| Periodontal disease is a chronic inflammatory and destructive disease involving thealveolar bone, periodontal ligament and cementum proved to be the prime cause of adulttooth loss. Currently, tissue reconstruction and regeneration of periodontal tissue lesions isstill the major bottleneck of periodontal therapy，tissue engineering technology is the bestway of periodontal tissue regeneration, which includes three elements: seed cells, growthfactors and scaffold materials. How to select the appropriate seed cells and growth factors,and ensure the best regulatory effect is the key to its success. Seed cells types range suchas bone marrow mesenchymal stem cells, adipose-derived stem cells, periodontal ligamentstem cells, induced pluripotent stem cells, etc., among which, periodontal ligament stem cells (PDLSCs) have become the first choice of seed cells in periodontal tissueregeneration due to the advantages of tissue origin and differentiation properties. Amongthe many growth factors, vascular endothelial growth factor (VEGF) and basic fibroblastgrowth factor (FGF-2) as two important growth factors have become the focus of people’sattention. FGF-2can promote mesenchymal stem cell proliferation and differentiation.However, its role in the osteogenic differentiation of stem cells is controversial, there arereports that the application of the long-term and high-dose of FGF-2can inhibit osteogenicdifferentiation, while endogenous FGF-2may play an important regulatory role forosteogenic differentiation. VEGF, secreted by mesenchymal stem cells, can enhance thedifferentiation capacity of adipose-derived stem cells, and promote the osteogenic andvascular differentiation of bone marrow derived mesenchymal stem cells as well as theproliferation and migration of the umbilical vein endothelial cells. In addition, in view ofthe affirmative properties of these two factors to promote the vascular differentiation ofmesenchymal stem cells, whether they can obtain satisfactory effects of bonedifferentiation so as to realize the total periodontal regeneration when applied to PDLSCsbecomes our concerns. In this study, we discussed the influence of different applicationmethods of the two growth factors to the PDLSCs for the first time.Objective:FGF-2and VEGF were applied in different application methods to the periodontalligament stem cells to observe the proliferation, migration, adhesion and osteogenicdifferentiation ability, in order to explore its mechanism for promoting periodontalregeneration and optimize the growth factor application methods aiming for betterpromoting effect to periodontal regeneration.Methods:1. Periodontal ligament stem cells were cultured using primary culture method,examine the cellular morphology and cloning formation with the optical microscopy, stemcell surface molecule markers were identified by flow cytometry, Osteoblasts and lipoblasts staining was used to verify the multipotent differentiation capacity.2. Measured cell growth curve using CCK-8, cell cycle was measured by flowcytometry, combined with the quantitative detection of alkaline phosphatase activity toidentify the effective concentration of the two factors to promote the proliferation andosteogenic differentiation of cells.3. Observed the proliferation, migration, adhesion ability changes by CCK-8, woundhealing assay, cell adhesion experiments.4. Alkaline phosphatase qualitative and quantitative analysis, alizarin red staining,real-time RT-PCR, and western blot were used to detect the osteogenic related genes andprotein expression to study their osteogenic differentiation capacity.5. The attachment and spreading properties of PDLSCs pretreated by the two factorson the surface of β-TCP and Bio-Oss bone meal were examined by the scanning electronmicroscope, then implanted subcutaneously into nude mice (n=10), eight weeks later，themice were killed and histological observation was made to evaluate the osteogenic abilityin vivo.Results:1. Primary periodontal ligament stem cells showed adherent growth and a typicalfibroblast-like morphology, possessing a certain colony forming ability; Cells showedremarkable mineralized nodules and lipid droplets after in vitro osteogenic and adipogenicinduction; Flow cytometry showed positive of STRO-1, CD146, CD105, CD90, andnegative of CD34, CD14.2. CCK-8showed that in the serum concentrations of2%, none of the two factorsshowed significant proliferation effect; In the serum concentration of10%,20μg/L FGF-2showed significant proliferation effect; Cell cycle results showed that10μg/L VEGFsignificantly increased the proportion of cells in the active proliferation phase (S+G2M):Alkaline phosphatase quantitative analysis proved that20μg/L FGF-2significantlyreduced ALP activity, while10μg/L VEGF significantly enhanced it. So that we chose theculture medium with10%serum concentration and20μg/L FGF-2and10μg/L VEGF as a working concentration used for subsequent experiments.3. CCK-8showed the combination of FGF-2and VEGF had a synergisticproliferation effect; Wound healing experiment indicated that VEGF can promote cellmigration to the wound site; Cell adhesion test indicated that FGF-2significantlypromoted initial adhesion and stretching of cells on the polystyrene material surface.4. Alkaline phosphatase staining and quantitative detection, alizarin red stainingexperiments, real-time RT-PCR and western blot results showed significantly inhibited cellosteogenic differentiation in FGF-2group, whereas VEGF significantly promotedosteogenic differentiation. The effect of FGF-2is strong enough to reverse the positiveregulation of the VEGF when they were applied in combination. Sequenced application ofFGF-2and VEGF showed greater promoting effects of osteogenic differentiationcompared with the single use of VEGF.5. Scanning electron microscopy proved that the adhesion and stretch of the cellspretreated by the two factors was better on the surface of Bio-Oss bone meal than that ofthe β-TCP material surface.6.8weeks after being implanted subcutaneously into nude mice, HE staining andimmunohistochemical staining of the cell-materials complex show that VEGF canpromote the formation of bone-like structures on the Bio-Oss bone meal surface andexpression of osteogenic-related protein, while the FGF-2group showed the oppositeresults, sequenced application had similar bone inducing effects as the in vitro results.Conclusion:FGF-2and VEGF could promote PDLSCs proliferation in a dose-dependent manner,influenced by the serum concentration, their combination showed synergistic effects;VEGF significantly enhanced the cell migration to the wound site; FGF-2could promotecell adhesion to the polystyrene surface and significantly inhibited cell osteogenicdifferentiation, VEGF could promote osteogenic differentiation, and their sequencedapplication showed obvious osteogenic differentiation effects. |
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