Bone Mass In Humans Research Articles

Validation of bone mineral density measurement using quantitative CBCT image based on deep learning

The bone mineral density (BMD) measurement is a direct method of estimating human bone mass for diagnosing osteoporosis, and performed to objectively evaluate bone quality before implant surgery in dental clinics. The objective of this study was to validate the accuracy and reliability of BMD measurements made using quantitative cone-beam CT (CBCT) image based on deep learning by applying the method to clinical data from actual patients. Datasets containing 7500 pairs of CT and CBCT axial slice images from 30 patients were used to train a previously developed deep-learning model (QCBCT-NET). We selected 36 volumes of interest in the CBCT images for each patient in the bone regions of potential implants sites on the maxilla and mandible. We compared the BMDs shown in the quantitative CBCT (QCBCT) images with those in the conventional CBCT (CAL_CBCT) images at the various bone sites of interest across the entire field of view (FOV) using the performance metrics of the MAE, RMSE, MAPE (mean absolute percentage error), R2 (coefficient of determination), and SEE (standard error of estimation). Compared with the ground truth (QCT) images, the accuracy of the BMD measurements from the QCBCT images showed an RMSE of 83.41 mg/cm3, MAE of 67.94 mg/cm3, and MAPE of 8.32% across all the bone sites of interest, whereas for the CAL_CBCT images, those values were 491.15 mg/cm3, 460.52 mg/cm3, and 54.29%, respectively. The linear regression between the QCBCT and QCT images showed a slope of 1.00 and a R2 of 0.85, whereas for the CAL_CBCT images, those values were 0.32 and 0.24, respectively. The overall SEE between the QCBCT images and QCT images was 81.06 mg/cm3, whereas the SEE for the CAL_CBCT images was 109.32 mg/cm3. The QCBCT images thus showed better accuracy, linearity, and uniformity than the CAL_CBCT images across the entire FOV. The BMD measurements from the quantitative CBCT images showed high accuracy, linearity, and uniformity regardless of the relative geometric positions of the bone in the potential implant site. When applied to actual patient CBCT images, the CBCT-based quantitative BMD measurement based on deep learning demonstrated high accuracy and reliability across the entire FOV.

The gut microbiota is a transmissible determinant of skeletal maturation.

Genetic factors account for the majority of the variance of human bone mass, but the contribution of non-genetic factors remains largely unknown. By utilizing maternal/offspring transmission, cohabitation, or fecal material transplantation (FMT) studies, we investigated the influence of the gut microbiome on skeletal maturation. We show that the gut microbiome is a communicable regulator of bone structure and turnover in mice. In addition, we found that the acquisition of a specific bacterial strain, segmented filamentous bacteria (SFB), a gut microbe that induces intestinal Th17 cell expansion, was sufficient to negatively impact skeletal maturation. These findings have significant translational implications, as the identification of methods or timing of microbiome transfer may lead to the development of bacteriotherapeutic interventions to optimize skeletal maturation in humans. Moreover, the transfer of SFB-like microbes capable of triggering the expansion of human Th17 cells during therapeutic FMT procedures could lead to significant bone loss in fecal material recipients.

Changes in bone mass associated with obesity and weight loss in humans: Applicability of animal models.

The implications of obesity and weight loss for human bone health are not well understood. Although the bone changes associated with weight loss are similar in humans and rodents, that is not the case for obesity. In humans, obesity is generally associated with increased bone mass, an outcome which is exacerbated by advanced age and menopause. In rodents, by contrast, bone mass decreases in proportion to severity and duration of obesity, and is influenced by sex, age and mechanical load. Despite these discrepancies, rodents are frequently used to model the situation in humans. In this review, we summarise the existing knowledge of the effects of obesity and weight loss on bone mass in humans and rodents, focusing on the translatability of findings from animal models. We then describe how animal models should be used to broaden the understanding of the relationship between obesity, weight loss, and skeletal health in humans. Specifically, we highlight the aspects of study design that should be considered to optimise translatability of the rodent models of obesity and weight loss. Notably, the sex, age, and nutritional status of the animals should ideally match those of interest in humans. With these caveats in mind, and depending on the research question asked, our review underscores that animal models can provide valuable information for obesity and weight-management research.

Postoperative Administration of the Acetylcholinesterase Inhibitor, Donepezil, Interferes with Bone Healing and Implant Osseointegration in a Rat Model.

