重组腺病毒BMP-9对牙囊干细胞成骨向诱导分化的研究
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摘要
牙周炎作为人类最普遍、最古老的疾病之一，是一种慢性感染性疾病；牙菌斑中的微生物引起牙齿支持组织形成炎症、产生深牙周袋、附着丧失与牙槽骨的垂直性或水平性吸收，最终导致牙齿松动和脱落；也是成年人牙齿缺失的主要原因[4]。牙周骨缺损修复和正常生理结构与生理功能的重建是牙周炎治疗的最终目的,目前对骨上袋缺损的再生性修复尚缺乏行之有效的治疗手段，不可能实现真正意义上的牙周生物学修复。牙源性干细胞与牙周组织工程技术为新的生物性修复再生牙周组织及其牙齿提供了可能，即用细胞工程和生物工程化的方法实现牙周组织再生，形成新附着。应用牙源性干细胞和组织工程技术，按照牙周组织生长与发育的基本原理和基本方法来模拟再现牙周组织整个发育过程才能真正地实现牙周组织再生。
再生组织工程中，种子细胞的选择具有决定性意义；细胞获取方便，移植入机体后细胞生物学活性稳定，分化增殖能力强等是理想种子细胞必备条件。发育形成牙周组织的牙囊是一种疏松结缔组织，起源于外胚间充间充质，包绕着牙胚期的成釉器和牙乳头。一般认为牙周分泌牙骨质的成牙骨质细胞由靠近正在形成中牙根的牙囊最内层细胞发育而成，而牙周组织中分泌骨基质、形成部分牙槽骨的成骨细胞是邻近牙槽骨的外层牙囊细胞发育而成；成纤维细胞由中间层牙囊细胞发育，产生牙周膜纤维。作为牙周组织前体细胞的牙囊细胞具有自我更新能力和多向分化能力的干细胞特性与异质性，如果能将牙囊干细胞（dental follicle stem cells，DFCs）分离出来，这对研究牙周组织的生长发育形成起着至关重要的作用；对牙齿移植、牙再植及牙再生均具有重要的指导意义；DFCs较强的增殖分化能力也使其成为一种值得考虑的组织工程候选种子细胞。
骨形成蛋白（Bone Morphogenetic Proteins，BMPs）是一类糖蛋白，在骨的发育和塑建中起着关键性作用，可通过多种途径多种方式促进细胞向成骨部位定向趋化、募集和促进成骨细胞定向分化，促进血管的生成和组织血管化形成，调节多种局部生长因子的活性，影响生长因子的形成，促进骨组织的形成。在已知对骨再生有促进作用的生长因子中，BMPs是最强的生长因子，目前已经分离出20余种，其中已报道具有成骨潜能是BMPs2、4、6、7、9；BMP9具有诱导和维持胚胎神经元的类胆碱分化功能，能够调节脂肪酸代谢和葡萄糖代谢，能够调节体内铁离子的动态平衡等多种重要生物学功能，但长期以来人们缺乏研究和了解BMP9在骨形成和骨再生中的作用。TC HE系统研究了在骨形成过程中的BMP2至BMP15共14种BMPs所起的生物学作用，结果表明这14种BMPs中，BMP9具有很强的促进软骨形成与成骨活性，作用强于BMP2和BMP7，也是BMPs家族中促进成骨分化的生长因子之一，极具研究潜力和应用前景[64,65]；但在口腔成骨方面和对DFCs的影响未见相关报道。
DFCs具有成脂肪、成骨、成神经等多向分化潜能的干细胞特征，在适当的诱导条件下，可以多向分化。然而，由于细胞因子网络性调控和信号通路的复杂性以及研究手段的限制，DFCs的具体分化调控机制尚不明确。本研究旨在着力探讨重组腺病毒BMP-9基因转染大鼠DFCs的可行性及其转染后的成骨向分化与作用机制，以获得可用于牙周骨组织再生工程的基因修饰的种子细胞，为后续牙周组织工程治疗口腔骨缺损奠定基础。研究内容和研究结果如下：
1.在解剖显微镜下，从SD大鼠下颌骨牙胚上剥离牙囊，组织块法和酶消化获取牙囊细胞原代培养、有限稀释法克隆纯化、细胞表型鉴定，成骨、成脂肪多向诱导分化，结果表明：DFCs具有成骨、成脂肪等多向分化潜能的干细胞特征，在定向成骨诱导分化过程中，没有出现成脂肪向分化。
2.腺病毒介导的BMP-9转染第三代DFCs，设立空白对照组，绿色荧光病毒组（GFP组）、BMP组，成骨诱导组、联合组。通过观察细胞形态和MTT生长曲线变化、荧光显微镜及RT-PCR检测转染后BMP-9基因mRNA的表达；碱性磷酸酶ALP活性、ALP染色及钙茜素红染色测定转染后DFCs的早晚期成骨活性。结果表明：转染的DFCs稳定表达BMP-9，说明转染后的BMP-9基因能成功被转录并表达于DFCs；与未转染对照组相比较，转染组ALP染色及茜素红钙结节染色为阳性；转染组显著高于未转染组，说明DFCs有良好的骨向分化能力，也提示了BMP-9对DFCs有成骨诱导作用。
