| Background and ObjectiveThe ultimate aim of periodontal treatment is to achieve complete periodontal regeneration, which means the regeneration of the soft (gingival connective tissue and periodontal ligament) and the hard tissue (cementum and alveolar bone) of the periodontium. Current therapeutics for periodontal disease fail to completely and reliably reconstitute all tissues destroyed through periodontal inflammation. The cell-based therapy has been a promising alternative for clinical trial in periodontal reconstitution. Seed cells, scaffold materials, growth factors are the main factors in the traditional tissue engineering research. It is the hotspot in periodontal tissue regeneration medicine to looking for the most effective stem cells with abundance and ready access, and an appropriate carrier for cell transplantation.To date, multiple mesenchymal stem cell lineages have been found in tissue engineering. Bone marrow stem cells (BMSCs) have been extensively studied in periodontal regeneration, because of their capacity of self-renewal and multiple differentiation potential. PDLSCs are identified as a promising seed cells in periodontal regeneration.In recent years, PDLSCs have been isolated from Periodontal Ligament and confirmed capable of forming clonogenic colonies, expressing a typical MSC surface marker profile and possessing multiple differentiatial capacity in vitro. Moreover, PDLSCs also have been demonstrated to possess the ability to form new bone in vivo. On account of having properties of stable morphology, uniformly homogenous and faster proliferation, PDLSCs is considered being superior to BMSCs and other dental stem cells for cell therapy in regenerative medicine. Thus, PDLSCs might be potentially a better alternative of cell-based approaches for periodontal regeneration.MSCs are believed to not only directly differentiate into different cell types including osteoblasts, adipocytes and chondroblasts, but also secrete trophic factors that exert chemotactic, mitotic, and differentiation-modulating effects. This effect has been interpreted as a major mechanism of MSCs in tissue repair. However, the process of periodontal or bone regeneration is involved in coordination of osteoblasts and osteoclasts and very little is known about the possible effect of PDLSCs on differentiation of their neighbouring cells, osteoblast-like cells and osteoclast precursors during periodontal tissue regeneration. In this study, the indirect co-culture approach was introduced to preliminarily elucidate the effects of PDLSCs on mature differentiation of osteoblast-like cells or osteoclast precursors.Methods:1. Isolation, cultivation and identification of human PDLSCs and PDLSCs1.1 Isolation, cultivation of human PDLSCsHuman periodontal ligament samples were achieved from healthy individuals as discarded tissues following conventional dental procedures. PDLSCs were isolated, expanded, and cultured. The 2rd-5th passage PDLSCs were used for the following experiments. Cloning of single cells was gotten by the limiting dilution method and monoclonal cells were harvested.1.2 Identification of human PDLSCsThe Colony-forming unit-fibroblasts assay and the Flow cytometric analysis were performed. PDLSCs plated in 24-well plates were cultured respectively in osteogenic medium (a-MEM containing 5% FBS,0.1μM dexamethasone,10 mM β-glycerophosphate, and 50 mg/ml ascorbate-2-phosphate). Cells cultured in a-MEM containing 5% FBS were as control. The mineralized nodules were characterized by 2% Alizarin red staining four weeks later. Cells were incubated adipogenic medium (a-MEM containing 10% FBS,0.1μM dexamethasone,60μM indomethacin, and 50 mg/ml ascorbate-2-phosphate). staining of Oil Red O was performed to identify the oil globules 2 weeks later.2. Effects of PDLSCs on osteogenic differentiation by Co-culture of MC3T3-E1 with PDLSCsTranswell cell culture insert (Corning Costar, Cambridge, MA) with a 0.4-μm pore-size filter was placed in individual well of six-well plates. Two groups were set up:(1) Control group; MC3T3-E1 (5×104 cells/ml) grown alone in six-well plate with an empty culture plate insert placed on top. (2) PDLSCs CC; PDLSCs (3×104 cells/ml) were grown on the inserts on top and MC3T3-E1 (5×104 cells/ml) grown on six-well plates. The cell culture medium was replaced every 3 days. Alkaline phosphatase (ALP) activity, staining, quantitation of ALP, bone sialoprotein (BSP),,osteopontin (OPN) mRNA by RT-PCR and BSP, OPN proteins by western-bloting and mineral matrix deposition by alizarin red staining were performed on MC3T3-E1 cells after incubation for different time.3. Effects of PDLSCs on osteoclastic differentiation by Co-culture of RAW264.7 with PDLSCsCell culture plate and cell culture insert were the same. Two groups were designed:(1) Control group; RAW264.7 (5×104 cells/ml) grown alone in six-well plate with an empty culture plate insert placed on top. (2) PDLSCs CC; PDLSCs (3× 104 cells/ml) were grown on the inserts on top and RAW264.7 (5x 104 cells/ml) grown on six-well plates. The cell culture medium supplemented with lOng/ml macrophage colony stimulating factor (M-CSF) and 20ng/ml receptor activator of nuclear factor-kappaB ligand (RANKL) was refreshed every 2 days. Tartrate-resistant acid phosphatase (TRAP) positive cells counting and quantification of TRAP, tumor necrosis factor receptor-associated factor 6 (TRAF6) and Cathepsin K (CTSK) mRNA by RT-PCR and TRAP, TRAF6 proteins by western-bloting were conducted on RAW264.7 