Donepezil is an acetylcholinesterase inhibitor commonly used to treat mild to moderate Alzheimer’s disease. Its use has been associated with increased bone mass in humans and animals. However, the effect of postoperative administration of donepezil on bone healing remains unknown. Therefore, this study aimed to assess the impact of postoperative injection of donepezil on bone healing, titanium-implant osseointegration, and soft tissue healing. Twenty-two Sprague-Dawley rats were randomly assigned to receive a daily dose of either donepezil (0.6 mg/kg) or saline as a control. In each rat, a uni-cortical defect was created in the right tibia metaphysis and a custom-made titanium implant was placed in the left tibiae. After two weeks, rats were euthanized, and their bones were analysed by Micro-CT and histology. The healing of bone defect and implant osseointegration in the rats treated with donepezil were significantly reduced compared to the saline-treated rats. Histomorphometric analysis showed lower immune cell infiltration in bone defects treated with donepezil compared to the saline-treated defects. On the other hand, the healing time of soft tissue wounds was significantly shorter in donepezil-treated rats compared to the controls. In conclusion, short-term administration of donepezil hinders bone healing whereas enhancing soft tissue healing.

Demonstration of a SiC Protective Coating for Titanium Implants.

To mitigate the corrosion of titanium implants and improve implant longevity, we investigated the capability to coat titanium implants with SiC and determined if the coating could remain intact after simulated implant placement. Titanium disks and titanium implants were coated with SiC using plasma-enhanced chemical vapor deposition (PECVD) and were examined for interface quality, chemical composition, and coating robustness. SiC-coated titanium implants were torqued into a Poly(methyl methacrylate) (PMMA) block to simulate clinical implant placement followed by energy dispersive spectroscopy to determine if the coating remained intact. After torquing, the atomic concentration of the detectable elements (silicon, carbon, oxygen, titanium, and aluminum) remained relatively unchanged, with the variation staying within the detection limits of the Energy Dispersive Spectroscopy (EDS) tool. In conclusion, plasma-enhanced chemical vapor deposited SiC was shown to conformably coat titanium implant surfaces and remain intact after torquing the coated implants into a material with a similar hardness to human bone mass.

Macrophage-lineage TRAP+ cells recruit periosteum-derived cells for periosteal osteogenesis and regeneration.

The periosteum, a thin tissue that covers almost the entire bone surface, accounts for more than 80% of human bone mass and is essential for bone regeneration. Its osteogenic and bone regenerative abilities are well studied, but much is unknown about the periosteum. In this study, we found that macrophage-lineage cells recruit periosteum-derived cells (PDCs) for cortical bone formation. Knockout of colony stimulating factor-1 eliminated macrophage-lineage cells and resulted in loss of PDCs with impaired periosteal bone formation. Moreover, macrophage-lineage TRAP+ cells induced transcriptional expression of periostin and recruitment of PDCs to the periosteal surface through secretion of platelet-derived growth factor-BB (PDGF-BB), where the recruited PDCs underwent osteoblast differentiation coupled with type H vessel formation. We also found that subsets of Nestin+ and LepR+ PDCs possess multipotent and self-renewal abilities and contribute to cortical bone formation. Nestin+ PDCs are found primarily during bone development, whereas LepR+ PDCs are essential for bone homeostasis in adult mice. Importantly, conditional knockout of Pdgfrβ (platelet-derived growth factor receptor beta) in LepR+ cells impaired periosteal bone formation and regeneration. These findings uncover the essential role of periosteal macrophage-lineage cells in regulating periosteum homeostasis and regeneration.

Growth spurts of the bone from infancy to puberty.

Growth spurts of the bone occur during infancy(1 to 4 years)and puberty(12 to 17 years). While, generally, pubertal spurts appear to draw more attention than infantile spurts, the latter constitute maximum growth spurts. Indeed, those during the first year of life lead to a 1.5-fold increase in height or a height increase of 25 cm, thus representing the greatest of all growth spurts that occur in humans during their lifetimes. Again, while height growth continues through the first 3 years of life, nutrition represents the single greatest contributing factor to height growth during this period. Again, while, as with other organ primordia, the bone primordium is formed during the organogenesis stage, calcification becomes most active during the third trimester of pregnancy. Thus, this review provides an overview of bone growth in humans, in relation to bone/calcium metabolism, which begins in the fetal stage before birth and continues through infancy and puberty, finally leading to attainment of peak bone mass in humans.