3.应用抑制剂p38蛋白激酶抑制剂SB203580和ERK1/2激酶抑制剂PD98059干扰BMP-9转染的DFCs，用来阻断p38丝裂原活化蛋白激酶（p38MAPK）和细胞外信号调节激酶（ERK1/2)胞内信号传递，通过ALP活性及染色检测；Western blot分析；钙茜素红染色实验分析MAPKs信号通路对BMP-9诱导的DFCs成骨向分化过程中的调控和影响。结果表明：抑制MAPKp38的表达后降低了Ad-BMP-9诱导的DFCs的ALP的活性和ALP染色，且减少了钙盐沉积与骨桥蛋白(osteopontin,OPN)和骨钙素（osteocalin,OCN）的表达；抑制ERK1/2的活性后则相反。同时，Western blot分析显示Smad和MAPK通路激活，表明BMP诱导DFCs成骨向分化涉及到两条或可能更多的信号通路激活。研究结论：
1.成功获得了大鼠DFCs克隆，合适诱导条件下能够多向诱导分化。
2.在腺病毒BMP-9转染DFCs实验中，通过RT-PCR可检测到转染后BMP-9基因mRNA的表达，碱性磷酸酶和钙茜素红染色测定转染后DFCs的早期和晚期成骨活性，说明BMP-9可以成功地转染大鼠DFCs，转染后DFCs高表达BMP-9，且具有明显的成骨作用。
3.应用MAPKp38蛋白激酶抑制剂和ERK1/2激酶抑制剂干涉BMP-9转染的DFCs，通过ALP活性及染色检测、Western blot分析、钙盐沉积实验观察BMP-9转染的DFCs成骨向分化可通过ERK1/2和p38信号通路进行调控，为后续试验奠定了基础。
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 cells
Briefly, 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 DFCs
The 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 pathway
The 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.
再生组织工程中，种子细胞的选择具有决定性意义；细胞获取方便，移植入机体后细胞生物学活性稳定，分化增殖能力强等是理想种子细胞必备条件。发育形成牙周组织的牙囊是一种疏松结缔组织，起源于外胚间充间充质，包绕着牙胚期的成釉器和牙乳头。一般认为牙周分泌牙骨质的成牙骨质细胞由靠近正在形成中牙根的牙囊最内层细胞发育而成，而牙周组织中分泌骨基质、形成部分牙槽骨的成骨细胞是邻近牙槽骨的外层牙囊细胞发育而成；成纤维细胞由中间层牙囊细胞发育，产生牙周膜纤维。作为牙周组织前体细胞的牙囊细胞具有自我更新能力和多向分化能力的干细胞特性与异质性，如果能将牙囊干细胞（dental follicle stem cells，DFCs）分离出来，这对研究牙周组织的生长发育形成起着至关重要的作用；对牙齿移植、牙再植及牙再生均具有重要的指导意义；DFCs较强的增殖分化能力也使其成为一种值得考虑的组织工程候选种子细胞。
骨形成蛋白（Bone Morphogenetic Proteins，BMPs）是一类糖蛋白，在骨的发育和塑建中起着关键性作用，可通过多种途径多种方式促进细胞向成骨部位定向趋化、募集和促进成骨细胞定向分化，促进血管的生成和组织血管化形成，调节多种局部生长因子的活性，影响生长因子的形成，促进骨组织的形成。在已知对骨再生有促进作用的生长因子中，BMPs是最强的生长因子，目前已经分离出20余种，其中已报道具有成骨潜能是BMPs2、4、6、7、9；BMP9具有诱导和维持胚胎神经元的类胆碱分化功能，能够调节脂肪酸代谢和葡萄糖代谢，能够调节体内铁离子的动态平衡等多种重要生物学功能，但长期以来人们缺乏研究和了解BMP9在骨形成和骨再生中的作用。TC HE系统研究了在骨形成过程中的BMP2至BMP15共14种BMPs所起的生物学作用，结果表明这14种BMPs中，BMP9具有很强的促进软骨形成与成骨活性，作用强于BMP2和BMP7，也是BMPs家族中促进成骨分化的生长因子之一，极具研究潜力和应用前景[64,65]；但在口腔成骨方面和对DFCs的影响未见相关报道。
