Mmp-13 deletion in cells of the mesenchymal lineage increases bone mass, decreases endocortical osteoclast number, and attenuates the cortical bone loss caused by estrogen deficiency in mice

The protective effect of estrogens against cortical bone loss is mediated via direct actions on mesenchymal lineage cells, but functional evidence for the precise molecular mechanism(s) and the mediators of these effects has only recently began to emerge. We report that the matrix metalloproteinase 13 (MMP-13) is the highest up-regulated gene in calvaria or bone marrow cells from mice lacking the estrogen receptor (ER) alpha in osteoprogenitors. We, therefore, generated mice with conditional Mmp-13 deletion in Prrx1 expressing cells ( Mmp-13 ?Prrx1 ) and compared the effect of estrogen deficiency on their bone phenotype to that of control littermates ( Mmp-13 f/f ). Femur and tibia length was decreased in sham-operated Mmp-13 ?Prrx1 mice as compared to Mmp1 3 f/f . Cortical thickness and trabecular bone volume in the femur and tibia were increased and osteoclast number at the endocortical surfaces was decreased in the sham-operated female Mmp13 ?Prrx1 mice; whereas bone formation rate was unaffected. Ovariectomy (OVX) caused a decrease of cortical thickness in the femur and tibia of Mmp-13 f/f control mice. This effect was attenuated in the Mmp-13 ? Prrx1 mice; but the decrease of trabecular bone caused by OVX was not affected. These results reveal that mesenchymal cell–derived MMP-13 regulates osteoclast number, bone resorption, and bone mass. We have recently reported that the loss of cortical, but not trabecular bone, caused by OVX is also attenuated in Cxcl12 ?Prrx1 mice. Together with the present report, this functional genetic evidence provides proof of principle that increased production of mesenchymal cell-derived factors, such as CXCL12 and MMP-13, are important mediators of the adverse effect of estrogen deficiency on cortical, but not trabecular, bone. Therefore, the mechanisms responsible for the protective effect of estrogens on these two major bone compartments are different.

Driving β2- While Suppressing α-Adrenergic Receptor Activity Suppresses Joint Pathology in Inflammatory Arthritis

ObjectiveHypersympathetic activity is prominent in rheumatoid arthritis, and major life stressors precede onset in ~80% of patients. These findings and others support a link between stress, the sympathetic nervous system and disease onset and progression. Here, we extend previous research by evaluating how selective peripherally acting α/β2-adrenergic drugs affect joint destruction in adjuvant-induced arthritis.MethodsComplete Freund’s adjuvant induced inflammatory arthritis in male Lewis rats. Controls received no treatment. Arthritic rats then received vehicle or twice-daily treatment with the α-adrenergic antagonist, phentolamine (0.5 mg/day) and the β2-adrenergic agonist, terbutaline (1200 µg/day, collectively named SH1293) from day (D) of disease onset (D12) through acute (D21) and severe disease (D28). Disease progression was assessed in the hind limbs using dorsoplantar widths, X-ray analysis, micro-computed tomography, and routine histology on D14, D21, and D28 post-immunization.ResultsOn D21, SH1293 significantly attenuated arthritis in the hind limbs, based on reduced lymphocytic infiltration, preservation of cartilage, and bone volume. Pannus formation and sympathetic nerve loss were not affected by SH1293. Bone area and osteoclast number revealed high- and low-treatment-responding groups. In high-responding rats, treatment with SH1293 significantly preserved bone area and decreased osteoclast number, data that correlated with drug-mediated joint preservation. SH1293 suppressed abnormal bone formation based on reduced production of osteophytes. On D28, the arthritic sparing effects of SH1293 on lymphocytic infiltration, cartilage and bone sparing were maintained at the expense of bone marrow adipocity. However, sympathetic nerves were retracted from the talocrural joint.Conclusion and SignificanceOur findings support a significant delay in early arthritis progression by treatment with SH1293. Targeting sympathetic neurotransmission may provide a strategy to slow disease progression.

