| Periodontitis is a chronic infectious disease affecting the tissues that surround and support the teeth and usually caused by the microorganism infection in the dental plaques. Periodontitis may cause the inflammation of the periodontal supporting tissues, formation of periodontal pocket, progressive attachment loss and alveolar bone absorption, which finally result in loose and loss of teeth. Periodontitis is one of immemorial and common diseases and a major cause of tooth loss in adults. The final goal of periodontitis treatment is to repair the periodontal defect and reconstruct normal structure and function of periodontal tissues. However, no effective strategy has been developed to promote the regeneration of periodontal tissues in the supragingival pocket and actual biological repair is lacking.Tooth related stem cells and tissue engineering technique makes biological repair of periodontal tissues and teeth possible. That is, biological engineering technique has been employed for regeneration of periodontal tissues. This is also a method to manually mimic the development of periodontal tissues. It is following the principles in the development of periodontal tissues that tissue regeneration be realize d.However, the selection of seed cells is determinant in realizing theperiodontal tissue regeneration with tooth related stems and tissue engineering technique. The ideal seed cells have the following requirements:ease collection, stable nature after transplantation and potent proliferation and differentiation. The dental follicle is loose connective tissues between the enamel in the pre-eruptive phase and tissues surrounding thedental papilla and can develop into periodontal tissues. Dental follicle isderived from ectodermal mesenchyma. The cementoblasts in the innerlayer of the root of the tooth can secret cementum, and the cells close to the alveolar bone can differentiate into osteoblasts which can secret bone matrix. The cells in the middle layer can differentiate into fibroblasts which may produce the periodontal membrane fiber. Numerous studies have confirmed that dental follicle is composed of heterogeneous cells.The dental follicle cells are considered as precursor cells of periodontal tissues and have the characteristics of stem cells: self-renewal and multilineage differentiation potential.Thus, to isolate DFCs may be criticalfor the investigation of development of periodontal tissues. In addition,investigations on DFCs may provide evidence for the dental implant, tooth replantation and tooth regeneration. These cells with potent proliferation and differentiation may serve as favorable seed cells for the tissueengineering of periodontal tissues. Bone morphogenetic proteins (BMPs) are a group of glycoproteinsplaying important roles in the development and remodeling of the bone, and they can promote the chemotaxis and aggregation of cells into osteogenic site in different ways and facilitate the differentiation into osteoblasts. In addition, these proteins can also promote the angiogenesis,regulate the activity of some growth factors and affect the production of these growth factors, which is helpful for the osteogenesis. BMPs have been considered as the most potent growth factors that can promote the bone regeneration. To date, more than20BMPs have been identified and BMP-2,-4,-6and-7have found to the osteogenic potential. BMP-9is one of proteins in the BMP family and less investigated. BMP-9is also known as growth differentiation factor2(GDF-2) and mainly expressed in the liver. BMP-9can induce and maintain the cholinergic differentiation of embryonic neurons, regulate the metabolism of glucoseand fatty acid, modulate the dynamic balance of iron and exert other important biological functions. However, the role of BMP-9in the osteogenesis and bone regeneration is poorly understood. TC HE systemically investigated the roles of14BMPs (BMP-2-15) in the oseogenesis. BMP-9is one of potent osteogenic growth factors and has been found topossess the capability to promote the osteogenesis and chondrogenesis.The results demonstrated the potent osteogenic activity of BMP-9.Theability of BMP-9to induce the osteogenic differentiation of stem cells was found to more potent than other BMPs such as BMP-2, and BMP-7. To date, no study has been conducted to investigate the effect of BMP-9on dental follicle cells and its role in the dental bone regeneration.Dental follicle is composed of heterogeneous cells. Dental folliclecells serve as precursor cells of periodontal tissues and have characteristics of stem cells: self-renewal and multilineage differentiation potential suggesting the presence of stem cells in the dental follicle cells. The rat dental follicle cells could be induced to differentiate into adipocytesand neurons in vitro, which further confirm that the dental follicle cellshave the mesenchyma derived cells which possess the potent differentiation potential.In the present study, adenovirus served as a vector mediating the transfection of BMP-9into DFCs. RT-PCR was employed to detect the transfection efficiency, and the early and late osteogenesis of these DFCswere identified. Moreover, the role of p38and ERK1/2MAPK signaling pathway in the BMP-9induced osteogenesis of rat DFC. Our resultsprovide evidence that DFCs may become promising seed cells for periodontal bone regeneration in tissue engineering.We study the differentiation of rat dental follicle cells mediated by adenovirus BMP9in vitro.The research works are as follows: 1）Primary culture of rat dental follicle stem cellsBriefly, the neonatal rats were sacrificed by decapitation, and the first and second molar germs