Background and Purpose: Muscle weakness and bone fragility are both associated with hip fracture. In general, muscle contractions create forces to the bone, and bone strength adapts to mechanical loading through changes in bone architecture and mass. However, the relationship between impairment of muscle and bone function remain unclear. In particular, the associations of muscle with properties of proximal femur cortical and trabecular bone are still not well understood. The aim of this study was to explore the associations of hip/thigh muscle density (CT attenuation value in Hounsfield units) and size with cortical and trabecular bone mineral density (BMD) of the proximal femur. Materials and Methods: Three-dimensional quantitative computed tomography (QCT) imaging of the lumber, hip and mid-thigh was performed in a total of 301 participants (mean age 68.4 ± 6.1 years, 194 women and 107 men) to derive areal BMD (aBMD) and volumetric BMD (vBMD). Handgrip strength (HGS) and the Timed Up and Go (TUG) test were also performed. From the CT images, cross-sectional area (CSA), and density were determined for the gluteus maximus muscle (G.MaxM), trunk muscle at the vertebrae L2 level, and mid-thigh muscle. Multivariate generalized linear models were applied to assess associations. Results: Total hip (TH) aBMD was associated significantly with G.MaxM CSA (men: P = 0.042; women: P < 0.001) and density (men: P = 0.012; women: P = 0.043). In women, 0.035 cm2 of mid-thigh CSA (95% CI, 0.014-0.057; P = 0.002) increased per SD increase in TH aBMD, but this significance was not observed in men (P = 0.095). Trunk muscle density and CSA were not associated with proximal femur BMD. The associations of hip/thigh muscle parameters with femoral neck BMD were weaker than those with trochanter and intertrochanter BMD. Furthermore, compared to muscle density, muscle CSA showed better associations with vBMD. G.MaxM CSA was associated with trochanter (TR) Cort. vBMD in men (β, 19.898; 95% CI, 0.924-38.871; P = 0.040) and in women (β, 15.426; 95% CI, 0.893-29.958; P = 0.038). Handgrip strength was only associated with TR aBMD (β, 0.038; 95% CI, 0.006-0.070; P = 0.019) and intertrochanter aBMD (β, 0.049; 95% CI, 0.009-0.090; P = 0.016) in men. Conclusions: We observed positive associations of the gluteus and thigh muscle size with proximal femur volumetric BMD. Specifically, the gluteus maximus muscle CSA was associated with trochanter cortical vBMD in both men and women.

Glucocorticoid-induced osteoporosis (GIO) is associated with an increase in bone marrow adiposity, which skews the differentiation of mesenchymal stem cell (MSC) progenitors away from osteoblastogenesis and toward adipogenesis. We have previously found that vanadate, a non-specific protein tyrosine phosphatase inhibitor, prevents GIO in rats, but it was unclear whether vanadate directly influenced adipogenesis in bone-derived MSCs. For the present study, we investigated the effect of vanadate on adipogenesis in primary rat MSCs derived from bone marrow (bmMSCs) and from the proximal end of the femur (pfMSCs). By passage 3 after isolation, both cell populations expressed the MSC cell surface markers CD90 and CD106, but not the hematopoietic marker CD45. However, although variable, expression of the fibroblast marker CD26 was higher in pfMSCs than in bmMSCs. Differentiation studies using osteogenic and adipogenic induction media (OM and AM, respectively) demonstrated that pfMSCs rapidly accumulated lipid droplets within 1 week of exposure to AM, while bmMSCs isolated from the same femur only formed lipid droplets after 3 weeks of AM treatment. Conversely, pfMSCs exposed to OM produced mineralized extracellular matrix (ECM) after 3 weeks, compared to 1 week for OM-treated bmMSCs. Vanadate (10 μM) added to AM resulted in a significant reduction in AM-induced intracellular lipid accumulation and expression of adipogenic gene markers (PPARγ2, aP2, adipsin) in both pfMSCs and bmMSCs. Pharmacological concentrations of glucocorticoids (1 μM) alone did not induce lipid accumulation in either bmMSCs or pfMSCs, but resulted in significant cell death in pfMSCs. Our findings demonstrate the existence of at least two fundamentally different MSC depots within the femur and highlights the presence of MSCs capable of rapid adipogenesis within the proximal femur, an area prone to osteoporotic fractures. In addition, our results suggest that the increased bone marrow adiposity observed in GIO may not be solely due to direct effect of glucocorticoids on bone-derived MSCs, and that an increase in femur lipid content may also arise from increased adipogenesis in MSCs residing outside of the bone marrow niche.

