牙髓、牙周组织中骨髓来源细胞特性的实验研究
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摘要
骨髓来源的细胞可以迁移到多种组织中并分化为组织特异性的细胞,如心肌细胞,骨骼肌细胞,脂肪细胞,神经细胞,肝脏细胞等。但对于其是否可以迁移到牙齿组织还不了解。牙齿的发育是由上皮与来自神经嵴的外胚间充质细胞相互作用而形成,以往研究发现牙源性间充质中存在非神经嵴来源的细胞,但其来源不明。另外,牙髓干细胞和牙周膜干细胞内均存在不同增殖能力的细胞克隆,推测,不同来源的细胞在增殖能力和功能上存在差异。同时在表型上牙源性间充质干细胞与骨髓来源的间充质干细胞相似。因此,我们采用骨髓移植的模型,研究了骨髓来源的细胞在牙髓牙周组织中的分布与特性;探讨了迁移来的细胞和组织自身的细胞之间的关系与差异;并将培养的嵌合鼠的牙髓干细胞与骨髓间充质干细胞进行了比较,最后观察了骨髓来源细胞在牙周组织修复中的作用。本实验研究有以下三部分:
第一部分骨髓来源细胞在牙源性间充质组织内的分布
实验一骨髓来源细胞在多种组织中的分布
采用全骨髓移植的方法,将绿色荧光(green flurencence protein,GFP)小鼠的单核细胞通过尾静脉移植入野生型小鼠体内。当外周血单核细胞的GFP阳性率达到85%以上时认为嵌合成功。结果发现,骨髓来源的细胞可以迁移到多种组织中,包括牙髓、牙周组织、心脏、肺脏、脂肪、皮肤、胰腺和胸腺。而迁移到牙源性间充质组织中的骨髓来源的细胞多于其他组织。对照组采用GFP+的成纤维细胞与野生型小鼠单核细胞混合移植后,观察到牙源性间充质组织中几乎没有GFP+细胞。说明骨髓来源的细胞具有迁移到牙齿组织中的能力。采用免疫组织化学方法,观察到迁移至下颌骨中的细胞位于CD31阳性的血管内皮细胞周围,说明骨髓来源细胞是通过循环迁移到牙源性间充质组织中来的。
实验二SDF-1和CXCR-4在牙源性间充质组织中的表达
采用免疫组织化学和聚合酶链式反应检测了基质细胞相关因子(stromal derived factor 1,SDF-1)在牙齿组织,肺脏,肝脏,以及脂肪组织中的蛋白和m RNA表达水平。结果发现,SDF-1蛋白在牙齿组织中的表达量明显高于其他三个组织,而SDF-1的m RNA表达顺序为牙髓>脂肪>肝脏。同时发现了骨髓间充质干细胞表达SDF-1的特异性受体CXCR-4。说明骨髓来源的细胞迁移到不同的组织内,是通过SDF-1/CXCR-4这个通路介导的,且SDF-1在组织内的表达量与迁移的细胞数量呈正相关。
第二部分骨髓来源的细胞在牙髓组织中的分布与特性
实验一骨髓来源细胞在牙髓中的分布和功能
骨髓移植后随着移植时间的延长,迁移入牙髓的细胞逐渐增多,而且分布逐渐变广。移植1个月后,骨髓来源的细胞主要位于牙髓的中央,未见细胞分布于成牙本质细胞层。移植3个月后,骨髓来源的细胞可见于牙髓中央,少细胞层,多细胞层,且有少量的成牙本质细胞为GFP阳性,同时表达牙本质涎蛋白和牙本质蛋白1。说明牙髓和骨髓间存在着持续、活跃的交流,迁入牙髓的细胞中少部分可以转化为成牙本质细胞。而将嵌合鼠的下颌切牙根尖蕾组织移植入野生型小鼠的肾被膜内,4周后可见移植物形成了牙本质样结构,而部分骨髓来源细胞位于牙本质的内侧成牙本质细胞层,说明迁移入牙髓的细胞具有参与牙髓再生的功能。
实验二牙髓中骨髓来源细胞的表型
采用免疫荧光技术,检测了骨髓来源细胞的间充质干细胞表型。发现牙髓中大部分骨髓来源细胞已经分化,少部分表达间充质干细胞标志物如CD106、CD105、CD90,但不表达造血细胞标志物CD45,说明迁移入牙髓的细胞具有间充质细胞的表型。同时发现牙髓内骨髓来源的间充质干细胞明显少于组织内源性的间充质干细胞。提示,组织内源性的和迁移来的间充质干细胞共同参与维持牙髓组织的稳态,其中内源性的干细胞起着更为主要的作用。
实验三体外培养的牙髓干细胞与骨髓间充质干细胞的比较
采用酶消化法培养嵌合鼠的小鼠牙髓干细胞,牙髓干细胞内含有不同表型的克隆。其中GPF+的克隆Ki67+的细胞明显多于GFP-的细胞克隆,提示不同来源的克隆在增殖能力上有所差异。而与嵌合鼠的骨髓间充质干细胞相比,牙髓内GFP+/CD106+,、CD105+、CD90+的细胞明显少于骨髓间充质干细胞。说明牙髓的微环境改变了部分骨髓来源细胞的特性。
第三部分骨髓来源细胞在牙周组织内的特性
实验一骨髓来源的细胞在牙周组织中的分布与特性
随着骨髓移植时间的延长,迁入牙周组织的细胞逐渐增多,其特征与牙髓相似。移植1个月后,骨髓来源的细胞主要位于微血管周围。移植3个月后,骨髓来源细胞可见于牙槽骨、牙周膜、牙骨质内。部分骨髓来源的细胞表达成骨及牙周膜相关的蛋白,如骨桥蛋白、碱性磷酸酶、纤维连接蛋白。说明骨髓来源的细胞迁移到了牙周组织内,并分化成了牙周组织特异性的细胞,而且骨髓来源的Ki67+的细胞多于内源性的Ki67+的细胞。说明骨髓来源的细胞增殖能力强于内源性的细胞。同时鉴定牙周组织中骨髓来源细胞的表型,我们发现牙周组织中CD106、CD105和CD90的表达状况与牙髓组织相似。进一步证实了迁移到牙周组织内的骨髓来源细胞的间充质特性。
