| Bone defect repair and mesenchymal stem cell induced osteogenesis are hot topics in current medical research.It's an important research field to choose suitable scaffold materials as carriers in which some drug components can be controlled releasing or slow releasing to induce osteogenesis.At present,the main scaffold materials at home and abroad include non absorbable polymethyl methacrylate and absorbable collagen,chitin,hydroxyapatite and so on.The former needs to be removed by the second operation and the latter has a single structure so the drug carrying capacity can not meet the ideal requirements.Therefore,it is necessary to modify or qualify the traditional scaffold material to achieve the drug loading requirements.The scaffold materials used in this study are nano modified hydroxyapatite(Nano-HAp)microspheres,which are bioactive ceramic materials composed of calcium,phosphorus and various of trace elements.They have the same compositions as human bone in inorganic substance of chemical composition and crystal structure.When the materials are combined with natural bone a strong chemical combination is formed with good biocompatibility and bone conductivity.At the same time,being conducive to drug absorption after the particle surface area is improved by micro nano hybrid modifying,the hydroxyapatite microspheres can be used as scaffold materials or drug delivery carriers to enhance the effect of osteoinductive capacity.Theaflavin-3,3'-gallate(TF3)is one kind of polyphenol substance extracted from black tea and it's one of the four main components of theaflavins(TFs).Its characteristic in the molecules of the benzene and ketone structure makes TF3 have a series of treatment effects,for example,antioxidant,inhibition of cell mutation,anti tumor cell proliferation,anti-infection,antiviral and other features.It has been found by foreign scholars that TF3 could inhibit DNA methylation of the osteoclast precursor cells and inhibit their differentiation to osteoclasts,so as to reduce bone absorption.However,the application of TF3 in osteogenesis,its effect on osteoblasts and whether it can be involved in the osteogenesis induction of bone marrow mesenchymal stem cells(BMSCs)has not been reported yet.The purpose of this study was to observe the repair effect on bone defects by TF3 loaded onto nano-modified hydroxyapatite microspheres.In the research,the bone marrow mesenchymal stem cells were primarily cultured by a whole bone marrow adherent method and the biological effect and mechanism of TF3 to induce osteogenic differentiation of BMSCs were discussed.Moreover,nano-structured HAp microspheres sustaining TF3release were fabricated,and the local delivery system for TF3 to promote bone regeneration in rat femoral defects was constructed.This slow-release system was applicated to evaluate in vivo effect on bone repair of TF3 and to explore new ideas and methods for the repair of bone defects.Part one Primary culture and inducing differentiation of rat BMSCsObjectives:To identify the purity and the multidirectional induced potential on osteogenic differentiation and adipogenic differentiation of BMSCs which were primarily cultured by a whole bone marrow adherent method.Methods:1 Rat BMSCs were isolated,primarily cultured and passaged by a whole bone marrow adherent method.The situation of cell growth and morphological changes were observed under a microscope.2 Growth curve was made according to the daily optical density measured for the activity of rat BMSCs at the 3~rdd passage.3 In order to identify the purity of rat BMSCs,surface antigens of CD29,CD34,CD45 and CD90 with PE markers were detected by Flow Cytometry.4 Rat BMSCs were cultured in osteogenic medium for osteogenic induction,and alkaline phosphatase(ALP)activities of the cells were detected.Meanwhile,cells were stained with Alizarin Red(AR)to identify the osteogenic capacity of rat BMSCs and the osteogenic progress was observed.5 Rat BMSCs at the 3~rdd passage were selected for lipid induction.After15 days,Oil Red O staining was carried out to identify their ability to differentiate into adipocytes.6 Statistical analysis:Variance analysis was used to compare the difference between the experimental results of each group.The statistical significance was considered when P<0.05.Results:1.The primary culture and passage of BMSCs were successfully performed.The primary cells were spherically shaped.However,after passaged,the morphology of cells changed to be in different forms:spindle polygonal,spherical,irregular shapes and so on.2.Growth curve of BMSCs was consistent with the characteristics of the growth curve of stem cells,and it was found that the cell growth period from the third to fifth days was the logarithmic growth period of rat BMSCs.3.Results of the identification of the surface antigens of rat BMSCs:The positive expression rate of CD29 was 100%while the positive expression rate of CD90 was 96.9%.Moreover,the positive expression rate of CD34 was 0.7%and the positive expression rate of CD45 was 0.8%.It was known from these results that the purity of rat BMSCs could meet the requirements of experiments behind.4.After osteogenic induction,there was significant difference among the ALP activities at different points of time.The ALP activities at day 7~10 were higher than those of day 3;the ALP activities at day 10 was higher than those of day 7(P<0.05).After osteogenic induction,there