Differentiation Of Pre-osteoblastic MC3T3-E1 Cells Research Articles

3D printed hetero-layered composite scaffold with engineered superficial zone promotes osteogenic differentiation of pre-osteoblast MC3T3-E1 cells

In bone tissue engineering, a new design of composite scaffold with various compositions by incorporating both natural and synthetic cues via dual nozzle 3D printing technique could efficiently accelerate the cell differentiation to osteogenic lineage. Based on the hypothesis that a biomimetic scaffold per se can supply the vital cues to provoke bone formation, 3D printed poly(ε-caprolactone) (PCL) scaffold loaded with synthetic hydroxyapatite (HAs) decorated on graphene oxide (GO) sheets (GO@HAs), was developed. To further improve its osteogenic potential, the upper and lower layer/s of scaffolds were 3D printed using another ink containing PCL and naturally allograft HA (HAa). The FE-SEM images of the 3D printed scaffolds revealed a highly regular architecture, demonstrating the successful printability of the developed materials. The compressive mechanical test confirmed the drastic role of GO@HAs on the enhancement of Young's modulus and mechanical strength of the PCL scaffold. Moreover, existence of HAa on the upper and lower sections of scaffold interestingly improved the cellular behavior of the 3D printed scaffold without having an adverse effect on mechanical features. The experimental results indicated that such engineered scaffold significantly facilitated the cell attachment and evoked higher levels of pre-osteoblast MC3T3-E1 differentiation than PCL/GO@HAs scaffold as demonstrated by alizarin red staining, ALP activity and osteo-related gene expression. Taken together, these results indicate that such fascinating scaffold comprising both mechanical and biological cues has a high potency for inducing bone repair and also open new avenues for future advancements in the realm of bone tissue engineering exploration.

Molecular mechanism of resveratrol promoting differentiation of preosteoblastic MC3T3-E1 cells based on network pharmacology and experimental validation

The purpose of this study was to investigate the mechanism by which resveratrol (Res) inhibits apoptosis and promotes proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells, laying the groundwork for the treatment of osteoporosis (OP). The TCMSP database was used to find the gene targets for Res. The GeneCards database acquire the gene targets for OP. After discovering the potential target genes, GO, KEGG, and Reactome enrichment analysis were conducted. Verifying the major proteins involved in apoptosis can bind to Res using molecular docking. CCK8 measured the proliferative activity of mouse pre-osteoblasts in every group following Res intervention. Alkaline phosphatase staining (ALP) and alizarin red staining to measure the ability of osteogenic differentiation. RT-qPCR to determine the expression levels of Runx2 and OPG genes for osteogenic differentiation ability of cells. Western blot to measure the degree of apoptosis-related protein activity in each group following Res intervention. The biological processes investigated for GO of Res therapeutic OP involved in cytokine-mediated signaling pathway, negative regulation of apoptotic process, Aging, extrinsic apoptotic signaling pathway in absence of ligand, according to potential therapeutic target enrichment study. Apoptosis, FoxO signaling pathway, and TNF signaling pathway are the primary KEGG signaling pathways. Recactome pathways are primarily engaged in Programmed Cell Death, Apoptosis, Intrinsic Apoptotic Pathway, and Caspase activation via extrinsic apoptotic signaling pathways. This research established a new approach for Res treatment of OP by demonstrating how Res controls the apoptosis-related proteins TNF, IL6, and CASP3 to suppress osteoblast death and increase osteoclastogenesis.

Isolation of Osteoblastic Differentiation-Inducing Constituents from Peanut Sprouts and Development of Optimal Extraction Method

