| Periodontal membrane is important to support the teeth organization.Stimulation of periodontal membrane with periodic light occlusal force in the state of masticatory function, thus has a strong ability of adaptive renovation. In 2004, with the periodontal ligament stem cells the success of the isolation and culture, a large number of studies have confirmed that the periodontal ligament stem cells in periodontal membrane proliferation differentiation ability strong cell clusters, stress stimuli periodontal health is directly related to the active hight or low. Studies have shown that PLDSCs in reimplantation of avulsed teeth can promoted the periondontal healing; however, the effect was still unsatisfied. The possible explanation for this observation includes three parts as follows: firstly, single cell suspension is easy to running off. Secondly, the lack of growth factor which may inhibit the proliferation and differentiation of PDLSCs. Thirdly, PDLSCs which cultivated in vitro lack the regulation of biomechanical environment in vivo. In view of the first two questions, our previous studies have induced single cell suspension of PDLSCs into cell membrane to avoid the loss of cells. And autologous platelet-rich fibrin(PRF) membrane was used in our previous studies as a growth factor-rich scaffold. Our previous research was constructed a cells transplant combined with cell sheet fragments of PDLSCs and plate-rich granules which has an obvious therapeutic effect on avulsed tooth reimplantation. Periodontal membrane is a kind of tissue which withstand a variety of stress stimuli, such as chew, bite, orthodontic forces and so on. Accordingly, the biomechanical environment is crucial in periodontal membrane regeneration. Previous studies have shown that no matter what kind of medical treatments for periodontal regeneration, it is necessary to maintain teeth physical mobility and chomp stimulation; however, the mechanism is unclear. Mechano-growth factor(MGF) is an insulin-like growth factor-1( IGF-1) variant of alternative splicing that is sensitive to mechanical stress.MGF is not expressed in most of normal tissues, but it is synthesized rapidly when tissue is injured or endured the mechanical stress. MGF has been found in muscles, nerves, heart and other tissue, but the expression and function of MGF in periodontal tissue has not been reported.This experiment is divided into three parts:Experiment 1: the separation of hPDLSCs cultivation and construction of the cell membrane.Application of the piece of separation method and enzyme digestion method to culture the periodontal ligament cells, and use different ways to identify its stem cell characteristics for example: the cells surface marker identification, determination of clone formation rate and osteogenesis induction. MTT methods to determine the growth curve of hPDLSCs, and use liquid film induced hPDLSCs into cell membrane. The results show that: separation of hPDLSCs has a good ability of proliferation and clone formation, and can express the ectomesenchymal stem cell surface marker, and have a potential into adipogenesis, osteogenesis, and has a wealth of extracellular matrix of the cell membrane.Experiment 2: The influence of dynamic pressure on hPDLSCs and mechanobiological effects of MGF.The hPDLSCs and hPDLSCs sheet wes treated with different pressure(0.1 Hz)in vitro, including 0-90 k Pa,0-120 k Pa,0-150 k Pa,﹣20-90 k Pa,﹣20-120 k Pa,﹣20-150 k Pa,﹣40-90 k Pa,﹣40-120 k Pa and﹣40-150 k Pa. The above 9 groups of BMSCs were treated with pressure 1h, while the control hPDLSCs were treated without pressure. CCK-8 was used for detecting the proliferation of hPDLSCs. Real-time PCR was used for detecting the m RNA expression level of MGF, IGF-1, IGF-1Ea, Col-Ⅰ, ALP, RUNX2 and PCNA. The results show that dynamic hydrostatic pressure 0-120 k Pa and ﹣ 20-120 k Pa significantly promote the proliferation of hPDLSCs. The m RNA expression level of MGF and IGF-1Ea were significantly promote when 12 h and 36 h after pressure(0-120 k Pa, ﹣20-120 k Pa) stimuli. The expression of MGF m RNA peak in 24 h after dynamic hydrostatic pressure ahead of IGF-1. The 0-120 k Pa pressure significantly promote expression of MGF m RNA which is highest expression level among different dynamic pressure, the second is﹣20-120 k Pa pressure. In addition, the study found that 0-120 k Pa pressure significantly promote expression of Col-Ⅰ、ALP、RUNX2 and PCNA m RNA. According to these results our study single out most appropriate pressure(0-120 k Pa, ﹣20-120 k Pa) that significantly promote expression of MGF m RNA.Experiment 3: The influence of dynamic pressure on hPDLSCs differentiation and MGF response of hPDLSCs/PRF co-culture system.hPDLSCs/PRF co-culture system load in 0-120 k Pa, ﹣20-120 k Pa periodic force. The Real-time PCR method to detect the expression of the MGF, IGF-1, IGF-1Ea, Col-Ⅰ, ALP, RUNX2 and PCNA m RNA in the cell membrane, and using the Western Blotting and immunohistochemistry method to detect the expression of MGF and IGF-1 protein in the cell membrane. The results show that: the highly expression of MGF, IGF-1,IGF-1Ea m RNA when under 0-120 k Pa and ﹣ 20-120 k Pa pressure in the hPDLSCs cell membrane.And compared with cultured hPDLSCs alone under pressure, MGF m RNA in the co-culture system express significant rise. Under the 0-120 k Pa, the Col-Ⅰ、ALP、RUNX2 and PCNA m RNA expression quantity increased significantly in hPDLSCs of co-culture system. However, only Col-Ⅰand PCNA m RNA expression significantly increased, when pression on ﹣20-120 k Pa. To the expression of MGF in hPDLSCs of co-culture system, whether in the 0-120 k Pa or ﹣20-120 k Pa pressure, is significantly increased.Summary: On the basis of previous research of the team, the research construct indirect co-culture system hPDLSCs/PRF. The pressure was produced by the novel developed cytomechanics loading device simulate the bite force of periodontal membrane in the body. To observe the influence of pressure on the hPDLSCs proliferation, differentiation and MGF expression in the hPDLSCs sheets and hPDLSCs/PRF co-culture system. The study found that dynamic pressure can effectively promote cell proliferation and differentiation, at the same time, it induced the expression of MGF m RNA. And the expression of MGF m RNA peak in 24 h after dynamic hydrostatic pressure. This study was the first to notice that dynamic hydrostatic pressure can be used promote expression of MGF m RNA in the hPDLSCs. In addition, our study also found that dynamic hydrostatic pressure can be used promote cell proliferation and differentiation, and PRF with the dynamic hydrostatic pressure promotes MGF m RNA expression in hPDLSCs. Centered on force sensitive factor MGF preliminary discusses the mechanical signal transmission mechanism in the periodontal ligament. Meanwhile, microenvironment combined with hPDLSCs/PRF co-culture system provides the experimental basis for the periodontal ligament tissue engineering applications and provides a new train of thought for the periodontal ligament tissue engineering in the clinical application of avulsed teeth replantation. |
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