The cathepsin K (CatK) inhibitor odanacatib (ODN) is currently being developed for the treatment of osteoporosis. In clinical trials, efficacy and resolution of effect of ODN treatment on bone turnover biomarkers and accrued bone mass have been demonstrated. Here, we examine the effects of continuing treatment and discontinuation of ODN versus alendronate (ALN) on osteoclast (OC) function. First, accessibility and reversible engagement of active CatK in intracellular vesicles and resorption lacunae of actively resorbing OCs were demonstrated by the selective and reversible CatK inhibitors, BODIPY-L-226 (IC50=39nM) and L-873,724 (IC50=0.5nM). Next, mature human OCs on bone slices were treated with vehicle, ODN, or ALN for 2days, followed by either continuing with the same treatment, or replacement of the inhibitors by vehicle for additional times as specified per experimental conditions. Maintaining OCs on ODN or ALN significantly reduced CTx-I release compared to vehicle controls. However, only the treatment of OCs with ODN resulted in the formation of small shallow discrete resorption pits, retention of intracellular vesicles enriched with CatK and other lysosomal enzymes, increase in 1-CTP release and number of TRAP(+) OCs. Upon discontinuation of ODN treatment, OCs rapidly resumed bone resorption activity, as demonstrated by a return of OC functional markers (CTx-I, 1-CTP), cell number and size, morphology and number of resorption pits, and vesicular secretion of CatK toward the respective vehicle levels. As expected, discontinuation of ALN did not reverse the treatment-related inhibition of OC activity in the time frame of the experiment. In summary, this study demonstrated rapid kinetics of inhibition and reversibility of the effects of ODN on OC bone resorption, that differentiated the cellular mechanism of CatK inhibition from that of the bisphosphate antiresorptive ALN.

High-bone-mass (HBM)-causing missense mutations in the low density lipoprotein receptor-related protein-5 (Lrp5) are associated with increased osteoanabolic action and protection from disuse- and ovariectomy-induced osteopenia. These mutations (e.g., A214V and G171V) confer resistance to endogenous secreted Lrp5/6 inhibitors, such as sclerostin (SOST) and Dickkopf homolog-1 (DKK1). Cells in the osteoblast lineage are responsive to canonical Wnt stimulation, but recent work has indicated that osteoclasts exhibit both indirect and direct responsiveness to canonical Wnt. Whether Lrp5-HBM receptors, expressed in osteoclasts, might alter osteoclast differentiation, activity, and consequent net bone balance in the skeleton, is not known. To address this, we bred mice harboring heterozygous Lrp5 HBM-causing conditional knock-in alleles to Ctsk-Cre transgenic mice and studied the phenotype using DXA, μCT, histomorphometry, serum assays, and primary cell culture. Mice with HBM alleles induced in Ctsk-expressing cells (TG) exhibited higher bone mass and architectural properties compared to non-transgenic (NTG) counterparts. In vivo and in vitro measurements of osteoclast activity, population density, and differentiation yielded significant reductions in osteoclast-related parameters in female but not male TG mice. Droplet digital PCR performed on osteocyte enriched cortical bone tubes from TG and NTG mice revealed that ~8-17% of the osteocyte population (depending on sex) underwent recombination of the conditional Lrp5 allele in the presence of Ctsk-Cre. Further, bone formation parameters in the midshaft femur cortex show a small but significant increase in anabolic action on the endocortical but not periosteal surface. These findings suggest that Wnt/Lrp5 signaling in osteoclasts affects osteoclastogenesis and activity in female mice, but also that some of the changes in bone mass in TG mice might be due to Cre expression in the osteocyte population.