Osteoporosis, one of the most common bone diseases, results from an imbalance between bone formation and resorption. Osteoblasts are primarily involved in bone formation, whereas osteoclasts are involved in bone resorption. Therefore, any substances that can increase osteoblastic differentiation would be beneficial for the prevention or treatment of osteoporosis. In this study, peanut sprouts, a germinated product of peanuts, significantly enhanced osteoblastic differentiation of pre-osteoblastic MC3T3-E1 cells, as determined by the alkaline phosphatase (ALP) assay. The ethyl acetate fraction of peanut sprout extract was subjected to diverse column chromatographies using silica gel, ODS, and Sephadex LH-20 as stationary phases, and three nucleobases, namely, adenine, uracil, and thymine, and three phenolic acids, including caffeic acid, coumaric acid, and ferulic acid, were isolated as active constituents. In particular, adenine at 4 μg/mL and caffeic acid at 1 μg/mL increased ALP activity by 40 and 30%, respectively, compared with the osteoblastic differentiation medium-treated group, and these two compounds were set as marker compounds. Furthermore, extraction of peanut sprouts grown for 14–17 days with 60% ethanol was the best condition to obtain a high quantity of extract for peanut sprouts by analyzing the contents of marker compounds through HPLC. Together, these results suggest that peanut sprouts and their isolated compounds significantly enhance osteoblastic differentiation. Therefore, peanut sprouts have the potential to be developed as functional foods against osteoporosis.

High mobility group AT-hook 2 regulates osteoblast differentiation and facial bone development

The mutation and deletion of high mobility group AT-hook 2 (Hmga2) gene exhibit skeletal malformation, but almost nothing is known about the mechanism. This study examined morphological anomaly of facial bone in Hmga2−/− mice and osteoblast differentiation of pre-osteoblast MC3T3-E1 cells with Hmga2 gene knockout (A2KO). Hmga2−/− mice showed the size reduction of anterior frontal part of facial bones. Hmga2 protein and mRNA were expressed in mesenchymal cells at ossification area of nasal bone. A2KO cells differentiation into osteoblasts after reaching the proliferation plateau was strongly suppressed by alizarin red and alkaline phosphatase staining analyses. Expression of osteoblast-related genes, especially Osterix, was down-regulated in A2KO cells. These results demonstrate a close association of Hmga2 with osteoblast differentiation of mesenchymal cells and bone growth. Although future studies are needed, the present study suggests an involvement of Hmga2 in osteoblast-genesis and bone growth.

Impact of Hydrolyzed Collagen from Defatted Sea Bass Skin on Proliferation and Differentiation of Preosteoblast MC3T3-E1 Cells.

Osteoporosis is a serious problem affecting health of the elderly. Drugs (bisphosphonates) applied for treatment are often accompanied by adverse side effects. Thus, fish byproduct-derived peptides, particularly hydrolyzed collagen (HC) from defatted sea bass skin, could be a safe source of anti-osteoporosis agents. This study aimed to examine the effects of HC on proliferation and differentiation of preosteoblast cells. HC prepared using papain before Alcalase hydrolysis was determined for molecular weight (MW) distribution. Thereafter, the resulting HC (50–800 µg/mL) was added to the cell. Proliferation, alkaline phosphatase activity (AP-A) and mineralization of cells were investigated. Moreover, the expression of runt-related transcription factor 2 (RUNX2) and the p-Akt/Akt pathway were also determined using Western blot. The results showed that HC had an MW < 3 kDa. HC (50–200 µg/mL) could promote cell proliferation. Nevertheless, HC at 100 µg/mL (HC-100) had enhanced AP-A and increased mineralization during the first 7 days of culture. Moreover, HC-treated cells had higher calcium depositions than the control (p < 0.05). Additionally, cells treated with HC-100 had higher levels of RUNX2 and p-Akt expressions than control (p < 0.05). Therefore, HC could be a promising functional ingredient to promote osteoblast proliferation and differentiation, which could enhance bone strength.

TMARg, a Novel Anthraquinone Isolated from Rubia cordifolia Nakai, Increases Osteogenesis and Mineralization through BMP2 and β-Catenin Signaling.