DFCs具有成脂肪、成骨、成神经等多向分化潜能的干细胞特征，在适当的诱导条件下，可以多向分化。然而，由于细胞因子网络性调控和信号通路的复杂性以及研究手段的限制，DFCs的具体分化调控机制尚不明确。本研究旨在着力探讨重组腺病毒BMP-9基因转染大鼠DFCs的可行性及其转染后的成骨向分化与作用机制，以获得可用于牙周骨组织再生工程的基因修饰的种子细胞，为后续牙周组织工程治疗口腔骨缺损奠定基础。研究内容和研究结果如下：
1.在解剖显微镜下，从SD大鼠下颌骨牙胚上剥离牙囊，组织块法和酶消化获取牙囊细胞原代培养、有限稀释法克隆纯化、细胞表型鉴定，成骨、成脂肪多向诱导分化，结果表明：DFCs具有成骨、成脂肪等多向分化潜能的干细胞特征，在定向成骨诱导分化过程中，没有出现成脂肪向分化。
2.腺病毒介导的BMP-9转染第三代DFCs，设立空白对照组，绿色荧光病毒组（GFP组）、BMP组，成骨诱导组、联合组。通过观察细胞形态和MTT生长曲线变化、荧光显微镜及RT-PCR检测转染后BMP-9基因mRNA的表达；碱性磷酸酶ALP活性、ALP染色及钙茜素红染色测定转染后DFCs的早晚期成骨活性。结果表明：转染的DFCs稳定表达BMP-9，说明转染后的BMP-9基因能成功被转录并表达于DFCs；与未转染对照组相比较，转染组ALP染色及茜素红钙结节染色为阳性；转染组显著高于未转染组，说明DFCs有良好的骨向分化能力，也提示了BMP-9对DFCs有成骨诱导作用。
3.应用抑制剂p38蛋白激酶抑制剂SB203580和ERK1/2激酶抑制剂PD98059干扰BMP-9转染的DFCs，用来阻断p38丝裂原活化蛋白激酶（p38MAPK）和细胞外信号调节激酶（ERK1/2)胞内信号传递，通过ALP活性及染色检测；Western blot分析；钙茜素红染色实验分析MAPKs信号通路对BMP-9诱导的DFCs成骨向分化过程中的调控和影响。结果表明：抑制MAPKp38的表达后降低了Ad-BMP-9诱导的DFCs的ALP的活性和ALP染色，且减少了钙盐沉积与骨桥蛋白(osteopontin,OPN)和骨钙素（osteocalin,OCN）的表达；抑制ERK1/2的活性后则相反。同时，Western blot分析显示Smad和MAPK通路激活，表明BMP诱导DFCs成骨向分化涉及到两条或可能更多的信号通路激活。研究结论：
1.成功获得了大鼠DFCs克隆，合适诱导条件下能够多向诱导分化。
2.在腺病毒BMP-9转染DFCs实验中，通过RT-PCR可检测到转染后BMP-9基因mRNA的表达，碱性磷酸酶和钙茜素红染色测定转染后DFCs的早期和晚期成骨活性，说明BMP-9可以成功地转染大鼠DFCs，转染后DFCs高表达BMP-9，且具有明显的成骨作用。
3.应用MAPKp38蛋白激酶抑制剂和ERK1/2激酶抑制剂干涉BMP-9转染的DFCs，通过ALP活性及染色检测、Western blot分析、钙盐沉积实验观察BMP-9转染的DFCs成骨向分化可通过ERK1/2和p38信号通路进行调控，为后续试验奠定了基础。
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 cells
Briefly, 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 DFCs
The 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 pathway
The 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.
引文
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