| BackgroundEndodontics, periodontal diseases, periapical diseases are common diseases causing tissue damage with high prevalence in the whole world.More and more clinical and basic researches show that dental periodontal injury is of a close bi-directional relationship with people’s systemic health or illness. For now, root canal and periodontal treatments are still the main treatment methods that can reduce the patient’s discomfort, prolong duration of the teeth, but these therapies can only construct teeth with poor biocompatibility and significantly shorten life compared with natural biological teeth. Therefore, with the use of cell biology and molecular biology techniques, stem cell-based tissue regeneration therapy become the hot spot of international and domestic study in recent years.Stem cells is kind of cells with strong proliferation ability and differentiation potential, under certain conditions, they can differentiate into many kinds of cells. Stem cells can be divided into embryonic stem cells and adult stem cells according to the source. In 2006,Japanese scholars reprogrammed somatic cells by transfection of specific gene combinations to induce differentiated somatic cell into a new type of multiple potential of stem cells known as induced pluripotent stem cells.In the field of tooth regeneration and repairation, adult stem cells, embryonic stem cells and induced pluripotent stem cells have been studied for few years.Embryonic stem cells derived from the blastocyst of embryonic period, which can be maintained in an undifferentiated state in vitro for a long term and have the potential to differentiate into any cell of the body. Somatic stem cell, which is derived from the mature stem tissue cells of the body, is a kind of multipotent stem cells or single potent stem cells. For the sake of ethics consideration and practical difficulties, the application of embryonic stem cells in clinical is restricted. The application of induced pluripotent stem cells is restricted in teeth regeneration because it may cause DNA damage, produce clinical malignant tumor and other reasons.Dental pulp stem cells (DPSCs) are mesenchymal stem cells among mature dental pulp cells. They are widely used for the study of dental defect odontogenic. DPSCs in caries and other dental pulp injury are substitute odontoblast precursor cells involved in pulp repair. Bone mesenchymal stem cells (BMSCs) are a class of non-hematopoietic origin multipotent differentiation capacity of stem cells. Because of its potential to differentiate into a variety of mesoderm-derived tissues and neuroectodermal cells, causing a small damage, easy to culture, and other advantages, BMSCs become the concern of the stem cells. BMSCs have the ability to migrate to a variety of organizations at physiological state, their ability to migrate to the damaged area are stronger and promote repair function. During remediation of injury in the pulp, BMSCs play an important role in repairing periodontal and pulp damage. Some scholars prove the ability of BMSCs involved in repair of periodontal and tooth tissue defects by transplanting BMSCs.Stem cell therapy involves the role of stem cell viability, regulating microenvironment and paracrine, mobilize and raise the host’s own stem cells in tissue regeneration. MSC therapeutic effect is not just affected by their secretion of biological factor, another important role of the transplanted MSCs reflected in the differentiation ability. Directed differentiation of stem cells play an important role in the way it functions. Studies have pointed out that the differentiation of stem cells not only involved in chemical microenvironment of the cells, the physical microenvironment to guide stem cells also plays an important role. BMSCs can actively transforming the environment in accordance with its corresponding cell types.In summary, BMSCs which have ability to promote pulp wound healing, may have an impact through physical contact or paracrine manner to promote the repair of injuried dental pulp, This reaserch explored the interaction between BMSCs and pulp cells by in vitro co-culture, aiming to provide a theoretical basis for the clinical application of BMSCs.ObjectiveIn this study, in vitro contact co-culture cells were studied to explore the interaction of two coexisting cells, to provide a theoretical basis for tissue engineering to choose appropriate seed cells and the application of bone marrow mesenchymal stem cells in clinical endodontics dental treatment.Materials and methodsPart I Isolation and culture of SD rat dental pulp cellsAfter Sodium pentobarbital anesthesia, SD rats were sacrificed and soaked in 75% alcohol for 15min. Surgically removed the mandible was taken in laminar flow cabinet and then washed 2 to 3 times with PBS containing double-antibody. Hard tissue around the teeth were crushed with pliers to sepatate the lower incisors. After getting the whole pulp tissue, cut off the apical 1/3. The pulp tissues were cut into approximately 0.5 mm x 0.5 mm×0.5 mm size, then digested with 3mg/mL collagenase I in 37℃ in constant temperature oscillation table for 10 min until when the tissues were loosely contacted. Wash out the collagenase before the tissue blocks were spread evenly on wet culture flask, and place the flask in 37℃,5% CO2 incubator with the tissues free of DMEM/F12 culture medium containing 10% FBS for 4h. Then medium was changed every 3 to 4 days regulatly. Subculture the cells when they grew to approximately 80% confluence.Part Ⅱ Isolation, culture and identification of SD rat bone marrow mesenchymal stem cellsAfter Sodium pentobarbital anesthesia, SD rats were sacrificed and soaked in 75% alcohol for 15min. Remove the femur and tibia under sterile conditions and then cut metaphyseal of the bones to expose the bone marrow cavity. Bone marrow cavities were then washed with a syringe for several times.Eluate was centrifuged at 800rpm for 3 minutes, the supernatant was discarded after centrifugation. After resuspending the cells with 1 mL DMEM/F12 medium containing 10% FBS, seed them in 25cm2 culture flasks and culture at 37℃,5% CO2 incubator. Replace the culture medium for the first time to remove adhesion fibriblast