Stress analysis prediction on screw orthopedic implant in trabecular bone

Abstract Trabecular bone fractures heal through intramembranous ossification. It is a highly porous biological tissue with an open-celled cellular structure, which accounts for up to 20% until 60% of the bone mass in humans for healthy bone while about 80% for an osteoporosis case. In bone healing, there is very little callus formation, woven bone forms directly without a cartilage intermediary, and the woven bone is remodelled to form trabecular bone. Trabecular bone has less mineral and porosity percentages compared to the compact bone. Thus, the stress distributions around the screw were affected by the amount of the porosity within the trabecular bone. In bone healing mechanism, stability may speed up the bone healing process. The interface contact between external fixator pins and screws of internal fixator influence the fixation stability for bone healing. This study aims to investigate the amount of stress distribution on the around screw implant through the trabecular bone porosity condition under pull out loading. Finite element analysis is employed to simulate the stress distribution associated to the screw implant and cortical-trabecular interface As a result, the area of screw head is found to be yielded as the porosity of trabecular bone is considered.

Conditional Deletion of Sost in MSC-Derived Lineages Identifies Specific Cell-Type Contributions to Bone Mass and B-Cell Development

Sclerostin (Sost) is a negative regulator of bone formation and blocking its function via antibodies has shown great therapeutic promise by increasing both bone mass in humans and animal models. Sclerostin deletion in Sost KO mice (Sost-/- ) causes high bone mass (HBM) similar to sclerosteosis patients. Sost-/- mice have been shown to display an up to 300% increase in bone volume/total volume (BV/TV), relative to age-matched controls. It has been postulated that the main source of skeletal sclerostin is the osteocyte. To understand the cell-type specific contributions to the HBM phenotype described in Sost-/- mice, as well as to address the endocrine and paracrine mode of action of sclerostin, we examined the skeletal phenotypes of conditional Sost loss-of-function (SostiCOIN/iCOIN ) mice with specific deletions in (1) the limb mesenchyme (Prx1-Cre; targets osteoprogenitors and their progeny); (2) midstage osteoblasts and their progenitors (Col1-Cre); (3) mature osteocytes (Dmp1-Cre); and (4) hypertrophic chondrocytes and their progenitors (ColX-Cre). All conditional alleles resulted in significant increases in bone mass in trabecular bone in both the femur and lumbar vertebrae, but only Prx1-Cre deletion fully recapitulated the amplitude of the HBM phenotype in the appendicular skeleton and the B-cell defect described in the global KO. Despite WT expression of Sost in the axial skeleton of Prx1-Cre deleted mice, these mice also had a significant increase in bone mass in the vertebrae, but the sclerostin released in circulation by the axial skeleton did not affect bone parameters in the appendicular skeleton. Also, both Col1 and Dmp1 deletion resulted in a similar 80% significant increase in trabecular bone mass, but only Col1 and Prx1 deletion resulted in a significant increase in cortical thickness. We conclude that several cell types within the Prx1-osteoprogenitor-derived lineages contribute significant amounts of sclerostin protein to the paracrine pool of Sost in bone. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Lrp1 in osteoblasts controls osteoclast activity and protects against osteoporosis by limiting PDGF\u2013RANKL signaling

Skeletal health relies on architectural integrity and sufficient bone mass, which are maintained through a tightly regulated equilibrium of bone resorption by osteoclasts and bone formation by osteoblasts. Genetic studies have linked the gene coding for low-density lipoprotein receptor-related protein1 (Lrp1) to bone traits but whether these associations are based on a causal molecular relationship is unknown. Here, we show that Lrp1 in osteoblasts is a novel regulator of osteoclast activity and bone mass. Mice lacking Lrp1 specifically in the osteoblast lineage displayed normal osteoblast function but severe osteoporosis due to highly increased osteoclast numbers and bone resorption. Osteoblast Lrp1 limited receptor activator of NF-κB ligand (RANKL) expression in vivo and in vitro through attenuation of platelet-derived growth factor (PDGF-BB) signaling. In co-culture, Lrp1-deficient osteoblasts stimulated osteoclastogenesis in a PDGFRβ-dependent manner and in vivo treatment with the PDGFR tyrosine kinase inhibitor imatinib mesylate limited RANKL production and led to complete remission of the osteoporotic phenotype. These results identify osteoblast Lrp1 as a key regulator of osteoblast-to-osteoclast communication and bone mass through a PDGF–RANKL signaling axis in osteoblasts and open perspectives to further explore the potential of PDGF signaling inhibitors in counteracting bone loss as well as to evaluate the importance of functional LRP1 gene variants in the control of bone mass in humans.