Quantification of Osteoclasts in Culture, Powered by Machine Learning

In vitro osteoclastogenesis is a central assay in bone biology to study the effect of genetic and pharmacologic cues on the differentiation of bone resorbing osteoclasts. To date, identification of TRAP+ multinucleated cells and measurements of osteoclast number and surface rely on a manual tracing requiring specially trained lab personnel. This task is tedious, time-consuming, and prone to operator bias. Here, we propose to replace this laborious manual task with a completely automatic process using algorithms developed for computer vision. To this end, we manually annotated full cultures by contouring each cell, and trained a machine learning algorithm to detect and classify cells into preosteoclast (TRAP+ cells with 1–2 nuclei), osteoclast type I (cells with more than 3 nuclei and less than 15 nuclei), and osteoclast type II (cells with more than 15 nuclei). The training usually requires thousands of annotated samples and we developed an approach to minimize this requirement. Our novel strategy was to train the algorithm by working at “patch-level” instead of on the full culture, thus amplifying by >20-fold the number of patches to train on. To assess the accuracy of our algorithm, we asked whether our model measures osteoclast number and area at least as well as any two trained human annotators. The results indicated that for osteoclast type I cells, our new model achieves a Pearson correlation (r) of 0.916 to 0.951 with human annotators in the estimation of osteoclast number, and 0.773 to 0.879 for estimating the osteoclast area. Because the correlation between 3 different trained annotators ranged between 0.948 and 0.958 for the cell count and between 0.915 and 0.936 for the area, we can conclude that our trained model is in good agreement with trained lab personnel, with a correlation that is similar to inter-annotator correlation. Automation of osteoclast culture quantification is a useful labor-saving and unbiased technique, and we suggest that a similar machine-learning approach may prove beneficial for other morphometrical analyses.

miR-325-3p, a novel regulator of osteoclastogenesis in osteolysis of colorectal cancer through targeting S100A4

Background To investigate effect of microRNA-325-3p (miR-325-3p) on bone metastasis of colorectal cancer (CRC) and the precise role on osteoclastogenesis. Methods CT-26 cells were injected into tibias to establish bone metastatic model of CRC in vivo. AgomiR-325-3p or antagomir-325-3p were injected in tail-veins of Balb/c mice to interfere the osteoclastogenesis and bone metastasis of CRC. Safranin O and Fast Green staining examined the changes of trabecular area and TRAP staining examined the osteoclast number in bone metastasis of CRC. Real-time PCR was conducted to test the RNA level of miR-325-3p and mRNA levels of TRAP and Cathepsin K in osteoclast precursors (OCPs). Dual-luciferase reporter system was utilized to identify the direct target of miR-325-3p. Conditioned medium from CT-26 cells was collected to stimulate the OCPs during osteoclastogenesis induced by RANKL and M-CSF in vitro. Western blot analysis was performed to examine the protein level of S100A4 in OCPs after interfered by agomiR-325-3p or antagomir-325-3p cultured in CM or not. Results miR-325-3p downregulated in OCPs in CRC microenvironment both in vivo and in vitro. By luciferase activity assay, S100A4 was the target gene of miR-325-3p and the protein level of S100A4 in OCPs upregulated in CRC microenvironment. Overexpression of miR-325-3p inhibited the osteoclastogenesis of OCPs and it can be reversed after transfection with plasmid containing S100A4. Treatment with miR-325-3p can preserve trabecular area in bone metastasis of CRC. Conclusion miR-325-3p can prevent osteoclast formation through targeting S100A4 in OCPs. Overexpression of miR-325-3p efficiently decreased the osteoclast number and attenuated bone resorption in bone metastasis of CRC.

The Effect of Strontium Ranelate on Fracture Healing: An Animal Study

Background. Strontium ranelate (StR) is an antiosteoporotic agent previously utilized for the enhancement of fracture union. We investigated the effects of StR on fracture healing using a rabbit model. Methods. Forty adult female rabbits were included in the study and were divided in 2 equal groups, according to StR treatment or untreated controls. All animals were subjected to osteotomy of the ulna, while the contralateral ulna remained intact and served as a control for the biomechanical assessment of fracture healing. Animals in the study group received 600 mg/kg/day of StR orally. All animals received ordinary food. At 2 and 4 weeks, all animals were euthanatized and the osteotomy sites were evaluated for healing through radiological, biomechanical, and histopathological studies. Results. The treatment group presented statistically significant higher callus diameter, total callus area, percentage of fibrous tissue ( p < 0.001 ), vessels/mm2, number of total vessels, and lower osteoclast number/mm2 ( p < 0.05 ) than the control group at 2 weeks. Additionally, the treatment group presented significantly higher percentages of new trabecular bone, vessels/mm2, osteoclast number/mm2, and lower values for callus diameter, as well as total callus area ( p < 0.05 ), than the control group at 4 weeks. At 4 weeks, in the treatment group, force applied ( p = 0.003 ), energy at failure ( p = 0.004 ), and load at failure ( p = 0.003 ) were all significantly higher in the forearm specimens with the osteotomized ulnae compared to those without. Radiological bone union was demonstrated for animals receiving StR at 4 weeks compared with controls ( p = 0.045 ). Conclusion. StR appears to enhance fracture healing but further studies are warranted in order to better elucidate the mechanisms and benefits of StR treatment.

