The Function And Mechanism Of Endothelial Progenitor Cells In Periodontal Tissue Remodeling During Tooth Movement

Endothelial progenitor cells (EPCs) are a kind of endothelial precursor cells, which have the potential of proliferation and differentiation and can also participate in the process of neovascularization. Since the isolation of EPCs from human peripheral blood by Asahara and colleagues, more and more people realized the importance of EPCs in vascularization.Endothelial progenitors normally reside in the adult bone marrow. They may become mobilized into circulation in condition of endogenous stimulus, such as tissue ischemic, blood vessel injury, and ectogenous stimulus such as cytokine or angiogenic growth factor signals from the periphery. They could enter extravascular tissue, and promote de novo vessel formation by virtue of physically integrating into vessels and/or supplying growth factors. For this reason, autologous endothelial progenitors, mobilized in situ or transplanted, has become a major target of therapeutic revascularization approaches to ischemic disease, and endothelial injury. Moreover, endothelial progenitors represent a potential target of strategies to block tumor growth. What's more, due to the important function of vascular formation in bone formation and bone remodeling, people found EPCs can promote fracture healing and distraction osteogenesis, showing its great potential as the seed cell for vascularization bone tissue engineering. Afte all, EPCs have a bright future in clinical applicantion.How to accelerate orthodontic tooth movement and periodontal tissue remodeling, and how to shorten the period of orthodontic therapy are always hot subject of research in orthodontics. Orthodontic tooth movement is a complicated biological process induced by mechanical force and involed by multiple signal pathways. Under persistence mechanical force, there will be periodontal bone formation ,bone resorption and blood vessel remodeling. At the same time, all kinds of cell factors, such as VEGF,bFGF,MMP-9,TGF,IL etc, will be regularly expressed and participate in periodontal blood vessel and bone remodeling. Now the orthodontic research have been mainly focused on cell, gene and molecular signaling.On basis of this, our research use cell culture technique to isolate EPCs from peripheral blood, and observe their proliferation, migration, the activity of vascularization, as well as their influence on osteoblast in vitro; and observe their effecton orthodontic periodontic bone and blood vessel remodeling in vivo. We try to elucidate the signal mechanism of EPCs mobilization, differentiation and migration during periodontal remodeling, which could provide us new concept on periodontal tissue remodeling in orthodontics, and a bright future on maxillofacial tussue remodeling.There are three parts in this research:1. Culture, induction and identification of EPCs from rat peripheral blood endothelial cells Rat tooth movement models were established according to references. Peripheral blood was harvested to make cell smear 1 day after undergoing force. Peripheral blood mononuclear cells were isolated by Percoll density gradient centrifugation. Immuocy- tochemistry and immunofluorescence chemical technique were used to identify surface markers of these cells. Fluorescence chemical technique was employed to measure acetylated low-density lipoprotein and ulex europaeus agglutinin-1 expression, which showed the increase of CD133 positive number cells. MTT assay was applied to test cell proliferation. The isolated cells could absorb Dil labeled acetylated low-density lipoprotein, FIFC labeled ulex europaeus agglutinin-1, and express CD133,Ⅷfactor, CD34 etc, they also exhibited lined growth pattern and grid disposal on matrigel. Studies have primarily confirmed that experimental tooth movement can induce rat bone marrow EPCs into peripheral blood.2.The effect of EPCs on blood remodeling in vivo.Firstly,EPCs were labelled with BrdU at different concentrations (5, 10, and 15μmol/L) and detected at different time points (12h,24h,48h,72h,96h)to identify the labeling index to get the optimal concentration and incubating time for EPCs labeling. Incubation of the EPCs with BrdU at 10μmol/L and for an optimal length of 72h appeared to get the highest labeling index with lowest cell toxicity. Secondly ,EPCs labelled by BrdU were injected into rat skin, using immunohistochemical and immunofluorescence dyeing to observe the movement of EPCs to the injured