| ObjectiveIn this study,hyperbranched hyaluronic acid was combined with hydroxyapatite and Irgacure 2959 was used as photocrosslinking agent to synthesize photocrosslinkable hyaluronic acid-hydroxyapatite composite hydrogel(Hyperbranched hyaluronic acid-hydroxyapatite composite hydrogels,HH-HA).The study aims to test the in vitro and in vivo osteogenic properties of HH-HA,thus provide theoretical basis for its application in bone defect repair.Materials and methods1.Primary culture and identification of human periodontal stem cells:Human periodontal ligament cells(hPDLCs)were isolated by tissue-enzyme digestion technique,and human periodontal ligament stem cells(hPDLSCs)were obtained by limiting dilution method,identified by morphological observation,cell proliferation ability,osteogenic and adipogenic differentiation potential and cell phenotype analysis.2.The biocompatibility and in vitro osteogenic property test of HH-HA:HH-HA extract was prepared and used to culture hPDLSCs.The biocompatibility of hPDLSCs was detected by CCK-8 and live/dead staining.The osteogenic differentiation media containing different concentrations of HH-HA extract,different concentrations of HA extract,and no extract were set as the experimental group(HHHA),the control group(HA),and the blank group(OM),respectively.Alizarin red staining,quantitative detection of alkaline phosphatase(ALP)activity,and real-time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR)were used to verify the effect of HH-HA on osteogenic differentiation of hPDLSCs.3.The in vivo osteogenic property test of HH-HA:The calvarial defect models of SD rats were established.HH-HA polymerized in situ by visible light,HA,and nothing were implanted in the defect area as experiment,control and blank group respectively.Micro-computed tomography(Micro-CT),hematoxylin-eosin(HE)staining,and Masson staining were used to detect the repair of calvarial defects in SD rats after 8 weeks.Results1.HPDLCs were successfully isolated and cultured by tissue-enzyme digestion technique,and were successfully isolated and purified by limited dilution method.Cell identification showed that the cells were spindle-shaped,uniform in morphology,slow in cell proliferation,with osteogenic and adipogenic differentiation ability.Besides,the cells were positive for bone marrow mesenchymal stem cell markers and negative for hematopoietic cell markers.The isolated and purified cells were confirmed to be hPDLSCs.2.CCK-8 assay presented that the relative growth rate of HH-HA group was more than 75%after 1,3,and 7 days of extract culturing.Compared with control group,live/dead cell staining confirmed that there were no obvious dead cells in HHHA group after 1,3,and 7 days of extract culturing.Alizarin red staining revealed that all groups formed obvious red precipitates,and the staining on 21 days was deeper than that on 14 days.Quantitative detection of ALP activity implied that there was no significant difference in ALP expression between HH-HA group and OM group(P>0.05).The qRT-PCR reported that the expressions of ALP and Runx2 in HH-HA group were lower than those in the blank at 7 days,and the expressions of Runx2 and OPN in HH-HA group were higher than those in the blank at 14 days(P<0.05).3.The results of Micro-CT three-dimensional reconstruction listed that in the blank group and HA group,a small amount of new bone tissue was formed but the calvarial defects were not healed completely at 8 weeks after operation.While the calvarial defects were basically healed in HH-HA group.Trabecular bone thickness(Tb.Th),number of trabecular bone(Tb.N),trabecular bone spacing(Tb.Sp)and new bone volume/total bone volume(BV/TV)were not significantly different between the HA group and the blank group(P>0.05),while Tb.N and BV/TV in HH-HA group were higher than those in blank group(P<0.05),Tb.Sp in HH-HA group was lower than blank group(P<0.05).HE staining and Masson staining noted that in HH-HA group,continuous bone tissue was formed at the defect site,which completely covered the implant,osteoblasts were more common,and mature bone tissue was observed in the center of the defect(Masson staining red).Continuous bone tissue was formed at the bone defects in the HA group,the thickness was smaller than that in the HH-HA group.In the blank group,only a small amount of discontinuous bone tissue was formed at the edge of the bone defect,and the defect was mainly fibrous connective tissue.ConclusionWith good biocompatibility,HH-HA can promote osteogenesis both in vitro and in vivo.It is a promising material to be used in clinical for bone defect repair. |
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