In mice and humans, the effect of genetic deficiency of cathepsin K (catK) is impaired bone resorption, or osteopetrosis. Inhibition of catK is therefore a promising strategy for the treatment of osteoporosis. The enzyme acts in an acid environment. This provides a further potential opportunity: if the inhibitor is basic it is more likely to accumulate in membrane-bound acidic compartments (lysosomotropism), so minimizing off-target effects. However, the resorptive hemivacuole is not membrane-bound, and so might not retain lysosomotropic compounds. We therefore elected to determine whether the osteoclastic resorptive apparatus supports such accumulation. First, we attempted to compare the persistence of a lysosomotropic dye in the hemivacuole versus intracellular vesicles. To our surprise the dye could not be detected in the ruffled border region by confocal microscopy. We found that this could be explained by the tight packing of the folds of the ruffled border, and their close apposition to the bone surface. We also found that the dye persisted similarly in resorbing osteoclasts and macrophages, consistent with the notion that resorbing osteoclasts support lysosomotropism. Next, we compared the ability of basic and non-basic inhibitors of catK to suppress bone resorption by human osteoclasts. We found that basic compounds were considerably more potent than non-basic compounds at suppression of osteoclastic resorption than would be anticipated from their potency as enzyme inhibitors. Also consistent with osteoclastic lysosomotropism, basic inhibitors suppressed resorption for substantially longer than a non-basic inhibitor after washout from cell cultures. Furthermore, selectivity of basic inhibitors for inhibition of catK versus other cathepsins persisted: concentrations that inhibited catK in osteoclasts had no detectable effect on cathepsin S (catS) in a cell-based assay. This data is consistent with accumulation and enrichment of such basic inhibitors in the resorptive apparatus of the osteoclast, allowing for prolonged efficacy at the intended site of action. Our results suggest a major advantage for lysosomotropic compounds as inhibitors of bone resorption by osteoclasts in osteoporosis and other diseases caused by excessive osteoclastic activity.

Osteoporosis and cardiovascular (CV) diseases are closely correlated. RANKL/RANK/OPG pathway and Wnt signalling pathway both implicated in the pathogenesis of osteoporosis and cardiovascular diseases. We aimed to investigate the effect of denosumab or romosozumab therapy on cardiovascular outcomes in patients with primary osteoporosis.

Survival of chronic diseases in childhood is often achieved utilizing glucocorticoids, but comes with significant side effects, including glucocorticoid-induced osteoporosis (GIO). Knowledge of the mechanism of GIO is limited to the adult skeleton. We explored the effect of genetic loss and inhibition of cathepsin K (Ctsk) as a potential treatment target in a young GIO mouse model as genetic loss of cathepsin K results in a mild form of osteopetrosis secondary to impaired osteoclast bone resorption with maintenance of bone formation. We first characterized the temporal osteoclast and osteoblast progenitor populations in Ctsk-/- and wild type (WT) mice in the primary and secondary spongiosa, as sites representative of trabecular bone modeling and remodeling, respectively. In the primary spongiosa, Ctsk-/- mice had decreased numbers of osteoclasts at young ages (2 and 4 weeks) and increased osteoblast lineage cells at later age (8 weeks) relative to WT littermates. In the secondary spongiosa, Ctsk-/- mice had greater numbers of osteoclasts and osteoblast lineage cells relative to WT littermates. We next developed a young GIO mouse model with prednisolone 10 mg/m2/day injected intraperitoneally daily from 2 through 6 weeks of age. Overall, WT-prednisolone mice had lower bone volume per tissue volume, whereas Ctsk-/--prednisolone mice maintained a similar bone volume relative to Ctsk-/--vehicle controls. WT-prednisolone mice exhibited a decreased number of osteoclasts, tartrate-resistant acid phosphatase and platelet-derived growth factor type BB (PDGF-BB) co-positive cells, type H endothelial cells, and osteoblasts relative to WT-vehicle mice in both the primary and secondary spongiosa. Interestingly, Ctsk-/--prednisolone mice demonstrated a paradoxical response with increased numbers of all parameters in primary spongiosa and no change in secondary spongiosa. Finally, treatment with a cathepsin K inhibitor prevented WT-prednisolone decline in osteoclasts, osteoblasts, type H vessels, and bone volume. These data demonstrate that cells in the primary and secondary spongiosa respond differently to glucocorticoids and genetic manipulation. Inhibition of osteoclast resorption that preserves osteoclast coupling factors, such as through inhibition of cathepsin K, may be a potential preventive treatment strategy against GIO in the growing skeleton.

