The Role Of Bone Marrow-derived Cells In Pulp Revascularization In Mouse Model

Background:Endodontics and periapical disease are the common diseases in dentistry. The main treatment is root canal treatment for these diseases. The procedure of root canal treatment is removing the pulp tissue and the infection of root canal and filling materials with the original closure the space occupied by the pulp tissue, in order to prevent bacterial invasion. As the pulp tissue is completely destroyed, the teeth lost the nutritional support, the original feeling and metabolic function. Because the teeth is discoloration, affect the appearance, brittle and easy to break, it the need restored by crowns.The long-term effect is not good. Meanwhile, the technology of the high sensitivity of root canal therapy requires sophisticated equipment and technical skills. The above treatments are non biological restoration. It can not form a healthy and natural tooth.As the anatomical characteristics of young permanent teeth:the root development is not complete, the apical foramen has not yet formed. When the pulp serious disease or periapical infection was happened in young permanent teeth, the root canal treatment may not routine for this situation; due to the root is not closure completely. At present, apexification or periapical surgical treatment are the conventional treatment to close the open apex for young permanent teeth and then filling materials in root canal. The tooth after surgery can not continue to promote root growth, and the resulting are shorter root, the ratio crown to root increasing, the long-term preservation of teeth is bad. Another treatment is Apexification that can form apical dentin bridge to close apical foramen.A series of clinical studies and case reports show that pulp necrosis of young permanent teeth after root canal disinfection, inducing blood into the tightly closed root canal and crown, root access to continue developping and new tissue was growth in root canals. The x-ray showed the root was becoming longer, the wall of root was becoming thickening, the root apical was becoming closure and apical lesions disappeared, and dental pulp regain vitality, this treatment method was called "pulp revascularization". However, to obtain good clinical efficacy, while the corresponding animals experiments have confirmed that the new tissue in the root canal is completely different with the normal pulp tissues, cementum by category, bone, periodontal ligament tissue and loose connective tissue thereof. How to make newborn "revascularization" tissue closer to normal pulp tissue structure and function, that is the most critical issues currently "pulp revascularization" study faced. In order to solve this problem, we need to process the pulp revascularization component sources of stem cells, to study the proliferation of in-depth systematic mechanism competition between different sources of stem cells.Root continued growth, the source of stem cells for new tissues and pulp revascularization and the exact mechanism remains unclear. Most scholars have speculated that their cells have a variety of sources, including the remnants of vital pulp tissue dental pulp stem cells (DPSCs), the root of the canine papilla stem cells (SCAPs), periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMMSCs). Indeed, pulp necrosis and severe periapical infection (apical lesions exist), and root canal disinfection and flushing drugs can significantly damage the root of the canine dental pulp stem cells and stem cell nipples, these two sets of dental pulp stem cells have few opportunity to involve in revascularization. Our Hypothesis is the bone marrow-derived cells may be involved in the process of pulp revascularization and it is the main source of cells in newborn tissues. ObjectivesThe experiment aimed to establish the molars pulp revascularization model in mice and investigating the process of pulp revascularization and its histological characteristics. Then through the establishment of GFP+bone marrow chimeric mice models were involved in the maintenance of bone marrow-derived cells jaw and dental tissue and systemic stability in the role of other organizations. And through the GFP+bone marrow chimeric mice revascularization molar endodontic treatment, to investigate the mechanism of bone marrow-derived cells in dental pulp tissue revascularization process. To lay the foundation for follow-up experiments how to regulate the proliferation and differentiation of bone marrow cells in dental pulp tissue revascularization process.Materials and Methods(1) Selectting the4-week-old C57BL/6mice and using intraperitoneal injection of1%sodium pentobarbital for anesthetized. Mice dedicated fixed by oral traction devices designed for oral surgery and mice were placed under microscope, and the surgical field was sterilized by iodine and then placed aspirator. The whole experimental process maintains aseptic technique. Experiments conducted on dental pulp revascularization treatment, using high-speed steel ball drill (#1/4) for opening pulp champ from occlusal surface, then remove pulp tissues with a6#K file, and flushing with Physiological saline,6#and8#K continue to clean the filing residual pulp in root canal space and appropriate expansion of the apical foramen, root canal irrigation with normal saline, sterile paper point dry,8#K file insert apical stimulation periapical tissue bleeding to enamel cementum community level, set a saline wet cotton ball, wait5minutes to allow the blood to form clots, MTA covered tightly closed light-curing glass ionomer marrow open hole. Respectively, the animals were killed after1week,4weeks,8weeks, radiologic and histologic observation of pulp revascularization situation.(2) Bone marrow cells from GFP+transgenic mice were transplanted into wild-type C57BL/6mice treated by radiation exposure. The GFP+bone marrow chimeric mice were formatted. Bone marrow-derived cells were observed in the pulp through a small animal imaging in vivo and histological methods distribution of periodontal tissue, heart, lungs, fat, skin, and kidney.