Bone Morphogenetic Protein 7 (BMP-7) Promotes Odontogenic Differentiation Of Dental Pulp Stem Cells

BackgroundDental caries have always been the most commonly and frequently diseases occurring in oral medicine. When bacteria and their toxic products invade into the deep side of dentin, dental pulp may degenerate or necrosis. When the pulp tissues destroy by external stimuli, dental pulp stem cells (DPSCs) in pulp tissues could proliferate and migrate into the injury lesions, then differentiate into odontoblast-like cells which could secrete reparative dentin for the sake of preventing further damage to the pulp tissues. However the self-repairing ability of pulp is so limited that the amount of reparative dentin is rare. Tooth defect would reduce the quality of life of patients of all ages and lead to an economic burden to society. The current therapy in clinical is to remove the lesions and to use artificial restorative materials to repair the missing tissues.In recent years materials in oral field have emerged rapid development and biological character or mechanical strength also can achieve a higher degree. However these materials cannot compare to dental tissues in biological performance and may affect the vitality of pulp. In the long-term of clinical observation microleakage or secondary caries may appear between synthetic materials and dental tissue, which will result in the failure of treatment.In recent years, research on stem cells reveals that they play a significant role in the area of wound repairing and tissue regeneration. In 1990, Fitzgerald discovered when dental pulp tissue were exposed, cells in dental pulp could migrate into the damaged area and form reparative dentin, which indicated the existence of dental pulp stem cells. Dental pulp stem cells with the potential of differentiate into osteoblasts, adipocytes, odontoblasts under different culture conditions, which belong to one of the odontogenic stem cells. In addition, unlike embryonic stem cells, postnatal DPSCs do not involve any ethical issues and can be easily acquired after tooth extraction treatment. The present study showed that dental pulp stem cells can be long-term cryogenic storage in vitro and their biological character keep stable. Therefore, these biological features of DPSCs provide an important basis for the study of dental pulp wound repairing and tooth tissue reconstruction.In the tissue engineering field, inductive factors are indispensable. Recently, more efforts have focused on growth factors. Bone morphogenetic protein 7 (BMP-7) is a safety factor, which belongs to the transforming growth factor (TGF)-beta superfamily. It was firstly detected in the 1870s.Because of its osteogenetic effect, it’s also named osteogenic protein 1 (OP-1). In orthopaedic, BMP-7 as security inducers has entered the stage of clinical application. Some study reported that BMP-7 has been used in the treatment of meniscus injury, which showed excellent results. When BMP-7 gene was transferred into MSCs, ossification activity of cells increased. In clinical study, Ashraf Ayoub applied rhBMP-7 on a type I collagen carrier to the treatment of alveolar cleft, which showed satisfactory results no matter from the radiographical or clinical view.Some study indicated that chitosan/collagen scaffold loading BMP-7 DNA enhanced DPSCs differentiation toward an odontoblast-like phenotype in vitro and found the formation of hard tissue in vivo. Although studies have reported that high concentrations of BMP-7 inhibits cell proliferation. The effect of BMP-7 alone on the growth and differentiation of DPSCs has not been investigated. The suitable concentration of protein on cells has not been established. Therefore, it is necessary to study the response of DPSCs to BMP-7 to predict their potential in tooth tissue engineering.Scaffold materials are the basis of tissue reconstruction. They not only need to build a spatial structure for cells adhesion, growth and differentiation in the defect area, but also favor the formation of new blood vessels. These characteristics require materials have three-dimensional structure, high mechanical strength and excellent biological performance. Currently, the materials for tissue engineering include calcium phosphate, Polylactic acid, biological ceramics and etc. Because of its excellent biocompatibility, strong mechanical strength and beautiful performance, biological ceramic materials have been widely applied in oral clinic, such as porcelain crown and porcelain implant for front teeth aesthetic area. In our former research we produced the three-dimensional nano-zirconia porous scaffolds which own high mechanical strength. Then three-dimensional nano-zirconia porous scaffold seeded with canine bone marrow mesenchymal stem cells was implanted into the mandibular defect area, with the result of bone regeneration.Therefore, we tried to find the appropriate BMP-7 protein concentration on DPSCs in vitro and evaluated the possibility of BMP-7 as growth factor applying to the repair of dental pulp tissues and tooth engineering. Objective:The aim of this study was to evaluate the effect of various doses of BMP-7 on DPSCs proliferation and differentiation. In particular we focused on the effect toward odontogenic differentiation and searched out an appropriate dose of BMP-7. To study the possibility of BMP-7 for use in dental tissue regeneration, three-dimensional nano-zirconia porous scaffolds with human dental pulp stem cells were implanted under immunocompromised mice subcutaneous.Methods:1. Isolation, culture and identification of human dental pulp stem cellsHealthy dental pulp tissues of third molars were collected from young patients aged 19-25 years. The tissue block and enzymatic digestion method were used to isolate and culture primary hDPSCs.By Observing the cell growth and clone formation, we examined the biological characteristics of hDPSCs. Then, we used osteogenic and adipogenic induction methods to identify the pluripotency of hDPSCs. 