Objective：To compare the reactive oxygen species (ROS) levels in young and aged bone marrow mesenchymal stem cells(BMSCs), explore whether redundant reactive oxygen species (ROS) inhibits osteogenic differentiation of aged BMSCs, and investigate the mechanism of ROS enhancement. Methods：Osteogenic differentiation of BMSCs was confirmed by ALP staining and realtime RT-PCR. Fluorescence analysis and Flow cytometry were performed to detect ROS levels. Realtime RT-PCR was performed to measure superoxide dismutase 2(SOD2) and catalase(CAT) expression. Results：Osteogenic differentiation of aged BMSCs was significantly decreased compared to young BMSCs. Redundant ROS inhibited osteogenic differentiation of aged BMSCs. ROS levels in aged BMSCs were enhanced by dysfunction of SOD2 and CAT. Conclusions：Dysfunction of enzymatic antioxidant system leads to oxidative damage, which inhibits osteogenic differentiation of aged BMSCs.

Objective： To evaluate the effect of TNF-α on the autophagy level in periodontal ligament stem cells (PDLSCs) and jaw bone marrow mesenchymal stem cell (JBMMSCs). Methods：The human PDLSCs/JBMMSCs were isolated and cultured by the method of trypsmization, the flow cytometry and real-time quantitative PCR screened the concentration of TNF-α (The maximum concentration that didn’t cause the apoptosis of cells in a short period). Western blot was applied to estimate the level of autophagy and apoptosis at different time points after PDLSCs/JBMMSCs co-culturing with TNF-α. Results： We have successfully screened the concentration of TNF-α. When co-culturing with TNF-α, activated autophagy reduced apoptosis of PDLSCs/JBMMSCs in short time, and vice versa in long time. Conclusions： The inflammatory cytokines TNF-α could activate the autophagy level, and avoid apoptosis of PDLSCs/JBMMSCs under certain conditions.

Objective： To evaluate the effect of TNF-α on the regulation of the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) through NF-κB signal pathways. Methods：hPDLSCs were isolated and cultured by the method of the limiting dilution technique. hPDLSCs multi-differentiation potential was determined by alizarin Red staining. The gene and protein expression of osteogenic was determined by real-time RT-PCR and Western blot. The expression of NF-κB and osteogenic gene of PDLSCs were determined after treatment by TNF-α (10 ng/mL) by real-time RT-PCR and Western blot. Results：TNF-α inhibits proliferation and differentiation potentials of hPDLSCs. TNF-α upregulated NF-κB protein expression and downregulated Runx2 and Osterix protein levels. Besides, BAY 11-7082 can inhibit the effect of TNF-α. Conclusions： TNF-α regulates the osteogenic differentiation of hPDLSCs through NF-κB signal pathways.

Objective: To investigate the inhibitory molecules repressing osteogenic differentiation of PDLSCs in inflammatory microenvironment.Methods:The osteogenic ability of H-PDLSCs and P-PDLSCs was investigated. The expressions of GSK3β/p-GSK3β and β-catenin in H-PDLSCs and P-PDLSCs were examined by Western blot.Luciferase assay was performedto studyβ-catenin/TCF transcriptional activity,using the Dual Luciferase Assay System kit. RNAinterference technique was used to suppressβ-catenin levels in PDLSCs.Human recombinant Wnt3aand lithium chloride (LiCl) was applied during osteogenesis and used Alizarin red stainingto investigate the osteogenic differentiation of H-PDLSCs.Results:PDLSCs from periodontitis patients had impaired differentiation capacity.More strikingly enhanced p-GSK3β and β-catenin were found in P-PDLSCs than H-PDLSCs.Inhibition of β-cateninpreserved the osteogenesis of TNF-α-stimulated PDLSCs. Alizarin red staining showed that the osteogenic differentiation of PDLSCs was decreased significantly after LiCl or Wnt3a stimulation, which had the same effect as TNF-α.Conclusions:GSK3β was required forTNF-α-mediated inhibition of osteogenicdifferentiation in PDLSCs, which may provide a novel potential approach tobone regeneration in inflammatory microenvironments.

Objective：Search for the key miRNA that regulate mouse bone marrow stromal stem cells (mBMSCs) osteogenesis in the microenviroment of estrogen-deficiency-induced osteoporosis.To investigate the key miRNA whether involved in the osteogenic differentiation of mBMSCs and the effect in this progress in estrogen-deficiency-induced osteoporosis.Methods：An ovariectomized animal model was employed. Screen the key miRNA through combination of bioinformatics methods and microRNA gene chip technology in the mBMSCs derived from OVX and Sham mice. Real-time RT-PCR was used to detect the levels of key miRNA in these types of mBMSCs. The miR-21 function was investigated by transfecting pre-miR-21 and anti-miR-21 into mBMSCs. And osteogenic gene and protein expression was determined by Alizarin Red S, Oil red O staining, real-time RT-PCR and Western blot analysis, respectively.Results：OVX model were sucessfully build. And miR-21 were screened. Real-time RT-PCR shows that miR-21 in OVX-mBMSCs decreased compared with Sham-mBMSCs. Upregulation miR-21 in OVX-mBMSCs. Real-time RT-PCR, Western blot, Alizarin Red S and ALP staining suggested the potential osteogenesis of mBMSCs were enhanced..Conclusions：miR-21 was the key miRNA that regulate mBMSCs osteogenesis in the microenviroment of estrogen-deficiency-induced osteoporosis. And miR-21could promote the potential of mBMSCs in nomal and estrogen deficiency-induced osteoporosis.

