| ObjectiveThe study aims to prepare injectable calcium alginate hydrogel and its nano-material composites that is conducive to regeneration of periodontal tissues,to detect the material's physical performance and in vivo and in vitro biological performance.Studying the effect of calcium alginate hydrogel on hPDLCs proliferation and inflammatory factor expression,observing the effect of calcium alginate hydrogel on the mineralization of BMSCs and the expression of osteogenic factors,monitoring the antibacterialeffect of calcium alginate/nano silver hydrogel on common bacteria Staphylococcus aureus,carrying out animal in vivo experiments to verify the in vivo effect of calcium alginate hydrogel on tissue inflammation and bone formation.The research is intended to provide the basis for the later study of 3D printing composite scaffold and the clinical application of calcium alginate hydrogel.Material and MethodReagent and instrument:Sodium alginate,calcium chloride and bovine serum albumin(BSA),DMEM(Dulbecco's modified eagle medium),fetal bovine serum(FBS),penicillin and streptomycin,collagenase,PBS solution,dimethyl sulfoxide(DMSO),MTT cell proliferation and cytotoxicity assay kit,nano silver,deionized water,LB nutrient culture medium,hematoxylin,eosin,xylene,freezing microtome,inverted microscope,Micro-CT.Method:Preparation of calcium alginate hydrogel and component detection and adopt ATR-FTIR to analyze the surface characteristics of the prepared samples;assay of calcium alginate hydrogel BSA protein release:Mix sodium alginate and BSA,dissolve them in deionized water,then add into calcium chloride solution for ion exchange,put the prepared calcium alginate gel/BSA in deionized water,monitor BSA release solubility by BSA kit for detection in a predetermined time.Draw BSA release curve;assay of water absorption of calcium alginate hydrogel:monitor the dry weight and wet weight of the prepared calcium alginate gel,and conduct water proportion assay;assay of calcium alginate hydrogel degradation rate:place prepared calcium alginate hydrogel in PBS solution,respectively measure the dry weight and wet weight at the scheduled time of 3rd day,7th day,14th day,28th day and 56th day,record the data,and according to the dry and wet weight loss rate,analyze calcium alginate hydrogel degradation;primary culture and identification of hPDLCs:use inverted phase contrast microscope to observe the primary and third generation cells and get the pictures,then identify the cells by cytokeratin and vimentin staining.MTT(methyl thagolyl tetragoliam)to detect the proliferation of hPDLCs and detect cytotoxicity and proliferation of calcium alginate hydrogel:groups:Control group,PLA group,12.5 mg/mL,25 mg/mL and 50 mg/mL calcium alginate hydrogel.Take the third generation hPDLCs in logarithmic growth phase to inoculate at a certain density on the 96-well plate,and respectively add PLA and calcium alginate gel above.Use MTT to detect the proliferation at 1st day,3rd day,5th day and 7th day,and then to determine their cytotoxicity;effect of calcium alginate hydrogel on the expression of inflammatory cytokines in human periodontal ligament cells:RT-PCR detect inflammation associated cytokines expression of[interleukin 8(IL-8),interleukin 6(IL-6),TLR-4,TNF-? and interleukin 1?(IL-1?)]mRNA expression level,compare significant difference.Preliminarily discuss the effect of calcium alginate hydrogel on the inflammatory reaction mechanism;affect of calcium alginate hydrogel on the osteogenesisrelated gene expression of bone marrow mesenchymal stem cells:RT-PCR detect the relative mRNA expression of OPG,OPN and RUNX2 in cells,and determine the effect of calcium alginate on osteogenesis related genes.Compare statistical difference;alizarin red staining to detect the affect of calcium alginate gel on BMSCs mineralization ability:calcium alginate hydrogel with BMSCs,after 14 days of induction mineralization,take alizarin red staining.Count the number of mineralized nodules;assay of minimum bacteriostasis concentration of nano silver particles:the minimum bacteriostasis concentration of nano silver particles can be determined by broth dilution method;preparation and composition analysis of calcium alginate/nano silver composite hydrogel:form the calcium alginate/nano silver gel with crosslinking method.Analyze surface composition by X ray photoelectron spectroscopy;antibacterial experiment of calcium alginate/nano silver composite hydrogel:adopt the inhibition zone method to detect the inhibitory effect of nano silver composite calcium alginate hydrogel