Combinatorial Surface Roughness Effects on Osteoclastogenesis and Osteogenesis.
ABSTRACT: Implant surface properties are a key factor in bone responses to metallic bone implants. In view of the emerging evidence on the important role of osteoclasts in bone regeneration, we here studied how surface roughness affects osteoclastic differentiation and to what extent these osteoclasts have stimulatory effects on osteogenic differentiation of osteoprogenitor cells. For this, we induced osteoclasts derived from RAW264.7 cell line and primary mouse macrophages on titanium surfaces with different roughness ( Ra 0.02-3.63 ?m) and analyzed osteoclast behavior in terms of cell number, morphology, differentiation, and further anabolic effect on osteoblastic cells. Surfaces with different roughness induced the formation of osteoclasts with distinct phenotypes, based on total osteoclast numbers, morphology, size, cytoskeletal organization, nuclearity, and osteoclastic features. Furthermore, these different osteoclast phenotypes displayed differential anabolic effects toward the osteogenic differentiation of osteoblastic cells, for which the clastokine CTHRC1 was identified as a causative factor. Morphologically, osteoclast potency to stimulate osteogenic differentiation of osteoblastic cells was found to logarithmically correlate with the nuclei number per osteoclast. Our results demonstrate the existence of a combinatorial effect of surface roughness, osteoclastogenesis, and osteogenic differentiation. These insights open up a new dimension for designing and producing metallic implants by considering the implant roughness to locally regulate osseointegration through coupling osteoclastogenesis with osteogenesis.
Project description:Osteoclastic bone resorption and osteoblastic bone formation/replenishment are closely coupled in bone metabolism. Anabolic parathyroid hormone (PTH), which is commonly used for treating osteoporosis, shifts the balance from osteoclastic to osteoblastic, although it is unclear how these cells are coordinately regulated by PTH. Here, we identify a serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI), as a critical mediator that is involved in the PTH-mediated shift to the osteoblastic phase. Slpi is highly upregulated in osteoblasts by PTH, while genetic ablation of Slpi severely impairs PTH-induced bone formation. Slpi induction in osteoblasts enhances its differentiation, and increases osteoblast-osteoclast contact, thereby suppressing osteoclastic function. Intravital bone imaging reveals that the PTH-mediated association between osteoblasts and osteoclasts is disrupted in the absence of SLPI. Collectively, these results demonstrate that SLPI regulates the communication between osteoblasts and osteoclasts to promote PTH-induced bone anabolism.
Project description:Coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption maintains both mechanical integrity and mineral homeostasis. Zinc is required for the formation, mineralization, growth, and maintenance of bones. We examined the effects of zinc sulfate on osteoblastic differentiation of human periosteum-derived cells (hPDCs) and osteoclastic differentiation of THP-1 cells. Zinc sulfate enhanced the osteoblastic differentiation of hPDCs; however, it did not affect the osteoclastic differentiation of THP-1 cells. The levels of extracellular signaling-related kinase (ERK) were strongly increased during osteoblastic differentiation in zinc sulfate-treated hPDCs, compared with other mitogen-activated protein kinases (MAPKs). Zinc sulfate also promoted osteogenesis in hPDCs and THP-1 cells co-cultured with the ratio of one osteoclast to one osteoblast, as indicated by alkaline phosphatase levels, mineralization, and cellular calcium contents. In addition, the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio was decreased in the zinc sulfate-treated co-cultures. Our results suggest that zinc sulfate enhances osteogenesis directly by promoting osteoblastic differentiation and osteogenic activities in osteoblasts and indirectly by inhibiting osteoclastic bone resorption through a reduced RANKL/OPG ratio in co-cultured osteoblasts and osteoclasts.
Project description:Corylin, a flavonoid isolated from the fruit of <i>Psoralea corylifolia</i>, has an osteogenic effect on osteoblasts in vitro and bone micromass ex vivo. However, the effect and mechanism of corylin in regulating osteoclastogenesis remain unknown. By using murine bone marrow macrophages as the osteoclast precursor, corylin was found to inhibit the receptor activator of nuclear factor (NF) κB ligand (RANKL)-induced osteoclast differentiation via down-regulating osteoclastic marker genes. In parallel, F-actin formation and osteoclast migration were diminished in corylin-treated cultured osteoclasts, and subsequently the expressions of osteoclastic proteins were suppressed: the suppression of protein expression was further illustrated by transcriptomic analysis. Furthermore, corylin inhibited the nuclear translocation of p65, giving rise to a restraint in osteoclastic differentiation through the attenuation of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor of activated T cells c1 (NFATc1). There was no obvious change in apoptosis when the RANKL-induce osteoclasts were cultured in the presence of corylin. The finding supports the potential development of corylin as an osteoclast inhibitor against osteoporosis.
Project description:In patients with prostate cancer (PCa), bone lesions appear osteoblastic in radiographs; however, pathological fractures frequently occur in PCa patients, and bone resorption is observed in all metastatic lesions under histopathologic assessment. The mechanisms that balance the activities of osteoblasts and osteoclasts in PCa patients remain unclear. We unexpectedly discovered that PCa exosomes are critical mediators in the regulation of bone homeostasis that results in osteoclastic lesions and thereby promotes tumor growth in bone. We evaluated how exosomes derived from osteoblastic, osteoclastic, and mixed PCa cell lines affect osteoblast and osteoclast differentiation, revealing that all three types of PCa exosomes promoted osteoclastogenesis in vitro and induced osteolysis in vivo. Mechanistically, microRNAs (miRNAs) delivered by PCa exosomes were found to play several key roles in bone homeostasis. Among the delivered miRNAs, miR-92a-1-5p, the most abundant miRNA, downregulated type I collagen expression by directly targeting <i>COL1A1</i>, and thus promoting osteoclast differentiation and inhibiting osteoblastogenesis. Furthermore, PCa exosomes also markedly reduced type I collagen expression in vivo. Our findings not only offer a novel perspective on tumor bone metastasis, where-contrary to our initial hypothesis-exosomes derived from an osteoblastic tumor induce osteoclast differentiation, but also suggest potential therapeutic targets for PCa bone metastasis.