Unlike other antiresorptive medications, bisphosphonate molecules accumulate in the bone matrix. Previous studies of side-effects of anti-resorptive treatment focused mainly on systemic effects. We hypothesize that matrix-bound bisphosphonate molecules contribute to the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). In this study, we examined the effect of matrix-bound bisphosphonates on osteoclast differentiation in vitro using TRAP staining and resorption assay, with and without pretreatment with EDTA. We also tested the effect of zoledronate chelation on the healing of post-extraction defect in rats. Our results confirmed that bisphosphonates bind to, and can be chelated from, mineralized matrix in vitro in a dose-dependent manner. Matrix-bound bisphosphonates impaired the differentiation of osteoclasts, evidenced by TRAP activity and resorption assay. Zoledronate-treated rats that underwent bilateral dental extraction with unilateral EDTA treatment showed significant improvement in mucosal healing and micro-CT analysis on the chelated sides. The results suggest that matrix-bound bisphosphonates are accessible to osteoclasts and chelating agents and contribute to the pathogenesis of BRONJ. The use of topical chelating agents is a promising strategy for the prevention of BRONJ following dental procedures in bisphosphonate-treated patients.

Decreased effectiveness of bones' adaptive response to mechanical loading contributes to age-related bone loss. In young mice, intermittent administration of parathyroid hormone (iPTH) at 20-80μg/kg/day interacts synergistically with artificially applied loading to increase bone mass. Here we report investigations on the effect of different doses and duration of iPTH treatment on mice whose osteogenic response to artificial loading is impaired by age. One group of aged, 19-month-old female C57BL/6 mice was given 0, 25, 50 or 100μg/kg/day iPTH for 4weeks. Histological and μCT analysis of their tibiae revealed potent iPTH dose-related increases in periosteally-enclosed area, cortical area and porosity with decreased cortical thickness. There was practically no effect on trabecular bone. Another group was given a submaximal dose of 50μg/kg/day iPTH or vehicle for 2 or 6weeks with loading of their right tibia three times per week for the final 2weeks. In the trabecular bone of these mice the loading-related increase in BV/TV was abrogated by iPTH primarily by reduction of the increase in trabecular number. In their cortical bone, iPTH treatment time-dependently increased cortical porosity. Loading partially reduced this effect. The osteogenic effects of iPTH and loading on periosteally-enclosed area and cortical area were additive but not synergistic. Thus in aged, unlike young mice, iPTH and loading appear to have separate effects. iPTH alone causes a marked increase in cortical porosity which loading reduces. Both iPTH and loading have positive effects on cortical periosteal bone formation but these are additive rather than synergistic.

Osteocytes are believed to be the primary mechanosensors of bone tissue, signaling to osteoblasts and osteoclasts by releasing specific proteins. Sclerostin, interleukin-6 (IL-6), and insulin-like growth factor-I (IGF-I) are osteocyte proteins that signal to osteoblasts. The primary objective of this study was to determine if osteocyte protein response to mechanical unloading is restricted to the unloaded bone using the hindlimb unloading (HU) rodent model. We also examined tumor necrosis factor-α (TNF-α) due to its interactions with all three osteocyte proteins. We hypothesized that unloaded hindlimb cancellous bone would have an altered osteocyte protein (sclerostin, IL-6, and IGF-I) response compared to controls, while the response in the weight-bearing forelimb would not differ from ambulating controls. Male Sprague Dawley rats (7-mo old) experienced either HU (n=7) or normal cage activity (CON; n=7) for 28days. The unloaded distal femur and the weight-bearing proximal humerus were compared in HU vs CON. Metaphyseal bone density was reduced in the HU rats' hindlimb, but not in the proximal humerus, compared to CON values. Osteocyte density was 30% lower in the HU distal femur, but not different from CON in the proximal humerus. %Sclerostin+osteocytes in the distal femur were higher in HU compared to CON, but lower in the proximal humerus. Both %IGF-I+ and %IL-6+ osteocytes were lower in the distal femur for HU, but higher in the proximal humerus for HU. Osterix surface, a marker of osteoblasts, was lower in HU in the distal femur; however, the proximal humerus had more %osterix+surface in HU. In HU %Cathepsin K+ surface, a marker of osteoclasts, was higher in the distal femur and lower in the proximal humerus. %TNF-α+osteocytes were no different from CON in either bone site. HU proximal humerus osteocyte protein responses of sclerostin, IL-6, and IGF-I changed in the opposite direction as observed in the distal femur within the same animal. The opposite response of osteocyte proteins and osteoblast surface in hind- and forelimb bones within the same animal suggests that, while osteocytes in the unloaded hindlimb sense a lack of mechanical strain, osteocytes in the weight-bearing forelimb in HU animals sense some increase in local strain and generate molecular signaling to osteoblasts.

