| Background:Bone defect is a major medical problem affecting human life and health.Although traditional bone scaffold materials can partially meet the needs of replacing the defect site and guiding tissue regeneration,it is difficult to achieve complete repair of bone tissue in terms of structure and function.In addition,bacterial infection is another major cause of implant treatment failure,and its resulting intraoperative and postoperative complications directly affect the prognosis of the disease course.Therefore,developing a material with suitable shape and both promoting bone integration and antibacterial properties,regulating cell behavior and promoting bone tissue regeneration under the premise of preventing infection,is one of the research hotspots in the field of prosthodontics.The structure of the hydrogel is similar to natural extracellular matrix,which exhibits desirable biocompatibility.The porosity and pore diameter of the hydrogels is suitable for bone growth,regulating cell proliferation and differentiation behavior.In recent years,the biomineralization-inspired hydrogel system has attracted the interest of scholars.mineralized hydrogels contain both inorganic mineralized components（hydroxyapatite,white phosphor,etc.）and organic components（collagen,fibrin,etc.）in natural bone tissue.Therefore,the chemical composition and structural characteristics of the mineralized hydrogels mimic natural bone tissue,improving the weakness of the traditional hydrogel scaffolds,giving it superior bone conductivity.Although the mineralized hydrogels are rich in various inorganic ions,the higher ion concentration will be generated at the initial stage of implantation,inhibiting cell proliferation and reducing the biological activities.Polydopamine（PDA）is rich in active functional groups,and the modified material exhibits superior biological properties.The modified mineralized hydrogel can effectively promote the adhesion and proliferation of cells around the defective tissue and improve the biocompatibility.Gallic acid（GA）is a kind of plant extract,which has the advantages of broad-spectrum antibacterial activity,wide source and low cost.GA can damage the integrity of bacterial cell wall by contacting with it,thus playing an antibacterial role and eliminating free radicals while inhibiting the growth of bacteria,so that local cells can reach a state of physiological balance and thus effectively promote the healing of infected wounds.After GA modification,the antibacterial properties of the hydrogel system can be obtained or improved.Compared with the traditional drug-loaded antibacterial hydrogel,it overcomes the difficulty that the release of antibacterial substances is difficult to control,so as to ensure the effective prevention of infection and improve the biocompatibility.Purpose:In this study,nano-hydroxyapatite（n-HAP）,PDA,chitosan（CS）and hyaluronic acid（HA）were used as raw materials.Firstly,GA was grafted onto CS surface.The CS-GA-HA（CGH）hydrogel was formed by Schiff base reaction,and polydopamine-modified hydroxyapatite nanoparticles（PDA@HAP）were loaded to form a novel composite hydrogel scaffold,abbreviated as‘CGH/PDA@HAP’.The bone regeneration and antibacterial properties of the hydrogel system were further investigated.Method:1.Prepare the CGH/PDA@HAP hydrogels and evaluate their characterization and physicochemical properties.2.In vitro biocompatibility of the CGH/PDA@HAP hydrogel and its ability to induce osteogenic differentiation were evaluated with rat bone marrow stem cells（BMSC）.3.The animal model of critical skull defects in rat were established,and the CGH/PDA@HAP hydrogel was implanted at the same time to evaluate the therapeutic effect of bone tissue defect in vivo.4.Staphylococcus aureus（S.aureus）and Escherichia coli（E.coli）to evaluate the antibacterial activity of the CGH/PDA@HAP hydrogel in vitro.A model of S.aureus and E.coli co-infection in vivo was established to evaluate the antibacterial and anti-inflammatory activity of the CGH/PDA@HAP hydrogel.Results:1.The CGH/PDA@HAP hydrogel can promptly form into gel in vitro.The CGH/PDA@HAP hydrogel is with good rheological properties,exhibiting injectable and self-healing properties.The CGH/PDA@HAP hydrogel can effectively achieve the filling effect of irregular defects.After PDA modification,HAP was uniform Ly distributed in the composite hydrogel system,which overcomes the disadvantage of easy agglomeration of nanoparticles.SEM results showed that the CGH/PDA@HAP hydrogel had a three-dimensional porous structure,and its porosity and pore diameter were similar to those of natural bone tissue.In addition,the CGH/PDA@HAP hydrogels showed good swelling and degradation properties,and the presence of Schiff base effectively slowed down the degradation of HA,making the degradation rate of the whole system match the growth of bone tissue.2.CGH/PDA@HAP hydrogel had very low cytotoxicity,and had no significant up-regulation effect on inflammation-related genes（TNF-αand IL-1β）.It effectively promoted the proliferation of BMSCs,illustrating good biocompatibility.In the early stage of osteogenesis,alkaline phosphatase（ALP）activity of BMSC was significantly enhanced.In the late stage of osteogenesis,it promoted the mineralization of the extracellular matrix of BMSCs.The expression of genes and proteins related to bone formation of BMSCs was significantly up-regulated.3.At 4 and 8 weeks after CGH/PDA@HAP hydrogel implantation,the skull defect healed well.Imaging images showed that a large number of new bones were formed around and in the middle of the defect,and the new bones had a dense trabecular structure.Histological images showed a large number of new bone tissues and active bone matrix secretion.In addition,CGH/PDA@HAP hydrogel had no histological toxicity to major organs including heart,liver,spleen,lung and kidney,and no significant pathological changes were observed under microscope.4.The CGH/PDA@HAP hydrogel can effectively inhibit the growth of S.aureus and E.coli,and the inhibition rates of suspended bacteria both reached more than95%,effectively preventing bacterial colonization in the defect site.On the other hand,the CGH/PDA@HAP hydrogel can effectively inhibit the formation of bacterial biofilms,kill bacteria attached to the surface,and reduce metabolic activity.Conclusions:1.The CGH/PDA@HAP hydrogel has good physical and chemical properties and surface structure.Gelatinized rapidly in vitro,showing injectable and self-healing properties,suitable for irregular bone defects.Appropriate pore diameter and porosity can regulate the biological behavior of BMSC and promote the proliferation and differentiation of BMSC.2.The Ca2+sustained release ability of CGH/PDA@HAP hydrogel improves the bone inductivity,and can effectively promote the osteogenic differentiation of BMSCs in vitro,providing favorable conditions for subsequent tissue regeneration.In vivo,the bio-mineralization of PDA and the osteogenic induction of HAP are synergistic to accelerate the healing process of bone defects.3.The CGH/PDA@HAP hydrogel effectively inhibit bacterial migration,colonization and biofilm formation in vitro.It shown antibacterial and anti-inflammatory properties in vivo,and achieved a dynamic balance with its osteogenic induction effect,so that it had both osteogenic and antibacterial effects,significantly reducing the risk of bacterial infection during bone regeneration. |
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