(3) To establish the pulp revascularization of the irst molar in GFP+bone marrow chimeric mouse model, and through small animal in vivo imaging, iconography, histology and immunohistochemistry to evaluation the pulp revascularization situation, and evaluate the role of bone marrow-derived cells in the pulp revascularization process, and to explore mechanisms of bone marrow-derived cells involved in the process of pulp revascularization.Results(1) By using a self-designed mouse oral fixation and microsurgery assisted traction device, exposed mice oral surgery area fully, the mandibular molar region of mice are fully exposed, The pulp revascularization can be done in the first mandibular molar in mouse. The growth of experimental animals was in good condition during the experiment without fracture or loosening of the tooth structure, the filling Materials were no loss, periodontal tissue without exception. No significant irritation of oral mucosa. Imaging results showed that first molars canal fillings of the experimental group were seen video coverage of dense root canal orifices, filling the top intact, with no loss; root space was visible, the apical1/3of root canal narrowing, there were new tissues formed, periapical tissue and periodontal tissue showed no abnormalities. In the control group (periapical disease) showed that the first molars canal open, root canals were visible, near and far apical1/3of the visible gap widened periodontal ligament, bone hard board disappeared periapical bone mass destruction. Furcation has a larger range of low-density; mouse mandibular first molar root canal may prompt deputy. Histological results showed that the experimental group root lumen1/2are covered with highly vascularized tissue near the apical see a large calcified tissue, calcified tissue surrounding the small number of high columnar cells, root canal wall visible hard tissue deposition, surface soma large eucalyptus stained cells, where high columnar odontoblast-like cell layer, the cells were fake complex layered arrangement, similar to the odontoblast layer. Intramedullary visible mesh-like tissue, irregular-shaped bone-like cells located in the lacuna, more number of cells were found in the bone-like tissue. Control group widened periodontal ligament, periodontal ligament fibers disordered apical alveolar bone destruction, vasodilatation, showing neutrophils, lymphocytes, macrophages and other chronic inflammatory cell infiltration, local see bone hyperplasia. Radiographic and histological results were prompt achievement of mice to establish marrow pulp revascularization animal models.(2) Bone marrow cells from GFP+transgenic mice transplanted into wild-type C57BL/6mice treated by radiation exposure, the mice within14days have appeared in weight loss, decreased appetite, mild diarrhea, hair loss, decreased activity, with gray hair depilation other symptoms. After weight loss in mice transplanted apathetic gradually recovered after two weeks, all survived more than120days. Flow cytometry showed that the proportion of green fluorescent mice transplanted with peripheral blood mononuclear cells were higher than90%, fitting success rate, the results of the establishment of GFP+bone marrow chimera models.3months after bone marrow transplantation, small animal in vivo imaging showed that the jaw area limbs and head were seen in mice after transplantation obvious green fluorescence, green fluorescence mandibular region than other regions stronger. Histological results showed that we can observe cells from GFP+bone marrow-derived cells in a variety of tissues, Including liver, lung, heart, kidney, skin, fat, dental pulp and periodontal tissues. GFP+bone marrow-derived cells migrated in dental pulp and periodontal tissues than in others.(3) After pulp revascularization endodontic treatment of the first molars in the chimeric mice, small animal imaging in vivo results showed that compared with the untreated mice in the experimental group (pulp regeneration) and control group (Apical periodontitis) where the jaw area of green fluorescent protein were increased in the experimental group increased the amount of green fluorescent protein expression was significantly higher, no two sets of green fluorescent protein expression to8weeks after surgery significantly different, but the control group compared to people at a higher level. Further immunohistochemistry results showed that visible GFP+bone marrow-derived cells in revascularization of the pulp tissue and differentiate into odontoblasts, vascular tissue, nerve tissue in case of tissue injury, bone marrow-derived cells can migrate to the lesion involved the formation of new tissue.Conclusions(1) Self-designed micro-surgery oral fixation and traction for mouse can be fully exposed mouse oral cavity. The pulp revascularization can be done in the first mandibular molar in mouse. The operation by using these devices can save operation time, and can effectively improve the success rate of dental pulp revascularization treatment. Radiographic and histological results showed that the pulp revascularization was successfully established in mouse model.(2) Bone marrow cells from GFP+transgenic mice were transplanted into wild-type C57BL/6mice treated by radiation exposure. The fluorescent flow cytometry showed that the proportion of green cells in peripheral blood mononuclear cells were higher than90%in transplanted mice. The results show that GFP+bone marrow chimeras models was established successfully.2months after bone marrow transplantation, bone marrow-derived cells can migrate to the jaw area and the limbs of mouse, which GFP+marrow-derived cells migrate to the chest pulp and periodontal tissues of bones than other tissues.(3) After pulp revascularization endodontic treatment of the first molars in the chimeric mice, small animal imaging in vivo results showed that bone marrow-derived cells can migrate to the lesion involved in the formation of new tissue. The histochemistry results showed that the bone marrow-derived cells expressing more in revascularization pulp-like tissue and can differentiate into odontoblasts, vascular tissues, nerve tissues.In summary, our study demonstrated that there are a variety of sources cells into the root canal tissue in pulp revascularization proces, including bone marrow-derived cells migrate into the root in pulp revascularization procedure to differ into odontoblasts, vascular tissues, and nerve tissues.