was significant difference among the AR staining results at different points of time.There was no significant difference between the semi-quantitative results at day 3 and day 7;the semi-quantitative results at day 14 and day 21 were higher than those of day 3 and day 7;and the semi-quantitative results at day 21 were higher than those of day 14(P<0.05).5.BMSCs was stained with Oil Red O after 15 days of lipid induction.Orange red lipid droplets could be observed in the cells,and the lipid differentiation appeared.Summaries:1.Growth curve of BMSCs was consistent with the characteristics of the growth curve of stem cells.2.Through Flow Cytometry,osteogenic and adipogenic differentiation,it was confirmed that the obtained BMSCs can meet the experimental requirements.Part two Study on the osteogenesis mechanism of theaflavin-3,3'-gallate on rat BMSCsObjectives:To detect the effects on the proliferation of rat BMSCs and the related mechanism of inducing BMSCs osteogenesis of TF3.Effects of TF3 on the expression levels of osteogenic gene,bone morphogenetic protein2,collagen type I,Runt related transcription factor 2 and osteocalcin,angiogenic gene,vascular endothelial growth factor and angiopoietin 1expressed in rat BMSCs would be dicussed.Methods:1 The purity of the TF3 sample was analyzed by HPLC and the structure was identified.2 L-929 cells were used to perform a cytotoxic reaction grading test on TF3.3 The proliferation of rat BMSCs dealt with different concentrations of TF3 solution were measured by CCK-8 method,and the suitable concentrations of TF3 solution were selected to be applied to the follow-up experiments.4 After the treatment of different concentrations of TF3 solution on rat BMSCs,ALP staining and quantitative detection were performed on the 3rd,7th,10th days respectively.5 After the treatment of different concentrations of TF3 solution on rat BMSCs,AR staining and AR sem-quantitative detection were performed on the 21st days.6 After the treatment of different concentrations of TF3 solution on rat BMSCs,the effects of TF3 on the expression of osteogenic and vascular genes were detected.7 After the treatment of different concentrations of TF3 solution on rat BMSCs,the expression of BMP-2 signaling pathway proteins were detected by Western Blotting.Then the BMP-2 signaling pathway was blocked,and the expression of BMP-2 signaling pathway proteins were detected after the action of TF3.8 After the treatment of different concentrations of TF3 solution on rat BMSCs,the expression of MAPK signaling pathway proteins were detected by Western Blotting.Then the MAPK signaling pathway was blocked,and the expression of downstream proteins Runx2 and OCN were detected after the action of TF3.9 Statistical analysis:Variance analysis was used to compare the difference between the experimental results of each group.The statistical significance was considered when P<0.05.Results:1.The purity of the TF3 sample purchased by HPLC was 98.04%.2.The relative growth rate of cells treated with 10?M TF3 was 111.49%and the cytotoxicity level was grade 0.The RGR value of cells treated with100?M TF3 was 87.64%and the cytotoxicity level was grade 1.The RGR value of cells treated with 1mM TF3 was 55.83%and the cytotoxicity level was grade 2.3.After osteogenic induction,the proliferation activity of BMSCs decreased.Compared with 0?M group,TF3 at concentrations of 0.1?M,1?M,5?M and 10?M concentration-dependently promoted BMSCs proliferation.However,cell proliferation inhibition occurred at TF3concentrations of 20?M and 40?M.4.After the treatment of different concentrations of TF3 solutionon on rat BMSCs,the results of ALP staining increased with the concentrations of TF3solution,and the quantitative results of ALP activity were positively correlated with the concentrations of TF3 solution and the time of treatment.5.After the treatment of different concentrations of TF3 solutionon on rat BMSCs,the results of AR staining and semi-quantitative analysis of AR staining were all increased with the concentrations of TF3 solution.6.After detection of RT-qPCR method,we knew that the expressions of osteogenic genes,BMP-2,COL-I,Runx2 and OCN,and the expressions of angiogenic genes,VEGF and ANG1 could be upregulated by TF3.The optimal concentration of TF3 to promote osteogenic differentiation was10?M,while the optimal concentration of TF3 to promote angiogenic differentiation was 5?M.7.TF3 significantly up-regulated the expression level of pathway proteins BMP-2,p-Smad1/5,Runx2 and its downstream protein OCN.The expression of each protein was increased with the concentration of TF3 and there were significant differences compared with 0?M group(P<0.05).The expression levels of BMP-2,p-Smad1/5,Runx2 and OCN protein were decreased in varying degrees after treatment with pathway blocker Noggin and there were significant differences compared with TF3 group(P<0.05).However,these values were higher than 0?M group with significant differences(P<0.05).8.TF3 up-regulated the phosphorylation level of extracellular signal regulated kinase protein(ERK)and p38 protein in MAPK signaling pathway,while it had no effect on the phosphorylation level of c-Jun amino terminal kinase protein(JNK).The expression levels of downstream proteins Runx2and OCN were decreased after ERK pathway and p38 pathway were blocked.These values were lower than the expression levels of TF3 group with significant