实验二骨髓来源细胞在牙周再生中的作用
将煅烧牛骨粉末植入到嵌合鼠的实验性牙周缺损处,观察骨髓来源的细胞在牙周组织再生过程中的作用。结果,术后5d,大量的GFP+的细胞进入缺损区域并表达CD105,随后进入缺损区域的细胞的速度减缓并有新骨形成。术后30d,可见GFP+的细胞位于新形成的牙槽骨内,而且这些细胞同时表达骨桥蛋白、碱性磷酸酶、Ki67、纤维连接蛋白。说明骨髓来源细胞可以迁移到缺损区域并分化为牙周组织相关的细胞。小部分骨髓来源细胞仍能保持间充质干细胞的特性,但数量上明显少于内源性的间充质干细胞。同时牙周缺损处的SDF-1的表达量明显高于正常牙周组织,这与迁入的骨髓来源的细胞数量呈正相关。提示在牙周损伤修复过程中,SDF-1参与了骨髓细胞的归巢。
总之,采用骨髓移植的模式,我们首次发现了骨髓来源细胞可以迁移到牙髓、牙周组织中,并且迁移来的细胞多于其他组织。迁移来的细胞大部分分化为了牙源性相关细胞,表达牙齿相关的标志物。少部分维持了间充质干细胞的表型,但其在数量明显少于内源性的间充质干细胞。说明骨髓和牙源性间充质组织间存在着活跃的联系,他们作为牙源性间充质前体/干细胞的储备库,在维持牙源性组织的稳态中发挥重要作用。
Bone marrow derived cells (BMDCs) are capable of homing to many tissues in response to injury signals and differentiating into tissue-specific cells, including muscle, liver, brain, pancreases islet, and even epithelial lineages, suggesting high developmental plasticity. However, the potential of the BMDC to contribute to the dental mesenchymal cells is unknown. It is traditionally considered that the dental mesenchymal stem cells originated from the cranial neural crest ectomesenchyme (CNC). It has been demonstrated that non-CNC-derived cells increased during the tooth morphogenesis, while the origin of the non-CNC-derived cells was still unknown. In addition, previous studies have suggested that the individual dental pulp stem cells (DPSC) colonies have distinguished differences in their proliferation and regeneration ability. To test that hypothesis that BMDCs could migrate into dental mesenchymal tissues, we used the GFP+ bone marrow chimeric mice to identify the characteristics of migrating cells in pulp and periodontium. We also determined the relationships and differences of the migrating and tissue-resident stem cells. In addition to the pulp tissue analysis, DPSCs were also isolated and compared with BMMSCs. We found that BMDCs can migrate into normal as well as damaged dental tissues and differentiate into the dental tissue specific cell type, while only a small fraction of migrating BMDCs maintain the stem cell phenotype.
