| PurposesPeriodontitis is a chronic progressive disease that mainly affects the supporting tissues around the teeth,including the gums,periodontal ligament,alveolar bone and cementum.It develops from gingivitis caused by plaque accumulation.Clinically,gingival bleeding,periodontal pocket formation,and loose of teeth are the main manifestations.In severe cases,the teeth may fall off.In terms of treatment,in addition to improving awareness of residents' oral hygiene to prevent periodontal disease,current researches are more focused on the use of tissue engineering techniques to achieve regeneration of periodontal tissue after the control of inflammation.In the three basic elements of the technology(scaffold material,seed cells,growth factors),periodontal ligament stem cells are considered the ideal source of seed tissue cells because they can be successfully induced to form periodontal ligament,alveolar bone and cementum in vitro.However,due to their limited source and spontaneous differentiation during in vitro culture,more researches are focused on how to achieve large-scale expansion of cells in shorter culture time before transplantation.And maintain its stem cell properties as much as possible.Existing studies have confirmed that the addition of basic fibroblast growth factor to culture medium during in vitro culture can promote the proliferation and adhesion of periodontal ligament stem cells and maintain their multi-lineage differentiation ability,while there is a certain inhibitory effect on its osteogenic differentiation ability in early culture stage.As a multifunctional cytokine,transforming growth factor beta regulates cell proliferation and differentiation,and as a receptor inhibitor of transforming growth factor,it has been reported that A83-01 can inhibit the differentiation of pluripotent stem cells and maintain self-renewal and increase its amplification efficiency.Therefore,the aim of this study was to investigate whether the combination of A83-01 and basic fibroblast growth factor in the in vitro culture of human periodontal ligament stem cells can promote their proliferative capacity and improve their biological properties.MethodsThe periodontal ligament tissue was harvested from the premolar and third molars obtained from orthodontic extraction patients(19-29 years old).The primary periodontal ligament cells were obtained by tissue block method,and the P1 cells were purified by limiting dilution method.After amplification,it can be frozen or passaged to the P3 generation for use.?The CCK8 assay detects cell proliferation ability and screens the optimal concentration of the drug that promotes cell proliferation.?The periodontal ligament stem cells were separately and combinedly pretreated by A83-01 and FGF-2 for 48 hours and then changed to normal culture medium for further culture.?Apoptosis detection assay detects the apoptosis rate of pretreated cells.?Alkaline phosphatase activity,alizarin red staining,Western blot and RT-PCR assay to detect changes in osteogenic ability in different periods.?RT-PCR assay for expression of sternness related genes c-Myc and Nanog.?Enzyme-linked immunosorbent assay(ELISA)was used to detect the paracrine efficacy of the cellular inflammation-related factor IL-6,the angiogenesis-related factors VEGF and TGF-?.ResultsCompared with the blank control group,combined pretreatment with A83-01 andbasic fibroblast growth factor significantly increased the proliferative capacity of periodontal ligament stem cells,and decreased the apoptosis rate,and amplified the expression of stemnss genes in osteogenesis.In the late induction stage,the osteogenic differentiation of the cells is promoted,and the paracrine effect of the cells is enhanced.In addition,the combination group also showed significant advantages in promoting cell proliferation,sternness maintenance,and paracrine effects compared to the alkaline fibroblast growth factor alone.ConclusionsCompared with the use of basic fibroblast growth factor alone,the combination of A83-01 and basic fibroblast growth factor has an advantage in promoting proliferation,dry maintenance and cytokine secretion of human periodontal ligament stem cells.This may be an effective strategy for the expansion and culture of human periodontal ligament stem cells and optimization of biological properties. |
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