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Conditional deletion of the transcription factor Runt-related transcription factor 1 (Runx1) in myeloid osteoclast precursors promotes osteoclastogenesis and subsequent bone loss. This study posits whether Runx1 regulates clastic cell-mediated bone and cartilage resorption in the fracture callus. We first generated mice, in which Runx1 was conditionally abrogated in osteoclast precursors (LysM-Cre;Runx1F/F ; Runx1 cKO). Runx1 cKO and control mice were then subjected to experimental mid-diaphyseal femoral fractures. Our study found differential resorption of bony and calcified cartilage callus matrix by osteoclasts and chondroclasts within Runx1 cKO calluses, with increased early bony callus resorption and delayed calcified cartilage resorption. There was an increased number of osteoclasts and chondroclasts in the chondro-osseous junction of Runx1 cKO calluses starting at day 11 post-fracture, with minimal woven bone occupying the callus at day 18 post-fracture. LysM-Cre;Runx1F/F mutant mice had increased bone compliance at day 28, but their strength and work to failure were comparable with controls. Taken together, these results indicate that Runx1 is a critical transcription factor in controlling osteoclastogenesis that negatively regulates bone and cartilage resorption in the fracture callus. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1007-1015, 2020.

Sexual dimorphism of the skeleton is well documented. At maturity, the male skeleton is typically larger and has a higher bone density than the female skeleton. However, the underlying mechanisms for these differences are not completely understood. In this study, we examined sexual dimorphism in the formation of osteoclasts between cells from female and male mice. We found that the number of osteoclasts in bones was greater in females. Similarly, in vitro osteoclast differentiation was accelerated in female osteoclast precursor (OCP) cells. To further characterize sex differences between female and male osteoclasts, we performed gene expression profiling of cultured, highly purified, murine bone marrow OCPs that had been treated for 3 days with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that 125 genes were differentially regulated in a sex-dependent manner. In addition to genes that are contained on sex chromosomes, transcriptional sexual dimorphism was found to be mediated by genes involved in innate immune and inflammatory response pathways. Furthermore, the NF-κB-NFATc1 axis was activated earlier in female differentiating OCPs, which partially explains the differences in transcriptomic sexual dimorphism in these cells. Collectively, these findings identify multigenic sex-dependent intrinsic difference in differentiating OCPs, which results from an altered response to osteoclastogenic stimulation. In humans, these differences could contribute to the lower peak bone mass and increased risk of osteoporosis that females demonstrate relative to males. © 2021 American Society for Bone and Mineral Research (ASBMR).

Due to deleterious side effects of currently available medications, the search for novel, safe, and effective preventive agents for improving bone health in aging continues and is urgently needed. This study aimed to determine whether dietary blackcurrants (BC), an anthocyanin-rich berry, can improve bone mass in a mouse model of age-related bone loss. Thirty-five female C57BL/6J mice, 3 months old (n = 20) and 18 months old (n = 15), were randomized to consume either a standard chow diet or a standard chow diet with 1% (w/w) BC for four months. Dual-energy X-ray absorptiometry, Micro computed tomography (&micro, CT), and histomorphometric analyses were conducted to assess bone parameters on femurs. Biochemical assays were conducted to determine bone resorption, antioxidant activity, and inflammation in humerus homogenates. Trabecular bone volume (BV/TV) was significantly lower in aged mice compared to young mice (young control, 3.7 &plusmn, 0.4% vs aged control, 1.5 &plusmn, 0.5%, mean &plusmn, SEM (standard error of mean), p &lt, 0.01, young BC, 5.3 &plusmn, 0.6% vs aged BC, 1.1 &plusmn, 0.3%, p &lt, 0.001). &micro, CT analysis revealed that BC supplementation increased trabecular BV/TV in young mice by 43.2% (p &lt, 0.05) compared to controls. Histomorphometric analysis revealed a 50% increase, though this effect was not statistically significant (p = 0.07). The osteoblast surface increased by 82.5% in aged mice with BC compared to controls (p &lt, 0.01). In humerus homogenates of young mice, BC consumption reduced C-telopeptide of type I collagen by 12.4% (p &lt, 0.05) and increased glutathione peroxidase by 96.4% (p &lt, 0.05). In humerus homogenates of aged mice, BC consumption increased catalase by 12% (p = 0.09). Aged mice had significantly elevated concentrations of tumor necrosis factor &alpha, (TNF-&alpha, ), a pro-inflammatory cytokine contributing to bone resorption, which was reduced by 43.3% with BC consumption (p = 0.06). These results suggest that early consumption of BC may protect from aging-associated bone loss.