after half an hour, replace the medium afer 24hours to remove unadhesion cells, then medium was changed every 3 to 4 days regulatly. When cells were grown to approximately 80% confluence, they were subcultured. Lift cells by incubation in trypsin for 1 minute at room temperature. The remaining adherent cells were discarded. The dislodged cells were again incubated in 37℃,5% CO2 incubator.To identify the expression of cell surface markers in mesenchymal stem cells, we used flow cytometry to evaluate the 3rd passage BMSCs; proliferation ability were evaluated with the MTT cell growth curve method; multilineage differentiation capability of BMSCs were examined.3×103 cells BMSCs were seeded in each hole of 6-well plate. After induction of 96h adding liquid mineralization DMEM/F12 (containing 10% FBS, O.luM dexamethasone,50uM vitamin C,10MM β-glycerophosphate), alizarin red staining were apllied at the 14th day of mineralization and captured under inverted microscope. Adipogenic solution (containing 10% fetal bovine serum medium was added 1μM dexamethasone,200μM indomethacin, 0.5mM IBMX, 10μg/mL insulin) were used to identify the adipogenic differentiation ability. A mixture of oil red O were apllied at the 21th day of adipogenesis and formation of lipid droplets were captured under inverted microscope.Part III Effects of in vitro contact co-culture on cell proliferation and mineralization abilityRespectively, the third generation of BMSCs, DPCs and 1:1 mixed cell were seeded by 5 x 103/hole in 96-well plates, cell growth curves were determined by MTT method to compare effects of contact co-culture on cell proliferation. Respectively, the third generation of BMSCs, DPCs and 1:1 mixed cells were seeded by 3×105/hole in 6-well plate. After mineralized induced for 14 d and 21 d, total RNA were collected and purified, then, use Real-time quantitative polymerase chain reaction (PCR) to detect mineralization related gene OCN, OPN, ALP, DSPP expression at mRNA level. Observe calcium nodule formation by alizarin red staining.Part IV Comparison of biological effects of LPS-stimulation produced by the co-cultured cellsRespectively, BMSCs, DPCs and 1:1 mixed ratio of the two cells were divided into three groups, with 3×105/hole cells, they were seeded in 6-well plates. The third day after inoculation till cell density growth was around 80%, replace the culture medium with DMEM/F12 medium containing 10ng/mL LPS or 100ng/mL LPS, the negative control group without LPS. The LPS-stimulated cells and control group RNA were extracted and performed with qRT-PCR detection after 2hours’stimulation to detect inflammation associated factor expression at mRNA level.RusultsPart I Dental pulp cells of rats were successfully isolated and culturedAround 3rd day of Modified tissue enzyme digestion of primary culture, there were cells moving out from the dental pulp tissue around the block. Rat dental pulp cells arranged the dental pulp tissue more concentrated form polygon-based, and relatively sparse at the edges. Overall dental pulp cells showed polygonal cell shape, cell clusters grow, abundant cytoplasm, round nuclei, center. After subculture of the cells, they were of rapid proliferation and growth, and showed uniform polygonal, cobblestone cells shape.Part II BMSCs of rats were successfully isolated, cultured and identifiedPrimary vaccination was full of round or oval mixed cell by whole bone marrow adherent culture method.24h later, adherent cells began to show up there, clumps forming cell clones was visible after the second medium change; about 10th day after, when the cell density reached about 80% confluence, they could be subcultured. After two or three passages, cells were uniform long spindle, arranged in spiral-shaped fibroblast-like morphology, intermingled with a small amount of light round cells. The cells were cultured with good proliferation ability and osteogenic, adipogenic capacity. Flow cytometry showed that hematopoietic stem cell surface marker CD34, CD45 expression was negative, the positive rate was less than 1%, mesenchymal stem cell surface markers CD44, CD29, CD90 were positive, bimodal.Part III Effects on cell proliferation and mineralization ability of vitro contact co-cultureThe mixed cells showed good condition under microscope. Co-cultured cell proliferation capacity was significantly enhanced than single culture. Real-time PCR results showed a good mineralization in each group, the mineralization-related genes RUNX2, ALP, OCN, OPN, were expressed with the increased growth trend for two weeks. RUNX2, ALP, OCN expression of BMSCs was significantly increased compared with other groups (P<0.05). OCN, OPN expressions were significantly increased in mixed cell group. After mineralization induced culture for 2 weeks, DPCs were significantly elevated odontogenic mineralization associated gene DSPP, mixed cell group tended to increase, but there was no statistical difference.Part IV Comparison of biological effects of LPS stimulation produced by the contact co-cultured cellsReal-time PCR showed that, in each group IL-1β expression increased with LPS dose increased. DPCs showed relatively low expression while and mixed cell group showed high expression.Conclusions1.Through modified tissue enzyme digestion method, we adopted rat primary dental pulp cells more easily than other methods. Passage 3-5 cells were chosen for further study.2. Through this study, we adopted BMSCs through relatively straightforward, simplified method. Stem cell surface markers and differentiation potential were identified, phenotypic and biological characteristics of the cells obtained were consistent with BMSCs, indicating that they were BMSCs and provided a stable and reliable source of cells for follow-up experiments.3. Co-cultured cells of BMSCs and DPCs showed significantly increased cell proliferation and didn’t weaken the odontogenic and osteogenic capacity, thereby promoting tissue repair ability and providing the basis for BMSCs application to the pulp tissue regeneration.4. Co-culture method did have an effect on the expression of IL-1β while detailed mechanism and effects need more researches. |
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