LRP5: From bedside to bench to bone

A role for low-density lipoprotein–related receptor 5 (LRP5) in human bone was first established by the identification of genetic alterations that led to dramatic changes in bone mass. Shortly thereafter, mutations that altered the function of the sclerostin (SOST) gene were also associated with altered human bone mass. Subsequent studies of LRP5 and sclerostin have provided important insights into the mechanisms by which these proteins regulate skeletal homeostasis. Sclerostin normally binds to LRP5 and the related LRP6 protein and prevents their activation by Wnts, the LRP5/LRP6 ligands. The interaction of sclerostin with LRP5 or LRP6 is facilitated by the LRP4 protein. Loss of LRP5 leads to defective osteoblast function and low bone mass, while loss of SOST or mutations in LRP5, which produce a protein that can no longer be bound by SOST, result in high bone mass. Insights gained from the use of genetically engineered mouse models are presented, as well as a brief summary of the status of antibodies in clinical trials that block the function of SOST as a mechanism to increase bone mass.

Role of the Cytokine-like Hormone Leptin in Muscle-bone Crosstalk with Aging.

The cytokine-like hormone leptin is a classic adipokine that is secreted by adipocytes, increases with weight gain, and decreases with weight loss. Additional studies have, however, shown that leptin is also produced by skeletal muscle, and leptin receptors are abundant in both skeletal muscle and bone-derived mesenchymal (stromal) stem cells. These findings suggest that leptin may play an important role in muscle-bone crosstalk. Leptin treatment in vitro increases the expression of myogenic genes in primary myoblasts, and leptin treatment in vivo increases the expression of microRNAs involved in myogenesis. Bone marrow adipogenesis is associated with low bone mass in humans and rodents, and leptin can reduce marrow adipogenesis centrally through its receptors in the hypothalamus as well as directly via its receptors in bone marrow stem cells. Yet, central leptin resistance can increase with age, and low circulating levels of leptin have been observed among the frail elderly. Thus, aging appears to significantly alter leptin-mediated crosstalk among various organs and tissues. Aging is associated with bone loss and muscle atrophy, contributing to frailty, postural instability, and the incidence of falls. Therapeutic interventions such as protein and amino acid supplementation that can increase muscle mass and muscle-derived leptin may have multiple benefits for the elderly that can potentially reduce the incidence of falls and fractures.

The Relationship of Serum Serotonin Levels to the Rate of Bone Loss and Fractures in Men

Recent genetic studies in rodents have revealed that circulating serotonin plays a key role in regulating bone formation and skeletal mass. However, the reported effects of circulating serotonin on bone mass in humans have been conflicting. We determined whether circulating serotonin levels influenced the rate of bone loss and fractures in men. We assessed the effect of serum serotonin on bone loss rate in a population-based cohort of 202 ambulatory men aged 56–70 years who were followed up for a median duration of 3.7 years. Serum serotonin levels were assayed, and the Timed Up and Go Test (TUGT) was performed, at baseline. Dual-energy X-ray absorptiometry was performed both at baseline and during follow-up. Fracture prevalence was assessed using questionnaires. The serotonin levels were inversely associated with the lumbar spine bone mineral density (r = −0.174, p = 0.028) at baseline. No association was evident between the bone mineral densities of the femoral neck or total hip and serotonin level. The annual rates of bone loss from the lumbar spine, the femoral neck, and the total hip were 0.01%, 0.46%, and 0.46%, respectively. The baseline serum serotonin level did not predict the bone loss rate in any skeletal site. Lower limb disability evident upon TUGT at baseline predicted bone loss from the total hip. No significant difference of serotonin level was observed between subjects with and without fractures. The serum serotonin level was not associated with the rate of bone loss in elderly men. Thus, the circulating serotonin level does not reliably predict bone loss.

Regulation of bone metabolism by Wnt signals.