Sex steroid deficiency plays critical roles in the pathophysiology of bone as the result of uncertain bone remodeling, i.e., increased bone resorption with equivocal bone formation. We have previously shown that GPR109A, a G protein coupled receptor, controls osteoclastogenesis and bone resorption, where global GPR109A deletion decreased osteoclast bone resorption and increased bone mass. Here, we used global GPR109A gene deletion, ovariectomized (OVX) and orchidectomized (ORX) mouse models to probe the role of GPR109A in gonadectomy-induced bone loss in female and male mice. Six months old GPR109A-/- mice and their wild type littermates were allocated to Sham or gonadectomized groups for six weeks. Using densitometric micro-CT confirmed by peripheral quantitative CT (pQCT) scans on tibia and spine, and three-point bending test on femur ex vivo, we found the bone volume, trabecular number, as well as bone mineral density and content in both trabecular and cortical sites were significantly decreased in wild type OVX and ORX compared with respective Sham groups. While bone mass in both male and female GPR109A-/- Sham groups were significantly higher compared with their respective wild type Sham groups, global GPR109A gene deletion ameliorated gonadectomy-induced bone loss. In GPR109A-/- females, most of bone mass and strength parameters measured by micro-CT, pQCT and three-point bending test were not different between Sham and OVX groups. In wild type but not in GPR109-/- mice, bone remodeling marker measurements indicated that both bone resorption (Cathepsin K) and bone formation (osteocalcin) markers were increased in gonadectomized mice compared to sham, with the exception of bone specific ALP, which was decreased in gonadectomized mice. Expression of bone resorption markers (Cathepsin K) were significantly lower, but β-catenin expression was higher in GPR109A-/- mice compared with their wild type littermates. Collectively, these data indicate that global GPR109A deletion ameliorates gonadectomy-induced bone loss through suppression of bone resorption.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of antiresorptive medications such as denosumab (humanized anti-RANKL antibody), yet its pathophysiology remains elusive. It has been posited that inhibition of osteoclastic bone resorption leads to the pathological sequelae of dead bone accumulation, impaired new bone formation, and poor wound healing in MRONJ, but this hypothesis has not been definitively tested. We previously engineered myeloid precursors with a conditional receptor activator of nuclear factor kappa-Β intracellular domain (iRANK cells), which differentiate into osteoclasts in response to a chemical inducer of dimerization (CID) independently of RANKL. In this study, we showed that CID-treated iRANK cells differentiated into osteoclasts and robustly resorbed mineralized surfaces even in the presence of anti-RANKL antibody in vitro. We then developed a tooth extraction-triggered MRONJ model in nude mice using anti-RANKL antibody to deplete osteoclasts. This model was used to determine whether reconstitution of engineered osteoclasts within sockets could prevent specific pathological features of MRONJ. Locally delivered iRANK cells successfully differentiated into multinucleated osteoclasts in response to CID treatment in vivo as measured by green fluorescent protein (GFP), tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, matrix metallopeptidase 9 (MMP-9), and cathepsin K staining. Sockets treated with iRANK cells + CID had significantly more osteoclasts and less necrotic bone than those receiving iRANK cells alone. These data support the hypothesis that osteoclast deficiency leads to accumulation of necrotic bone in MRONJ.

Recent reports have described the interactions of muscle and bone. Various muscle-derived humoral factors, known as myokines, affect bone. Although extracellular vesicles (EVs) play a vital role in physiological and pathophysiological processes by transferring their contents to distant tissues during bone metabolism, the roles of EVs in the muscle-bone interactions remain unknown. In the present study, we investigated the effects of EVs secreted from mouse muscle C2C12 cells on mouse bone cells and mitochondrial biogenesis. EVs secreted from C2C12 cells (Myo-EVs) were isolated from the conditioned medium of C2C12 cells by ultracentrifugation. Myo-EVs suppressed osteoclast formation as well as the expression of tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cells cytoplasmic 1 and dendritic cell-specific transmembrane protein induced by receptor activator of nuclear factor κB ligand (RANKL) in mouse bone marrow cells and preosteoclastic Raw264.7 cells. Moreover, Myo-EVs suppressed oxygen consumption and mRNA expression of the mitochondrial biogenesis markers enhanced by RANKL in these cells. However, Myo-EVs did not affect the phenotypes or mitochondrial biogenesis of mouse primary osteoblasts. In conclusion, the present study showed for the first time that Myo-EVs suppress osteoclast formation and mitochondrial energy metabolism in mouse bone marrow and Raw264.7 cells. EVs secreted from skeletal muscles might be a crucial mediator of muscle-bone interactions.