The genetic epidemiology of variation in bone mineral density (BMD) and osteoporosis is not well studied in Iranian populations and needs more research. We report a candidate gene association study of BMD variation in a healthy cross-sectional study of 501 males and females sampled from the Iranian Multi-Centre Osteoporosis Study, Shiraz, Iran. We selected to study the association with 21 single nucleotide polymorphisms (SNPs) located in the 7 candidate genes LRP5, RANK, RANKL, OPG, P2RX7, VDR, and ESR1. BMD was measured at the three sites L2-L4, neck of femur, and total hip. Association between BMD and each SNP was assessed using multiple linear regression assuming an allele dose (additive effect) on BMD (adjusted for age and sex). Statistically significant (at the unadjusted 5% level) associations were seen with seven SNPs in five of the candidate genes. Two SNPs showed statistically significant association with more than one BMD site. Significant association was seen between BMD at all the three sites with the VDR SNP rs731246 (L2-L4 p = 0.038; neck of femur p = 0.001; and total hip p < 0.001). The T allele was consistently associated with lower BMD than the C allele. Significant association was also seen for the P2RX7 SNP rs3751143, where the G allele was consistently associated with lower BMD than the T allele (L2-L4 p = 0.069; neck of femur p = 0.024; and total hip p = 0.045).

Purpose: The association between bone mineral density (BMD), bone turnover markers, and serum cholesterol in healthy population has already been proved. However, in patients with type 2 diabetes mellitus (T2D), it has not been adequately analyzed. In this study, we investigated the correlation between BMD, bone turnover markers, and serum cholesterol levels in people with T2D. Methods: We enrolled 1,040 men and 735 women with T2D from Zhongshan Hospital between October 2009 and January 2013. Their general condition, history of diseases and medication, serum markers, and BMD data were collected. We used logistic regression analysis to identify the association between serum cholesterol levels and BMD as well as bone turnover markers. Results: In multivariate regression analysis, we observed that in men with T2D, high high-density lipoprotein-cholesterol and total cholesterol levels were significantly associated with low total lumbar, femur neck, and total hip BMD, while low-density lipoprotein-cholesterol level was only inversely associated with total lumbar and femur neck BMD. Total cholesterol and low-density lipoprotein-cholesterol levels were also negatively associated with osteocalcin, procollagen type I N-terminal propeptide, and β-crosslaps. In women with T2D, high-density lipoprotein-cholesterol level was observed to be negatively correlated with total lumbar, femur neck, and total hip BMD, while total cholesterol and low-density lipoprotein-cholesterol levels were only associated with BMD at the total lumbar. Furthermore, total cholesterol was also negatively associated with osteocalcin, procollagen type I N-terminal propeptide, and β-crosslaps; high-density lipoprotein-cholesterol was only related to osteocalcin and parathyroid hormone, while low-density lipoprotein-cholesterol was only related to β-crosslaps in women. Conclusion: Our study suggests a significantly negative correlation between serum cholesterol levels and BMD in both men and women with T2D. The associations between serum cholesterol levels and bone turnover markers were also observed in T2D patients.

