| Research background and objectiveBone tissue is a dynamic remodeling tissue known for its inherent self-healing potential.However,when the size of the bone defect is larger than the critical size,the unique selfhealing and regeneration abilities of this complex tissue will decrease significantly.Especially bone defects caused by trauma or degenerative diseases,such as large-scale jaw defects caused by oral and maxillofacial tumors or trauma,cannot be repaired by self-healing ability.In clinical restoration,the autologous bone sampling is limited by the shape,quantity,and volume,and obvious bone resorption is prone to occur after transplantation,and the donor site is discomfortable due to trauma.Processed allogeneic bone transplantation may causes immunological rejection,and osteogenic capacity is limited.Therefore,there is a considerable demand for regenerative bone grafts contributing to promoting the healing of large bone defects.The unique physiological characteristic of bone tissue is the largest challenge in the clinical development of mimetic synthetic substitutes for natural bones.In recent decades,a large number of clinical studies have been carried out to meet the huge demand for ideal treatment strategies on promoting bone growth.Bioactive polymer scaffolds play a central role in bone tissue engineering by providing a suitable microenvironment for cell attachment and proliferation,and then the scaffolds are degraded slowly to promote the formation of new bone.However,when exploring various available polymers and inorganic biological materials,most failures occur because the following conditions cannot be met,for example,the bone graft does not comply with the requirements of compressive strength,and there is no or poor osteoinductive ability.The hopeful method of bone tissue engineering is a therapeutic strategy on the basis of stem cells,in which the self-renewal and differentiation capabilities of mesenchymal stem cells can promotethe formation of new bone more quickly,and the combined method of stem cells embedded in biodegradable scaffolds is an effective tissue engineering structure.Dental pulp stem cells(DPSCs),were first separated from pulp tissue through enzyme digestion,and they are derived from the ectoderm and migratory neural crest cells and have the characteristics of mesenchymal stem cells(MSCs).In the past ten years,because of ability of self-renewal and differentiation to various cell phenotypes,DPSCs have attracted wide attention from the field of tissue engineering and regenerative medicine.Studies have shown that DPSCs have stronger cloning and proliferation capabilities than bone marrow stem cells(BMSCs).Hence,DPSCs are a potential and ideal cell source in bone tissue engineering.However,DPSCs are adult stem cells with limited proliferation and differentiation potencies.In order to meet the requirements of bone tissue engineering,the proliferation and osteogenic differentiation abilities of DPSCs must be improved as soon as possible.At present,there are three methods in promoting the differentiation and osteogenesis of stem cells:gene transfection,osteogenesis-related factors,and small molecular compounds with biological activity extracted from Chinese herbal medicines.In addition,some biological materials also have a good effect of promoting the osteogenesis and differentiation of stem cells.The separation and identification of small molecule compounds from Chinese herbal medicines has opened up a new demonstration in regenerative medicine and tissue engineering.Phytochemicals have received significant attention worldwide because of the advantages of low cost,high benefit,small side effects,and strong healing ability.Therefore,economical Chinese herbal medicine-derived small molecule compounds with effective identification for the induction of the proliferation and differentiation of DPSCs is an urgent task in bone tissue engineering.Chrysin is a natural flavonoid compound that can be found in various plants and foods,such as passionflower,chamomile,propolis and honey.Many studies have shown that small molecular compounds of flavonoids,including quercetin and epimedium,can induce the differentiation of mesenchymal stem cells.Some scholars have found that chrysin can induce the osteogenic differentiation of pre-osteoblasts MC3T3-E1 by preferentially activating ERK1/2.Hence,studies have proved that chrysin has a great potential to prevent and treat osteoporosis by promoting the osteoblast differentiation.However,the effect of chrysin on the proliferation and differentiation of DPSCs is still not known.Proteomics has become a basic field of biological research and is considered to be a key tool for discovering drug targets.There are many advantages for proteomics applying a large number of sophisticated data analysis methods,because it can describe molecular reactions more directly than gene-level analysis.Therefore,proteomics analysis and differential expression analysis of gap-associated