Effect Of Resveratrol/hypoxia Pretreatment On Periodontal Ligament Stem Cells Derived From Aged Individuals

Mesenchymal stem cells derived from periodontal ligament tissues(PDLSCs)have been suggested as one of the most important resource of human stem cells.It is also believed that PDLSCs play an essential role in maintaining the homeostasis of periodontal microenvironment,balancing the physiological remodeling of support tissues,and regulating the root resorption during the course of teeth replacement.What's more,with capacities of proliferation,differentiation and immune regulation,the use of autologous PDLSCs for periodontal regenerative medicine have attracted more and more attentions.It is suggested that functional regeneration of periodontal tissues(Alveolar bone,periodontal ligament,cementum)could be achieved by the use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets in vivo.Although PDLSCs have been considered as the most favorable candidate for use in periodontal regenerative medicine,but an age-related decline in overall cell functionality hinders the use of autologous aged PDLSCs for cell-based therapies.For chronic periodontitis is typically observed in a population of 40-60 years old,and studies have indicated that there are age-related changes in PDLSCs which derived from that population(aged PDLSCs).Comparing with PDLSCs derived from population under the age of 30(young PDLSCs),the capacities of proliferation,colony forming,migration and osteogenesis are declined in aged PDLSCs.In vivo studies also support those foundings,hence,the age-related changes in PDLSCs with respect to proliferation,differentiation and loss of regenerative potential must be considered when autologous aged PDLSCs are used for clinical therapies.For successful cell therapy,there is considerable interest in cell pretreatment/preconditioning prior to in vivo delivery in attempts to combat cell dysfunction as a result of aging or an adverse microenvironment.Recently,resveratrol(trans-3,5,4'-trihydroxystilebene)is found exert various pharmacological activities related to inflammation,the cardiovascular system and cancer development;moreover,this compound possesses many health-benefiting effects such as the promotion of bone formation and combating of aging processes.Subsequent studies have shown that resveratrol can effectively reduce the production of acetylated P53 in stem cells by activating the expression of SIRT1(member of the SIRTUIN family),and reversing the age-related changes of cells.Meanwhile,Hypoxic preconditioning is also considered to be an effective way to enhance the stemness of PDLSCs.It is reported the proliferation potential and migration ability of PDLSCs have been improved by hypoxic preconditioning.It has also been shown that hypoxic enhances the expression of HIF1-? protein,of which increases cementum protein(CEMP)-1 expression in human PDLSCs.However,less works have been done to rescue human PDLSCs from age-associated dysfunction.Therefore,the identification of effective strategies that can rescue human PDLSCs from age-associated dysfunction is critical for these cells to progress from animal studies to the clinical arena.AimTo investigate the differences in proliferation,colony forming and osteogenesis capacities between PDLSCs derived from young individuals and PDLSCs derived from aged individuals.Further,to explore the feasibility of reversing the age-related decline in aged PDLSCs by pretreating with resveratrol or by resveratrol pretreatment followed hypoxia preconditioning,to identify an effective strategy for clinical use.Methods1.Isolation,culture and characterization of PDLSCs: Enzymatic method was used to acquire the prime PDLSCs,limiting dilution method was utilized to obtain single cell-derived colonies.PDLSCs were determined by their surface markers and cell morphology.2.Comparison of PDLSCs derived from young and aged donors: Cell proliferation ability was determined by MTT assay,colony forming capacity was clarified by crystal violet staining.Capacity of osteogenesis was determined by Western Blot,RT-PCR,ALP staining and alizarin staining.Further,Western Blot and RT-PCR were used to determine the expression of Sirt1.3.The effect of resveratrol pretreatment on PDLSCs derived from aged individuals: After pretreated by resveratrol for 3 d,the capacities of proliferation,colony forming,and osteogenesis of aged PDLSCs were determined.Meanwhile,protein and gene expressions were verified by Western Blot and RT-PCR.4.The effect of hypoxia precondition on aged PDLSCs after pretreated by resveratrol: PDLSCs derived from aged individuals pretreated by resveratrol for 3d and with further hypoxia preconditioning for 24 h.Subsequently,the same methods,as we mentioned above,were used to verify the potential of proliferation,colony forming and osteogenesis of PDLSCs.5.Statistical analysis: All data were analyzed by SPSS 19.0,student's t-test and One-Way ANOVA was used for comparison between groups,and results were presented as mean ± standard deviation(Mean ± SD).A p value < 0.05 was defined as statistically significant.Figures were output by GraphPad Prism 5.Results1.PDLSCs isolated from PDL tissues,respectively derived from young or aged individual,were negative for the hematopoietic markers CD31 and CD34 expression,while positive for the mesenchymal-associated marker CD44 CD90 and CD105.However,compared with PDLSCs derived from young individuals,aged PDLSCs showed an age-related declines in the proliferation,and colony formation potentials with statistical significance(p <0.001,p <0.01).What's more,PDLSCs from young donors exhibited a greater potential toward osteogenic differentiation(p < 0.001),and higher expressions of RUNX-2,ALP,SP7 and SIRT1(p < 0.05,p < 0.01,p < 0.001).2.Pretreatment of aged PDLSCs with resveratrol led to,at least to a certain degree,enhanced cell proliferation,increased hypoxia-inducible factor-1alpha(HIF1-?)and sirtuin-1(SIRT1)protein/gene expression,and augmented cell osteogenesis.3.Short-term(24 h)hypoxia preconditioning applied following cell pretreatment did lead to an increasing in cell proliferation compared to age PDLSCs without any treatments(p < 0.05).The potential of osteogenesis in aged PDLSCs tended to be impaired by further hypoxia preconditioning,while compared with PDLSCs only pretreated by resveratrol,and with statistical significant(p < 0.001).Conclusions1.PDLSCs derived from aged individuals exhibit impaired potentials of proliferation,colony forming and osteogenesis compared with young PDLSCs.And the expression of SIRT exists an age-related change in PDLSCs.It can be indicated that aged PDLSCs with resveratrol pretreatment enhance the expression of SIRT1,which resulted in reversing the impaired stem cell functions.Although short-term hypoxia preconditioning applied following resveratrol pretreatment did not necessarily lead to a synergistic effect,our findings suggest that with further hypoxia preconditioning after resveratrol pretreatment has positive effect on cell proliferation capacity,but negative effect on cell osteogenesis potential.2.Although we demonstrated that resveratrol pretreatment tend to be an effective way of reverse the age related decline in age PDLSCs,the concentration of resveratrol is still worthy study.What's more,further studies should be undertaken to identify the relationship between SIRT1 and HIF1-?.