAP-SN nanocomposite hydrogel prepolymer with nanoparticles of optimal concentrations was prepared and subsequently injected into the cranial defect area via a syring,and finally irradiated in situ by UV light.The rats were all sacrificed after 8 weeks of feeding,and the calvaria samples were harvested for Micro-CT scanning and hard tissue section with H&E and Goldner staining to analyze the in vivo osteogenesis of the MSC encapsulated GelMA-HAP-SN nanocomposite hydrogel samples.ResultsMicro-CT scanning with three-dimensional reconstruction analysis indicated that the MSC encapsulated GelMA-HAP-SN nanocomposite hydrogel promoted a significantly more bone regeneration for healing the critical cranial defect.The H&E and Goldner staining results also indicated more mineralized bone,osteoid and neovascularized tissue formation after the treatment of the MSC encapsulated GelMA-HAP-SN nanocomposite hydrogel samples.ConclusionThe MSC-encapsulated GelMA-HAP-SN nanocomposite hydrogel possesses superior in vivo osteogenesis.