proteins may contribute to clarifying the potential mechanism of chrysin-induced osteogenic of DPSCs.In this study,relatively purified DPSCs cells were cultivated and screened,and the effects of chrysin on the proliferation and osteogenic differentiation of DPSCs were investigated through in vitro and in vivo experiments.Through proteomics analysis,the mechanism of chrysin affecting the osteogenic differentiation of DPSCs was discussed,which provides a theoretical basis for the application of chrysin in bone tissue engineering.Research methods1.In vitro separation,culture and identification of dental pulp stem cellsThe method of tissue mass combined with enzyme digestion was used to treat the third molar pulp tissue(young patients aged 18-22 years old),cultivate primary dental pulp stem cells and detect cell clonality.The expression situation of cell surface markers CD73,CD44,CD90,CD 105,CD34 and CD45 were detected by flow cytometry(FCM).The cells were subjected to osteogenic,adipogenic and chondrogenic induction respectively to identify their multi-differentiation potential.2.The effect of chrysin on the proliferation and osteogenic differentiation of dental pulp stem cells(1)The human dental pulp stem cells were cultivated for 1?5 days with complete culture solution containing chrysin 0,10-6,10-5,10-4,10-3 and 10-2mmol/L,and cell proliferationtoxicity detection kit(cell counting kit-8 CCK-8)was used to detect the effect of chrysin on the proliferation of DPSCs at different time(1d,2d,3d,4d,5d).The mineralized induction solution containing the above different concentrations of chrysin was used to induce DPSCs for 7 days,so as to detect the alkaline phosphatase(ALP)activity of each group.Continued to use the mineralized induction solution containing the above different concentrations of chrysin to induce DPSCs for 14 days,selected Alizarin Red staining to evaluate the degree of mineralization of the cellular calcium matrix in each group,and the quantity real-time polymerase chain reaction was used(QRT-PCR)to detect the mRNA expression levels of DPSCs osteogenesis-related genes ALP,collagen type I(Colla I),Runt-related transcription factor 2(Runx2)and osteocalcin(OCN).The optimal concentration of chrysin(10-5 mmol/L)was screened out.(2)To further confirm that the ability of chrysin on promoting the osteogenic differentiation of DPSCs has been enhanced.The experiment was divided into two groups:the control group was osteogenic medium,and the experimental group was chrysin osteogenic medium.The two groups of DPSCs were subjected to osteogenic induction for 7days,14days and 21 days respectively,and Alizarin Red staining was used to evaluate the degree of mineralization of the cellular calcium matrix of each group.The qRT-PCR technology was used to detect the mRNA expression level of ALP,Colla I,Runx2,OCN related to cellular osteogenesis genes in each group.Western blots technology was used to detect the expression levels of the osteogenic differentiation-related proteins ALP,Runx2,and osteopontin(OPN)in each group,so as to further analyze the effect of chrysin on the osteogenic differentiation potential of DPSCs.3.Osteogenic differentiation of dental pulp stem cells induced by chrysin in vivo(1)In this experiment,a model of ectopic osteogenesis model was established under the skin of nude mouse.?-tricalcium phosphate(?-TCP)with a thickness of 2 mm,a diameter of 5 mm,and a porosity of 75%is used as the scaffold material.The experiment was randomly divided into 3 groups:the first group(n=8),?-TCP;the second group(n=8),?-TCP+DPSCs;the third group(n=8),?-TCP+DPSCs+Chrysin.DPSCs were induced in conventional osteogenic induction solution and osteogenic medium containing chrysin(10-5 mmol/L)for 10 days,and then mixed with ?-TCP.All specimens were transplanted into the back skin of the nude mouse aged 8 weeks old.All samples were collected 12 weeks after surgical implantation,and hematoxylin-eosin staining(HE)staining and Masson staining were carried out.(2)In this experiment,an orthotopic osteogenesis model was established in the rat skull.Sixteen male SD rats aged 8w were selected.And two symmetrical full-thickness critical skull defects(5mm in diameter)were formed on the parietal bone of each rat's skull.32 cases of skull defects were randomly divided into 4 groups:Group 1(n=8),Blank;Group 2(n=8),?-TCP;Group 3(n=8),?-TCP+DPSCs;Group 4(n=8),?-TCP+DPSCs+Chrysin.DPSCs were induced in conventional osteogenic induction solution and osteogenic medium containing chrysin(10-5 mmol/L)for 10 days,and then mixed with ?