Study On Osteogenic Differentiation Capacity Of PDLSCs In Type 2 Diabetes Mellitus With Periodontitis

ackground and objectivePeriodontitis is closely relating to diabetes mellitus.Nowadays, periodontitis have been gradually taken into important sight as the Sixth Complication of diabetes mellitus, but the relationships between type 2 diabetes mellitus(T2DM)and periodontitis remains unclear. The aim of our research is to investigate the advanced glycation end products and inflammatory cytokines whether affects on the osteogenic capacity of human periodontal ligament stem cells or not in the occurrence and development of T2 DM with periodontitis.Material and methods:1.The culture and identificaton of PDLSCs :These three kinds of PDLSCs were separately isolated and cultured by tissue piece anchorage primary and limited dilution technique in vitro, including the PDLSCs from 20 healthy tissues samples ware cultured(H- PDLSCs), from 20 inflammatory tissue samples of patients with periodontitis were cultured(P- PDLSCs)and from 20 inflammatory tissue samples of T2 DM patients with periodontitis ware cultured(D- PDLSCs). Preliminary identification of PDLSCs was assayed by the cloning efficiency, immunofluorescence cytochemistry staining, flow cytometry, phenotype of surface markers and cellular proliferation etc.2.The healthy PDLSCs were stimulated separately with(1ug/ml、10ug/ml、100ug/ml、200ug/ml)AGEs and(10ug/ml/1ng/ml)AGEs/TNF-αin vitro,the cellularproliferation, cellular morphology,ALP activity, mineralized capacity and genes protein expression were tested.3. The relative genes protein expression were tested separately with real-time PCR and Western Blot after the canonical Wnt signal pathway of PDLSCs was regulated by Dkk-1/Licl and β-catenin si RNA.4. The relative genes protein levels of PDLSCs and AGE/RAGE were detected separately after The NF-κB pathway of PDLSCs was regluted by PTDC.5.The three groups of PDLSCs,the PDLSCs induced with DKK-1/Wnt3 a and CBB were implanted into subcutaneous tissue of nude mice.their osteogenic capacity was investigated with histological observation.Results :1, Isolation, proliferation and differentiation of PDLSCs(1) Isolation, culture and identification of PDLSCs:We successfully isolated and cultured the H-PDLSCs, P-PDLSCs and D-PDLSCs, all of them were exhibited their fibroblastic spindle shape, and the shape was not significant difference. Immunofluorescence chemistry staining results showed that the expression of vimentin of three groups of periodontal ligament stem cells was positive, and their cytokeratin was negative. By the Image-Pro Plus, the percentage positivity for STRO-1 and CD146 from D-PDLSC was lower than that of those from H-PDLSC and P-PDLSC(p<0.05). The positivity of STRO-1: 62.6±1.6%(H-PLDSC), 40.6±2.5%(P-PDLSC) and 27.2±1.6 %(D-PDLSC); CD146: 69.6±3.4%(H-PLDSC), 48.9±1.6%(P-PDLSC)and32.1±0.9 %(D-PDLSC), there were statistically significant difference. At the same time, we used the flow cytometry technology to detect CD14, CD34, CD29, CD90 and STRO-1, further to clear the source and characterization of these stem cells.(2) The ability of Self-renew and proliferation of PDLSCs:Three group of H-PDLSCs, P-PDLSCs and D-PDLSCs have a certain self-renewal capacity, the results showed there were statistically significant differences: D-PDLSC showed lower clonal formation unit(CFU) ability than P-PDLSCs and H-PDLSCs, the CFU rates of each group were 17.8±2.1%(D-PDLSC), 25.0±2.3%(P-PDLSC), and 36.8±4.9%(H-PDLSC). MTT showed that the proliferative ability of D-PDLSC was weaker than those of H-PDLSC and P-PDLSC with the same induction condition after 7 days(p<0.01). In addition, D-PDLSC presented a lower population in cell S+M phases than those of H-PDLSC and P-PDLSC by FCM for cell phenotype analysis.(3) Comparison of Osteogenic and adipogenic differentiation:After 21 days conditional induction, we used alizarin red staining and oil red O staining to observe formation of mineralized nodule and Lipid drops, the results showed that the amount of mineralized nodule was the most for H-PDLSCs, while the least for D-PDLSCs by IPP software analysis, there were statistically significant differences. In addition, after seven- day osteogenic or adipogenicinductionin in vitro, RT-PCR(Real time-PCR) was performed to investigate the expression of genes responsible for H-PDLSC, P-PDLSC, D-PDLSC osteogenitic differentiation, the results showed that the m RNA expressions of Runx-2, ALP, Col-I, BSP, OCN in D-PDLSC were lower than those of H-PDLSC and P-PDLSC, but the expression of adipogenic relative genes has not obvious change. And then, we used western blot to clarify these results.2, Simulating the microenvironment of PDLSCs derived from diabetes mellitus associated with periodontitis in vitro:(1) MTT: Different consequences on the PDLSCs proliferative capacity was result from different concentrations of AGEs, high concentrations(100ug/ml, 200ug/ml) dramatically decreased the proliferation of PDLSCs, and the differences were statistically significant(p<0.05). The lower concentration(1ug/ml, 10ug/ml) almost did not effect on the proliferative capacity of PDLSCs(p>0.05).(2) Calcified nodules: After three weeks induction, cetylpyridinium quantitation and the alizarin red staining displayed that the mineralization capacity of experimental groups was less than that in the control group(p<0.05). With the increase of the AGEs concentration, the mineralization capacity of the periodontal ligament stem cells was reduced(p<0.05).