Section 1 BMDCs migrat into multi-organs
In experiment 1, in order to identify the capacity of BMDC to engraft into multiple organs under normal conditions, the frozen sections of several organs from the chimeric mouse were evaluated by fluorescence microscopy. The results indicated that the bone marrow derived cells have the ability to migrate into multiple organs including dental pulp, periodontium, liver, lung, heart, pancreas, skin, and thymus at 3 month post BMT. More importantly, we found that the GFP+ BMC cells homed to the dental tissues to a greater degree than in other organs, except for the thymus which was expected and considered as a positive control. And we also found that the delivery of BMDC into the dental tissue may be through the circulation by the immunostaining of CD31 in the mandible. Interestingly, we found that there was a passage connecting the bone marrow cavity and the periodontal ligament, through which the GFP+ cells were detected to pass.
In experiment 2, to further dissect the mechanism and to identify the signal that controls the stem cell recruitment, we detected SDF-1 protein expression in the dental, lung, liver, and pancreatic tissues, which were measured by the area of SDF-1+/Hoechst staining. The SDF-1 expression was significantly higher in dental than in other tissues. And also the m RNA expression in the dental pulp cells were higher than in the liver and adipose cells by realtime PCR. We also determination the characteristics of the bone marrow derived mesenchymal stem cells form chimeric mice. We found these cells expresse the CXCR-4 which was the unique receptor. Thus, the results provide preliminary evidence suggesting that SDF-1/CXCR-4 pathway may involve in the homing of the BMDCs in the dental tissues.
Section 2 characterization of the BMDCs in the dental pulp tissue
In experiment 1, in order to understand the interaction between the bone marrow and dental pulp, we serially analyzed the engraftment of the BMDC in the dental tissue after BMT. We found that the migration of the GFP+ BMDC into dental pulp was gradually increased with time after BMT. One month after BMT, the GFP+ BMC were mainly located in the cell-rich layer and the center part of pulp. The cells then gradually appeared in both the cell-free layer and the cell-rich layer at 3 month post-BMT. Moreover, the GFP+ cells could be observed, although rarely, in the odontoblast which were also positive for the DSP and DMP-1. In order to confirm the founction of BMDCs in the pulp, we transplanted the apical bud from chimiric mice into the WT mice and found that the BMDCs could involve the regeneration of the dental pulp. These results above demonstrated that the bone marrow and dental pulp have vivid communication, and the BMDCs could differentiation into the odontoblast in normal and regeneration conditions.
In experimental 2, to characterize the phenotype of the engrafting cells in the dental pulp, the mandible sections were stained for CD106, CD105, CD90, and CD45. Actually, most of the GFP+ cells were not positive for the mesenchymal stem cells’surface markers in the pulp and only small fraction of cells maintain the mesenchymal stem/progenitor cells phenotype, which was positive for the surface marlers of CD105, CD106, CD90 and negative for CD45. And we also found the GFP+/CDs+ cells were less than GFP-/CDs+ cells significantly. This experiment indicated that the BMDCs would migrate into the denal pulp and convert into the dental mesenchymal progenitor cells.
In experiment 3, to further identify the characteristics of the dental pulp cells, we isolated and cultured the dental pulp stem cells (DPSC) from the chimeric mice at 4 month post-BMT. The colonies with more GFP+ cells expressed stronger Ki67 signals than the colonies without GFP signals, suggesting that the BMDCs have stronger ability in proliferation than the tissue resident cells. Further investigation of the mesenchymal phenotype of the migrating cells in dental pulp and comparison with BMSC showed that most of the GFP+ migrating cells did not stain positively for CD105, CD106, and CD90 however, most of the GFP+ BMSCs were also positive for those markers, indicating that the microenviroment of dental pulp educated the BMDCs and change the phenotype of these cells.