Wnt ligands play a central role in the development and homeostasis of various organs through β-catenin-dependent and -independent signalling. The crucial roles of Wnt/β-catenin signals in bone mass have been established by a large number of studies since the discovery of a causal link between mutations in the low-density lipoprotein receptor-related protein 5 (Lrp5) gene and alternations in human bone mass. The activation of Wnt/β-catenin signalling induces the expression of osterix, a transcription factor, which promotes osteoblast differentiation. Furthermore, this signalling induces the expression of osteoprotegerin, an osteoclast inhibitory factor in osteoblast-lineage cells to prevent bone resorption. Recent studies have also shown that Wnt5a, a typical non-canonical Wnt ligand, enhanced osteoclast formation. In contrast, Wnt16 inhibited osteoclast formation through β-catenin-independent signalling. In this review, we discussed the current understanding of the Wnt signalling molecules involved in bone formation and resorption.

Missense Mutations in LRP5 Associated with High Bone Mass Protect the Mouse Skeleton from Disuse- and Ovariectomy-Induced Osteopenia.

The low density lipoprotein receptor-related protein-5 (LRP5), a co-receptor in the Wnt signaling pathway, modulates bone mass in humans and in mice. Lrp5 knock-out mice have severely impaired responsiveness to mechanical stimulation whereas Lrp5 gain-of-function knock-in and transgenic mice have enhanced responsiveness to mechanical stimulation. Those observations highlight the importance of Lrp5 protein in bone cell mechanotransduction. It is unclear if and how high bone mass-causing (HBM) point mutations in Lrp5 alter the bone-wasting effects of mechanical disuse. To address this issue we explored the skeletal effects of mechanical disuse using two models, tail suspension and Botulinum toxin-induced muscle paralysis, in two different Lrp5 HBM knock-in mouse models. A separate experiment employing estrogen withdrawal-induced bone loss by ovariectomy was also conducted as a control. Both disuse stimuli induced significant bone loss in WT mice, but Lrp5 A214V and G171V were partially or fully protected from the bone loss that normally results from disuse. Trabecular bone parameters among HBM mice were significantly affected by disuse in both models, but these data are consistent with DEXA data showing a failure to continue growing in HBM mice, rather than a loss of pre-existing bone. Ovariectomy in Lrp5 HBM mice resulted in similar protection from catabolism as was observed for the disuse experiments. In conclusion, the Lrp5 HBM alleles offer significant protection from the resorptive effects of disuse and from estrogen withdrawal, and consequently, present a potential mechanism to mimic with pharmaceutical intervention to protect against various bone-wasting stimuli.

Effect of swimming exercise on three-dimensional trabecular bone microarchitecture in ovariectomized rats.

Swimming is generally considered ineffective for increasing bone mass in humans, at least compared with weight-bearing sports. However, swimming exercise has sometimes been shown to have a strong positive effect on bone mass in small animals. This study investigated the effects of swimming on bone mass, strength, and microarchitecture in ovariectomized (OVX) rats. OVX or sham operations were performed on 18-wk-old female Fisher 344 rats. Rats were randomly divided into four groups: sham sedentary (Sham-CON), sham swimming exercised (Sham-SWI), OVX sedentary (OVX-CON), and OVX swimming exercised (OVX-SWI). Rats in exercise groups performed swimming in a water bath for 60 min/day, 5 days/wk, for 12 wk. Bone mineral density (BMD) in right femurs was analyzed using dual-energy X-ray absorptiometry. Three-dimensional trabecular architecture at the distal femoral metaphysis was analyzed using microcomputed tomography (μCT). Geometrical properties of diaphyseal cortical bone were evaluated in the midfemoral region using μCT. The biomechanical properties of femurs were analyzed using three-point bending. Femoral BMD was significantly decreased following ovariectomy. This change was suppressed by swimming. Trabecular bone thickness, number, and connectivity were decreased by ovariectomy, whereas structure model index (i.e., ratio of rod-like to plate-like trabeculae) increased. These changes were also suppressed by swimming exercise. Femurs displayed greater cortical width and maximum load in SWI groups than in CON groups. Together, these results demonstrate that swimming exercise drastically alleviated both OVX-induced decreases in bone mass and mechanical strength and the deterioration of trabecular microarchitecture in rat models of osteoporosis.

The regulation of osteoclast differentiation by Wnt signals.

