Oligodeoxynucleotid Induced Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells To Osteoblastic Cells

Periodontal disease is one of the two main types of oral diseases. The late 20th century, more and more clinical and research data indicate that periodontal disease has a two-way close relationship with the systemic health and systemic disease. The defect of the periodontal tissue is the most common signs of periodontal disease which is caused by various kinds of reasons.Regeneration of periodontal tissue defects has been the nodu and hot spot in clinical and basic research. Finding effective biological or chemical treatment method to promote the regeneration of periodontal tissue defects, can effectively reduce the periodontal tissue defects due to periodontal disease and some systemic diseases like the cardiovascular which is caused by periodontal disease.The main manifestations of periodontal disease include the destruction and reconstruction of periodontal soft and hard tissue. The rebuilding of periodontal tissue assumes cyclicity, including the cycle of destruction and reconstruction. Both of the two modality determine the character and processes of the disease. The regeneration of bone occurrs in the healing of alveolar bone, its mechanism is that the immune system could prevent the formation of osteoclasts and increase the death of osteoclasts, by this way the activity of osteoclasts would been significantly reduced. The other mechanism is activating osteoblasts to induce reproducibility bone healing. Understanding the healing process will help us to develop the healing of periodontal tissue into the direction of regeneration during periodontal treatment.The capacity of periodontal tissue regeneration is limited. In recent years, a considerable number of studies dedicated to the development of technology and materials which can increase the capacity of periodontal tissue regeneration. The treatment of periodontal tissue defects caused by periodontal disease includes that the application of guided tissue regeneration , tissue engineering methods to obtain new attachment of pericementum and repairing the natural anatomy of periodontal tissue.At present, many studies focus on the activation of osteoblasts, periodontal ligament cells and bone marrow-derived mesenchymal stem cells, as a way of promoting regeneration healing. Because the extraction and culture of osteoblasts and periodontal ligament cells are difficult, more researches select the bone marrow-derived mesenchymal stem cells. The bone marrow derived mesenchymal stem cells has the characteristics of mesenchymal stem cell, shows strong proliferative ability and variety of cell differentiation potential. Under certain inducing conditions, the bone marrow derived mesenchymal stem cells can differentiate to osteoblasts. Bone marrow derived mesenchymal stem cells can be easily abstracted and cultured, it is one of the most ideal seed cells for tissue engineering research.ODN (oligodeoxynucleotides, ODN) refers to the multi-nucleotide chains containing 50 or less nucleotide monomers. ODN exists in some inferior organism's genomic DNA, It can simulate the immune stimulating effect of bacterial DNA to activate the various types of immune cells to produce proinflammatory cytokines and other inflammatory chemical mediator, to participate the anti-infection immunity in the body. DNA or RNA degradation products frequently contain ODN. ODN can be absorbed into cells through the form of receptor-mediated without the need of constructing a vector, thio-modified phosphate may enhance their cellular uptake capability and may resist nuclease digestion in vivo, these modifications of ODN are good for keeping stable in vivo and increasing the intracellular uptake rate. In addition, ODN also has the advantages of high efficiency, low toxicity, easy-synthesis and stability. Therefore, ODN becomes the hot spot in basic medical field over the past decade. ODN has been applied to the current pre-clinical studies to evaluate the clinical results of local application to the treatment of mastitis, allergic rhinitis and conjunctivitis. Some researches show that specific ODN sequences can regulate osteoclast differentiation through different signaling pathways, which provide a theoretical basis for the application of ODN to regulate the rebuilding of periodontal bone.Present studies about ODN focus on the CG-rich motif ODN (CpG ODN). In recent studies showed that CpG ODN could regulate osteoclast formation and differentiation in TLR9 dependent individual through Toll-like receptor 9 (Toll like receptor 9, TLR9)-mediated molecular pathway. CpG ODN can inhibit osteoclast differentiation by upregulating the expression of IL-12 to inhibited RANKL, which suggests the possibility of its use of the inhibition to prevent the possibility of pathological bone resorption. CpG ODN via TLR9 produces effective TNF-α, which mediated the expression of RANKL reduced in co-culture of bone marrow stromal cells and osteoblasts, which was specificity inhibited by chloroquine. The results suggested that CpG ODN regulated osteoblast-osteoclast balance through its classical mode of action. Jae-Ho Chang's research shows that the CpG-KSK13 inhibites the activation of osteoclasts via TLR9 is mediated by the down expression of TREM-2. In addition, a specific sequence of CpG ODN can induce dephosphorylation reaction to hinder the need of sustained phosphorylation of extracellular signal kinase (ERK) in order to maintain a high level expression of c-fos, as an essential condition in the procedure of osteoclast differentiation. The above studies have shown that a specific sequence of ODN can be effected osteoclast differentiation and regulation of osteoblast - osteoclast balance through different signaling pathways, which provided a possibility for the further research of ODN.In view of the above studies, aiming at how to regulate periodontal tissue repair and regeneration in periodontal disease, according to the specific sequence of ODN could regulate the osteoblast-osteoclast balance and the research of bone remodeling and bone marrow-derived mesenchymal stem cell, we propose