cells after incubation for different time.Results1. Isolation, identification of human PDLSCs and PDLSCsPrimary cultures of the single-cell suspensions obtained from the healthy gingiva formed adherent, clonogenic cell clusters, and the cultured PDLSCs and PDLSCs retained fibroblastic phenotype. Flow cytometry analysis revealed that both cells were uniformly positive for CD44, CD29, CD90, CD105 markers and Stro-1, and did not express hematopoietic stem markers CD45 and endothelial cells markers CD34. To investigate the multiple differentiation potential of PDLSCs, the cells were cultured in the osteogenic medium for 4 weeks. Extensive amounts of mineralized nodules were found in the experimental groups, but the mineralized nodules formed by PDLSCs were scattered. Moreover, after cultured in the adipogenic medium for 2 weeks, PDLSCs were found to differentiate towards adipocytes as indicated by the accumulation of lipid droplets. These findings indicates that the single colony-derived Periodontal Ligament Stem Cells had the basic characteristics of mesenchymal stem cells.2. Effects of PDLSCs on osteogenic differentiation of MC3T3-E12.1 PDLSCs increased ALP activity in MC3T3-E1 cellsALP activity has been widely used as a marker of the early differentiation of osteoblast-like cells. In our research, ALP activity of MC3T3-E1 was measured at day 7 and day 14 of incubation with or without PDLSCs CC and the result showed that ALP activity increased in the presence of PDLSCs.2.2 PDLSCs increased the gene expressions of osteogenic parameters in MC3T3-E1 cellsTo further determine the effects of PDLSCs on osteogenic differentiation of MC3T3-E1, total RNA was extracted from MC3T3-E1 after different time of incubation with or without PDLSCs CC and RT-PCR was performed to measure the gene expressions of osteogenic markers. It was found that the gene expression levels of ALP and BSP were significantly up-regulated but no significant difference in the gene expression of OPN was detected when cells were co-cultured with PDLSCs for 7 days. At day 14 and day 21 the gene expression levels of ALP, BSP and OPN were all significantly higher in the co-culture group than those in the control group. In addi-tion, the results also indicated that the highest expression level of ALP was on day 14 while the highest expression levels of BSP and OPN were on day 21 in the co-culture group.2.3 PDLSCs increased the protein expressions of BSP & OPN in MC3T3-E1 cellsWestern bloting was used to detect the protein expressions of osteogenic markers in MC3T3-E1 cells after different time of incubation with or without PDLSCs. The results demonstrated that the protein expression of BSP increased at day 7, day 14 and day 21, while that of OPN, an osteogenic marker in the later stage, increased at day 14 and day 21 in co-culture group compared with the control group.2.4.PDLSCs increased the mineral deposition in MC3T3-E1 cellsSome studies indicated mineralization by MC3T3-E1 cells occurred in a time-dependent manner and can be easily observed after 3 weeks of culture. PDLSCs significantly enhanced the mineralization in MC3T3-E1 cells when compared with the control group on day 21. The size of mineralization nodule was increased in co-culture group by Alizarin red staining.3. Effects of PDLSCs on osteoclastic differentiation of RAW264.7 cells3.1 PDLSCs improved the osteoclasts-like cell formation from RAW264.7The effect of PDLSCs on osteoclastic differentiation of RAW264.7 cells in co-culture system was examined using TRAP staining. We examined whether PDLSCs had any effect on multinucleated osteoclast-like cell formation in RANKL-stimulated RAW264.7 cells. Result of TRAP staining revealed that PDLSCs markedly increased the osteoclast-like cell formation in RANKL-stimulated RAW264.7 cells.3.2 PDLSCs increased the gene expressions of osteoclastogenic parameters in RAW264.7 cellsTo further elucidate the role of PDLSCs play in osteoclastic differentiation, the expressions of osteoclastic marker genes of RAW264.7 cells with or without PDLSCs co-culture were detected. Result showed that expressions of TRAP, TRAF6 and CTSK were significantly up-regulated upon co-culture with PDLSCs.3.3 PDLSCs increased the protein expressions of TRAP & TRAF6 in RAW264.7 cellsWestern bloting was used to detect the protein expressions of osteoclastic markers in RAW264.7 cells with or without PDLSCs co-culture. Results demonstrated that the protein expressions of TRAP and TRAF6 were increased on day 3 and day 7 when compared with the control group.Conclusion1. Both PDLSCs and PDLSCs share the similar characteristics with other stem cells, including self-renewability, expression of specific surface marker of stem cells and multi-lineage differentiation potential.2. PDLSCs enhanced ALP activity, expressions of ALP, Runx2, BSP, OPN mRNA and BSP, OPN proteins and mineralization matrix deposition in MC3T3-E1, osteoblasts precursors.3. PDLSCs improved maturation of osteoclasts and expressions of TRAP, CSTK, TRAF6 mRNA and TRAP, TRAF6 proteins in RAW264.7, osteoclast precursors.4. These data suggest that PDLSCs regulate both osteoblastic and osteoclastic differentiation, at least partially, in a paracrine fasion. Further investigation on regulation mechanisms of MSCs including PDLSCs on mature differentiation and functions of osteoblast and osteoclast precursors will help in more roundly understanding the mechanisms of MSCs in bone tissue engineering. |
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