| Objective:Hydroxyapatite（HA）,Ca10（PO4）6（OH）2,an inorganic mineral with chemical properties similar to natural bone salts,has been widely used in the preparation of scaffold materials in bone tissue engineering.Previously,we constructed porous HA scaffolds with grooves performing good bone induction,while the bone induction mechanism was not clear.MicroRNAs（miRNAs）,a class of non-coding RNA molecules consisting of approximately 19-25 nucleotides,have shown to be involved in regulating a wide range of biological processes including bone regeneration.They inhibited gene or protein expression in cells by targeting complementary sequences of target genes.In this dissertation,miRNA expression sequencing was used to analyze the differential expression of miRNA during the in vivo and in vitro osteogenic process of HA scaffold,and the related mechanism of bone induction of differential miRNA was further studied.The results will provide ideas and basic theoretical basis for the screening of bone-related miRNAs and the construction of miRNA-loaded composite scaffolds.Methods:1.Preparation,characterization of HA scaffolds and study of their osteogenic differentiation induction.The porous HA scaffold with grooves was prepared by the sugar ball pore-making method and the sugar ball mold wet treatment technology.The microstructure and surface morphology of the scaffold were observed by scanning electron microscope.In vitro,the cells without scaffold group were used as control group.HA scaffolds were co-cultured with human placenta-derived mesenchymal stem cells,rat bone marrow mesenchymal stem cells and mouse embryonic osteoblast progenitor cells,respectively.After 7 days of osteogenic differentiation induction,alkaline phosphatase activity and expression of osteogenic genes were detected,and the ability of HA scaffolds to induce osteogenic differentiation in vitro was evaluated.In vivo,the ectopic osteogenic model of rat back muscle pocket was constructed,and the performance of HA scaffold induced osteogenic differentiation was evaluated by histological staining.2.MiRNA expression profile analysis during in vivo and in vitro osteogenic differentiation of HA scaffold.MicroRNA expression profiles of samples in vitro and in vivo were sequenced using Illumina high-throughput sequencing platform.After sequencing data analysis,filtration and verification,among the miRNAs with high expression during the induction process of HA scaffold both in vivo and in vitro,miR-129-5p was screened.Then,the target genes were further analyzed by GO and KEGG bioinformatics methods to study the possible relationship between miR-129-5p and genes or signaling pathways related to osteogenic differentiation.3.The function and mechanism of miR-129-5p expression in osteogenic differentiation of cells were studied at the cellular level.MiR-129-5p negative control（NC）,mimics and inhibitor were synthesized and transfected into cells.The expression of osteogenic genes was detected by RT-q PCR,immunofluorescence and western blot.Cell migration ability was detected by scratch assay and Transwell assay.Cell proliferation capacity,alkaline phosphatase（ALP）activity,calcium nodule formation,and protein kinase A（PKA）activity were detected by related kits.ENCORI database was used to predict miRNA targets,and dual luciferase reporting systems were used for interaction verification.The relationship between the expression of PKA-catenin/creb and miR-129-5p expression was detected by western blot and immunofluorescence.Under the condition of overexpression of miR-129-5p,the ability of cell migration and osteogenic differentiation of H89 cells with and without the addition of PKA small molecule inhibitor was compared,and the relationship between the influence of miR-129-5p overexpression on cell phenotype and PKA activity was further determined.4.The effect of miR-129-5p expression on ectopic osteogenesis of BMSCs in vivo was studied at the animal level.Four groups of scaffolds（blank HA,HA+BMSCs-NC,HA+BMSCs-mimcs,HA+BMSCs-inhibitor）were implanted into the back muscle pocket of 4 groups of 8-week-old rats to construct the ectopic osteogenesis model.Histological,immunological and imaging methods were used to evaluate the difference of ectopic osteogenesis ability among the 4 groups of animals after 1 and 2 months,respectively.Results:1.The HA porous scaffolds with groove structure（porosity of 82.6%,pore size of 750-900μm and groove structure of 25-30μm）were prepared successfully.HA scaffolds performed good inductivity of osteogenic differentiation in vivo and in vitro.2.Differential expression of miRNA during in vivo and in vitro osteogenic differentiation induction of HA scaffold（compared with the control group,58 miRNA expressions were up-regulated in ectopic osteogenesis HA samples,41 miRNA expressions in HA+BMSCs samples,and 43 miRNA expressions in HA+MC3T3-E1 samples）.Through sequencing data analysis,filtration and verification,it was found that only one miRNA,miR-129-5p,was highly expressed both in vivo and in vitro osteogenic differentiation induced by HA scaffold.3.The osteogenic differentiation potential and cell migration ability of BMSCs and MC3T3-E1 cells overexpressed with miR-129-5p were significantly increased,while those with the inhibited miR-129-5p decreased.Mechanically,miR-129-5p targeted the CAMP-dependent protein kinase inhibitorα（Pkia）and activated PKA activity.Moreover,the phosphorylation levels ofβ-catenin（Ser-675）and CAMP-reactive element-binding protein（CREB）were dose-dependent with the overexpression of miR-129-5p.The preculture of H89（a PKA inhibitor）significantly weakened the promotion effect of miR-129-5p overexpression on cell osteoblast differentiation and cell migration.4.In vivo experiments showed that compared with the control group,new bone area,bone morphogenetic protein-2（BMP2）expression and surface bone mineral density were significantly increased in the miR-129-5p overexpressed BMSCs group,while converse results in miR-129-5p silenced group.Conclusions:1.HA scaffolds with grooves have good osteogenic differentiation induction both in vivo and in vitro.2.MiR-129-5p is the only miRNA that is highly expressed both in vivo and in vitro during osteogenic differentiation induced by HA scaffolds.3.miR-129-5p targets Pkia to activate PKA,and then promotes the phosphorylation ofβ-catenin（Ser-675）and CREB to help it enter the nucleus and enhance the transcriptional expression of osteogenic genes.4.The inductivity of osteogenic differentiation of HA scaffolds modified with miR-129-5p overexpression was significantly enhanced in vivo. |