To define their gene expression and function, osteocytes are commonly isolated and purified by fluorescence-activated cell sorting (FACS) from mice expressing GFP directed by the dentin matrix protein 1 (Dmp1) promoter (DMP1-GFP). These cells express mRNA for osteocyte genes, including sclerostin (Sost) and Dmp1, and genes associated with the osteoclast phenotype: Dcstamp, Oscar, Cathepsin K (Ctsk), tartrate resistant acid phosphatase (TRAP/Acp5) and calcitonin receptor (Calcr). This suggests either that osteoclasts and osteocytes share genes and functions or that DMP1-GFP(+) preparations contain haematopoietic osteoclasts. To resolve this we stained DMP1-GFP cells for haematopoietic lineage (Lin) surface markers (CD2, CD3e, CD4, CD45, CD5, CD8, CD11b, B220, Gr1, Ter119) and CD31. Lin(-)CD31(-) (Lin(-)) and Lin(+)CD31(+) (Lin(+)) populations were analysed for GFP, and the four resulting populations assessed by quantitative real-time PCR. Lin(-)GFP(+) cells expressed mRNAs for Sost, Dmp1, and Mepe, confirming their osteocyte identity. Dcstamp and Oscar mRNAs were restricted to haematopoietic (Lin(+)) cells, but Calcr, Ctsk and Acp5 were readily detected in purified osteocytes (Lin(-)GFP(+)). The capacity of these purified osteocytes to support osteoclastogenesis was assessed: no TRAP+ cells with >2 nuclei were formed when purified osteocytes were cultured with bone marrow macrophages and stimulated with 1,25-dihydroxyvitamin-D3/prostaglandin E2. Lin(-)GFP(+) osteocytes also expressed lower levels of Tnfsf11 (RANKL) mRNA than the osteoblast-enriched population (Lin(-)GFP(-)). This demonstrates the importance of haematopoietic depletion in generating highly purified osteocytes and shows that osteocytes express Acp5, Ctsk and Calcr, but not other osteoclast markers, and do not fully support osteoclast formation in vitro.

Musculoskeletal diseases and disorders, including osteoporosis and rheumatoid arthritis are diseases that threaten a healthy life expectancy, and in order to extend the healthy life expectancy of elderly people, it is important to prevent bone and joint diseases and disorders. We previously reported that alymphoplasia (aly/aly) mice, which have a loss-of-function mutation in the Nik gene involved in the processing of p100 to p52 in the alternative NF-κB pathway, show mild osteopetrosis with a decrease in the osteoclast number, suggesting that the alternative NF-κB pathway is a potential drug target for ameliorating bone diseases. Recently, the novel NF-κB-inducing kinase (NIK)-specific inhibitor compound 33 (Cpd33) was developed, and we examined its effect on osteoclastic bone resorption in vitro and in vivo. Cpd33 inhibited the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis accompanied by a decrease in the expression of nfatc1, dc-stamp, and cathepsin K, markers of osteoclast differentiation, without affecting the cell viability, in a dose-dependent manner. Cdp33 specifically suppressed the RANKL-induced processing of p100 to p52 but not the phosphorylation of p65 or the degradation or resynthesis of IκBα in osteoclast precursors. Cpd33 also suppressed the bone-resorbing activity in mature osteoclasts. Furthermore, Cdp33 treatment prevented bone loss by suppressing the osteoclast formation without affecting the osteoblastic bone formation in ovariectomized mice. Taken together, NIK inhibitors may be a new option for patients with a reduced response to conventional pharmacotherapy or who have serious side effects.