(3) ALP staining: After one-week induction, there was the deepest color in the control group, with the increase of AGEs concentration, the staining was lighter.(4) We used 10ug/ml AGEs with 1ng/ml TNF-a to simultaneously stimulate PDLSCs for seven days, and found that the color was lighter in the PDLSCs treated with AGEs and TNF-a group via ALP staining, and the expression of osteogenic relative genes & proteins was lower in the simultaneous stimulation of PDLSCs through RT-PCR and western blot. After three-group PDLSCs were cultured with the osteogenic medium for 21 days, we utilized alizarin red staining to detect the amount of mineralized nodule formation,the amounts was lower in the PDLSCs treated AGEs and TNF-a group.3, Effects of Wnt signal pathway on the osteogenic ability of PDLSCs(1) After three-groups cells osteogenic induction, the results showed that both the H-PDLSCs group and the P-PDLSCs group was higher expression of DKK-1, but the D-PDLSCs group did not increase. Three-group cells were changed with the osteogenic medium treated with 50ng/ml DKK-1, the number of mineralized nodule formation and the expression of osteogenic relative genes & proteins was higher in the D-PDLSCs+DKK-1 group, as compared with the control, and there were statically significant tdifference.(p<0.05)(2)osteogenic capacity of cells treated with AGEs+DKK-1 group was higher than those in the sole AGEs group using ALP staining and ALP activity In vitro, the expression of osteogenic relative m RNA & protein was in accordance with the staining, there were statically significant difference.(p<0.05)the expression of RAGE in the AGEs+DKK-1 group was lower than in the AGEs group, and the difference was statically significant.(p<0.05)(3) We used β-catenin si RNA to transfect PDLSCs for seventy-two hours, and conformed the transfection efficiency. In the β-catenin si RNA group, the osteogenic ability was observed using with ALP staining and activity, and the expression of osteogenic relative genes and proteins were higher than those of the control group. Moreover, β-catenin si RNA could reduce the expression of RAGE which was the production of PDLSCs treated with AGEs, but invalid for H-PDLSCs or D-PDLSCs.4, Effects of NF-k B signal pathway on the osteogenic ability of PDLSCsWith regards to the P-PDLSCs and D-PDLSCs groups, activation of NF-k B expression was higher than in the H-PDLSCs group. PDTC could reduce the activation of NF-k B expression in the P-PDLSCs and D-PDLSCs group, and only improve the expression of osteogenic relative genes in the P-PDLSCs group, but not in the D-PDLSCs group. PDTC also could decrease the expression of osteogenic relative genes in the H/P-PDLSCs treated with AGEs group, but could not to up-regulate Runx2. Moreover, There was no effect on the AGEs and RAGE by ALP staining and ALP activity.5, histological resultsThe results of histological observation showed that after D-PDLSCs were implanted into nude mice subcutaneously, blue area was less than P-PDLSCs and H-PDLSCs successively. But after P-PDLSCs were treated with DKK-1, blue area was significantly increased.Conclusion:1, We isolated and cultured PDLSCs derived from diabetes mellitusassociated with periodontitis successfully, and found the proliferative ability, osteogenic ability and stemness of D-PDLSCs were lower than those of H-PDLSCs and P-PDLSCs, but there was no difference in the adipogenic differentiation of PDLSCs.2, We used AGEs/TNF-a to successfully create the microenvironment of D-PDLSCs in vitro, The proliferation and osteogenic ability of PDLSCs decreased with the increase of AGEs concentration, and AGEs could deteriorate the effect of TNF-a on PDLSCs. TNF-a could promote the expression of RAGE in PDLSCs, but there was no change on RAGE after PDLSCs were treated with AGEs.3, DKK-1 could rescue the osteogenic ability of D-PDLSCs and PDLSCs treated with AGEs, and drop the expression of RAGE. The inhibition of β-catenin si RNA could promote the expression of osteogenic genes in the D-PDLSCs group, and decrease the expression of RAGE in the PDLSCs treated with AGEs group. These results showed that activation of wnt signal pathway was negatively correlated with the osteogenic ability of D-PDLSCs and PDLSCs treated with AGEs/TNF-a.4, Based on the test of the NF-k B relative indicators and PDTC regulation in three groups, it was no direct correlation between the osteogenic ability of D-PDLSCs and the expression of RAGE and NF-k B.5, Animal experiments showed the osteogenic differentiation of PDLSCs in type2 diabets with periodontitis was obviously lower than those of other two kind PDLSCs in vivo, but the DKK-1 could enhance the osteogenic differentiation of D-PDLSCs.In a word,these results showed that in the occurrence and development of diabetes mellitus associated with periodontitis, the wnt signaling pathway might be mediated AGEs and TNF-a to regulate the osteogenic ability of PDLSCs, and inhibition of wnt signal pathway could improve the osteogenic ability of PDLSCs. Our results suggested that regulation of wnt signal pathway could improve the osteogenic ability of D-PDLSCs, and DKK-1 or small molecular compounds, as the effective drugs, might cure diabetes mellitus associated with periodontitis. Therefore, we hope that we seek for new therapeutic strategies of diabetes mellitus associated with periodontitis, and provide new theoretical and experimental basis.