Signaling lipid mediators released from 5 lipoxygenase (5LO) pathways influence both bone and muscle cells, interfering in their proliferation and differentiation capacities. A major limitation to studying inflammatory signaling pathways in bone and muscle healing is the inadequacy of available animal models. We developed a surgical injury model in the vastus lateralis (VL) muscle and femur in 129/SvEv littermates mice to study simultaneous musculoskeletal (MSK) healing in male and female, young (3 months) and aged (18 months) WT mice compared to mice lacking 5LO (5LOKO). MSK defects were surgically created using a 1-mm punch device in the VA muscle followed by a 0.5-mm round defect in the femur. After days 7 and 14 post-surgery, the specimens were removed for microtomography (microCT), histopathology, and immunohistochemistry analyses. In addition, non-injured control skeletal muscles along with femur and L5 vertebrae were analyzed. Bones were microCT phenotyped, revealing that aged female WT mice presented reduced BV/TV and trabecular parameters compared to aged males and aged female 5LOKO mice. Skeletal muscles underwent a customized targeted lipidomics investigation for profiling and quantification of lipid signaling mediators (LMs), evidencing age, and gender related-differences in aged female 5LOKO mice compared to matched WT. Histological analysis revealed a suitable bone-healing process with osteoid deposition at day 7 post-surgery, followed by woven bone at day 14 post-surgery, observed in all young mice. Aged WT females displayed increased inflammatory response at day 7 post-surgery, delayed bone matrix maturation, and increased TRAP immunolabeling at day 14 post-surgery compared to 5LOKO females. Skeletal muscles of aged animals showed higher levels of inflammation in comparison to young controls at day 14 post-surgery; however, inflammatory process was attenuated in aged 5LOKO mice compared to aged WT. In conclusion, this new model shows that MSK healing is influenced by age, gender, and the 5LO pathway, which might serve as a potential target to investigate therapeutic interventions and age-related MSK diseases. Our new model is suitable for bone-muscle crosstalk studies.

Bariatric surgery is associated with a positive effect on the progress of non-alcoholic associated fatty liver disease (NAFLD). Although weight loss is the obvious mechanism, there are also weight-independent mechanisms.

Extracorporeal shock wave therapy (ESWT) has been identified to accelerate bone formation. However, detailed mechanism has not been fully explained. In this study, we found that ESWT promoted osteoblast formation in vitro. Local ESW treatment of femur increased bone formation in vivo. Furthermore, changing the density or frequency of energy, there was no statistical difference in osteogenic differentiation. Therapeutically, local ESW therapy relieved bone loss and increased the number of bone trabecular in a rabbit osteoporosis model and promoted endogenous levels of SMAD2 protein expression. Thus, ESWT may be a potential therapy by promoting osteoblast maturation through TGF-β/SMAD2 pathway.

Chordin-like 1 (CHRDL1) is a secreted glycoprotein with repeated cysteine-rich domains, which can bind to BMPs family ligands. Although it has been reported to play important roles in several systems, the exact roles of CHRDL1 on human bone mesenchymal stem cells (hBMSCs) osteogenesis remain to be explored. The present study aimed to investigate the roles of CHRDL1 on the osteogenic differentiation of hBMSCs and the underlying molecular mechanisms. We found that CHRDL1 was upregulated during hBMSCs osteogenesis, and rhBMP-4 administration could enhance CHRDL1 mRNA expression in a dose and time dependent manner. Knockdown of CHRDL1 did not affect hBMSCs proliferation, but inhibited the BMP-4-dependent osteogenic differentiation, showing decreased mRNA expression levels of osteogenic markers and reduced mineralization. On the contrary, overexpression of CHRDL1 enhanced BMP-4 induced osteogenic differentiation of hBMSCs. Moreover, in vivo experiments by transplanting CHRDL1 gene modified hBMSCs into nude mice defective femur models displayed higher new bone formation in CHRDL1 overexpression groups, but lower new bone formation in CHRDL1 knockdown groups, compared with control groups. In consistent with the bone formation rate, there were increased CHRDL1 protein expression in new bone formation regions of defective femur in CHRDL1 overexpression groups, while reduced CHRDL1 protein expression in CHRDL1 knockdown groups compared with control groups. These indicate that CHRDL1 can promote osteoblast differentiation in vivo. Furthermore, the mechanisms study showed that CHRDL1 improved BMP-4 induced phosphorylation of SMAD1/5/9 during osteogenic differentiation of hBMSCs. Besides, promotion of osteogenic differentiation and the activation of SMAD phosphorylation by CHRDL1 can be blocked by BMP receptor type I inhibitor LDN-193189. In conclusion, our results suggested that CHRDL1 can promote hBMSCs osteogenic differentiation through enhancing the activation of BMP-4-SMAD1/5/9 pathway.

