Surface electric fields increase human osteoclast resorption through improved wettability on carbonate-incorporated apatite

Osteoclast-mediated bioresorption can be of an efficient means of incorporating the dissolution of biomaterials in the bone remodeling process. Because of compositionally and structurally close resemblance of biomaterials with the natural mineral phases of the bone matrix, synthetic carbonate-substituted hydroxyapatite (CA) is considered as an ideal clinical biomaterial. The present study therefore investigated the effects of electrical polarization on the surface characteristics and interactions with human osteoclasts of hydroxyapatite (HA) and CA. Electrical polarization was found to improve the surface wettability of these materials by increasing the surface free energy, and this effect was maintained for one month. Analyses of human osteoclast cultures established that CA subjected to a polarization treatment accelerated osteoclast resorption but did not affect the early differentiation phase or the adherent morphology of the osteoclasts as evaluated by staining. These data suggest that the surface characteristics of the CA promoted osteoclast resorption. The results of this work are expected to contribute to the design of cell-mediated biomaterials that can be resorbed by osteoclasts after fulfilling their primary function as a scaffold for bone regeneration.

Acid-Induced Inflammatory Cytokines in Osteoblasts: A Guided Path to Osteolysis in Bone Metastasis

Bone metastasis (BM) is a dismal complication of cancer that frequently occurs in patients with advanced carcinomas and that often manifests as an osteolytic lesion. In bone, tumor cells promote an imbalance in bone remodeling via the release of growth factors that, directly or indirectly, stimulate osteoclast resorption activity. However, carcinoma cells are also characterized by an altered metabolism responsible for a decrease of extracellular pH, which, in turn, directly intensifies osteoclast bone erosion. Here, we speculated that tumor-derived acidosis causes the osteoblast–osteoclast uncoupling in BM by modulating the pro-osteoclastogenic phenotype of osteoblasts. According to our results, a low pH recruits osteoclast precursors and promotes their differentiation through the secretome of acid-stressed osteoblasts that includes pro-osteoclastogenic factors and inflammatory mediators, such as RANKL, M-CSF, TNF, IL-6, and, above the others, IL-8. The treatment with the anti-IL-6R antibody tocilizumab or with an anti-IL-8 antibody reverted this effect. Finally, in a series of BM patients, circulating levels of the osteolytic marker TRACP5b significantly correlated with IL-8. Our findings brought out that tumor-derived acidosis promotes excessive osteolysis at least in part by inducing an inflammatory phenotype in osteoblasts, and these results strengthen the use of anti-IL-6 or anti-IL-8 strategies to treat osteolysis in BM.

Cobalt-containing calcium phosphate induces resorption of biomineralized collagen by human osteoclasts

Background Biomineralized collagen, consisting of fibrillar type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previously shown to stimulate resorptive activity of osteoclasts. Methods To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Upon fluorescent staining of nuclei, F-actin and tartrate-resistant alkaline phosphatase, the cells were analyzed by laser confocal microscopy. Their resorption capacity was assessed by scanning electron microscopy (SEM), as well as indirectly quantified by measuring the release of calcium ions into cell culture medium. Results The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release in water of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and induced formation of resorption lacunae, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded. Conclusions This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, although the extent of osteoclast resorption was limited, and the method needs further optimization. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

Chondrogenesis mediates progression of ankylosing spondylitis through heterotopic ossification

Ankylosing spondylitis (AS) is chronic inflammatory arthritis with a progressive fusion of axial joints. Anti-inflammatory treatments such as anti-TNF-α antibody therapy suppress inflammation but do not effectively halt the progression of spine fusion in AS patients. Here we report that the autoimmune inflammation of AS generates a microenvironment that promotes chondrogenesis in spine ligaments as the process of spine fusion. Chondrocyte differentiation was observed in the ligaments of patients with early-stage AS, and cartilage formation was followed by calcification. Moreover, a large number of giant osteoclasts were found in the inflammatory environment of ligaments and on bony surfaces of calcified cartilage. Resorption activity by these giant osteoclasts generated marrow with high levels of active TGF-β, which induced new bone formation in the ligaments. Notably, no Osterix+ osteoprogenitors were found in osteoclast resorption areas, indicating uncoupled bone resorption and formation. Even at the late and maturation stages, the uncoupled osteoclast resorption in bony interspinous ligament activates TGF-β to induce the progression of ossification in AS patients. Osteoclast resorption of calcified cartilage-initiated ossification in the progression of AS is a similar pathologic process of acquired heterotopic ossification (HO). Our finding of cartilage formation in the ligaments of AS patients revealed that the pathogenesis of spinal fusion is a process of HO and explained why anti-inflammatory treatments do not slow ankylosing once there is new bone formation in spinal soft tissues. Thus, inhibition of HO formation, such as osteoclast activity, cartilage formation, or TGF-β activity could be a potential therapy for AS.