on Staphylococcus aureus;monitoring of calcium alginate hydrogel for the initial inflammatory response of soft tissue:capture view of the H-E staining tissue sections,and analyze the images in the visual field,compare the inflammatory response of each group;the establishment of mandibular defect model;evaluation of calcium alginate hydrogel for bone repair:adopt Micro-CT scan,three-dimensional reconstruction,software analysis of the new bone.Statistical analysis:All quantitative data presented in this research were expressed as the meanąstandard deviation.We used one-way ANOVA as statistical analysis method.All statistical tests were analyzed with SPSS 19.0.p<0.05 was considered statistically significant for this research.ResultThe injectable drug-loaded calcium alginate hydrogel was successfully prepared.In this study,the drug loaded calcium alginate hydrogel was successfully prepared by ion exchange method,and the FTIR result showed that the material was calcium alginate hydrogel.The determination of water absorption shows the good water absorption performance of calcium alginate hydrogel,and the water absorption of 12.5 mg/mL is the best,but its forming ability is poor.The results of BSA release show that the initial release amount of 12.5 mg/mL is larger,but the sustained release ability in not as good as that of 25 mg/mL and 50 mg/mL;for 50 mg/mL,its initial release ability is poor.The degradation experimental results show that the degradation time of calcium alginate hydrogel with 25 mg/mL concentration matches more with the time of bone tissue regeneration.Biological properties of calcium alginate hydrogel:the three concentrations of calcium alginate hydrogel prepared in this study all have more than 75%relative growth rate((RGR)on hPDLCs,according to ISO 10993-5,which are in the 1st grade,which is to say,for human periodontal ligament cells calcium alginate hydrogel is biologically non-toxic,has good safety performance.The calcium alginate hydrogel and PLA scaffold could promote the expression of mRNA of hPDLCs in TLR4,IL-6,IL-8,TNF-? and IL-1?.The inflammatory reaction induced by calcium alginate hydrogel group is milder than PLA group,with statistical difference(p<0.05).The mineralized nodules formed by bone marrow mesenchymal stem cells co-cultured with calcium alginate were significantly higher than those in the control group and the PLA group,and along with statistical difference(p<0.05).Compared with the control group,calcium alginate hydrogel could promote the mRNA expression of RUNX2,OPN and OPG(p<0.05)in bone marrow mesenchymal stem cells,and the effect is the best when the concentration is 25mg/mL(p<0.05).Antibacterial properties of calcium alginate hydrogel loaded nanosilver material:calcium alginate hydrogel loaded nano silver material was successfully prepared.The antibacterial experiment shows that calcium alginate hydrogel loaded nano silver material has antibacterial effect on Staphylococcus aureus.Calcium alginate hydrogel in vivo monitoring:compared with the PLA group,the inflammatory reaction induced by calcium alginate hydrogel was lighter,and it was well combined with the tissue.The regeneration effect of calcium alginate hydrogel on bone tissue defect is better than that of PLA.ConclusionIn this study,a drug-loaded hydrogels material using in oral bone tissue regeneration was produced by ion cross-linking method.Calcium alginate hydrogel has good water absorption capacity and BSA release ability.The concentration of 25 mg/mL has better degradation time which is in good match with bone regeneration.Prepared calcium alginate hydrogel meets the requirements of bio-compatibility using in oral bone tissue regeneration.Although it promotes the expression of inflammatory cytokines,it has less inflammatory reaction compared with the traditional PLA scaffolds.Calcium alginate hydrogel could promote mineralization of BMSCs and the expression of osteogenesis related cytokines.The calcium alginate/nano silver composite materials were successfully prepared,which has certain antibacterial effect on common bacterias Staphylococcus aureus and could help to reduce wound infection.In vivo study results correlated with the in vitro study results.The inflammatory response of calcium alginate hydrogel in soft tissue was lighter than that of PLA,but the osteoinductive bone ability of calcium alginate hydrogel was better than PLA.Therefore,the drug-loaded calcium alginate hydrogel is a potential bone defect-repair material for dental clinical application. |
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