Section 3 characterization of the BMDCs in the periodontal tissue
In experiment 1, the dynamic distribution of the BMDCs in the periodontal tissue was determined. We found that the migration of the GFP+ BMDC into periodontal tissue was gradually increased. 1 month after BMT, the GFP+ cells were detected predominantly near and surrounding the endothelial cells in the periodontal ligament, and at 3 month post-BMT, the GFP+ cells were more widely distributed in the tissues, including the periodontal ligament, alveolar bone, and cementum. And we also analyze the function of BMDCs in the periodontal tissues. We found that there were some OPN+/GFP+ cells in the pulp, gingival, and periodontal ligament. In the periodontal area, ALP+/GFP+ cells were observed along the alveolar bone with many ALP+/GFP- cells along the root surface, indicating the BMDCs near the alveolar bone may have converted into the periodontal ligament cells or osteoblasts, and most of the fibronectin + cells were in the periodontal ligament and were positive for GFP. Most of the Ki67 positive cells were also GFP+, and only a few Ki67+/GFP- cells were seen in the periodontal ligament, suggesting the BMDCs could proliferate and have more of a potential to do so in the dental tissue than the tissue-resident cells. In addition, the expression pattern of CD106, CD105, CD90, CD45 were similar with pulp.
In experiment 2, five days after the experimental alveolar bone defect(EABD) operation, and the distribution of CD105+ cells was similar with that of the GFP+ cells. Within 15 days of the operation, the newly formed alveolar bone was filled with the GFP+ osteoblasts. At 30 days, the new alveolar bone and the cells in the bone marrow cavity were positively stained with anti-GFP. Then we found that OPN positive cells were observed around the newly forming bone and periodontal ligament, and most GFP+ cells were also OPN positive. There were ALP+/GFP+ cells in the periodontal region and around the bone. Additionally, we found the expression of SDF-1 was elevated in the EABD compared with the healthy tissues. The results demonstrated that BMDCs could participate into the regeneration of the periodontal tissues and differentiate into tissue-spccific founctional cells.
Conclusion: Our findings suggest that the bone marrow and dental tissue are closely related and actively exchanged. BMDCs preferentially migrate into the pulp and periodontium over other organs,and can engraft into dental tissue and become tissue-specific mesenchymal progenitor cells. Furthermore, although the migrating stem cells were fewer in number compared with the tissue-resident stem cells, they have greater proliferative potential. The resident and migrating stem cells collectively play crucial roles in maintenance and regeneration.
第一部分骨髓来源细胞在牙源性间充质组织内的分布
实验一骨髓来源细胞在多种组织中的分布
采用全骨髓移植的方法,将绿色荧光(green flurencence protein,GFP)小鼠的单核细胞通过尾静脉移植入野生型小鼠体内。当外周血单核细胞的GFP阳性率达到85%以上时认为嵌合成功。结果发现,骨髓来源的细胞可以迁移到多种组织中,包括牙髓、牙周组织、心脏、肺脏、脂肪、皮肤、胰腺和胸腺。而迁移到牙源性间充质组织中的骨髓来源的细胞多于其他组织。对照组采用GFP+的成纤维细胞与野生型小鼠单核细胞混合移植后,观察到牙源性间充质组织中几乎没有GFP+细胞。说明骨髓来源的细胞具有迁移到牙齿组织中的能力。采用免疫组织化学方法,观察到迁移至下颌骨中的细胞位于CD31阳性的血管内皮细胞周围,说明骨髓来源细胞是通过循环迁移到牙源性间充质组织中来的。