skin blood vessel. The results showed that the allogeneil graft of EPCs can integrate into vessel wall and participate injured endodermis repair, The number of microvessels in experimental group was significantly higher than that in control group(P﹤0.05),which meant there was no evident immunological rejection of allogeneil graft of rat EPCs, and the transplanted EPCs had involved in endodermis repair and promoted neovascularization. Thirdly,the EPCs labelled by BrdU were transplanted back into the rats'body through tail vein with the model of rat tooth movement, analyzes influence of EPCs on VEGF and MVD of periodontal tissue uses by immunity fluorescence and immunityhistochemistry.We found the EPCs can move to periodontal tissue, which could reach the highest level on third day of movement. On the 7th day, VEGF level on the tensity side of experimental group was significantly higer than that in control group. (P<0.05), however, there is no difference between both groups on other time points. MVD in experimental group was significantly higer than that in control group(P<0.05)after 2 weeks'movement, with no difference after 4 weeks. All the results showed that the transplanted EPCs could affect VEGF expression on early stage by relatively promoting neovas- cularization.Finally, TNOS level in peripheral blood and VEGF mRNA expression in EPCs were detected through chemical colorimetry and RT-PCR respectively. The effect of VEGF on EPCs's proliferation, adhering, and chemotactic function was detected with MTT, scarification test and transwell migration assay. Immunohistochemistry staining was used to detect the VEGF and the eNOS expression in vivo. All these results were used to elucidate the mechanism of EPCs mobilization, chemotactic and differentiation during tooth movement. The result showed the level of TNOS in peripheral blood, TNOS and VEGF mRNA expression reached highest level after 24 hours'movement. VEGF can promoted EPCs's proliferation, adhering and chemotactic function. The expression of eNOS,VEGF reached peak after 3 day and 14 days'movement.The result indicated that VEGF and eNOS maybe involved in the mobilization of EPCs.3.The influence of EPCs on rat periodontal tissue bone remodelingWe established the EPCs, ECs and MSCs co-culture system. The effect of EPCs on the proliferation and differentiation of MSC into osteoblast-like cells were detected with alkaline phosphatase and chinalizarin staining in vitro. The effect of transplanted EPCs on ALP,BMP-2 expression and bone remodeling in periodontal tissue were detected with immunohistochemistry and tissue chemiluminescence staining. Results showed that compared with ECs ,EPCs can promote MSCs alkaline phosphatase secretion and the formation of mineralization nodal. After in vivo transplantation of EPCs, the expression of ALP and BMP-2 in experiment groups was highest after 7 and 5 days'movement. ALP expression in experiment group was significantly higher than that in control group, but there was no significant difference in BMP-2 expression. Fluorescein Sodium and tetracycline hydrochloride labeling bone cement lines showed there was no difference between experimental and control groups. The results showed that EPCs can promote osteogenesis activity induced by MSCs. Although there was no significant effect on bone remodeling during tooth movement after transplantation of EPCs, EPCs can influence ALP and BMP-2 on early stage of periodontal remodeling, indicating EPCs can promote early stage bone remodeling.On basis of the above results, we may reach the conclusion that periodontal remodeling induced by mechanical force has the tissue and sigaling basis of EPCs motivation and differentiation. EPCs can be isolated and cultured from peripheral blood during experimental tooth movement. EPCs can promote blood vessel remodeling in periodontal tissue on early stage of tooth movement. EPCs could promote the secretion and calcification function of BMCs by paracrine secretion, which can positively affect periodontal bone remodeling.The innovation of this research is that we have successfully isolated and cultured EPCs from rat peripheral blood during experimental tooth movement for the first time. On basis of this, we investigated the effect of EPCs on periodontal blood vessel and bone remodeling, trying to elucidate its signaling mechanism of motivation, chemotacsis and differentiation. The results showed that EPCs could have a bright future on periodontal remodeling and maxillofacial regeneration.