Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands.
Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands.
Bone. 2018 Apr;109:168-177. doi: 10.1016/j.bone.2017.07.007. Epub 2017 Jul 10.
Fibrodysplasia Ossificans Progressiva (FOP) is a progressive disease characterized by periods of heterotopic ossification of soft connective tissues, including ligaments. Though progress has been made in recent years in unraveling the underlying mechanism, patient-derived cell models are necessary to test potential treatment options. Periodontal ligament fibroblasts (PLF) from extracted teeth can be used to study deviant bone modeling processes in vitro since these cells are derived from genuine ligaments. They further provide a tool to study the hitherto unknown role of the bone morphogenesis protein receptor type 1 (BMPR-1) Activin A type 1 receptor ACVR1-R206H mutation in osteoclastogenesis. To further validate this potential model, osteogenesis and osteoclastogenesis was studied in the presence of TGF-β/activin receptor inhibitor GW788388. Control and FOP fibroblasts (n=6 of each) were used in osteogenesis and osteoclastogenesis assays in the absence or presence of TGF-β/activin receptor inhibitor GW788388. For osteogenesis, alkaline phosphatase (ALP) activity, alizarin red staining for mineralization and qPCR for expression of osteogenic markers was assessed. TRACP staining, multinuclearity and expression of osteoclastogenesis markers were used as a measure of osteoclast formation. FOP fibroblasts cultured in osteogenic medium displayed a trend of higher ALP activity at 7days. Gene expression of ALP from FOP fibroblasts was significantly higher at 3days. Mineralization was similar at 21days for both groups. GW788388 did not influence mineral deposition in both groups. Osteoclast formation was inhibited by GW788388 on plastic for both controls and FOP. On cortical bone slices, however, osteoclast formation was significantly lowered by GW788388, only in FOP cultures. qPCR revealed strong expression of RANKL at 7days and a significant decline at 14 and 21days in both FOP and control cultures. In contrast to the osteoclastogenesis results, the RANKL/OPG ratio was higher in the presence of GW788388, only in FOP cultures. TGF-β expression was significantly higher at 14 and 21days compared to 7days, possibly signifying a role in later stages of osteoclast formation. Addition of GW788388 strongly decreased TGF-β expression. Our study shows that periodontal ligament fibroblasts from FOP patients displayed at most slightly enhanced in vitro osteogenesis and osteoclastogenesis. This model could be useful to elucidate molecular mechanisms leading to heterotopic ossification in FOP such as in the presence of specific ACVR1-R206H activators as Activin A.
进行性骨化性纤维发育不良（FOP）是一种以异位骨化的软连接组织（包括韧带）为特征的进行性疾病。尽管近年来在揭示潜在的发病机制方面取得了进展，但仍需要患者来源的细胞模型来测试潜在的治疗选择。从提取的牙齿中分离出来的牙周韧带成纤维细胞（PLF）可用于体外研究异常的骨建模过程，因为这些细胞源自真正的韧带。它们进一步提供了一种工具来研究骨形态发生蛋白受体 1（BMPR-1）激活素 A 型 1 受体 ACVR1-R206H 突变在破骨细胞形成中的未知作用。为了进一步验证这种潜在的模型，在存在 TGF-β/激活素受体抑制剂 GW788388 的情况下研究了成骨作用和破骨细胞形成。在不存在或存在 TGF-β/激活素受体抑制剂 GW788388 的情况下，使用对照和 FOP 成纤维细胞（每组 6 个）进行成骨作用和破骨细胞形成测定。对于成骨作用，评估碱性磷酸酶（ALP）活性、茜素红染色的矿化和骨形成标志物的 qPCR。使用破骨细胞形成的 TRACP 染色、多核性和骨形成标志物的表达作为破骨细胞形成的衡量标准。在成骨培养基中培养的 FOP 成纤维细胞在第 7 天显示出 ALP 活性升高的趋势。FOP 成纤维细胞的 ALP 基因表达在第 3 天显著升高。两组在第 21 天的矿化情况相似。GW788388 对两组的矿化沉积均无影响。GW788388 在塑料上抑制了破骨细胞的形成，对对照组和 FOP 均如此。然而，在皮质骨切片上，GW788388 仅在 FOP 培养物中显著降低了破骨细胞的形成。qPCR 显示，RANKL 在第 7 天表达强烈，在第 14 天和第 21 天，FOP 和对照培养物中的表达均显著下降。与破骨细胞形成结果相反，只有在 FOP 培养物中，GW788388 存在时，RANKL/OPG 比值更高。与第 7 天相比，TGF-β 的表达在第 14 天和第 21 天显著升高，这可能表明其在破骨细胞形成的后期阶段发挥作用。添加 GW788388 可强烈降低 TGF-β 的表达。我们的研究表明，来自 FOP 患者的牙周韧带成纤维细胞在体外显示出最多稍微增强的成骨作用和破骨细胞形成。该模型可用于阐明导致 FOP 中异位骨化的分子机制，例如在存在特定的 ACVR1-R206H 激活剂（如激活素 A）的情况下。
