Cell-cell Interactions Between Osteoclasts And Fibroblasts In Vitro

Osteoclastic is the only cells with the function of bone resorption in vivo, which is derived from hematopoietic precursor cells, monocytes - macrophages. Osteoclasts plays a key role in remodeling and bone metabolism. Osteoclast-mediated bone resorption and osteoblast-mediated bone formation are coupling with a complex series of mechanisms, constitute the dynamic balance of bone mass. However, now the roles of fibroblasts in ossification are increasingly taken by the people. Fibroblasts (FB) is the most common cells in animal connective tissue, derived from embryonic mesenchymal cells. The main function of fibroblasts is to secrete extracellular matrix and collagen, maintaining the connective tissue structure the network. And they play a key role in angiogenesis and wound healing. In addition, fibroblasts involved in the pathogenesis of many fibrotic diseases, such as pulmonary fibrosis, kidney fibrosis, and scleroderma. Fibroblasts also have some osteogenic ability to participate in fracture healing. They also play an important role in the heterotopic ossification process in some disease ankylosing spondylitis. The mechanism osteogenic differentiation of fibroblasts and ossification are uncertain. Fibroblasts and osteoblasts are from a same lineage. It is widely accepted that osteoclasts can promote the differentiation of osteoblasts. Conditioned medium derived from human osteoclasts induces preosteoblasts to form bone-like nodules. Gene array analysis, in combination with loss-of-function studies, revealed that mature osteoclasts secrete Wnt10b, sphingosine 1-phosphate, and bone morphogenetic protein-6 (BMP6) might promote osteoblasts mineralization. So osteoclasts may play a key role in the process of inducing heterotopic ossification mediated by fibroblasts.The objective of the present study is to evaluate the effects of osteoclasts to inducing the osteogenic differentiation of fibroblasts, and investigate the mechanism initially, and investigate the effects of fibroblasts conditioned medium to induce the osteoclast precursors different into mature osteoclasts. Methods: Primary culture fibroblasts from human spinal ligament. And isolate mononuclear cells from human peripheral blood. Immunomagnetic beads were used to separate CD14~+ cells from mononuclear cells. CD14~+ cells were induced to OLC by M-CSF and RANKL. The osteoclastic characteristics were identified by TRAP staining and bone resorption lacuna observation by canning electron microscope. Fibroblasts were cultured in OLC conditioned medium for 14 days, then alkaline phosphatase(ALP) expression was detected by BCIP/NBT Alkaline Phosphatase Color Development Kit. Cultures were fixed and stained for mineralization with Alizarin red. Microarray analyses were performed to evaluate the mRNAs and miRNAs expression profilings of fibroblasts cultured in OLC conditioned medium compared with cultured in control medium. The conditioned medium treating and microarray experiments were repeated once with fibroblasts generated from ligament of another patient.To investigate the effects of fibroblasts conditioned medium to induce the osteoclastogenesis of osteoclast precursors, CD14~+ cells, were cultured in fibroblast conditioned medium. The osteoclastic differentiation was detected by TRAP staining, and RT-PCR was performed to detect genes related to osteoclastogenesis.Results: The ALP positive rate of fibroblasts was significantly higher that cultured in control medium. There are significant differences of mRNAs and miRNAs expression between fibroblasts cultured in OLC conditioned medium and that cultured in the control medium. 28 miRNAs and 522 mRNAs were up-regulated more than 2 folds, and 59 miRNAs and 415mRNAs were down-regulated more than 2 folds. In the repeat experiments, there are 3707 mRNAs regulated and 3251 genes down regulated; There are 23 miRNAs up regulated and 36 miRNAs down regulated. After 21d of culture in fibroblast conditioned medium, CD14~+ cells generated characters of osteoclasts. There is a high TRAP positive rate in fibroblast conditioned medium treated CD14~+ cells than control. Conclusions: Osteoclasts have the effects to induce the osteogenic differentiation of fibroblasts in vitro, and fibroblasts conditioned medium can induce the osteoclastogenesis of CD14~+ cells. And miRNA may play an important role in regulation of the interaction between osteoclasts and fibroblasts.