实验二SDF-1和CXCR-4在牙源性间充质组织中的表达
采用免疫组织化学和聚合酶链式反应检测了基质细胞相关因子(stromal derived factor 1,SDF-1)在牙齿组织,肺脏,肝脏,以及脂肪组织中的蛋白和m RNA表达水平。结果发现,SDF-1蛋白在牙齿组织中的表达量明显高于其他三个组织,而SDF-1的m RNA表达顺序为牙髓>脂肪>肝脏。同时发现了骨髓间充质干细胞表达SDF-1的特异性受体CXCR-4。说明骨髓来源的细胞迁移到不同的组织内,是通过SDF-1/CXCR-4这个通路介导的,且SDF-1在组织内的表达量与迁移的细胞数量呈正相关。
第二部分骨髓来源的细胞在牙髓组织中的分布与特性
实验一骨髓来源细胞在牙髓中的分布和功能
骨髓移植后随着移植时间的延长,迁移入牙髓的细胞逐渐增多,而且分布逐渐变广。移植1个月后,骨髓来源的细胞主要位于牙髓的中央,未见细胞分布于成牙本质细胞层。移植3个月后,骨髓来源的细胞可见于牙髓中央,少细胞层,多细胞层,且有少量的成牙本质细胞为GFP阳性,同时表达牙本质涎蛋白和牙本质蛋白1。说明牙髓和骨髓间存在着持续、活跃的交流,迁入牙髓的细胞中少部分可以转化为成牙本质细胞。而将嵌合鼠的下颌切牙根尖蕾组织移植入野生型小鼠的肾被膜内,4周后可见移植物形成了牙本质样结构,而部分骨髓来源细胞位于牙本质的内侧成牙本质细胞层,说明迁移入牙髓的细胞具有参与牙髓再生的功能。
实验二牙髓中骨髓来源细胞的表型
采用免疫荧光技术,检测了骨髓来源细胞的间充质干细胞表型。发现牙髓中大部分骨髓来源细胞已经分化,少部分表达间充质干细胞标志物如CD106、CD105、CD90,但不表达造血细胞标志物CD45,说明迁移入牙髓的细胞具有间充质细胞的表型。同时发现牙髓内骨髓来源的间充质干细胞明显少于组织内源性的间充质干细胞。提示,组织内源性的和迁移来的间充质干细胞共同参与维持牙髓组织的稳态,其中内源性的干细胞起着更为主要的作用。
实验三体外培养的牙髓干细胞与骨髓间充质干细胞的比较
采用酶消化法培养嵌合鼠的小鼠牙髓干细胞,牙髓干细胞内含有不同表型的克隆。其中GPF+的克隆Ki67+的细胞明显多于GFP-的细胞克隆,提示不同来源的克隆在增殖能力上有所差异。而与嵌合鼠的骨髓间充质干细胞相比,牙髓内GFP+/CD106+,、CD105+、CD90+的细胞明显少于骨髓间充质干细胞。说明牙髓的微环境改变了部分骨髓来源细胞的特性。
第三部分骨髓来源细胞在牙周组织内的特性
实验一骨髓来源的细胞在牙周组织中的分布与特性
随着骨髓移植时间的延长,迁入牙周组织的细胞逐渐增多,其特征与牙髓相似。移植1个月后,骨髓来源的细胞主要位于微血管周围。移植3个月后,骨髓来源细胞可见于牙槽骨、牙周膜、牙骨质内。部分骨髓来源的细胞表达成骨及牙周膜相关的蛋白,如骨桥蛋白、碱性磷酸酶、纤维连接蛋白。说明骨髓来源的细胞迁移到了牙周组织内,并分化成了牙周组织特异性的细胞,而且骨髓来源的Ki67+的细胞多于内源性的Ki67+的细胞。说明骨髓来源的细胞增殖能力强于内源性的细胞。同时鉴定牙周组织中骨髓来源细胞的表型,我们发现牙周组织中CD106、CD105和CD90的表达状况与牙髓组织相似。进一步证实了迁移到牙周组织内的骨髓来源细胞的间充质特性。
实验二骨髓来源细胞在牙周再生中的作用
将煅烧牛骨粉末植入到嵌合鼠的实验性牙周缺损处,观察骨髓来源的细胞在牙周组织再生过程中的作用。结果,术后5d,大量的GFP+的细胞进入缺损区域并表达CD105,随后进入缺损区域的细胞的速度减缓并有新骨形成。术后30d,可见GFP+的细胞位于新形成的牙槽骨内,而且这些细胞同时表达骨桥蛋白、碱性磷酸酶、Ki67、纤维连接蛋白。说明骨髓来源细胞可以迁移到缺损区域并分化为牙周组织相关的细胞。小部分骨髓来源细胞仍能保持间充质干细胞的特性,但数量上明显少于内源性的间充质干细胞。同时牙周缺损处的SDF-1的表达量明显高于正常牙周组织,这与迁入的骨髓来源的细胞数量呈正相关。提示在牙周损伤修复过程中,SDF-1参与了骨髓细胞的归巢。
总之,采用骨髓移植的模式,我们首次发现了骨髓来源细胞可以迁移到牙髓、牙周组织中,并且迁移来的细胞多于其他组织。迁移来的细胞大部分分化为了牙源性相关细胞,表达牙齿相关的标志物。少部分维持了间充质干细胞的表型,但其在数量明显少于内源性的间充质干细胞。说明骨髓和牙源性间充质组织间存在着活跃的联系,他们作为牙源性间充质前体/干细胞的储备库,在维持牙源性组织的稳态中发挥重要作用。
Bone marrow derived cells (BMDCs) are capable of homing to many tissues in response to injury signals and differentiating into tissue-specific cells, including muscle, liver, brain, pancreases islet, and even epithelial lineages, suggesting high developmental plasticity. However, the potential of the BMDC to contribute to the dental mesenchymal cells is unknown. It is traditionally considered that the dental mesenchymal stem cells originated from the cranial neural crest ectomesenchyme (CNC). It has been demonstrated that non-CNC-derived cells increased during the tooth morphogenesis, while the origin of the non-CNC-derived cells was still unknown. In addition, previous studies have suggested that the individual dental pulp stem cells (DPSC) colonies have distinguished differences in their proliferation and regeneration ability. To test that hypothesis that BMDCs could migrate into dental mesenchymal tissues, we used the GFP+ bone marrow chimeric mice to identify the characteristics of migrating cells in pulp and periodontium. We also determined the relationships and differences of the migrating and tissue-resident stem cells. In addition to the pulp tissue analysis, DPSCs were also isolated and compared with BMMSCs. We found that BMDCs can migrate into normal as well as damaged dental tissues and differentiate into the dental tissue specific cell type, while only a small fraction of migrating BMDCs maintain the stem cell phenotype.