Hdac3 regulates bone modeling by suppressing osteoclast responsiveness to RANKL

Hdac3 is a lysine deacetylase that removes acetyl groups from histones and additional proteins. Although Hdac3 functions within mesenchymal lineage skeletal cells are defined, little is known about Hdac3 activities in bone-resorbing osteoclasts. In this study we conditionally deleted Hdac3 within Ctsk-expressing cells and examined the effects on bone modeling and osteoclast differentiation in mice. Hdac3 deficiency reduced femur and tibia periosteal circumference and increased cortical periosteal osteoclast number. Trabecular bone was likewise reduced and was accompanied by increased osteoclast number per trabecular bone surface. We previously showed that Hdac3 deacetylates the p65 subunit of the NF-κB transcriptional complex to decrease DNA-binding and transcriptional activity. Hdac3-deficient osteoclasts demonstrate increased K310 NF-κB acetylation and NF-κB transcriptional activity. Hdac3-deficient osteoclast lineage cells were hyper-responsive to RANKL and showed elevated ex vivo osteoclast number and size and enhanced bone resorption in pit formation assays. Osteoclast-directed Hdac3 deficiency decreased cortical and trabecular bone mass parameters, suggesting that Hdac3 regulates coupling of bone resorption and bone formation. We surveyed a panel of osteoclast-derived coupling factors and found that Hdac3 suppression diminished sphingosine-1-phosphate production. Osteoclast-derived sphingosine-1-phosphate acts in paracrine to promote bone mineralization. Mineralization of WT bone marrow stromal cells cultured with conditioned medium from Hdac3-deficient osteoclasts was markedly reduced. Expression of alkaline phosphatase, type 1a1 collagen, and osteocalcin was also suppressed, but no change in Runx2 expression was observed. Our results demonstrate that Hdac3 controls bone modeling by suppressing osteoclast lineage cell responsiveness to RANKL and coupling to bone formation.

Effects of ginsenoside Rg3 on bone loss, bone mineral density and osteoclast number in glucocorticoid-induced osteoporosis rats, and the likely michanism of action

Purpose: To investigate the effect of ginsenoside Rg3 on bone loss, bone mineral density (BMD) and osteoclast number in glucocorticoid-induced osteoporosis (GIOP) rats, and the mechanism of action involved.Methods: Sixty female Wistar rats were assigned to control, model group, ginsenoside Rg3, and alendronate sodium groups, comprised of 15 rats per group. The osteoporosis rat model was established via intramuscular injection of dexamethasone. Changes in bone mineral content (BMC), BMD trabecular thickness and area, osteoblasts and osteoclasts in femurs and lumbar vertebrae were measured after 3 months of treatment.Results: There were significantly higher BMC and BMD levels in ginsenoside Rg3 group than in alendronate rats (p < 0.05). The thickness and trabecular area in femur and lumbar vertebrae in the ginsenoside Rg3 group were significantly higher than those in the model group (p < 0.05), but were comparable with those in the alendronate sodium group (p > 0.05). There were marked increases in osteoblasts, and marked decreases in osteoclasts in the ginsenoside Rg3 group, alendronate sodium and control rats, relative to model rats (p < 0.05).Conclusion: Ginsenoside Rg3 arrests bone loss, and enhances bone density, trabecular thickness and area, bone microstructure, osteoblast activity and population of osteoclasts number in glucocorticoidinduced osteoporotic rats. This provides a new research direction for the clinical treatment ofosteoporosis. Keywords: Ginseng soap, Rg3, Glucocorticoid, Osteoporosis, Bone loss, Bone mineral density, Osteoclast population