Experimental Study On Preparation Of Multifunctional Photothermal Hydrogel And Its Application On Orchestrating Osteoimmune For Accelerating Bone Regeneration

Part Ⅰ Preparation and Characterization of Multifunctional Photothermal HydrogelsObjective In this study,a composite hydrogel based on Alginate methacrylate(AlgMA)and Dopamine-grafted-alginate(Alg-DA)was synthesized and used as the primary constituent of the hydrogel.The Titanium carbide(Ti3C2Tx)powder was then etched to synthesize the original Titanium carbide nanosheets(MXene),which were subsequently modified with Polydopamine(PDA)to obtain PDA-functionalized MXene nanosheets(MXene@PDA or MP).The next step involved incorporating a certain quantity of MXene@PDA into the AMAD hydrogel to obtain a multifunctional photothermal hydrogel(AMAD/MP).Finally,the composite material was characterized and tested for its physicochemical properties.These findings provide insights into the development of advanced hydrogels with diverse applications in biomedical engineering,drug delivery,and tissue engineering.Methods To prepare the hydrogel precursor solution,a mass ratio of 4 wt%Alg-MA,2 wt%Alg-DA,and 0.3 wt%Lithium phenyl-2,4,6-trimethylbenzoylphosphinate(LAP)was utilized.The MXene@PDA was then added to the hydrogel precursor solution at different masses(0.5 wt%,1 wt%,2 wt%)and mixed thoroughly by vortexing and sonication.The resulting solution was poured into specific molds and cured by exposing it to 405 nm ultraviolet light(5 W/cm2)for three minutes,producing the final hydrogel(AMAD/MP).The chemical characterization of Alg-MA and Alg-DA was performed using 1H nuclear magnetic resonance(NMR)and ultraviolet-visible(UV-vis)spectroscopy,while the morphology and microstructure of MXene@PDA were analyzed using field-emission scanning electron microscopy(FE-SEM)and highresolution Micro-CT.Additionally,the chemical structures and compositions of different samples were analyzed using techniques such as energy-dispersive X-ray spectroscopy(EDS),X-ray photoelectron spectroscopy(XPS),Fourier-transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),Raman spectroscopy,zeta potential,thermal gravimetric analysis(TGA),and UV-vis-NIR absorption spectroscopy.Macroscopic,microscopic,and nanoscale morphology characteristics of the hydrogel were analyzed using digital cameras,optical microscopy,SEM,Micro-CT,and transmission electron microscopy(TEM).Moreover,the porosity,swelling behavior,in vitro degradation characteristics,rheological properties,in vitro mineralization performance,and photothermal properties of different samples were determined.Results In this study,Alg-MA,Alg-DA,and MXene@PDA were successfully synthesized,and based on this,AMAD/MP hydrogel was further synthesized.These materials presented an ideal surface appearance and microstructure,with evenly distributed MXene@PDA nanosheets within the AMAD/MP hydrogel.Chemical characterization experiments confirmed the successful preparation of MXene@PDA nanosheets and AMAD/MP hydrogel,and the non-covalent bonds,such as hydrogen bonding,between the various components enhanced the stability of the system.In vitro swelling experiments and mechanical performance testing demonstrated that MXene@PDA nanosheets significantly increased the mechanical strength of the hydrogel material,reduced the water absorption property,and contributed to the longterm integrity and stability of the scaffold.The in vitro enzyme degradation experiment revealed that AMAD/MP hydrogel exhibited a slow degradation characteristic under the action of protease,thus providing long-term mechanical support and structural stability to the defect area in vivo,contributing toward the creation of a stable microenvironment for subsequent tissue repair.Photothermal experiments demonstrated that AMAD/MP hydrogel possessed high photothermal conversion efficiency and stability.Among them,the temperature of AMAD/1%MP hydrogel was kept at 43.2±0.3℃ after 3 minutes of near infrared light(808 nm,1 W/cm2)irradiation,which is a very suitable temperature range for photothermal treatment.Therefore,we chose AMAD/1%MP hydrogel for subsequent experimental research.Conclusion The AMAD/MP hydrogel has ideal porosity,appropriate swelling property,excellent mechanical strength,delayed enzyme degradation,and outstanding photothermal effects.These superior properties pave the way for us to conduct cell biology experiments and in vivo bone defect repair experiments.Part Ⅱ The biological function of multifunctional photothermal hydrogel in vitro was studied by cell experimentPurpose This study aims to investigate the in vitro cellular compatibility