Bone marrow adipose tissue has brown fat characteristics. Several studies have demonstrated that total flavonoids of Epimedium (TFE) could prevent bone loss and reduce the white adiposity in bone marrow induced by ovariectomy (OVX) in rats. However, the effects of TFE on marrow brown fat in OVX rats remain unclear. In this word, we addressed this question expected to provide a new target for preventing and treating osteoporosis. Thirty-six 3-month-old female Sprague-Dawley rats were equally divided into Sham controls, OVX controls, and OVX treated with TFE. Chemical shift coding magnetic resonance was performed to detect marrow fat fraction at the left femur at baseline, 6 and 12 weeks post-OVX. Bone mineral density at the lumbar spine and femur was measured by dual-energy x-ray absorptiometry. Serum bone biomarkers by ELISA, trabecular bone microarchitecture at the proximal tibia by micro-CT, quantitative parameters of marrow adipocyte by hematoxylin, and eosin staining were evaluated. The marrow adipocyte gene and protein expressions profile were determined by real-time quantitative PCR and immunostaining in whole tibiae. We found that TFE treatment could decrease bone turnover rate and improved bone mineral density and trabecular microarchitecture in OVX rats. OVX resulted in marrow adipogenesis as evidenced by increased marrow fat fraction, larger marrow adipocyte size, increased adipocyte number and percentage of adipocyte area, marrow white adipocyte gene, and protein expression, including PPARγ2 and FABP4. These pathological changes induced by estrogen deficiency were restored by TFE treatment. TFE also increased brown adipocyte expressions of the transcription factor Ucp1 and Prdm16 in whole tibiae. There was no detectible protein expression of brown adipocyte markers in the proximal tibia. Taken together, TFE regulation of bone marrow adiposity in OVX rats is mediated, at least in part, via maintaining the reciprocity of white and brown adipose tissue.

Postmenopausal Osteoporosis (PMOP) is the most prevalent primary osteoporosis, attributable to an imbalance in osteoblast and osteoclast activity. Modified You-Gui-Yin (MYGY), a traditional Chinese herbal formula, is able to effectively treat PMOP, while the critical components and pharmacological mechanisms of MYGY are still unclear. In this study, we aimed to investigate the therapeutic effects and underlying mechanisms of N-butanol extract of MYGY (MYGY-Nb) in ovariectomized (OVX)-induced osteoporosis mice. Histological staining and micro-computed tomography (μCT) analysis showed that MYGY-Nb was more effective in the suppression of OVX-induced bone loss than MYGY original formula. Subsequently, liquid chromatography and mass spectrometry analysis identified 16 critical compounds of MYGY-Nb and some of them are reported to affect osteoclast functions. Furthermore, in vivo and in vitro experiments demonstrated that MYGY-Nb significantly attenuated osteoclastogenesis by down-regulating RANKL-mediated NF-κB signaling. In conclusion, our study indicated that MYGY-Nb suppresses NF-κB signaling and osteoclast formation to mitigate bone loss in PMOP, implying that MYGY-Nb and its compounds are potential candidates for development of anti-PMOP drugs.

Growing female mice housed at room temperature (22°C) weigh the same but differ in body composition compared to mice housed at thermoneutrality (32°C). Specifically, mice housed at room temperature have lower levels of white adipose tissue (WAT). Additionally, bone marrow adipose tissue (bMAT) and cancellous bone volume fraction in distal femur metaphysis are lower in room temperature-housed mice. The metabolic changes induced by sub-thermoneutral housing are associated with lower leptin levels in serum and higher levels of Ucp1 gene expression in brown adipose tissue. Although the precise mechanisms mediating adaptation to sub-thermoneutral temperature stress remain to be elucidated, there is evidence that increased sympathetic nervous system activity acting via β-adrenergic receptors plays an important role. We therefore evaluated the effect of the non-specific β-blocker propranolol (primarily β1 and β2 antagonist) on body composition, femur microarchitecture, and bMAT in growing female C57BL/6 mice housed at either room temperature or thermoneutral temperature. As anticipated, cancellous bone volume fraction, WAT and bMAT were lower in mice housed at room temperature. Propranolol had small but significant effects on bone microarchitecture (increased trabecular number and decreased trabecular spacing), but did not attenuate premature bone loss induced by room temperature housing. In contrast, propranolol treatment prevented housing temperature-associated differences in WAT and bMAT. To gain additional insight, we evaluated a panel of genes in tibia, using an adipogenesis PCR array. Housing temperature and treatment with propranolol had exclusive as well as shared effects on gene expression. Of particular interest was the finding that room temperature housing reduced, whereas propranolol increased, expression of the gene for acetyl-CoA carboxylase (Acacb), the rate-limiting step for fatty acid synthesis and a key regulator of β-oxidation. Taken together, these findings provide evidence that increased activation of β1 and/or β2 receptors contributes to reduced bMAT by regulating adipocyte metabolism, but that this pathway is unlikely to be responsible for premature cancellous bone loss in room temperature-housed mice.