‘Educated’ Osteoblasts Reduce Osteoclastogenesis in a Bone-Tumor Mimetic Microenvironment

Breast cancer (BC) metastases to bone disrupt the balance between osteoblasts and osteoclasts, leading to excessive bone resorption. We identified a novel subpopulation of osteoblasts with tumor-inhibitory properties, called educated osteoblasts (EOs). Here we sought to examine the effect of EOs on osteoclastogenesis during tumor progression. We hypothesized that EOs affect osteoclast development in the bone-tumor niche, leading to suppressed pre-osteoclast fusion and bone resorption. Conditioned media (CM) was analyzed for protein expression of osteoclast factors receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα) via ELISA. EOs were co-cultured with pre-osteoclasts on a bone mimetic matrix to assess osteoclast resorption. Pre-osteoclasts were tri-cultured with EOs plus metastatic BC cells and assessed for tartrate-resistance acid phosphatase (TRAP)-positive, multinucleated (≥3 nuclei), mature osteoclasts. Tumor-bearing murine tibias were stained for TRAP to determine osteoclast number in-vivo. EO CM expressed reduced amounts of soluble TNFα and OPG compared to naïve osteoblast CM. Osteoclasts formed in the presence of EOs were smaller and less in number. Upon co-culture on a mimetic bone matrix, a 50% reduction in the number of TRAP-positive osteoclasts formed in the presence of EOs was observed. The tibia of mice inoculated with BC cells had less osteoclasts per bone surface in bones with increased numbers of EO cells. These data suggest EOs reduce osteoclastogenesis and bone resorption. The data imply EOs provide a protective effect against bone resorption in bone metastatic BC.

Cobalt-containing Calcium Phosphate Induces Resorption of Biomineralized Collagen by Human Osteoclasts

Background: Biomineralized collagen, consisting of fibrillary type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previous shown to stimulate resorptive activity of osteoclasts.Methods: To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Their morphology was assessed by laser confocal microscopy and their capacity for resorption observed by scanning electron microscopy (SEM), as well as indirectly quantified by calcium release into cell culture supernatant. Results: The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and caused resorption lacunae to form, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded.Conclusions: This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, despite the fact that the extent of osteoclast resorption was limited, and the method needs further optimization,. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

Mineral density differences between femoral cortical bone and trabecular bone are not explained by turnover rate alone

Bone mineral density distributions (BMDDs) are a measurable property of bone tissues that depends strongly on bone remodelling and mineralisation processes. These processes can vary significantly in health and disease and across skeletal sites, so there is high interest in analysing these processes from experimental BMDDs. Here, we propose a rigorous hypothesis-testing approach based on a mathematical model of mineral heterogeneity in bone due to remodelling and mineralisation, to help explain differences observed between the BMDD of human femoral cortical bone and the BMDD of human trabecular bone. Recent BMDD measurements show that femoral cortical bone possesses a higher bone mineral density, but a similar mineral heterogeneity around the mean compared to trabecular bone. By combining this data with the mathematical model, we are able to test whether this difference in BMDD can be explained by (i) differences in turnover rate; (ii) differences in osteoclast resorption behaviour; and (iii) differences in mineralisation kinetics between the two bone types. We find that accounting only for differences in turnover rate is inconsistent with the fact that both BMDDs have a similar spread around the mean, and that accounting for differences in osteoclast resorption behaviour leads to biologically inconsistent bone remodelling patterns. We conclude that the kinetics of mineral accumulation in bone matrix must therefore be different in femoral cortical bone and trabecular bone. Although both cortical and trabecular bone are made up of lamellar bone, the different mineralisation kinetics in the two types of bone point towards more profound structural differences than usually assumed.