differences(P<0.05),but higher than that of 0?M group with significant differences(P<0.05).After JNK pathway was blocked,the expression levels of Runx2 and OCN remained unchanged.Summaries:1.Low concentration of TF3 promoted the proliferation of rat BMSCs,while high concentration of TF3 inhibited the proliferation of rat BMSCs.2.TF3 could promote the expressions of osteogenic and angiogenic genes of BMSCs.3.BMP-2 signaling pathway participated in the osteogenic differentiation of rat BMSCs induced by TF3.4.ERK pathway and p38 pathway in the MAPK signaling pathway participated in the osteogenic differentiation of rat BMSCs induced by TF3.Part threeStudy of nano modified hydroxyapatite loaded theaflavin-3,3'-gallate to promote the femur defect repair in ratsObjectives:To fabricate nano-structured HAp microspheres sustaining TF3 release and to construct the local delivery system for TF3 to promote bone regeneration in rat femoral defects.This slow-release system was applicated to evaluate in vivo effect on bone repair of TF3.Methods:1 The Nano-HAp microspheres were synthesized by the precursor water heat conversion technique,and their phenotypic characteristics were observed under the electron microscope.The Nano-HAp powder was tested by X-ray diffractometry to characterize its phase and crystallinity.2 The slow-release system of TF3 was constructed using Nano-HAp loaded TF3 whose solution concentration was 100?M and 1000?M,respectively.Meanwhile,the slow-release performance of TF3 sustained release system was detected by high performance liquid chromatography(HPLC),and the sustained release curve was made.3 Animal experiment:Male SD rats(8 weeks old)were anaesthetized with sodium pentobarbital by intraperitoneal injection.Then,medial defects(diameter,3.5 mm;depth,4 mm)were generated in the left and right femoral condyles using a dental micro-drill.Bone defects in bilateral femoral condyles were filled with Nano-HAp microspheres containing TF3 or not.Rat femurs were assigned to 3 groups administered Nano-HAp microspheres without TF3(group A,Nano-HAp,n=6),loaded with TF3 at 100?M(group B,Nano-HAp/TF3-100,n=6),and TF3 at 1000?M(group C,Nano-HAp/TF3-1000,n=6).4 The rats were sacrificed at 8 weeks after operation and the femurs were taken for Micro-CT detection.Moreover,the microstructural indices of the newly generated bone,such as bone mineral density(BMD)of the trabecular bone,bone volume/tissue volume(BV/TV),trabecular number(Tb.N),and trabecular space(Tb.Sp),in the defect location were determined and compared between each group.5 The specimens were fixed in 0.5%paraformaldehyde at 4°C,and decalcified with 10%EDTA.This was followed by paraffin embedding,sectioning,and staining with hematoxylin and eosin(H&E)for histological assessment under the microscope.The area of new bone formation in the bone defect was analyzed on Image Pro 5.0 analysis system.6 Statistical analysis:Variance analysis was used to compare the difference between the experimental results of each group.The statistical significance was considered when P<0.05.Results:1.Low magnification imaging showed that Nano-HAp products had 3D architecture with comparable morphologies;diameters were 120~150?m.High magnification imaging further revealed that the spherical nano HAp were composed of nanowires with diameters of 50~80 nm and lengths of1~2?m.The results of XRD atlas analysis showed that the synthesized products were completely in line with the pure HAp phase.2.Nano-HAp microsphere particles had sustained drug delivery ability to release TF3,and the release rate of group Nano-HAp/TF3-1000 in initial 3 h was higher than that of group Nano-HAp/TF3-100.After 3 h,the release rate of group Nano-HAp/TF3-1000 was lower than that of group Nano-HAp/TF3-100.3.Micro-CT was applicated to the reconstruction of the new bone morphology of the injured part in the femurs.Compared with group Nano-HAp,significantly increased BMD,BV/TV,Tb.N and Tb.Sp reduction were found in the group Nano-HAp/TF3-1000 and group Nano-HAp/TF3-100.A single factor analysis of variance was carried out and there were statistical differences in the comparisons between each group(P<0.05).4.Analysis of HE staining results:Some empty lacunae existed after decalcification of Nano-HAp microspheres and there were different degrees of new bone formation around the lacunar.The percentage of new bone formation in group Nano-HAp/TF3-1000 and group Nano-HAp/TF3-100was higher than that of group Nano-HAp.A single factor analysis of variance was carried out and there were statistical differences in the comparisons between each group(P<0.05)Summaries:1.Nano-HAp/TF3 slow-release system was successfully fabricated.2.TF3 loaded onto Nano-HAp had enhanced effect for the repair of femur defects.Conclusions:1.Low concentration of TF3 promoted the proliferation of rat BMSCs,while high concentration of TF3 inhibited the proliferation of rat BMSCs.2.TF3 could promote the expressions of osteogenic and angiogenic genes of BMSCs.3.BMP-2 signaling pathway participated in the osteogenic differentiation of rat BMSCs induced by TF3.4.ERK pathway and p38 pathway in the MAPK signaling pathway participated in the osteogenic differentiation of rat BMSCs induced by TF3.5.Nano-HAp/TF3 slow-release system was successfully fabricated.6.TF3 loaded onto Nano-HAp had enhanced effect for the repair of femur defects. |
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