Calcitonin is a small peptide hormone secreted from the parafollicular cells of the thyroid gland in response to an increase in serum calcium. The inhibition of osteoclastic resorption is the main mechanism by which calcitonin quickly decreases circulating calcium levels. Although calcitonin pharmacologically acts on osteoclasts to prevent bone resorption, the results of studies on genetically modified animals have shown that the physiological effect of calcitonin is in the inhibition of osteoblastic bone formation. Because the calcitonin receptor is only expressed in osteoclasts, the effect of calcitonin on osteoblasts maybe indirect and mediated via osteoclasts. Wnt ligands are involved in various aspects of skeletal biology, including bone remodeling and endochondral bone formation. Wnt10b has recently been recognized as a clastokine, and is potentially a therapeutic target for treating bone disorders. However, the extent to which Wnt signaling is involved in bone physiology and disease is not yet fully understood. We hypothesize that calcitonin indirectly increases osteoblastic bone formation by inducing Wnt10b expression in osteoclasts. Micro-CT analysis revealed reduced bone loss in calcitonin-treated ovariectomized rats. The serum of animals treated with calcitonin had decreased TRAP5b and CTX-1 but increased osteocalcin, P1NP, and Wnt10b. Immunohistochemistry staining showed that the level of Wnt10b in the femur was increased in calcitonin-treated groups as compared with control groups. Hematopoietic mononuclear cells were separated from rat femur and tibia bone marrow, and were induced into osteoclasts following treatment with M-CSF and RANKL. In these cells, immunoconfocal microscopy and Western blot analysis showed that calcitonin induced an increase in Wnt10b expression. In a culture of osteoblasts isolated from neonatal rat calvariae, the calcitonin-treated osteoclast supernatant showed an increase in mineralization, as indicated by ALP and alizarin red staining. Taken together, these results indicate that calcitonin induces bone formation by increasing the expression of Wnt10b in osteoclasts in ovariectomy-induced osteoporotic rats. The present study provides in-depth information about the effects of calcitonin on bone remodeling and will thus help in the development of future potential therapeutic strategies for postmenopausal osteoporosis.

Evidence shows that sympathetic nervous system (SNS) activation inhibits bone formation and activates bone resorption leading to bone loss. Because thyroid hormone (TH) interacts with the SNS to control several physiological processes, we raised the hypothesis that this interaction also controls bone remodeling. We have previously shown that mice with double-gene inactivation of α2A- and -adrenoceptors (α2A/2C-AR-/-) present high bone mass (HBM) phenotype and resistance to thyrotoxicosis-induced osteopenia, which supports a TH-SNS interaction to control bone mass and suggests that it involves α2-AR signaling. Accordingly, we detected expression of α2A-AR, α2B-AR and α2C-AR in the skeleton, and that triiodothyronine (T3) modulates α2C-AR mRNA expression in the bone. Later, we found that mice with single-gene inactivation of α2C-AR (α2C-AR-/-) present low bone mass in the femur and HBM in the vertebra, but that both skeletal sites are resistant to TH-induce osteopenia, showing that the SNS actions occur in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss. To further dissect the specific roles of α2-AR subtypes, in this study, we evaluated the skeletal phenotype of mice with single-gene inactivation of α2A-AR (α2A-AR-/-), and the effect of daily treatment with a supraphysiological dose of T3, for 4 or 12 weeks, on bone microarchitecture and bone resistance to fracture. Micro-computed tomographic (μCT) analysis revealed normal trabecular and cortical bone structure in the femur and vertebra of euthyroid α2A-AR-/- mice. Thyrotoxicosis was more detrimental to femoral trabecular bone in α2A-AR-/- than in WT mice, whereas this bone compartment had been previously shown to present resistance to thyrotoxicosis in α2C-AR-/- mice. Altogether these findings reveal that TH excess depends on α2C-AR signaling to negatively affect femoral trabecular bone. In contrast, thyrotoxicosis was more deleterious to femoral and vertebral cortical bone in WT than in α2A-AR-/- mice, suggesting that α2A-AR signaling contributes to TH actions on cortical bone. These findings further support a TH-SNS interaction to control bone physiology, and suggest that α2A-AR and α2C-AR signaling pathways have key roles in the mechanisms through which thyrotoxicosis promotes its detrimental effects on bone remodeling, structure and resistance to fracture.