Complement C3a Not C4a Activates Osteoclasts By Regulating the PI3K/PDK1/SGK3 Pathway in Patients with Multiple Myeloma

Objective: Myeloma bone disease (MBD) is the most common complication of multiple myeloma (MM). We found that the serum levels of C3/C4 in MM patients were significantly positive correlated with the severity of bone disease in our previously study. Thus, we conduct detailed studies to explore the effect and potential mechanism of C3a/C4a on osteoclasts in patients with MM. Methods: By adding C3a/C4a to culture system of osteoclasts induced from mononuclear cells in vitro, and the expression of related gene, number and function of osteoclasts were detected. RNA-Seq analysis was used to detect the differentially expressed genes on osteoclasts between the complement group and control group, and the possible signal pathways were analyzed. Quantitative real-time PCR (qRT-PCR), Western blot and pathway inhibitors were used for further validation. R esults: In vitro, the osteoclasts area per view induced by C3a 1μg/ml (52.794±13.027 %) and 10μg/ml (51.797±12.464 %) was significantly increased than control group (0μg/ml) (33.668±8.427 %) (P<0.001; P<0.001), the mRNA relative expression of osteoclasts related genes OSCAR/TRAP/RANKL/Cathepsin K induced by C3a 1μg/ml (median 5.041; 3.726; 1.638; 4.752) and 10μg/ml (median 5.140; 3.702; 2.250; 5.172) was significantly increased than control group (0μg/ml) (median 3.137; 2.004; 0.573; 2.257) (1μg/ml P=0.001; P=0.003; P<0.001; P=0.008; 1μg/ml P<0.001; P=0.019; P<0.001; P=0.002), and the absorption area of osteoclast resorption pit per view induced by C3a 1μg/ml (51.464±11.983 %) and 10μg/ml (50.219±12.067 %) was also significantly increased than control group (0μg/ml) (33.845±8.331 %) (P<0.001; P<0.001) in NDMM patients. There was no difference among the osteoclasts area, relative expression of osteoclasts related genes and absorption area of osteoclast resorption pit between C4a (1μg/ml and 10μg/ml) group and control group (0μg/ml). RNA-Seq was performed on total RNA of osteoclasts induced by C3a in 1μg/ml group and 0μg/ml group of 4 patients with NDMM. There were 184 differentially expressed genes that were detected by RNA-Seq analysis. KEGG Pathway enrichment bubble chart shows C3a may through Phosphoinositide 3-kinase (PI3K) signaling pathways (including PI3K-Akt pathway and AKT-independent signaling pathway) promotes the proliferation of osteoclast. Upregulated differentially expressed genes in this pathway among at least 3 patients with sequencing were validated by qRT-PCR and Western Blot. It was found that the relative expression level of Phosphoinositide dependent kinase-1 (PDK1) / Serum and glucocorticoid inducible protein kinases (SGK3) genes (median 2.078; 4.428) in C3a group (1μg/ml) was significantly higher than control group (0μg/ml) (median 1.336; 1.714) (P<0.001; P=0.001). The relative grayscale levels of PDK1/ P-SGK3 protein (1.785±0.323; 2.190±0.274) in C3a group (1μg/ml) was significantly stronger than control group (0μg/ml) (0.8653±0.588; 0.176±0.152) (P=0.034; P<0.001). Under the action of C3a in patients with NDMM, osteoclasts area per view in SGK inhibitor (EMD638683) 1μM group (39.244±9.089 %) and 10μM group (39.299±9.587 %) significantly reduced than control group (0μM) (54.884±12.837 %) (P<0.001; P<0.001), the relative expression of osteoclast related genes OSCAR/RANKL/TRAP/Cathepsin K in EMD638683 1μM group (median 0.869; 1.097; 0.902; 1.328) and 10μM group (median 0.703; 1.391; 0.843; 1.418) significantly decreased than control group (0μM) (median 2.270; 3.024; 2.208; 3.237) (1μM P=0.015; P=0.002; P=0.003; P=0.015; 10μM P=0.012; P=0.006; P<0.001; P=0.017), and the absorption area per view of osteoclast resorption pit in EMD638683 1μM (35.383±7.794 %) group and 10μM group (32.886±8.993 %) significantly reduced than control group (0μM) (49.358±11.856 %) (P < 0.001; P < 0.001). Conclusions:ComplementC3a activates osteoclasts by regulating the PI3K/PDK1/SGK3 pathway in patients with MM, thus leading to the occurrence of bone diseases. SGK inhibitor has a significant inhibitory effect on osteoclasts in patients with MM. This study provide important evidences for the search for new therapeutic targets and strategies for myeloma bone disease patients. Disclosures No relevant conflicts of interest to declare.