Wnt ligands activate β-catenin-dependent canonical and -independent noncanonical signaling pathways. Wnt regulates many physiological events such as the development of organs and bone metabolism. In contrast, failed signaling leads to pathological conditions including cancer and osteoporosis. Analyses of loss-of-function mutations in the low-density lipoprotein receptor-related protein (Lrp) 5 gene revealed that Lrp5 acted as a co-receptor of Wnt/β-catenin signals and positively regulated bone mass in humans and mice. Many players in Wnt signals including sclerostin, an osteocyte-derived Wnt antagonist, also have since been found to influence bone mass. Bone mass is regulated by the activities of bone-forming osteoblasts, -resorbing osteoclasts and matrix-embedded osteocytes. The roles of Wnt/β-catenin signals in osteoblastogenesis and osteoclastogenesis have been established by the findings of a large number of in vitro and in vivo studies. In contrast, the roles of noncanonical Wnt signals in bone metabolism are only now being examined. In this review, we introduced and discussed recent information on the roles of Wnt signals in bone resorption.

Cortical and trabecular bone in pediatric end-stage kidney disease.

Cortical bone represents nearly 80% of human bone mass and is the major determinant of bone strength; however, cortical bone parameters and their relationship to trabecular bone in the pediatric chronic kidney disease (CKD) population have not been evaluated. Biochemical values and cortical and trabecular bone parameters were assessed in 22 pediatric dialysis patients: 12 with high and 10 with normal to low trabecular bone turnover. Trabecular bone turnover and osteoid volume correlated with parathyroid hormone (PTH) levels (r = 0.86, p < 0.01 and r = 0.93, p < 0.01, respectively). Internal cortical osteonal bone formation rate was directly related to alkaline phosphatase (r = 0.45, p < 0.05) and inversely related to insulin-like growth factor (IGF)-1 values (r = -0.55, p < 0.01), and internal cortical porosity was also related to serum alkaline phosphatase levels (r = 0.57, p < 0.01). A similar relationship was not found between external cortical bone formation rate and parameters of bone turnover and porosity, however. No relationship was found between trabecular and cortical bone formation rates. Secondary hyperparathyroidism was associated with increased external cortical, relative to internal cortical, osteonal activity in pediatric dialysis patients. The clinical consequences of these changes and their response to therapy for secondary hyperparathyroidism remain to be defined.

Cytokine-mediated bone destruction in rheumatoid arthritis.

Bone homeostasis, which involves formation and resorption, is an important process for maintaining adequate bone mass in humans. Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation and bone loss, leading to joint destruction and deformity, and is a representative disease of disrupted bone homeostasis. The bone loss and joint destruction are mediated by immunological insults by proinflammatory cytokines and various immune cells. The connection between bone and immunity has been intensely studied and comprises the emerging field of osteoimmunology. Osteoimmunology is an interdisciplinary science investigating the interplay between the skeletal and the immune systems. The main contributors in osteoimmunology are the bone effector cells, such as osteoclasts or osteoblasts, and the immune cells, particularly lymphocytes and monocytes. Physiologically, osteoclasts originate from immune cells, and immune cells regulate osteoblasts and vice versa. Pathological conditions such as RA might affect these interactions, thereby altering bone homeostasis, resulting in the unfavorable outcome of bone destruction. In this review, we describe the osteoclastogenic roles of the proinflammatory cytokines and immune cells that are important in the pathophysiology of RA.

Canonical Wnt signaling activates miR-34 expression during osteoblastic differentiation

The canonical Wnt signaling pathway is crucial for the regulation of bone mass in humans and for the development of osteoblasts. MicroRNAs (miRs) represent a class of non‑coding RNAs, ~22 nucleotides in length, that regulate gene expression by targeting mRNAs for cleavage or translational repression. Several previous studies have demonstrated the involvement of miRNAs in modulating gene expression in osteoblasts and regulating osteoblast differentiation. In the present study, microRNA profiling was conducted using Wnt3a‑C2C12 cells; C2C12 cells were transfected with a Wnt3a expression plasmid to activate canonical Wnt signaling. miR‑34b‑5p and miR‑34c were identified to be upregulated by the activation of canonical Wnt signaling in C2C12 cells. Expression of mature miR‑34b/c increased from low levels at day 0 to maximum levels at day 28 of MC3T3‑E1 cell differentiation. To analyze the effects of these miRNAs on osteoblast differentiation, an antisense inhibitor was transfected into MC3T3‑E1 cells and osteoblast‑related gene expression was investigated. Knockdown of miR34b/c enhanced osteocalcin mRNA expression; however, alkaline phosphatase mRNA expression and activity were decreased by miR34b/c inhibition. These results indicated that miR‑34b/c regulates gene expression by targeting regulators of the osteogenic pathways and thereby contributes to osteoblast differentiation.