Background: Plant extracts have long been regarded as useful medicines in the treatment of human diseases. Rubia cordifolia Nakai has been used as a traditional medicine, as it has pharmacological properties such as antioxidant and anti-inflammatory activity. However, the biological functions of TMARg, isolated from the roots of R. cordifolia, in osteoblast differentiation remain unknown. This study was performed to investigate the pharmacological effects and intracellular signaling of TMARg in the osteoblast differentiation of pre-osteoblast MC3T3-E1 cells and mesenchymal precursor C2C12 cells. Methods: Cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP), and by staining it with Alizarin red S (ARS). Cell migration was determined by using migration assays. Western blot analysis and immunocytochemical analysis were used to examine the intracellular signaling pathways and differentiation proteins. Results: In the present study, TMARg showed no cytotoxicity and increased the osteoblast differentiation in pre-osteoblasts, as assessed from the alkaline phosphate (ALP) staining and activity and ARS staining. TMARg also induced BMP2 expression and increased the p-smad1/5/8-RUNX2 and β-catenin pathways in both MC3T3-E1 and C2C12 cells. Furthermore, TMARg activated mitogen-activated protein kinases (MAPKs) and increased the cell migration rate. In addition, the TMARg-mediated osteoblast differentiation was suppressed by BMP and Wnt inhibitors with the downregulation of BMP2 expression. Conclusion: These findings demonstrate that TMARg exerts pharmacological and biological effects on osteoblast differentiation through the activation of BMP2 and β-catenin signaling pathways, and suggest that TMARg might be a potential phytomedicine for the treatment of bone diseases.

The proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells from Vietnamese drug formulations

A public heath problem, osteoporosis, is recognized as the prevalent disease, and mainly causes for impairment and loss mass of bone. It closely related the balance between bone formation by osteoblasts and resorption by osteoclasts during the remodeling cycle of bone. Hence, pharmaceutical therapies are looking for the potential agents to stimulate osteoblastic bone formation, as well as inhibit osteoclastic processes.

A novel electron emission-based cell culture device promotes cell proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells.

In this report we demonstrate the effect of a novel electron emission-based cell culture device on the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells. Our device has an electron emission element that allows, for the first time, stable emission of electrons into an atmosphere. Atmospheric electrons react with gas molecules to generate radicals and negative ions, which induce a variety of biochemical reactions in the attached cell culture system. In this study, we demonstrated the effect of this new electron emission-based cell culture device on cell proliferation and differentiation using pre-osteoblastic MC3T3-E1 cells. Electron emission stimulation (EES) was applied directly to culture medium containing plated cells, after which the number of living cells, the mRNA levels of osteogenesis-related genes, and the alkaline phosphatase (ALP) activity were evaluated. The growth rate of EES-exposed cells increased by approximately 20% in comparison with unexposed control cells. We also found the mRNA levels of osteogenic specific genes such as collagen type I α-1, core-binding factor α-1, and osteocalcin to be up-regulated following EES. ALP activity, a marker for osteogenic activity, was significantly enhanced in EES-treated cells. Furthermore, reactive oxygen species generated by EES were measured to determine their effect on MC3T3-E1 cells. These results suggest that our new electron emission-based cell culture device, while providing a relatively weak stimulus in comparison with atmospheric plasma systems, promotes cell proliferation and differentiation. This system is expected to find application in regenerative medicine, specifically in relation to bone regeneration.

SPIO-Au core-shell nanoparticles for promoting osteogenic differentiation of MC3T3-E1 cells: Concentration-dependence study.

This work aims to explore the concentration-dependence of SPIO-Au core-shell nanoscale particles (NPs) (17.3 ± 1.2 nm in diameter) on biocompatibility and osteogenic differentiation of preosteoblast MC3T3-E1 cells. The stability of NPs was first investigated by UV-vis absorption spectra and zeta potential measurement. Then concentration effects of NPs (1-80 μg/mL) were evaluated on viability, morphology, proliferation, cellular uptake, and alkaline phosphate (ALP) activity levels. Results have shown strong stability and no acute toxicity (viability > 93%) or morphological difference at all concentration levels of NPs. The proliferation results indicated that the concentration of NPs below 40 μg/mL does not affect the cell proliferation for 7 days of incubation. Transmission electron microscopy images revealed the successful internalization of NPs into MC3T3-E1 cells and the dose-dependent accumulation of NPs inside the cytoplasm. The ALP level of MC3T3-E1 cells was improved by 49% (of control) after treated with NPs at 10 μg/mL for 10 days, indicating their positive effect on early osteogenic differentiation. This study confirmed the excellent biocompatibility of SPIO-Au NPs and their great potential for promoting osteogenic differentiation and promised the future application for these NPs in bone engineering including drug delivery, cell labeling, and activity tracking within scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3350-3359, 2017.