Faulty bony repair causes dysrepair of injured growth plate cartilage and bone growth defects in children; however, the underlying mechanisms are unclear. Recently, we observed the prominent induction of neurotrophin‑3 (NT-3) and its important roles as an osteogenic and angiogenic factor promoting the bony repair. The current study investigated its roles in regulating injury site remodelling. In a rat tibial growth plate drill-hole injury repair model, NT-3 was expressed prominently in osteoblasts at the injury site. Recombinant NT-3 (rhNT-3) systemic treatment enhanced, but NT-3 immunoneutralization attenuated, expression of cartilage-removal proteases (MMP-9 and MMP-13), presence of bone-resorbing osteoclasts and expression of osteoclast protease cathepsin K, and remodelling at the injury site. NT-3 was also highly induced in cultured mineralizing rat bone marrow stromal cells, and the conditioned medium augmented osteoclast formation and resorptive activity, an ability that was blocked by presence of anti-NT-3 antibody. Moreover, NT-3 and receptor TrkC were induced during osteoclastogenesis, and rhNT-3 treatment activated TrkC downstream kinase Erk1/2 in differentiating osteoclasts although rhNT-3 alone did not affect activation of osteoclastogenic transcription factors NF-κB or NFAT in RAW264.7 osteoclast precursor cells. Furthermore, rhNT-3 treatment increased, but NT-3 neutralization reduced, expression of osteoclastogenic cytokines (RANKL, TNF-α, and IL-1) in mineralizing osteoblasts and in growth plate injury site, and rhNT-3 augmented the induction of these cytokines caused by RANKL treatment in RAW264.7 cells. Thus, injury site osteoblast-derived NT-3 is important in promoting growth plate injury site remodelling, as it induces cartilage proteases for cartilage removal and augments osteoclastogenesis and resorption both directly (involving activing Erk1/2 and substantiating RANKL-induced increased expression of osteoclastogenic signals in differentiating osteoclasts) and indirectly (inducing osteoclastogenic signals in osteoblasts).

This study aimed to clarify whether elcatonin (EL) has a preventive action on bone dynamics in skeletal unloading. Seven-week-old male C57BL/6J mice with either ground control (GC) or tail suspension (TS) were administered EL 20U/kg or a vehicle (veh) three times per week and assigned to one of the following four groups: GCEL, GCveh, TSEL, and TSveh. Blood samples and bilateral femurs and tibias of the mice were obtained for analysis. After 7days of unloading, the trabecular bone mineral density in the distal femur obtained via peripheral quantitative computed tomography and the trabecular bone volume were significantly higher in the TSEL group than in the TSveh group. The bone resorption histomorphometric parameters, such as the osteoclast surface and osteoclast number, were significantly suppressed in the TSEL mice, whereas the number of preosteoclasts was significantly increased. The plasma level of tartrate-resistant acid phosphatase-5b (TRACP-5b) was significantly lower in the TSEL group than in all other groups. In the bone marrow cell culture, the number of TRACP-positive (TRACP(+)) multinucleated cells was significantly lower in the TSEL mice than in the TSveh mice, whereas the number of TRACP(+) mononucleated cells was higher in the TSEL mice. On day 4, the expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), cathepsin K and d2 isoform of vacuolar ATPase V0 domain (ATP6V0D2) mRNA in the bone marrow cells in the TSEL mice was suppressed, and the expression of calcitonin receptor (Calcr) mRNA on day 1 and Calcr antigen on day 4 were significantly higher in the TSveh mice than in the GCveh mice. EL prevented the unloading-induced bone loss associated with the high expression of Calcr in the bone marrow cells of mouse hindlimbs after tail suspension, and it suppressed osteoclast development from preosteoclasts to mature osteoclasts through bone-resorbing activity. This study of EL-treated unloaded mice provides the first in vivo evidence of a physiological role of EL in the inhibition of the differentiation process from preosteoclasts to osteoclasts.