-TCP.Rat skull specimens were collected 12 weeks after surgery.Then micro computed tomography(MicroCT)scanning and HE staining were performed.4.Gap-associated proteomics analysis of chrysin promoting the osteogenic differentiation of dental pulp stem cells(1)A,B,C are respectively from three different samples of DPSCs.A0,B0,C0 are the control group:DPSCs;A1,B1,and C1 are the experimental group:Chrysin+DPSCs.The two groups of samples were sent to the proteomics platform of the Institute of Human Phenotype Group of Fudan University after osteogenic induction respectively for 10 days.In order to explore the differentially expressed proteins of DPSCs between the experimental group and the control group,a proteomics analysis using liquid chromatography-mass spectrometry(LC-MS/MS)was carried out by us.In this project,the protein quantity of 6 samples was identified,and the difference protein with over twice up regulation and down regulation of A,B,C,in the experimental group and the control group was compared.The Gene Ontology analysis(GO analysis)was carried out on differentially expressed proteins and the interactive analysis was conducted on the differential proteins.Among them,the enrichment of Smad3 protein related to osteogenesis increased the most.(2)In order to further explore the changes of Smad3 expression and activity in DPSCs treated with chrysin,Western blot was used by us to detect the effect of chrysin on DPSCs at different time(0min,15min,30min,60min,and 120min),and to test the expression of protein Smad 3 and P-Smad 3.To determine whether Smad3 activation promotes chrysin-mediated osteogenic differentiation of DPSCs,the effect of Smad3 phosphorylation inhibitor(SIS3)on the osteogenic differentiation of DPSCs was evaluated by us.Alizarin Red Staining was used to evaluate the degree of mineralization of cellular calcium matrix in each group.The qRTPCR technology was used to detect the mRNA expression levels of ALP,Collagen I,Runx2,OCN related to cellular osteogenesis genes in each group.Western blot was used to detect the expression of protein Smad3 and P-Smad3 in each group.Research results1.In vitro separation,culture and identification of dental pulp stem cellsAfter the fresh dental pulp tissue was cultured in vitro by the tissue block combined with enzyme digestion for about 7-10 days,it could be seen that a large number of primary cells crawled out of the tissue block with a fusion degree of 80%.Most of the cells were mostly fibroblast-like cells,with a standard long spindle morphology,and the cell body was full.The cells show a regular directional arrangement.The nucleus is typically oval,composed of 2?3 nucleoli.The nucleoli were clear and located in the center of the cell.Flow cytometry revealed that DPSCs highly expressed the surface markers CD73,CD44,CD90 and CD 105 of mesenchymal stem cells with positive rates of 99.92%,99.98%,99.97%and 99.71%respectively.They did not express the surface markers CD45 and CD34 of hematopoietic stem cells.The DPSCs was stained with Alizarin Red after osteogenic induction for 21 days,and the mineralized nodules in the osteogenic induction group were stained red through macroscopic observation,while those in the control group were colorless.DPSCs was stained by red oil O after adipogenic induction for 28 days,and under the microscope,it could seen that there were obvious red lipid droplets between the cells.DPSCs was stained by alesund after chondrogenic induction for 28 days,and a blue cartilage-like matrix could be seen under the microscope.2.The effect of chrysin on the proliferation and osteogenic differentiation of dental pulp stem cells(1)CCK-8 cell proliferation-toxicity detection kit was used to detect the changes of chrysin with different concentrations(10-6,10-5,10-4,10-3 and 10-2mmol/L)on DPSCsstimulated proliferation at different time(1d,2d,3d,4d,5d).the results showed that there was no statistical difference in cell activity of each group(P>0.05).Chrysin has no obvious effect on the proliferation of DPSCs and has no cytotoxic effect.After culturing DPSCs by chrysin osteogenic solution with different concentrations for 7 days,the test results for alkaline phosphatase activity showed that DPSCs had the highest ALP activity under the action of 10-5mmol/L chrysin.After culturing DPSCs by chrysin osteogenic solution with different concentrations for 14 days,the results of Alizarin Red staining and CPC quantification showed that,compared with other concentrations of chrysin,10-5 mmol/L chrysin induced the most extensively mineralized nodules.qRT-PCR was used to detect the expression level of DPSCs osteogenic-related genes,and the results showed that the mRNA expression of osteogenic-related genes ALP,Colla I,Runx2,OCN was most significantly upregulated when chrysin was 10-5 mmol/L.