Objective：To investigate the effect of in vitro extended passaging on the phenotype, proliferation and differentiation characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs).Methods: Methods of enzymatic digestion, tissue explantation and combination of the two were compared for isolation of hUCMSCs. Cell morphology, growth curve, CFU-F forming ability, cell cycle, phenotype and differentiation activity of hUCMSCs after expanding over eighteen passages in vitro were comprehensively assessed. Results：Combination of enzymatic digestion and tissue explantation was the best way to successfully isolate abundant hUCMSCs. After long time expansion in vitro, morphology of hUCMSCs changed from elongated and spindled-shape with narrow cytoplasm to flattened-shape with more lamellopoid. Population doubling time (PDT) increased to more than 60 hours and CFU-F forming ability significantly decreased at P18 suggesting the replicative senescence of hUCMSCs. Mitotic frequency showed more cells in G0/G1-phase, indicating the existence of lower percentage of proliferating cells fraction after long time subsequent passages. MSCs markers were consistently expressed during passaging, while pluripotent stem cells markers Oct-4 and SSEA-4 were negatively expressed at late passages. Specific staining and gene expression detection showed attenuated differentiation viability of hUCMSCs at P18.Conclusions：HUCMSCs were MSCs subpopulation with high proliferation and differentiation viability. Although cellular senescence of hUCMSCs was noted after large amount of expansion in vitro, they still possess stable expression of MSCs markers and the ability of multi-differentiation.

Objective: to compare the periodontal regeneration of different specific-tissue derived mesenchymal stem cells. Methods: human periodontal ligament stem cells (hPDLSCs), human jaw-derived bone marrow-derived mesenchymal stem cells (hJBMMSCs) and human iliac-derived bone marrow-derived mesenchymal stem cells (hJBMMSCs) were cultured in vitro. After osteogenic induction, relative osteogenic tests were performed. PDLSCs induced by JBMMSCs/IBMMSCs were tested osteogenic expression by RTPCR. Cell aggregates (CAs) of PDLSCs and JBMMSCs/IBMMSCs were compound. The osteogenic genes expression were tested; PDL regeneration in nude mice ectopic transplantation was observed. Results: After osteogenic induction, relative osteogenic tests of JBMMSCs were higher than those of IBMMSCs and PDLSCs. PDLSCs induced by JBMMSCs expressed stronger osteogenic genes than by IBMMSCs. CAs of PDLSCs/JBMMSCs expressed stronger osteogenic genes, made more regular and vertical inserting fibers into the surface of CBB, regenerated more new bone matrix than CAs of PDLSCs/IBMMSCs. Conclusions: JBMMSCs and PDLSCs are specific-tissue MSCs and more appropriate for PDL regeneration.

Objective： To compare the level of acetyltransferase KAT2A in periodontal ligament stem cells derived from healthy individuals (H-PDLSCs) with periodontal ligament stem cells derived from periodontitis individual (P-PDLSCs). And to determine the effect of acetyltransferase KAT2A on osteogenic differentiation potential of PDLSCs. Methods： Human PDLSCs were cultured and cloned with limited diluted method. Quantitative RT-PCR and Western Blot were used to examine different expression of acetyltransferase KAT2A in two kinds of PDLSCs. Quantitative RT-PCR, Western Blot and Alizarin Red Staining were used to determine osteogenic differentiation potential of PDLSCs with gene KAT2A knockdown. Results： Quantitative RT-PCR and Western Blot showed that lower expression of KAT2A was detected in P-PDLSCs compared with that of H-PDLSCs, with statistical significance(P<0.05). Quantitative RT-PCR, Western Blot and Alizarin Red Staining indicated that osteogenic differentiation potential was inhibited in H-PDLSCs with KAT2A knockdown and the effect was statistically significant(P<0.05). Conclusions： Periodontitis might repress expression of KAT2A in PDLSCs, which associates with osteogenic differentiation potential.

Objective: To investigate the effect of advanced glycation end products ( AGEs) on the adipogenic differentiation of human periodontal ligament stem cells ( HPDLSCs) and detect the C/EBPβ、PPAR-γ gene expression．Methods: HPDLSCs were isolated by limited dilution of culture cells for single cell clone, The osteogenic differentiation capacity of HPDLSCs was evaluated by Alizarin red staining, the adipogenic differentiation capacity of HPDLSCs was evaluated by oil red staining. HPDLSCs were induced with AGEs, Real time quantitative reverse transcription polymerase chain reaction (real time PCR) was performed to detect the differences of gene expression between the control group and experimental group. Results:After 21 days induction，Alizarin red staining showed mineralization nodules were formation, oil red staining showed lipid droplets were formation. After AGEs stimulated,during HPDLSCs adipogenic differentiation,the formation of lipid droplets increased in the experimental group, the expressions of CCAAT enhancer binding protein β(C/EBP β)、peroxisome proliferator activated receptorsγ (PPAR-γ) in the experimental group were higher than those in the control group,the differences were statistically significant (p<0.05). Conclusions:AGEs promote the adipogenic differentiation capacity of HPDLSCs,and changed the expressions of C/EBP-β、PPAR-γ mRNA levels.