Ginkgolide B enhances the differentiation of preosteoblastic MC3T3-E1 cells through VEGF: Involvement of the p38 MAPK signaling pathway.

Ginkgolide B (GB) is one of the ginkgolides isolated from the leaves of the Ginkgo biloba tree. Our previous study indicated that GB promotes the proliferation, migration and adhesion of endothelial progenitor cells, and the induction of angiogenesis through vascular endothelial factor (VEGF). In the present study, the effects of GB on the differentiation of MC3T3‑E1 cells and the signaling pathway involved were investigated invitro. The MC3T3‑E1 cell viability activities were assessed using an MTS assay. Measurements of alkaline phosphatase activity and Alizarin Red staining were used to identify osteoblastic differentiation of the MC3T3‑E1 cells. The mRNA and secretion levels of VEGF were detected using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis and enzyme-linked immunosorbent assays, respectively. The protein expression levels of phosphorylation‑associated markers were detected using western blot analysis and associated gene expression was determined using RT‑qPCR analysis. It was found that GB significantly promoted alkaline phosphatase activity and osteoblastic mineralization in the MC3T3‑E1 cells. In addition, the mRNA expression and secretion levels of VEGF in the MC3T3‑E1 cells were significantly increased in MC3T3‑E1 cells treated with GB. SB203580, a specific inhibitor of p38 mitogen‑activated protein (MAP) kinase, markedly suppressed the GB‑induced p38 kinase phosphorylation and GB‑induced synthesis of VEGF. PD98059, an inhibitor of the upstream kinase, which activates p44/p42 MAP kinase, had minimal effect on the GB‑induced phosphorylation of p44/p42 MAP kinase or the GB‑induced synthesis of VEGF. Taken together, these results indicated that GB promoted osteoblastic differentiation of the MC3T3‑E1 cells through VEGF, and that the p38, but not the p44/p42 MAP kinase signaling pathway, was involved in the GB‑induced synthesis of VEGF.

Nanoporous anodic aluminum oxide tube encapsulating a microporous chitosan/collagen composite for long-acting drug release

The objective of this study is to develop a long-acting and implantable drug-release device that can effectively control the release rate and concentration of the loaded drug. The proposed device consists of a tubular nanoporous anodic aluminum oxide (AAO) encapsulating a microporous chitosan/collagen composite. The nanopore size of the AAO tube can be modified by adjusting the anodization parameters, which in turn adjust the release rate and concentration, while the microporous chitosan/collagen composite provides the long-acting release feature. Fabrication results indicate that the AAO tube has a uniform pore arrangement with a pore size around 50 nm. The synthesized microporous chitosan/collagen composite containing 90% chitosan yielded the highest moisture content and was therefore used as the drug carrier. Release experiments demonstrate that the proposed long-acting drug release device had released less than 65% of the loading drug on the 17th release day. We then applied the proposed long-acting drug-release scheme as a recombinant human bone morphogenetic-protein 2 (rh-BMP2) release device to induce differentiation of pre-osteoblast MC3T3-E1 cells into osteoblasts. Results from alkaline phosphate and alizarin red S assays demonstrate that the total amount of rh-BMP2 consumed by the proposed AAO tube is much less than that consumed using the conventional culture approach. Furthermore, our approach has the advantage of requiring only one-time dosing, whereas the conventional approach requires the periodic renewal of rh-BMP2. AAO’s one-time dosing feature combined with its biocompatibility and biodegradability can be beneficial in real implant applications.

Effects of red light-emitting diode irradiation on the proliferation and differentiation of preosteoblastic MC3T3-E1 cells

Effects of red light-emitting diode irradiation on the proliferation and differentiation of preosteoblastic MC3T3-E1 cells

Antiosteoporotic and antioxidant activities of diterpenoids from the Vietnamese soft corals Sinularia maxima and Lobophytum crassum