(2)The control group was the osteogenic medium,and the experimental group was the osteogenic medium containing 10-5 mmol/L chrysin.In the two groups,DPSCs were induced for 7days,14days and 21 days,respectively.The Alizarin Red staining is deeper in the experimental group than that in the control group,and the osteoblasts induction effect is significantly increased.Western blots technology was used to detect the expression quantity of representative proteins related to osteogenic differentiation.Chrysin up-regulated the expression of osteogenic proteins such as ALP,Runx2,and OPN.In the DPSCs treated with chrysin,the mRNA expression levels of ALP,Collal,Runx2 and OCN were increased significantly than that in the control group.However,the increase in gene expression was different at different time during the induction period.Chrysin up-regulated the mRNA expression of early markers ALP and Colla1 for osteoblast differentiation,after 7d and 14d induction.From 14d to 21d,the mRNA expression level of OCN continued to increase.3.Osteogenic differentiation of dental pulp stem cells in vivo induced by chrysin(1)HE staining of subcutaneous ectopic osteogenesis model specimens in the skin of nude mouse showed that the pores of the scaffold material in the ?-TCP group were clear,and the sparse fibrous tissues were arranged disorderly.?-TCP+DPSCs,?-TCP+DPSCs+Chrysin fibrous-like osteoid was formed on the edge of holes of the support materials.The formation of fibrous-like osteoid is the strongest in the ?-TCP+DPSCs+Chrysin group,which is significantly better than ?-TCP+DPSCs,and a wider osteoid formation area could be observed.In addition,the Masson staining results showed that ?-TCP+DPSCs+Chrysin formed the most osteoid.Calculating the percentage of new osteoid in the pore area shows that the ?-TCP+DPSCs+Chrysin group is significantly higher than the ?-TCP+DPSCs group.(2)Micro-CT scanning and HE staining was carried out on rat skull defect model samples.Micro-CT results showed that the bone defect in the Blank group could not be repaired.Compared with the ?-TCP group and ?-TCP+DPSCs group,the ?TCP+DPSCs+Chrysin group showed the richest bone formation,allowing the skull defect in this group to be completely repaired,and the bone volume(BV)and bone mineral density(BMD)of new bone is highest.HE staining showed that pale pink new fibrous osteoid can be observed to grow towards the center of the pore area at the edge of the pore areas of the scaffold material.In ?-TCP group,there were pores and voids,and fibrous bone tissue was adhered to the edge of the scaffold.In ?-TCP+DPSCs group,the pore space was decreased,and fibrous osteoid was loosely arranged.In the ?-TCP+DPSCs+Chrysin combined group,the gap of the new bone-like substance was filled,and the fiber bone mass was the densest,which was significantly higher than that in the Blank group,?-TCP group and ?-TCP+DPSCs group.4.Proteomic analysis of chrysin promoting the osteogenic differentiation of dental pulp stem cells(1)Proteomics analysis.A total of 35,975 proteins were identified in 6 samples.Among the identified proteins,there were 157 proteins whose expression levels were more than twice different between the two groups,86 were up-regulated proteins and 71 were down-regulated proteins.157 differentially expressed proteins were analyzed by GO.In biological processes(BP),39.8%of enriched differential proteins belong to metabolic and energy pathways.In the cell component(CC),60.4%of the enriched differential proteins belong to the cytoplasmic component.In differential protein interaction network nodes,it could be seen that the protein Smad3 was significantly enriched and increased.The above-mentioned proteomic analysis and the findings in previous reports indicated that the differentially expressed protein Smad3 was closely related to osteoblast differentiation.It was speculated that Smad3 may be involved in the chrysin-induced osteogenic differentiation of DPSCs.(2)In order to further investigate the changes of Smad3 expression and activity in the chrysin-treated DPSCs,Western blot was used by us to to detect the expressions of Smad3 and P-Smad3 of chrysin-affected DPSCs at different time(0min,15min,30min,60min,120min),and it was found that chrysin increased the protein expression and phosphorylation of Smad3 in a time-dependent manner.The treatment of Smad3 phosphorylation inhibitor(SIS3)significantly inhibited the osteogenic differentiation of chrysin-mediated DPSCs,the mineralized nodules stained with Alizarin Red were significantly reduced,and the mRNAs expression of ALP,Collal,Runx2 and OCN related to osteogenic differentiation were significantly reduced.The test results of Western blot showed that the expression of Smad3 and P-Smad3 decreased after SIS3 treatment.Research conclusion1.DPSCs with characteristics of mesenchymal stem cells were successfully obtained in this experiment,which provided a good in vitro cell model for the subsequent experiments.2.The chrysin with appropriate concentration has no cytotoxic effect on DPSCs and can promote the osteogenic differentiation of DPSCs.3.When the DPSCs and ?-TCP under the action of chrysin are applied in the body,it can promote the repairment of bone defects and provide a new solution for the treatment of critical bone defects.4.Through proteomics analysis of differential proteins and verification,it is shown that chrysin can further enhance the expression of osteogenic-related genes and proteins for the induction of osteogenic differentiation of DPSCs by increasing the expression of Smad3 and promoting its phosphorylation. |
PDF Full Text Request