A Chinese family was identified to have two patients with rickets, an adult female and a male child (proband), both exhibiting signs related to X-linked hypophosphatemic rickets (XLH). Gene sequencing analysis revealed a deletion of adenine at position 1985 (c.1985delA) in the PHEX-encoding gene. To investigate the relationship between this mutation and the pathogenicity of XLH, as well as analyze the effects of different dosages of PHEX gene mutations on clinical phenotypes, we developed a rat model carrying the PHEX deletion mutation. The CRISPR/Cas9 gene editing technology was employed to construct the rat model with the PHEX gene mutation (c.1985delA). Through reproductive procedures, five genotypes of rats were obtained: female wild type (X/X), female heterozygous (-/X), female homozygous wild type (-/-), male wild type (X/Y), and male hemizygous (-/Y). The rats with different genotypes underwent analysis of growth, serum biochemical parameters, and bone microstructure. The results demonstrated the successful generation of a stable rat model inheriting the PHEX gene mutation. Compared to the wild-type rats, the mutant rats displayed delayed growth, shorter femurs, and significantly reduced bone mass. Among the female rats, the homozygous individuals exhibited the smallest body size, decreased bone mass, shortest femur length, and severe deformities. Moreover, the mutant rats showed significantly lower blood phosphorus concentration, elevated levels of FGF23 and alkaline phosphatase, and increased expression of phosphorus regulators. In conclusion, the XLH rat model with the PHEX gene mutation dosage demonstrated its impact on growth and development, serum biochemical parameters, and femoral morphology.

Tea (Camellia sinensis) has several reported health benefits, including that on bone health attributed to catechins of which the most abundant is epigallocatechin-3-gallate (EGCG). However, several preclinical and clinical studies raise safety concerns about EGCG in tea extract causing acute liver failure. Tea also contains kaempferol, albeit scanty, and it has hepatoprotective and osteogenic effects. Here, we utilized a novel extraction procedure of acid hydrolysis to enhance the osteogenic effect of tea extract while reducing its hepatotoxicity. The resultant extract (USKECSE) has a ~40-fold increase in kaempferol and a 2.5-fold reduction in EGCG content compared with the hydroethanolic extract (USCSE). In a female Sprague Dawley (SD) rat femur osteotomy model, USKECSE (100 mg/kg) but not USCSE promoted bone regeneration. In a rat postmenopausal osteoporosis model induced by bilateral ovariectomy (OVX), USKECSE through an osteogenic mechanism maintained bone mass, strength, and microarchitecture to the levels of ovary-intact rats with no hepatotoxic effect. After a single oral dose (100 mg/kg) of USKECSE to adult rats, kaempferol was detectable for 48 hours, suggesting its significant absorption and distribution in plasma. Peak kaempferol concentration in plasma (Cmax) was 483 ng/ml (2 μM), and at this concentration, kaempferol induces osteoblast differentiation. USKECSE had no genotoxicity, and its safety index assessed by preclinical toxicity studies, including safety pharmacology, was >20-fold. Taken together, we report a novel extraction process that enhanced the osteogenicity and concomitantly reduced hepatotoxicity of tea extract with significant kaempferol bioavailability and a favorable systemic safety profile. Based on these data, we propose assessing the USKECSE effect for postmenopausal osteoporosis treatment.