Marine organisms possess bioactive potential with tremendous pharmaceutical promise. The purpose of the present study was to investigate the antioxidant (peroxyl radical-scavenging and reducing capacities) and antiosteoporotic activities of isolated constituents (1–26) from the soft corals Sinularia maxima and Lobophytum crassum on pre-osteoblastic MC3T3-E1 cells and pre-osteoclastic RAW 264.7 cells. Among the isolated compounds, 7, 9, 17, 18, and 20 (10.0 μM) exhibited significant peroxyl radical-scavenging capacity, with TE values ranging from 4.00 ± 0.14 to 9.06 ± 0.33 μM. In addition, compounds 1, 5, 7, 20, and 24 significantly stimulated the differentiation of pre-osteoblastic MC3T3-E1 cells by increasing collagen synthesis and/or mineralization functions of osteoblasts, while compounds 1, 5–8, 12, 14–22, 24, and 26 showed significant inhibition of TRAP in NF-κB ligand-induced osteoclastic RAW 264.7 cells, with values ranging from 99.11 ± 1.36 to 61.54 ± 1.61 %. These results indicate that the soft corals S. maxima and L. crassum are excellent sources among the antioxidant and antiosteoporotic marine invertebrates.

Design of an osteoinductive extracellular fibronectin matrix protein for bone tissue engineering.

Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05).

Effect of Fermented Red Ginseng Extract Enriched in Ginsenoside Rg3 on the Differentiation and Mineralization of Preosteoblastic MC3T3-E1 Cells.

In this study, red ginseng extract (RGE) was converted into high-content minor ginsenosides by fermenting with Bgp1 enzymes at 37°C for 5 days. Compared to the RGE, the minor ginsenoside contents were increased in fermented red ginseng extract (FRGE). Moreover, the amount of minor ginsenosides such as Rh1 (11%) and Rg2 (16%) was slightly augmented, while the level of Rg3 (33%) was significantly increased after bioconversion. Furthermore, we also examined and compared the effect of RGE and FRGE on the differentiation and mineralization of preosteoblastic MC3T3-E1 cells. Similarly, the level of mRNA expression of intracellular alkaline phosphatase (ALP) activity, type-1 collagen (Col-I) was also increased. Based on the comparison, it is clear that the FRGE has improved effects on bone formation and differentiation of preosteoblastic MC3T3-E1 cells.

In vitro studies on regulation of osteogenic activities by electrical stimulus on biodegradable electroactive polyelectrolyte multilayers.

In this study, a novel electroactive tetreaniline-containing degradable polyelectrolyte multilayer film (PEM) coating [(poly(l-glutamic acid)-graft-tetreaniline/poly(l-lysine)-graft-tetreaniline)n, (PGA-g-TA/PLL-g-TA)n] was designed and fabricated by layer-by-layer (LbL) assembly method. Compared with the nongrafted PEMs, the tetreaniline-grafted PEMs showed higher roughness and stiffness in micro/nanoscale structures. The special surface characteristics and the typical electroconductive properties were more beneficial for adhesion, proliferation, and differentiation of preosteoblast MC3T3-E1 cells. Moreover, the enhanced effects were observed on the modulation of MC3T3-E1 cells that differentiated into maturing osteoblasts, when the electroactive PEMs were coupled with electrical stimulus (ES), especially in the early phase of the osteoblast differentiation. The alkaline phosphatase (ALP) activity, calcium deposition, immunofluorescence staining, and RT-qPCR were evaluated on the differentiation of preosteoblast. These data indicate that the comprehensive effects through coupling electroactive scaffolds with electrical stimulus are better to develop bioelectric strategies to control cell functions for bone regeneration.

Evaluation of the antioxidant and anti-osteoporosis activities of chemical constituents of the fruits of Prunus mume

The present study investigated the antioxidant and anti-osteoporosis activities of phytochemicals in the fruits of Prunus mume. From the methanol extract, three new acylated sucroses, mumeoses P–R (1–3), were isolated together with 20 known compounds (4–23). Compounds 1–3 showed potent peroxyl radical-scavenging activities and 12–19 showed both potent peroxyl radical-scavenging and reducing activities. The anti-osteoporosis activity was evaluated using murine pre-osteoblastic MC3T3-E1 cells and pre-osteoclastic RAW 264.7 cells. Compounds 2 and 3 (cis–trans isomers), 5, 7, 8, and 10 significantly stimulated the differentiation of pre-osteoblastic MC3T3-E1 cells to increase collagen synthesis or mineralization functions of osteoblasts, while compounds 5, 6, 9, 10, 12, 14–16, 18, 20, and 22 significantly suppressed tartrate-resistant acid phosphatase activity in receptor activator of nuclear factor-κB ligand-induced osteoclastic RAW 264.7 cells. These results indicated that the fruits of P. mume are an excellent source of antioxidant and anti-osteoporosis phytochemicals.