Long-term glucocorticoid therapy is known to induce increased bone fragility and impaired skeletal regeneration potential. Growing evidence suggests that pulsed electromagnetic fields (PEMF) can accelerate fracture healing and increase bone mass both experimentally and clinically. However, how glucocorticoid-treated bone and bone cells respond to PEMF stimulation remains poorly understood. Here we tested the effects of PEMF on bone quantity/quality, bone metabolism, and porous implant osseointegration in rabbits treated with dexamethasone (0.5 mg/kg/day, 6 weeks). The micro-CT, histologic and nanoindentation results showed that PEMF ameliorated the glucocorticoid-mediated deterioration of cancellous and cortical bone architecture and intrinsic material properties. Utilizing the new porous titanium implant (Ti2448) with low toxicity and low elastic modulus, we found that PEMF stimulated bone ingrowth into the pores of implants and enhanced peri-implant bone material quality during osseous defect repair in glucocorticoid-treated rabbits. Dynamic histomorphometric results revealed that PEMF reversed the adverse effects of glucocorticoids on bone formation, which was confirmed by increased circulating osteocalcin and P1NP. PEMF also significantly attenuated osteocyte apoptosis, promoted osteoblast-related osteocalcin, Runx2 and Osx expression, and inhibited osteocyte-specific DKK1 and Sost expression (negative regulators of osteoblasts) in glucocorticoid-treated skeletons, revealing improved functional activities of osteoblasts and osteocytes. Nevertheless, PEMF exerted no effect on circulating bone-resorbing cytokines (serum TRAcP5b and CTX-1) or skeletal gene expression of osteoclast-specific markers (TRAP and cathepsin K). PEMF also significantly upregulated skeletal gene expression of canonical Wnt ligands (Wnt1, Wnt3a and Wnt10b), whereas PEMF did not alter non-canonical Wnt5a expression. This study demonstrates that PEMF treatment improves bone mass, strength and porous implant osseointegration in glucocorticoid-treated rabbits by promoting potent bone-anabolic action, which is associated with canonical Wnt-mediated improvement in osteoblast and osteocyte functions. This study provides a new treatment alternative for glucocorticoid-related bone disorders in a convenient and non-invasive manner.

Odanacatib (ODN) a selective and reversible cathepsin K inhibitor, inhibits bone resorption, increases bone mass and reduces fracture risk in women with osteoporosis. A 16-month (~7-remodeling cycles) study was carried out in treatment mode to assess the effects of ODN versus ALN on bone mass, remodeling status and biomechanical properties of lumbar vertebrae (LV) and femur in ovariectomized (OVX) rabbits. This study also evaluated the impact of discontinuing ODN on these parameters. Rabbits at 7.5months post-OVX were dosed for 16-months with ODN (7.5μM·h0-24, in food) or ALN (0.2mg/kg/wk, s.c.) and compared to vehicle-treated OVX- (OVX+Veh) or Sham-operated animals. After 8months, treatment was discontinued in half of the ODN group. ODN treatment increased in vivo LV aBMD and trabecular (Tb) vBMD until reaching plateau at month 12 by 16% and 23% vs. baseline, respectively, comparable levels to that in Sham and significantly above OVX+Veh. LV BMD was also higher in ALN that plateaued around month 8 to levels below that in ODN or Sham. ODN treatment resulted in higher BMD, structure and improved biomechanical strength of LV and central femur (CF) to levels similar to Sham. ALN generally showed less robust efficacy compared to ODN. Neither ODN nor ALN influenced material properties at these bone sites following ODN or ALN treatment for 7 remodeling cycles in rabbits. ODN and ALN persistently reduced the bone resorption marker urinary helical peptide over study duration. While ALN reduced the bone formation marker BSAP, ODN treatment did not affect this marker. ODN also preserved histomorphometry-based bone formation indices in LV trabecular, CF endocortical and intracortical surfaces, at the levels of OVX+Veh. Discontinuation of ODN returned bone mass, structure and strength parameters to the comparable respective levels in OVX+Veh. Together, these data demonstrate efficacy and bone safety profile of ODN and suggests the potential long-term benefits of this agent over ALN with respect to accrued bone mass without long-term effects on bone formation.

