Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands.
Amsterdam University College, University of Amsterdam and Vrije Universiteit, Science Park 113, 1098 XG Amsterdam, The Netherlands.
Int J Mol Sci. 2023 Apr 20;24(8):7574. doi: 10.3390/ijms24087574.
Sclerostin is a bone formation inhibitor produced by osteocytes. Although sclerostin is mainly expressed in osteocytes, it was also reported in periodontal ligament (PDL) fibroblasts, which are cells that play a role in both osteogenesis and osteoclastogenesis. Here, we assess the role of sclerostin and its clinically used inhibitor, romosozumab, in both processes. For osteogenesis assays, human PDL fibroblasts were cultured under control or mineralizing conditions with increasing concentrations of sclerostin or romosozumab. For analyzing osteogenic capacity and alkaline phosphatase (ALP) activity, alizarin red staining for mineral deposition and qPCR of osteogenic markers were performed. Osteoclast formation was investigated in the presence of sclerostin or romosozumab and, in PDLs, in the presence of fibroblasts co-cultured with peripheral blood mononuclear cells (PBMCs). PDL-PBMC co-cultures stimulated with sclerostin did not affect osteoclast formation. In contrast, the addition of romosozumab slightly reduced the osteoclast formation in PDL-PBMC co-cultures at high concentrations. Neither sclerostin nor romosozumab affected the osteogenic capacity of PDL fibroblasts. qPCR analysis showed that the mineralization medium upregulated the relative expression of osteogenic markers, but this expression was barely affected when romosozumab was added to the cultures. In order to account for the limited effects of sclerostin or romosozumab, we finally compared the expression of SOST and its receptors LRP-4, -5, and -6 to the expression in osteocyte rich-bone. The expression of SOST, LRP-4, and LRP-5 was higher in osteocytes compared to in PDL cells. The limited interaction of sclerostin or romosozumab with PDL fibroblasts may relate to the primary biological function of the periodontal ligament: to primarily resist bone formation and bone degradation to the benefit of an intact ligament that is indented by every chew movement.
骨硬化蛋白是一种由骨细胞产生的骨形成抑制剂。虽然骨硬化蛋白主要在骨细胞中表达，但也有报道称其在牙周韧带（PDL）成纤维细胞中表达，这些细胞在成骨和破骨细胞形成中都发挥作用。在这里，我们评估了骨硬化蛋白及其临床应用抑制剂罗莫佐单抗在这两个过程中的作用。对于成骨实验，将人牙周韧带成纤维细胞在控制或矿化条件下培养，同时加入不同浓度的骨硬化蛋白或罗莫佐单抗。通过茜素红染色法检测矿物质沉积和骨形成标志物的 qPCR 分析，评估成骨能力和碱性磷酸酶（ALP）活性。在存在骨硬化蛋白或罗莫佐单抗的情况下研究破骨细胞形成，在存在与外周血单核细胞（PBMC）共培养的牙周韧带成纤维细胞的情况下研究破骨细胞形成。用骨硬化蛋白刺激牙周韧带-PBMC 共培养物不会影响破骨细胞形成。相反，在高浓度下，添加罗莫佐单抗会轻微减少牙周韧带-PBMC 共培养物中的破骨细胞形成。骨硬化蛋白或罗莫佐单抗均不影响牙周韧带成纤维细胞的成骨能力。qPCR 分析显示，矿化培养基上调了成骨标志物的相对表达，但当向培养物中添加罗莫佐单抗时，这种表达几乎没有受到影响。为了说明骨硬化蛋白或罗莫佐单抗的有限作用，我们最终将 SOST 及其受体 LRP-4、-5 和 -6 的表达与富含骨的骨细胞中的表达进行了比较。与牙周韧带细胞相比，骨细胞中 SOST、LRP-4 和 LRP-5 的表达更高。骨硬化蛋白或罗莫佐单抗与牙周韧带成纤维细胞的有限相互作用可能与牙周韧带的主要生物学功能有关：主要抵抗骨形成和骨降解，以有利于每个咀嚼运动都会嵌入的完整韧带。
