| The traditional treatment methods for periodontitis in clinical include periodontal flap,guided tissue regeneration,material transplantation and the use of recombinant growth factors.These methods can partly control inflammation and repair periodontal tissue.For the regeneration of complete functional periodontal tissue,the traditional treatments are limited.Since stem cells in human periodontal ligament with multiple differentiation,generally termed periodontal ligament stem cells(PDLSCs),were isolated in 2004,a great deal of evidence has indicated that PDLSCs were more suitable as seed cells for periodontal cytotherapy than stem cells derived from other sources.However,the way to harvest PDLSCs is invasive and the amplification procedure in vitro is cumbersome.Therefore,it is a challenge to promote the proliferation and differentiation of PDLSCs in periodontal tissue engineering.Human urine-derived stem cells(USCs)were first isolated from human urine in 2008;USCs are noninvasive,easy to collect and stable in culture with multiple differentiation.USCs have been used as seed cells in various regenerative medicine,including bladder and urethral reconstruction,nerve regeneration and wound healing.Through paracrine effects,USCs efficiently attenuate severe hind-limb ischemic injury and particularly strengthen the chondrogenic differentiation of bone marrow stromal cells(BMSCs)after passaging in vitro by releasing trophic factors.The trophic factors of USCs include a variety of growth,inflammatory,and immunomodulatory factors.Given the above data,the goal of this study was to investigate the paracrine effects of USCs on PDLSCs through noncontact coculture.In vitro experimentMaterials and methods: Primary PDLSCs and USCs were isolated in vitro and identified by flow cytometry and multidirectional induction.Transwell chambers with 0.4 ?m membrane pores were used to set up a noncontact coculture system,PDLSCs and USCs were indirectly coculture at different ratios(PDLSCs/USCs,1/0.5,1/1 and 1/2).CCK8 assay was used to measure the effect of USCs on the proliferation of PDLSCs on the 1st,3rd,5th and 7th days.Alkaline phosphatase(ALP)staining was performed in PDLSCs after 7 days osteogenic induction,and the expression of ALP,RUNX2,OCN,POSTN and CEMP1 in PDLSCs was detected by RT-PCR and Western blot.Alizarin red staining and quantitative analysis were performed after 21 days osteogenic induction.The levels of VEGF and b-FGF in USCs serum-free medium and indirect co culture system were detected by ELISA.After 10 days osteogenic induction,PDLSC sheets were harvest and the surface structure was observed by scanning electron microscopy(SEM)and Hematoxylin and eosin(H&E)and immunohistochemical staining.Results: 1.PDLSCs and USCs could form mineralized nodules and lipid droplets by alizarin red staining and oil red O staining.In addition,flow cytometry analysis showed that PDLSCs and USCs were positive for CD90,CD105 and CD146 and negative for CD31,CD34 and CD45.2.The results of CCK8 showed that USCs could promote the proliferation of PDLSCs through indirect coculture.3.RT-PCR and Western blot showed that USCs could significantly promote the expression of ALP,RUNX2,OCN,POSTN and CEMP1 of PDLSCs.4.ELISA showed that the USCs serum-free medium contained VEGF and b-FGF,and USCs could increase the expression of VEGF and b-FGF in the medium in the indirect co culture system.5.The results of SEM and H&E staining showed that the PDLSC sheets cocultured with showed more cellular layers,extracellular matrix and denser reticular structure.Immunohistochemical staining showed that the PDLSC sheets cocultured with USCs had stronger positive expression of OCN,POSTN and CEMP1 than the corresponding control group.Conclusion: USCs can promote the osteogenic and cementogenic differentiation and proliferation of PDLSCs and accelerate cell sheet formation and differentiation.In vivo experimentsMaterials and methods: Ten 6-week-old immunodeficient mice were used in this experiment.Thirty milligrams of nano hydroxyapatite(nano-HA)was wrapped into a ball surrounded by three-layered cell sheets and then implanted into the subcutaneous of nude mice.Divided into experimental group and control group,experimental group: thirty milligrams of nano-HA and PDLSC sheets cocultured with USCs at a PDLSCs/USCs ratio of 1/2;control group: thirty milligrams of nano-HA and PDLSC sheets without USCs.Six weeks after transplantation,the mice were euthanized,and the grafts were removed for histological analysis.Results: 1.H&E and Masson's trichrome staining showed that a quantity of dense and notable collagenous fibers had formed in both groups,while regenerated tissues in the corresponding control group were few in number and scattered on the surface of nano-HA.2.Immunohistochemical staining showed that the expression of POSTN and CEMP1 in the experimental group was more positive than control group.Conclusion: USCs can promote the differentiation of PDLSCs in vivo,which has the potential to be used in periodontal tissue engineering. |
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