Despite years of extensive investigation, the cellular origin of heterotopic ossification (HO) has not been fully elucidated. We have previously shown that circulating bone marrow-derived osteoblast progenitor cells, characterized by the immunophenotype CD45-/CD44+/CXCR4+, contributed to the formation of heterotopic bone induced by bone morphogenetic protein (BMP)-2. In contrast, other reports have demonstrated the contribution of CD45+ hematopoietic derived cells to HO. Therefore, in this study, we developed a novel triple transgenic mouse strain that allows us to visualize CD45+ cells with red fluorescence and mature osteoblasts with green fluorescence. These mice were generated by crossing CD45-Cre mice with Z/RED mice that express DsRed, a variant of red fluorescent protein, after Cre-mediated recombination, and then crossing with Col2.3GFP mice that express green fluorescent protein (GFP) in mature osteoblasts. Utilizing this model, we were able to investigate if hematopoietic derived cells have the potential to give rise to mature osteoblasts. Analyses of this triple transgenic mouse model demonstrated that DsRed and GFP did not co-localize in either normal skeletogenesis, bone regeneration after fracture, or HO. This indicates that in these conditions hematopoietic derived cells do not differentiate into mature osteoblasts. Interestingly, we observed the presence of previously unidentified DsRed positive bone lining cells (red BLCs) which are derived from hematopoietic cells but lack CD45 expression. These red BLCs fail to produce GFP even under in vitro osteogenic conditions. These findings indicate that, even though both osteoblasts and hematopoietic cells are developmentally derived from mesoderm, hematopoietic derived cells do not contribute to osteogenesis in fracture healing or HO.

Among the interventions used to prevent osteoporosis in female organisms, strength training (ST) and oxytocin (OT) stand out, as a promising hormone with anabolic action on bone. This study aimed to verify whether the combined action of OT and ST, compared to isolated interventions, potentiates the bone remodeling process of the femoral neck of Wistar rats during periestropause. Forty Wistar rats (18 months) with irregular estrous cycle were randomly distributed into groups: 1-Vehicle (Veh; NaCl 0.15 mol/L ip); 2-Oxytocin (Ot; 134 μg/kg/ip); 3-Strength training (St); 4-Ot + St. The animals of the 1, 2 and 4 groups received two intraperitoneal injections with an interval of 12 h every 30 days, totaling 8 injections at the end of the experimental period (18 to 21 months). The animals in the St and Ot + St groups performed ST on a ladder 3 times a week, maximal voluntary carrying capacity (MVCC) test monthly. After 120 days, the animals were euthanized; the femur was collected for analysis of biomechanical testing, densitometry, bone microtomography, Raman spectroscopy, tissue PCR, and blood for analysis of bone biomarkers, liver damage, and oxidative stress. The main effects in the Ot group were observed in the maximum load and energy in the compression testing (femoral head), and stiffness and energy in the three-points bending testing (femur diaphysis). In addition, the main effects occurred on the bone mineral density (BMD), cortical thickness (Ct.Th), number of pores (Po.N), polar moment of inertia (J), trabecular thickness (Tb.Th), and connectivity density (Conn.Dn), Bone alkaline phosphatase (Alp), Tumor necrosis factor receptor superfamily member 11b (Opg), Tumor necrosis factor ligand superfamily member 11 (Rankl) and Cathepsin K (Ctsk) expression. There was an effect in the tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP). In the St group, the main effect was observed on the energy (compression and the three-points bending), stiffness, aBMD, BMD, cortical bone area (Ct.Ar), Po.N, trabecular bone volume (BV/TV), Tb.Th and in the mineralization ratio (ѵ1PO4/proline), Runt-related transcription factor 2 (Runx2), Bone morphogenetic protein 2 (Bmp2), Alp, Osteopontin/secreted phosphoprotein 1 (Opn/Spp1), Opg, Tumor necrosis factor receptor superfamily member 11ª (Rank), Rankl, Ctsk expression. There was an effect in the TRAP and ALP. The interaction in the combination of therapies in the Ot + St group was verified in energy to maximum load (compression and three-points bending testing), stiffness, BMD, Ct.Th, J, Tb.Th and ѵ1PO4/proline. In the gene analysis there was interaction in the Runx2, Osterix/Sp7 transcription factor (Osx/Sp7), Bmp2, Alp, Osteocalcin/Bone gamma-carboxyglutamate protein (Ocn/Bglap), Opg, Rankl and Acid phosphatase 5, tartrate resistant (Trap/Acp5) expression. In addition, the combination of OT and ST resulted in a higher maximum load compared to the Veh group, with higher BV/TV than the Ot group, higher Rankl and Ctsk expression than Veh and Ot groups, and lower Po.N and lower activity of TRAP than the other groups. In oxidative stress, total antioxidant capacity (TAC) was lower. These results showed that the combination of interventions is a promising anabolic strategy for the prevention of osteoporosis in the period of periestropause, standing out from the effects of isolated interventions.