Objective: The study aimed to explore the associations of rs4988300 and rs634008 in the low-density lipoprotein receptor-related protein 5 (LRP5) gene with bone mineral density (BMD), bone turnover markers (BTM), and fractures in elderly patients with osteoporosis (OP). Methods: Our study included 328 unrelated OP patients with or without fractures. Genomic DNA was extracted for genotyping. BTM levels were assessed by electrochemiluminescence (ECL). Dual-energy X-ray absorptiometry (DXA) was employed to measure BMD in the lumbar spine (LS) and proximal femur. Basic features between the OP and fracture groups were analyzed using the t-test. The Chi-square test was performed to analyze the differences in allele and genotype frequencies. The associations of single-nucleotide polymorphisms (SNPs) with BMD and BTM in the subgroups were investigated by the analysis of covariance (ANCOVA) adjusted for confounding factors. Results: In both females and males, individuals with fractures exhibited higher BTM levels and lower BMD values than those with OP (P < 0.05). The allele and genotype frequencies of rs4988300 in the subgroups were significantly different (P < 0.05). In both females and males suffering from OP, participants with rs4988300 GG or rs634008 TT presented lower procollagen I N-terminal propeptide (PINP) levels (P < 0.05). Women with OP carrying rs4988300 GG exhibited lower BMD values at FN and TH (P < 0.05). In both females and males with fractures, individuals carrying rs4988300 GG genotype or rs634008 TT genotype exhibited lower PINP levels and BMD values at FN and TH than those with other genotypes (P < 0.05). Conclusions: Rs4988300 and rs634008 polymorphisms in the LRP5 gene are associated with bone phenotypes in the elderly with OP or fractures.

Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a condition characterized by paucity of white adipose tissue, with eventual diabetes, and steatosis. We applied a mouse model of global deficiency of Bscl2 (SEIPIN), required for lipid droplet formation. Male twelve-week-old B6 knockouts (KO) and wild type (WT) littermates were assigned six-weeks of voluntary, running exercise (E) versus non-exercise (N=5-8). KO weighed 14% less than WT (p=0.01) and exhibited an absence of epididymal adipose tissue; KO liver Plin1 via qPCR was 9-fold that of WT (p=0.04), consistent with steatosis. Bone marrow adipose tissue (BMAT), unlike white adipose, was measurable, although 40.5% lower in KO vs WT (p=0.0003) via 9.4T MRI/advanced image analysis. SEIPIN ablation's most notable effect marrow adiposity was in the proximal femoral diaphysis (-56% KO vs WT, p=0.005), with relative preservation in KO-distal-femur. Bone via μCT was preserved in SEIPIN KO, though some quality parameters were attenuated. Running distance, speed, and time were comparable in KO and WT. Exercise reduced weight (-24% WT-E vs WT p<0.001) but not in KO. Notably, exercise increased trabecular BV/TV in both (+31%, KO-E vs KO, p=0.004; +14%, WT-E vs WT, p=0.006). The presence and distribution of BMAT in SEIPIN KO, though lower than WT, is unexpected and points to a uniqueness of this depot. That trabecular bone increases were achievable in both KO and WT, despite a difference in BMAT quantity/distribution, points to potential metabolic flexibility during exercise-induced skeletal anabolism.

Objective: Studies suggest that matrix Gla protein (MGP) is associated with osteoporosis. However, the precise mechanism through which MGP regulates bone metabolism is not fully understood. The purpose of this study was to clarify the role of MGP in bone metabolism. Methods: The MGP gene in MG63 cell line was knocked down using shRNA. Cell Counting Kit-8 assay was used to detect the proliferation of MG63 cells. Moreover, the differentiation and mineralization of MG63 cells were measured through alkaline phosphatase staining and Alizarin Red S staining. Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to detect the protein and mRNA levels of components of the Wnt/β-catenin signaling pathway, such as Wnt3a, β-catenin, and Runx2. Transgenic (MGP+) mice were used to detect the effects of MGP in vivo. Results: The Cell Counting Kit-8 assay suggested that upregulated MGP could promote the proliferation of MG63 cells, whereas its downregulation inhibited proliferation. The alkaline phosphatase assay and Alizarin Red S staining showed that overexpressed MGP led to prominently upregulated differentiation and mineralization of MG63 cells. Conversely, knockdown of MGP decreased the levels of differentiation and mineralization. Western blotting and quantitative reverse transcription-polymerase chain reaction showed that overexpression of MGP upregulated Wnt3a, β-catenin, and Runx2. In contrast, knocking down MGP reduced their transcriptional levels. In vivo, overexpression of MGP inhibited the decrease in bone mineral density induced via ovariectomy in the femur, and significantly prevented bone volume fraction, trabecular number, BV/TV, and TbTh to decrease. In addition, it increased the levels of estradiol in sera. Conclusion: The findings of this study suggest that the promotion of osteoblast proliferation, differentiation, and mineralization by MGP may be a mechanism to prevent osteoporosis. Furthermore, the results show that MGP promoted the osteogenic effects via the Wnt/β-catenin signaling pathway.