2. The effect of doses of BMP-7 on cell morphologyCells were seeded in culture flask and cultured in α-MEM with 10% FBS. One day after cell adhesion being observed, medium was changed with a fresh medium containing BMP-7 at various concentrations (0,25,50, and 100 ng/ml, respectively). Adherent cells were then incubated at 37℃ with 5% CO2, with BMP-7-containing medium changed every 3-4 days. Cell morphological features after 7 days of culture were recorded by inverted microscope.3. The effect of BMP-7 on the proliferation of human dental pulp stem cellsThe cells proliferation viability was measured by Cell Counting Kit-8 assay. Human dental pulp stem cells were cultured with different concentrations of BMP-7 (0,25,50, and 100 ng/ml, respectively). After 1,3,7 days of incubation, CCK-8 assay was used to measure cell viability in each well according to the manufacturer’s protocol.4. The effects of BMP-7 on differentiation of human dental pulp stem cellsTo determine the potential of odontogenic differentiation, immunocytochemistry, real-time polymerase chain reaction, and western blot analysis were used to evaluate some odontogenic differentiation-related markers in DPSCs after 7 and 14 days of induction by different doses of BMP-7.5. The effect of cooling rate on the structure of three-dimensional nano-zirconia porous scaffoldsIn the forming process of sintering in the original stents, by slowing down the cooling rate, we observed whether the temperature influence the integrity of scaffolds.6. In vivo study of three-dimensional nano-zirconia porous scaffolds with human dental pulp stem cellsHuman dental pulp stem cells after the induction of 100ng/ml BMP-7 were inoculated onto the scaffolds. Then three-dimensional nano-zirconia porous scaffolds were implanted under immunocompromised mice subcutaneous for2 months. Results:1. Isolation, culture and identification of human dental pulp stem cellsUsing tissue block and enzymatic digestion method we obtained young healthy adult pulp which proliferate and clone formation quickly. After culturing in osteogenic and adipogenic induction assay cells can differentiate into osteblats and adipocytes, which indicated that cells possess pluripotency potential. Therefore, these features illustrated the presence of dental pulp stem cells.2. The effect of doses of BMP-7 on cell morphologyAfter 7 days of BMP-7 induction, no obvious morphological changes were observed in any groups. Cells presented spindle、polygon and fibroblast-like morphology. Small cell clusters were observed.3. The effect of BMP-7 on the proliferation of human dental pulp stem cellsIn all groups, the cell viability increased gradually within the culture time. There were significant differences between the untreated and BMP-7 treated cells at any time point. As expected, the untreated cells had the highest levels of cell variability throughout the 7-day period. There were no significant differences among BMP-7-treated cells, but at day 7, a dose effect was found, showing diminished cell viability in DPSCs treated with 25,50, and 100 ng/ml of BMP-7. The experimental results showed that a high concentration of BMP-7 can inhibit DPSCs proliferation over time.4. The effects of BMP-7 on differentiation of human dental pulp stem cellsImmunocytochemical staining showed the expression of DSPP, DMP-1 and ALP in the induction medium was positive expression. However, in contrast, DSPP, DMP-1 and ALP in the control medium were negative or little expression.MRNA expression levels of dspp, runx2 and ocn were significantly present in 50ng/ml BMP-7 treatment group in comparison to control group. DSPP mRNA expression level in BMP-7 (50 ng/ml)-treated cells was also significantly higher than other dose of BMP-7-treated groups. However, with regards to the Col I, Col I was strongly expressed in 100ng/ml BMP-7 treatment group as compared to control group.Although DSPP was slightly expressed in untreated cells at day 14, DSPP expression was increased in response to BMP-7 treatment, showing a significant increase at 50 ng/ml and 100 ng/ml. Consistent with the pattern of mRNA expression, DSPP protein expression was highest in BMP-7 (50 ng/ml)-treated cells.5. The effect of cooling rate on the structure of three-dimensional nano-zirconia porous scaffoldsBy slowing down the cooling rate of scaffold, the structure of scaffold kept intact and collapse phenomenon didn’t happen. SEM revealed that scaffold presented three dimensional network structures and the distribution of internal pore size was uniform. However, micro cracks on the surface and fracture phenomenon still existed. Comparing with the original sintering process, the form of tomic structure didn’t see any obvious differences.6. In vivo study of three-dimensional nano-zirconia porous scaffolds with human dental pulp stem cellsThe surface and internal structure of scaffold were covered by cells after seeding. After two months all specimens were surrounded by blood vessels and fibrous connective tissue and did not see obvious inflammatory tissue around the scaffolds. We did not see obvious differences between the four groups. HE staining showed in the groupswithout any cells presented fiber-like tissue and groupswith cellshad odontoblasts-likecells and small blood vessels. Howevermature dentin structure didn’t been observed.Conclusions:1. BMP-7 regulates DPSCs proliferation in a time and dose-dependent manner in vitro.50ng/ml and 100ng/ml BMP-7 alone are capable to induce DPSCs toward odontogenic differentiation and not dramatically halt cell proliferation in vitro.2. The suitable protein concentration may induce DPSCs differentiating into odontoblast. It’s necessary to control dosage and duration time of protein, in order to benefit cells proliferation and differentiation.3. Three-dimensional nano-zirconia porous scaffolds couldprovide good extracellularmicroenvironment for DPSCs and showed good biological properties.4. DPSCs adherent to the three-dimensional nano-zirconia porous scaffolds is expected to regenerate dentin-liketissue and it needs further study.