We examined the ability of 1,25 (OH)2 vitamin D3 (Vit D) to stimulate osteoclast-like cell (OCL) formation in cocultures of spleen cells and primary calvarial osteoblasts from wild-type (WT) and IL-1R type 1-deficient (knockout; KO) mice. Vit D dose dependently increased OCL in cocultures containing WT osteoblasts. In contrast, there was a 90% reduction in OCL numbers in cocultures containing KO osteoblasts. In cocultures with either WT or KO osteoblasts, treatment with Vit D increased receptor activator of NF-κB ligand mRNA by 17-, 19-, or 3.5-fold, respectively. Vit D decreased osteoprotegerin mRNA to undetectable in all groups. Intracellular IL-1α protein increased after Vit D treatment in cocultures containing WT, but not KO osteoblasts. We also examined direct effects of Vit D, IL-1α, and their combination on gene expression in primary osteoblasts. In WT cells, Vit D and IL-1 stimulated receptor activator of NF-κB ligand mRNA expression by 3- and 4-fold, respectively, and their combination produced a 7-fold increase. Inhibition of osteoprotegerin mRNA in WT cells was partial with either agent alone and greatest with their combination. In KO cells, only Vit D stimulated a response. IL-1 alone increased IL-1α protein expression in WT osteoblasts. However, in combination with Vit D, there was a synergistic response (100-fold increase). In KO cultures, there were no effects of IL-1, Vit D, or their combination on IL-1α protein. These results demonstrate interactions between IL-1 and Vit D in primary osteoblasts that appear important in both regulation of IL-1α production and the ability of Vit D to support osteoclastogenesis.

We investigated the ability of murine bone organ cultures and osteoblast-like bone cells to produce 72- and 92-kDa gelatinase. 4-6 day newborn mouse calvaria cultures were found to release gelatinase activity into their conditioned medium (CM). This activity was increased by four stimulators of resorption, tumor necrosis factor alpha (TNF), interleukin-1 alpha (IL-1), parathyroid hormone (PTH) and the active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the 72- and 92-kDa forms of gelatinase were produced by murine bone cultures. In unstimulated bones 72-kDa gelatinase activity was approximately equal to that of the 92-kDa enzyme. IL-1, TNF, PTH and TPA all increased 92-kDa gelatinase activity in the CM of the bone cultures by about 2- to 2.5-fold. In addition TPA and IL-1 also increased 72-kDa gelatinase activity. In unstimulated osteoblast-like MC3T3-E1 cell cultures 72-kDa gelatinase enzyme activity was much greater than 92-kDa activity and was not substantially regulated (less than 40% change) by IL-1, TNF or PTH. In contrast, these agents stimulated 92-kDa gelatinase activity by 2- to 5-fold. As with the MC3T3-E1 cells, primary cells constitutively produced both 72-kDa and 92-kDa gelatinase. This was true for cells with both the most differentiated osteoblast-like phenotype (populations 3 and 4) and the least osteoblast-like phenotype (populations 1 and 2). In unstimulated cultures of all 4-primary populations, 92-kDa gelatinase production was less than 72-kDa and IL-1, TNF and PTH had only small effects on 72-kDa production in any of the populations (less than 60% change).(ABSTRACT TRUNCATED AT 250 WORDS)