Catecholamine signaling is known to influence bone tissue as reuptake of norepinephrine released from sympathetic nerves into bone cells declines with age leading to osteoporosis. Further, β-adrenoceptor-blockers like propranolol provoke osteoprotective effects in osteoporotic patients. However, besides systemic adrenal and sympathetic catecholamine production, it is also known that myeloid cells can synthesize catecholamines, especially under inflammatory conditions. To investigate the effects of catecholamines produced by CD11b+ myeloid cells on bone turnover and regeneration, a mouse line with specific knockout of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis, in CD11b+ myeloid cells (THflox/flox/CD11b-Cre+, referred to as THCD11b-Cre) was generated. For bone phenotyping, male mice were sacrificed at eight and twelve weeks of age and harvested bones were subjected to bone length measurement, micro-computed tomography, fluorescence-activated cell sorting of the bone marrow, gene expression analysis, histology and immunohistochemistry. Support for an age-dependent influence of myeloid cell-derived catecholamines on bone homeostasis is provided by the fact that twelve-week-old, but not eight-week-old THCD11b-Cre mice, developed an osteopenic phenotype and showed increased numbers of neutrophils and T lymphocytes in the bone marrow, while CCL2, IL-6, IL-4 and IL-10 mRNA expression was reduced in sorted myeloid bone marrow cells. To investigate the influence of myeloid cell-derived catecholamines on fracture healing, mice received a diaphyseal femur osteotomy. Three days post-fracture, immunohistochemistry revealed an increased number of macrophages, neutrophils and cytotoxic T lymphocytes in the fracture hematoma of THCD11b-Cre mice. Micro-computed tomography on day 21 showed a decreased tissue mineral density, a reduced bone volume and less trabeculae in the fracture callus indicating delayed fracture healing, probably due to the increased presence of inflammatory cells in THCD11b-Cre mice. This indicates a crucial role of myeloid cell-derived catecholamines in immune cell-bone cell crosstalk and during fracture healing.

Bone marrow adipose tissue (BMAT) is increased in both obesity and anorexia. This is unique relative to white adipose tissue (WAT), which is generally more attuned to metabolic demand. It suggests that there may be regulatory pathways that are common to both BMAT and WAT and also those that are specific to BMAT alone. The central nervous system (CNS) is a key mediator of adipose tissue function through sympathetic adrenergic neurons. Thus, we hypothesized that central autonomic pathways may be involved in BMAT regulation. To test this, we first quantified the innervation of BMAT by tyrosine hydroxylase (TH) positive nerves within the metaphysis and diaphysis of the tibia of B6 and C3H mice. We found that many of the TH+ axons were concentrated around central blood vessels in the bone marrow. However, there were also areas of free nerve endings which terminated in regions of BMAT adipocytes. Overall, the proportion of nerve-associated BMAT adipocytes increased from proximal to distal along the length of the tibia (from ~3-5 to ~14-24%), regardless of mouse strain. To identify the central pathways involved in BMAT innervation and compare to peripheral WAT, we then performed retrograde viral tract tracing with an attenuated pseudorabies virus (PRV) to infect efferent nerves from the tibial metaphysis (inclusive of BMAT) and inguinal WAT (iWAT) of C3H mice. PRV positive neurons were identified consistently from both injection sites in the intermediolateral horn of the spinal cord, reticular formation, rostroventral medulla, solitary tract, periaqueductal gray, locus coeruleus, subcoeruleus, Barrington's nucleus, and hypothalamus. We also observed dual-PRV infected neurons within the majority of these regions. Similar tracings were observed in pons, midbrain, and hypothalamic regions from B6 femur and tibia, demonstrating that these results persist across mouse strains and between skeletal sites. Altogether, this is the first quantitative report of BMAT autonomic innervation and reveals common central neuroanatomic pathways, including putative "command" neurons, involved in coordinating multiple aspects of sympathetic output and facilitation of parallel processing between bone marrow/BMAT and peripheral adipose tissue.