Abstract：
One of the deficits of knowledge on bone remodelling, is to what extent cells that are driven towards osteogenic differentiation can contribute to osteoclast formation. The periodontal ligament fibroblast (PdLFs) is an ideal model to study this, since they play a role in osteogenesis, and can also orchestrate osteoclastogenesis.when co-cultured with a source of osteoclast-precursor such as peripheral blood mononuclear cells (PBMCs). Here, the osteogenic differentiation of PdLFs and the effects of this process on the formation of osteoclasts were investigated. PdLFs were obtained from extracted teeth and exposed to osteogenic medium for 0, 7, 14, or 21 out of 21 days. After this 21-day culturing period, the cells were co-cultured with peripheral blood mononuclear cells (PBMCs) for an additional 21 days to study osteoclast formation. Alkaline phosphatase (ALP) activity, calcium concentration, and gene expression of osteogenic markers were assessed at day 21 to evaluate the different stages of osteogenic differentiation. Alizarin red staining and scanning electron microscopy were used to visualise mineralisation. Tartrate-resistant acid phosphatase (TRAcP) activity, TRAcP staining, multinuclearity, the expression of osteoclastogenesis-related genes, and TNF-α and IL-1β protein levels were assessed to evaluate osteoclastogenesis. The osteogenesis assays revealed that PdLFs became more differentiated as they were exposed to osteogenic medium for a longer period of time. Mineralisation by these osteogenic cells increased with the progression of differentiation. Culturing PdLFs in osteogenic medium before co-culturing them with PMBCs led to a significant decrease in osteoclast formation. qPCR revealed significantly lower DCSTAMP expression in cultures that had been supplemented with osteogenic medium. Protein levels of osteoclastogenesis stimulator TNF-α were also lower in these cultures. The present study shows that the osteogenic differentiation of PdLFs reduces the osteoclastogenic potential of these cells. Immature cells of the osteoblastic lineage may facilitate osteoclastogenesis, whereas mature mineralising cells may suppress the formation of osteoclasts. Therefore, mature and immature osteogenic cells may have different roles in maintaining bone homeostasis.
摘要：
关于骨骼重塑的知识之一，是被驱动向成骨分化的细胞可以在多大程度上促进破骨细胞形成。牙周膜成纤维细胞(PdLFs)是研究这一点的理想模型,因为它们在成骨中起作用，还可以协调破骨细胞生成。当与破骨细胞前体来源如外周血单核细胞(PBMC)共培养时。这里,研究了PdLFs的成骨分化以及该过程对破骨细胞形成的影响。从拔出的牙齿获得PdLF，并在21天中暴露于成骨培养基中0、7、14或21天。在这21天的培养期之后,将细胞与外周血单核细胞(PBMC)共培养另外21天,以研究破骨细胞的形成.碱性磷酸酶(ALP)活性,钙浓度，在第21天评估成骨标志物的基因表达，以评估成骨分化的不同阶段。使用茜素红染色和扫描电子显微镜观察矿化。抗酒石酸酸性磷酸酶(TRAcP)活性,TRAcP染色,多核化,破骨细胞生成相关基因的表达，评估TNF-α和IL-1β蛋白水平以评估破骨细胞生成。成骨测定显示,随着PdLFs暴露于成骨培养基更长的时间,它们变得更加分化。这些成骨细胞的矿化作用随着分化的进展而增加。在与PMCs共培养之前在成骨培养基中培养PdLFs导致破骨细胞形成的显着减少。qPCR显示在补充有成骨培养基的培养物中显著较低的DCSTAMP表达。在这些培养物中，破骨细胞生成刺激物TNF-α的蛋白质水平也较低。本研究表明，PdLFs的成骨分化降低了这些细胞的破骨细胞潜能。成骨细胞谱系的未成熟细胞可能促进破骨细胞生成，而成熟的矿化细胞可能抑制破骨细胞的形成。因此,成熟和不成熟的成骨细胞在维持骨稳态方面可能有不同的作用。