were obtained. The dental follicle tissues were added to the serum-free DMEM For the evaluation of DFSCs ability to form mineralized nodules in vitro, The result is that we confirmed the existence of dental follicle stem cells (DFCs), and the rat dental follicle cells could be induced to differentiate into osteogenics, adipocytes and neurons in vitro, which further confirm that the dental follicle cells have the mesenchyma derived cells which possess the potent differentiation potential. Thus, to isolate DFCs may be critical for the investigation of development of periodontal tissues.2）Adenovirus mediated transfection of DFCsThe medium for osteogenesis included vitamin C and glycerophosphate. The rat DFCs with good growth were used to prepare single cellsuspension one day before transfection and then seeded into24-well ata density of5×10~3/ml/well.After adenovirus mediated transfection of BMP-9, RT-PCR showeda band at275bp, and the optical density of this band increase over time. In the untransfection group, this band was not found, which suggested no mRNA expression of BMP-9. At12h after Ad-BMP-9mediated transfection, fluorescence was found in the DFCs, especially at the perinuclear area, and reached a maximal level at48h (Fig2B). After transfection, the quiescent phase was prolonged, the cell number slightly r educed, and the doubling time was about72h. The growth curve of DFCs was S-shaped. In the first3days after transfection, these cells were in the quiescent phase and then entered the logarithmic phase. At8days after transfection, the cell growth was suppressed and the cell number remained stable.Under a light microscope, the cell morphology remained unchanged in the transfection group and non-transfection group. Detection of ALP activity: In the osteogenic induction group, BMP-9group and osteogenic induction+BMP-9group, the ALP activity was markedly increased at3,5and7days after Ad-BMP-9mediated transfection, when compared with GFP group and blank control group (Each experiment was repeated three times, with the average value as the experimental results,P=0.0079<0.01), and the increase in ALP activity was the most evidenton day7. Immunohistochemistry for ALP also showed the expressionof ALP in the BMP-9group was significantly increased when compared with the GFP group and blank control group.At14d after Ad-BMP-9mediated transfection, Alizarin Red staining showed some calcified nodules which were large and red, suggestingthe increased osteogenesis of DFCs following transfection (Fig5A). Inthe non-transfection group, the calcified nodules were occasionally found and lightly stained. 3) Regulation of osteogenic differentiation of BMP-9transfected DFCs by MAPK signaling pathwayThe BMP-9transfected DFCs were independently treated with2μmol/L,5μmol/L and10μmol/L SB203580, an inhibitor of p38MAPK.Seven days later, detection of ALP activity and ALP staining were performed to study the osteogenesis. Results showed treatment with SB203580at different concentrations reduced the ALP activity of BMP-9transfected cells, which suggested a concentration dependent manner. The inhibition was the most evident after treatment with10μmol/L SB203580. Significant difference in ALP activity was noted between BMP9/DMSO group and other groups (P<0.05). ALP staining revealed the staining intensity of ALP reduced with the increase in SB203580concentration, which was consistent with findings in detection of ALP activity.When compared with the GFP/DMSO group, the number of calcified nodules was markedly increased in the BMP9/DMSO group. In theBMP9/SB group, the calcified nodules significantly reduced as compared to the BMP9/DMSO group. In addition, the number of calcified nodules in the BMP9/PD was significantly higher than that in the BMP9/DMSO group.Effect of MAPK signaling pathway on expression of markers for osteogenesis in BMP-9transfected DFCs: The BMP-9transfected DFCsshowed the expression of OPN and OCN in the BMP9/DMSO group was significantly increased when compared with GFP/DMSO group. The expression of OPN and OCN in the BMP9/SB group was markedly lower than that in the BMP9/DMSO group. In the BMP9/PD group, the OPN and OCN expression was increased when compared with the BMP9/DMSO group.Our results showed, following BMP-9transfection, the phosphorylated MAPKp38and ERK1/2increased, suggesting that BMP-9can directly or indirectly activate MAPK signaling pathway. BMP-9activates P38MAPK signaling pathway to promote the osteogenic differentiation of dental follicle cells. However, BMP-9inhibits the ERK1/2, which then suppresses the osteogenic differentiation of dental folicle cells. After administration of P38MAPK inhibitor (SB203580) and/or ERK1/2specificinhibitor (PD98059) to suppress the MAPK signaling pathway, the ALP activity, calcium deposition and expression of OPN and OCN were determined. Results showed1) Inhibition of p38MAPK expression reduced the ALP activity and calcium deposition in BMP-9transfected cells,and also reduced the expression of OPN and OCN;2) inhibition of ERK1/2activity increased the ALP activity and calcium deposition in BMP-9transfected cells, and also elevated the expression of OPN and OCN.Conclusion: our results reveal BMP-9can be used as a factor to induce the osteogenesis of DFCs in a time dependent manner in which MAPK signaling pathway involves. Dental follicle cells can serve as seed cells in the tissue engineering of periodontal tissues. Our findings provide evidence for future studies on local application of BMP-9aiming tocontrol the healing of periodontal bone defect and regeneration of periodontal tissues. |
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