the hypothesis that the specific sequence of ODN has some influence on the process of bone marrow-derived mesenchymal stem cells differentiate to osteoblasts. Around this assumption, we set the following experiment:Research 1: Filtration experiments in vitro. There has not yet seen the research on the effects of ODN to the bone marrow-derived mesenchymal stem cells, so we carried out a large number of initial screening in the beginning of this experiment. First of all, we choose 12 different sequences of ODN (in which partial sequence has applied for National Science and invention patents) which were designed and built by Jilin University, college of Basic Medical, department of Molecular Biology. According to the biological characteristics of ODN that could be absorbed into cells through the form of receptor-mediated without the need of constructing a vector, we took ODN and bone marrow-derived mesenchymal stem cells co-culture. By MTT colorimetric assay different ODN effects to the proliferation of BMSCs, and mapping dose-response curves and time curves about ODN impact on proliferation of different sources of rat BMSCs at different time points. The result showed that there are four different sequences ODN which can significantly stimulate the proliferation of BMSCs, initially confirmed that ODN has a certain effect on the biological characteristics of BMSCs. Second, different sequences of ODN were added in the BMSCs which were induced to osteoblast at the same time, the alkaline phosphatase kit was applicated to detect the expression of alkaline phosphatase in BMSCs in which with different concentrations of ODN at different time, the results show that there were two ODN can significantly enhance the osteoblastic differentiation of BMSCs, we successfully screened out the ODN which can promote the osteoblastic differentiation of BMSCs, the ODN has an impact in the process of osteoblastic differentiation of BMSCs was further confirmed.Research 2, the mechanism research of the specific ODN sequences has an impact in the process of osteoblastic differentiation of BMSCs. The previous experiments have confirmed that a specific sequence of ODN can promote the osteoblastic differentiation of BMSCs. And then, the cells with ODN added in whether have abnormal expression of the relevant factors in the process of osteoblastic differentiation of BMSCs? We undertook the following experiment. First, rat BMSCs were isolated and cultured, the third generation of BMSCs were taken for osteoblastic induction, while adding the specific sequence of ODN co-cultured for 1d, 3d and 5d. Real-time quantitative PCR were used to analysis the mRNA expression of bone-related factors OPG, RANKL, Osterix, Runx2 and typeⅠcollagen in experimental group and control group BMSCs. The results showed that in the early time(3d and 5d), the ODN can significantly increase the mRNA expression of Osterix-related factors such as RANKL, OPG, Runx2 and the RANKL / OPG ratio had an evident declining trend while the action time of ODN increased. The result suggested that the mechanism that the ODN could promote the osteoblastic differentiation of BMSCs may be related to the abnormal expression of osteoblast-related factors.Research 3, the purpose of this study is to find the active substance which can promote the osteoblastic differentiation of BMSCs and have the characteristics like high-performance, low-cost, easy synthesis and stable.That would provide an experimental basis for the future application in the field of periodontal tissue remodeling. Therefore, we used the ODN which was screened out from the vitro experiment for the studies in vivo to confirm whether the ODN has an impact in tissue repairing and regeneration of alveolar bone in vivo. First of all, the animal models of experimental periodontitis in rats was established by using the silk ligation which was currently accepted, after that, the ODN was injected into the local periodontal tissue of rats. In this process, experimental zoology, histopathology, immunohistochemistry chemistry and imaging technology were used in vivo studies to research the absorbed dose of alveolar bone, the histopathological changes and the expression of RANKL, OPG, Osterix, Runx2 and typeⅠcollagen in protein level. The results showed that the resorption of alveolar bone had no significant difference between the experimental group and the control group when 2 weeks after injection, however, when 4 weeks after injection, the resorption of alveolar bone in the experimental group was reduced than the control group (p<0.05), especially the resorption of palatal bone reduced significantly. We also detected that some protein expression of bone-associated factor appeared different expression in the experimental group and control group, the expression of osteoblast associated protein RANKL, OPG, etc. had increased.Integrated the experimental results, we believe that the specific sequence of ODN has significant influence on the biological characteristics of bone marrow-derived mesenchymal stem cells, especially it plays an important role in the process of osteoblast differentiation. In the process, bone remodeling regulatory factors were abnormally expressed; in vivo, ODN promoted the bone rebuilding, we can infer that ODN should have a certain effect on the periodontal bone remodeling. Therefore, the results of this study show that: ODN may has the influence on periodontal alveolar bone remodeling caused by periodontal infection. The results may provide new treatment ideas to block absorption of alveolar bone caused by periodontal infection, and to promote the periodontal tissue repairing and regeneration.ODN is a prominent achievement in the field of immunization scientific research in recent years. The innovative point of this study is to apply ODN to the field of oral medicine research, successfully screened and exploited the specific ODN which have a role in the process of bone marrow-derived mesenchymal stem cells differentiated to osteoblast. We made a preliminary exploration of its effect, mechanism and expected to provide a new treatment ideas on reparation and regeneration of periodontal tissue.