Project description:Osteoporosis is a reduction in skeletal mass due to the decrease of osteogenic ability and the activation of the osteoclastic function. Inhibiting bone resorption and accelerating the new bone formation is a promising strategy to repair the bone defect of osteoporosis. In this study, we first systematically investigated the roles of Chinese medicine Asperosaponin VI (ASP VI) on osteogenic mineralization of BMSCs and osteoclastogenesis of BMMs, and then explored the synergistic effect of ASP VI and BS (BMP-2 immobilized in 2-N, 6-O-sulfated chitosan) on bone formation. The result showed that ASP VI with the concentration lower than 10-4 M contributed to the expression of osteogenic gene and inhibited osteoclastic genes RANKL of BMSCs. Simultaneously, ASP VI significantly reduced the differentiation of mononuclear osteoclasts in the process of osteoclast formation induced by M-CSF and RANKL. Furthermore, by stimulating the SMADs, TGF-β1, VEGFA, and OPG/RANKL signaling pathways, ASBS (ASP VI and BS) substantially enhanced osteogenesis, greatly promoted angiogenesis, and suppressed osteoclastogenesis. The findings provide a new perspective on osteoporosis care and prevention.
Project description:Controlling osteoclastogenesis is critical to maintain physiological bone homeostasis and prevent skeletal disorders. Although signaling activating nuclear factor of activated T cells 1 (NFATc1), a transcription factor essential for osteoclastogenesis, has been intensively investigated, factors antagonistic to NFATc1 in osteoclasts have not been characterized. Here, we describe a novel pathway that maintains bone homeostasis via two transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte-induced maturation protein-1 (Blimp1). We show that Bcl6 directly targets 'osteoclastic' molecules such as NFATc1, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP), all of which are targets of NFATc1. Bcl6-overexpression inhibited osteoclastogenesis in vitro, whereas Bcl6-deficient mice showed accelerated osteoclast differentiation and severe osteoporosis. We report that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts exhibit osteopetrosis caused by impaired osteoclastogenesis resulting from Bcl6 up-regulation. Indeed, mice doubly mutant in Blimp1 and Bcl6 in osteoclasts exhibited decreased bone mass with increased osteoclastogenesis relative to osteoclast-specific Blimp1-deficient mice. These results reveal a Blimp1-Bcl6-osteoclastic molecule axis, which critically regulates bone homeostasis by controlling osteoclastogenesis and may provide a molecular basis for novel therapeutic strategies.
Project description:CypA (Cyclophilin A) is a peptidyl-prolyl isomerase previously shown to be required for chondrogenic differentiation and endochondral ossification. However, the effects of CypA on osteoclast activity and bone maintenance are entirely unknown. Here, we show that Ppia(-/-) mice demonstrate low bone mineral density, reduced osteoblast numbers, and increased osteoclast numbers. When isolated from the calvaria, Ppia(-/-) osteoblasts demonstrate decreased osteogenic differentiation, whereas Ppia(-/-) osteoclasts derived from the long bones showed increased osteoclastic activity. Overexpression and gene silencing of CypA verified osteogenic and anti-osteoclastic effects. In osteoblasts, CypA is necessary for BMP-2 (Bone Morphogenetic Protein-2)-induced Smad phosphorylation. In osteoclasts, loss of CypA activates BtK (Bruton's tyrosine kinase) and subsequently integrates with TRAF6 (TNF receptor-associated factor 6) and/or c-fos signaling to induce NFATc1 (nuclear factors of activated T cells, cytoplasmic 1). Collectively, CypA dually exerts pro-osteogenic and anti-osteoclastic effects. Thus, modulation of CypA may be useful in future efforts targeting osteoporosis.
Project description:Hypoxia and the hypoxia-inducible factor (HIF) transcription factor regulate angiogenic-osteogenic coupling and osteoclast-mediated bone resorption. To determine how HIF might coordinate osteoclast and osteoblast function, we studied angiopoietin-like 4 (ANGPTL4), the top HIF target gene in an Illumina HumanWG-6 v3.0 48k array of normoxic vs. hypoxic osteoclasts differentiated from human CD14(+) monocytes (14.3-fold induction, P<0.0004). ANGPTL4 mRNA and protein were induced by 24 h at 2% O(2) in human primary osteoclasts, monocytes, and osteoblasts. ANGPTL4 protein was observed by immunofluorescence in osteoclasts and osteoblasts in vivo. Normoxic inducers of HIF (CoCl(2), desferrioxamine, and l-mimosine) and 100 ng/ml ANGPTL4 stimulated osteoclastic resorption 2- to 3-fold in assays of lacunar dentine resorption, without affecting osteoclast viability. Isoform-specific HIF-1? small interfering RNA ablated hypoxic induction of ANGPTL4 and of resorption, which was rescued by addition of exogenous ANGPTL4 (P<0.001). In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P<0.01, 100 ng/ml) and, at lower doses (1-25 ng/ml), mineralization. These results demonstrate that HIF is sufficient to enhance osteoclast-mediated bone resorption and that ANGPTL4 can compensate for HIF-1? deficiency with respect to stimulation of osteoclast activity and also augments osteoblast proliferation and differentiation.