Suda N, Kitahara Y, Hammond V E, Ohyama K
Department of Maxillofacial Reconstruction and Function, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8549 Tokyo, Japan.
Bone. 2003 Jul;33(1):38-45. doi: 10.1016/s8756-3282(03)00172-8.
Osteoclasts are multinucleated cells with the specialized function of resorbing calcified tissues. These cells develop from hemopoietic cells of the monocyte-macrophage lineage with the support of osteoblasts/stromal cells. Tooth eruption is a vertical movement of teeth via creation of an eruption pathway in and through the alveolar bone. The precise cellular and molecular determinants of tooth eruption are not yet clear, and a cell culture system that can reproduce the activity of osteoclast formation during tooth eruption is expected to be a useful tool to clarify the mechanism of eruption pathway formation. To this end, mandibular bodies, including incisors and molars, were isolated from 9- to 11-day-old mice undergoing active tooth eruption. Primary cells were obtained from mandibular bodies by enzymatic digestion and cultured in alphaMEM containing 15% FBS without any cytokine or growth factor or hormone in the culture (AFT culture, for alveolar bone, dental follicle, and tooth). A progressive increase in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells was observed in AFT culture. The osteoclastic cells generated were immunopositive for cathepsin K and calcitonin receptor, and formed resorption pits when cultured on dentine slices. Parathyroid hormone-related protein (PTHrP), expressed by the enamel organ of tooth, is reported to be an essential factor in creation of the eruption pathway. To verify this point, cells were isolated from mandibular bodies from which all teeth and dental follicles had been removed and cultured similarly (A culture, for alveolar bone). Osteoclastic cells were not formed and PTHrP production was hardly detected in the medium of A culture, in contrast to the high level of PTHrP in AFT culture. Since our previous study demonstrated that neonatal homozygous PTHrP-knockout mice show impaired osteoclastogenesis around tooth germs, AFT culture was performed by using this sample to examine whether this culture system can reproduce the status of osteoclastogenesis observed in vivo. The result showed that none of the osteoclastic cells were generated from cells of homozygous mice. We here report a novel mouse osteoclast culture system that reproduces the activity of osteoclast formation around erupting teeth without addition of any cytokine or growth factor or hormone to the medium. Histological examination of various transgenic and mutant mice now offers valuable findings on studies of tooth eruption and the present culture system using these animals would be a powerful tool in clarifying the cellular and molecular mechanisms of eruption pathway formation.
破骨细胞是具有重吸收钙化组织特殊功能的多核细胞。这些细胞在成骨细胞/基质细胞的支持下，由单核细胞-巨噬细胞谱系的造血细胞发育而来。牙齿萌出是牙齿通过在牙槽骨内及穿过牙槽骨形成萌出通道而进行的垂直移动。牙齿萌出的确切细胞和分子决定因素尚不清楚，一种能够重现牙齿萌出过程中破骨细胞形成活性的细胞培养系统有望成为阐明萌出通道形成机制的有用工具。为此，从9至11日龄正在进行活跃牙齿萌出的小鼠中分离出包括切牙和磨牙的下颌骨体。通过酶消化从下颌骨体获得原代细胞，并在含有15%胎牛血清的αMEM中培养，培养过程中不添加任何细胞因子、生长因子或激素（AFT培养，用于牙槽骨、牙囊和牙齿）。在AFT培养中观察到抗酒石酸酸性磷酸酶阳性多核破骨细胞数量逐渐增加。所产生的破骨细胞对组织蛋白酶K和降钙素受体呈免疫阳性，并且在牙本质切片上培养时形成吸收陷窝。据报道，由牙齿釉质器官表达的甲状旁腺激素相关蛋白（PTHrP）是形成萌出通道的关键因素。为了验证这一点，从已去除所有牙齿和牙囊的下颌骨体中分离细胞并进行类似培养（A培养，用于牙槽骨）。与AFT培养中高水平的PTHrP相比，A培养的培养基中未形成破骨细胞且几乎检测不到PTHrP的产生。由于我们之前的研究表明新生纯合PTHrP基因敲除小鼠在牙胚周围的破骨细胞生成受损，因此使用该样本进行AFT培养，以检查该培养系统是否能够重现体内观察到的破骨细胞生成状态。结果表明，纯合小鼠的细胞未产生任何破骨细胞。我们在此报告一种新型的小鼠破骨细胞培养系统，该系统在培养基中不添加任何细胞因子、生长因子或激素的情况下，能够重现正在萌出牙齿周围破骨细胞形成的活性。目前对各种转基因和突变小鼠的组织学检查为牙齿萌出研究提供了有价值的发现，并且使用这些动物的当前培养系统将成为阐明萌出通道形成的细胞和分子机制的有力工具。