Section 1 BMDCs migrat into multi-organs
In experiment 1, in order to identify the capacity of BMDC to engraft into multiple organs under normal conditions, the frozen sections of several organs from the chimeric mouse were evaluated by fluorescence microscopy. The results indicated that the bone marrow derived cells have the ability to migrate into multiple organs including dental pulp, periodontium, liver, lung, heart, pancreas, skin, and thymus at 3 month post BMT. More importantly, we found that the GFP+ BMC cells homed to the dental tissues to a greater degree than in other organs, except for the thymus which was expected and considered as a positive control. And we also found that the delivery of BMDC into the dental tissue may be through the circulation by the immunostaining of CD31 in the mandible. Interestingly, we found that there was a passage connecting the bone marrow cavity and the periodontal ligament, through which the GFP+ cells were detected to pass.
In experiment 2, to further dissect the mechanism and to identify the signal that controls the stem cell recruitment, we detected SDF-1 protein expression in the dental, lung, liver, and pancreatic tissues, which were measured by the area of SDF-1+/Hoechst staining. The SDF-1 expression was significantly higher in dental than in other tissues. And also the m RNA expression in the dental pulp cells were higher than in the liver and adipose cells by realtime PCR. We also determination the characteristics of the bone marrow derived mesenchymal stem cells form chimeric mice. We found these cells expresse the CXCR-4 which was the unique receptor. Thus, the results provide preliminary evidence suggesting that SDF-1/CXCR-4 pathway may involve in the homing of the BMDCs in the dental tissues.
Section 2 characterization of the BMDCs in the dental pulp tissue
In experiment 1, in order to understand the interaction between the bone marrow and dental pulp, we serially analyzed the engraftment of the BMDC in the dental tissue after BMT. We found that the migration of the GFP+ BMDC into dental pulp was gradually increased with time after BMT. One month after BMT, the GFP+ BMC were mainly located in the cell-rich layer and the center part of pulp. The cells then gradually appeared in both the cell-free layer and the cell-rich layer at 3 month post-BMT. Moreover, the GFP+ cells could be observed, although rarely, in the odontoblast which were also positive for the DSP and DMP-1. In order to confirm the founction of BMDCs in the pulp, we transplanted the apical bud from chimiric mice into the WT mice and found that the BMDCs could involve the regeneration of the dental pulp. These results above demonstrated that the bone marrow and dental pulp have vivid communication, and the BMDCs could differentiation into the odontoblast in normal and regeneration conditions.