and biological effects of a multifunctional photothermal hydrogel at the cellular level.Method Sterile hydrogel precursor solution was injected into a cell culture plate,followed by UV irradiation to form a hydrogel.Subsequently,cells were seeded onto the hydrogel for cultivation.After co-culturing and appropriate photothermal treatment,the hydrogel’s impact on cell viability was analyzed through CCK-8 assay,live/dead cell staining,and apoptosis detection.Additionally,the hydrogel’s effects on cell adhesion,proliferation,spreading,migration,and other biological functions were examined using cell skeleton staining,EdU cell proliferation assay,and Vinculin immunofluorescence staining.Subsequently,the hydrogel’s influence on immune regulation and osteogenic properties was further assessed,including macrophage polarization(morphological observation,iNOS/CD206 immunofluorescence staining,CD86/CD206 flow cytometry,and qRT-PCR),as well as in vitro osteogenic differentiation(ALP activity,extracellular matrix mineralization,and osteogenic factor expression).Finally,its antioxidant capacity was evaluated through assays involving reactive oxygen species(ROS)fluorescent probes,DPPH free radical scavenging,hydroxyl radical scavenging,singlet oxygen fluorescent probes,and hydrogen peroxide clearance.The hydrogel’s antibacterial effect was assessed using turbidity and plate assays.Results The AMAD/1%MP hydrogel combined with photothermal therapy exhibits excellent biocompatibility and significantly enhances the in vitro adhesion,proliferation,migration,and spreading of MC3T3-E1 cells.Further evaluation revealed that the AMAD/1%MP hydrogel,in combination with mild photothermal treatment,synergistically promotes osteogenic differentiation in MC3T3-E1 cells,effectively increasing the expression levels of osteogenic-related genes and enzyme activity.Furthermore,the hydrogel promotes RAW 264.7 differentiation towards an antiinflammatory phenotype,while exhibiting excellent antioxidant capacity and antibacterial activity.Conclusion The combination of AMAD/1%MP hydrogel with mild photothermal therapy can effectively enhance the cell compatibility of MC3T3-E1 in vitro,promote cell differentiation towards osteogenesis,shape an anti-inflammatory microenvironment,clear intracellular and extracellular reactive oxygen species,and inhibit bacterial infection.This provides a foundation for further evaluation of the application of AMAD/1%MP hydrogel in bone defect repair in vivo.Part Ⅲ Biological function of multifunctional photothermal hydrogel in vivoPurpose Further investigate the feasibility of the multifunctional photothermal hydrogel for bone defect repair in vivo and evaluate its ability to regulate bone immunity,promote new bone formation,and angiogenesis in vivo.Method We selected 48 male SD rats(mean age 8 weeks)to construct the cranial defect model.Bilateral critical-sized circular defects with a diameter of 5 mm were prepared on each rat’s skull,and they were randomly divided into four groups,including blank,AMAD,AMAD/1%MP,and AMAD/1%MP+NIR groups.Samples were collected at three time points:2,4,and 8 weeks after implantation of the materials or after photothermal treatment.Gross morphology,Micro-CT scanning reconstruction,tissue section staining,immunohistochemistry,and immunofluorescence staining were used to investigate the ability of the multifunctional photothermal hydrogel to regulate bone immunity,promote new bone formation,and facilitate angiogenesis in vivo.Result The immunohistochemistry and immunofluorescence results confirmed the excellent performance of the AMAD/1%MP hydrogel combined with photothermal therapy in vivo in shaping the immune microenvironment,recruiting stem cells,and inducing angiogenesis.Furthermore,the results of Micro-CT scanning and reconstruction analysis showed that AMAD/1%MP+NIR had a strong ability to promote bone defect healing.Results from H&E,Masson,and Goldner staining also indicated that the AMAD/1%MP+NIR group significantly promoted bone tissue regeneration in the defect area,while improving the mineralization and maturation of new bone.All implanted hydrogel materials were degraded in vivo without adverse effects on the maj or organs of rats,demonstrating excellent biosafety.Conclusion The combination of AMAD/1%MP hydrogel and mild photothermal therapy exhibits various excellent biological activities,which can significantly promote bone tissue healing in the rat skull defect area.In summary,AMAD/1%MP+NIR has strong multidimensional abilities to promote bone defect healing and high biosafety,representing its potential for clinical treatment of bone defects.