In experimental 2, to characterize the phenotype of the engrafting cells in the dental pulp, the mandible sections were stained for CD106, CD105, CD90, and CD45. Actually, most of the GFP+ cells were not positive for the mesenchymal stem cells’surface markers in the pulp and only small fraction of cells maintain the mesenchymal stem/progenitor cells phenotype, which was positive for the surface marlers of CD105, CD106, CD90 and negative for CD45. And we also found the GFP+/CDs+ cells were less than GFP-/CDs+ cells significantly. This experiment indicated that the BMDCs would migrate into the denal pulp and convert into the dental mesenchymal progenitor cells.
In experiment 3, to further identify the characteristics of the dental pulp cells, we isolated and cultured the dental pulp stem cells (DPSC) from the chimeric mice at 4 month post-BMT. The colonies with more GFP+ cells expressed stronger Ki67 signals than the colonies without GFP signals, suggesting that the BMDCs have stronger ability in proliferation than the tissue resident cells. Further investigation of the mesenchymal phenotype of the migrating cells in dental pulp and comparison with BMSC showed that most of the GFP+ migrating cells did not stain positively for CD105, CD106, and CD90 however, most of the GFP+ BMSCs were also positive for those markers, indicating that the microenviroment of dental pulp educated the BMDCs and change the phenotype of these cells.
Section 3 characterization of the BMDCs in the periodontal tissue
In experiment 1, the dynamic distribution of the BMDCs in the periodontal tissue was determined. We found that the migration of the GFP+ BMDC into periodontal tissue was gradually increased. 1 month after BMT, the GFP+ cells were detected predominantly near and surrounding the endothelial cells in the periodontal ligament, and at 3 month post-BMT, the GFP+ cells were more widely distributed in the tissues, including the periodontal ligament, alveolar bone, and cementum. And we also analyze the function of BMDCs in the periodontal tissues. We found that there were some OPN+/GFP+ cells in the pulp, gingival, and periodontal ligament. In the periodontal area, ALP+/GFP+ cells were observed along the alveolar bone with many ALP+/GFP- cells along the root surface, indicating the BMDCs near the alveolar bone may have converted into the periodontal ligament cells or osteoblasts, and most of the fibronectin + cells were in the periodontal ligament and were positive for GFP. Most of the Ki67 positive cells were also GFP+, and only a few Ki67+/GFP- cells were seen in the periodontal ligament, suggesting the BMDCs could proliferate and have more of a potential to do so in the dental tissue than the tissue-resident cells. In addition, the expression pattern of CD106, CD105, CD90, CD45 were similar with pulp.
In experiment 2, five days after the experimental alveolar bone defect(EABD) operation, and the distribution of CD105+ cells was similar with that of the GFP+ cells. Within 15 days of the operation, the newly formed alveolar bone was filled with the GFP+ osteoblasts. At 30 days, the new alveolar bone and the cells in the bone marrow cavity were positively stained with anti-GFP. Then we found that OPN positive cells were observed around the newly forming bone and periodontal ligament, and most GFP+ cells were also OPN positive. There were ALP+/GFP+ cells in the periodontal region and around the bone. Additionally, we found the expression of SDF-1 was elevated in the EABD compared with the healthy tissues. The results demonstrated that BMDCs could participate into the regeneration of the periodontal tissues and differentiate into tissue-spccific founctional cells.
Conclusion: Our findings suggest that the bone marrow and dental tissue are closely related and actively exchanged. BMDCs preferentially migrate into the pulp and periodontium over other organs,and can engraft into dental tissue and become tissue-specific mesenchymal progenitor cells. Furthermore, although the migrating stem cells were fewer in number compared with the tissue-resident stem cells, they have greater proliferative potential. The resident and migrating stem cells collectively play crucial roles in maintenance and regeneration.
引文
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