Calcium phosphate deposition rate, structure and osteoconductivity on electrospun poly(l-lactic acid) matrix using electrodeposition or simulated body fluid incubation

Mineralized nanofibrous scaffolds have been proposed as promising scaffolds for bone regeneration due to their ability to mimic both nanoscale architecture and chemical composition of natural bone extracellular matrix. In this study, a novel electrodeposition method was compared with an extensively explored simulated body fluid (SBF) incubation method in terms of the deposition rate, chemical composition and morphology of calcium phosphate formed on electrospun fibrous thin matrices with a fiber diameter in the range ∼200–1400nm prepared using 6, 8, 10 and 12wt.% poly(l-lactic acid) (PLLA) solutions in a mixture of dichloromethane and acetone (2:1 in volume). The effects of the surface modification using the two mineralization techniques on osteoblastic cell (MC3T3-E1) proliferation and differentiation were also examined. It was found that electrodeposition was two to three orders of magnitude faster than the SBF method in mineralizing the fibrous matrices, reducing the mineralization time from ∼2weeks to 1h to achieve the same amounts of mineralization. The mineralization rate also varied with the fiber diameter but in opposite directions between the two mineralization methods. As a general trend, the increase of fiber diameter resulted in a faster mineralization rate for the electrodeposition method but a slower mineralization rate for the SBF incubation method. Using the electrodeposition method, one can control the chemical composition and morphology of the calcium phosphate by varying the electric deposition potential and electrolyte temperature to tune the mixture of dicalcium phosphate dihydrate and hydroxyapatite (HAp). Using the SBF method, one can only obtain a low crystallinity HAp. The mineralized electrospun PLLA fibrous matrices from either method similarly facilitate the proliferation and osteogenic differentiation of preosteoblastic MC3T3-E1 cells as compared to neat PLLA matrices. Therefore, the electrodeposition method can be utilized as a fast and versatile technique to fabricate mineralized nanofibrous scaffolds for bone tissue engineering.

OBIF, an osteoblast induction factor, plays an essential role in bone formation in association with osteoblastogenesis

In vertebrate bone formation, the functional mechanisms of transcription factors in osteoblastic differentiation have been relatively well elucidated; however, the exact roles of cell-extrinsic molecules are less clear. We previously identified human and mouse Obif, an osteoblast induction factor, also known as Tmem119, which encodes a single transmembrane protein. OBIF is predominantly expressed in osteoblasts in mouse. While exogenous Obif expression stimulated osteoblastic differentiation, knockdown of Obif inhibits the osteoblastic differentiation of pre-osteoblastic MC3T3-E1 cells. In order to investigate an in vivo role of OBIF in bone formation, we generated Obif-deficient mice by targeted gene disruption. Analyses of micro-computed tomography (mCT) revealed that Obif(-/-) mice exhibit significantly reduced cortical thickness in the mid-shaft of the femur at postnatal day 14 (P14). Furthermore, progressive bone hypoplasia is observed after 8 weeks. The expression levels of osteoblast marker genes, Collagen 1a1, Osteopontin, Runx2, and Osterix, in the calvaria were decreased in Obif(-/-) mice at P4. These data indicate that Obif plays an essential role in bone formation through regulating osteoblastogenesis.

Microtubule assembly affects bone mass by regulating both osteoblast and osteoclast functions: Stathmin deficiency produces an osteopenic phenotype in mice

Cytoskeleton microtubules regulate various cell signaling pathways that are involved in bone cell function. We recently reported that inhibition of microtubule assembly by microtubule-targeting drugs stimulates osteoblast differentiation and bone formation. To further elucidate the role of microtubules in bone homeostasis, we characterized the skeletal phenotype of mice null for stathmin, an endogenous protein that inhibits microtubule assembly. In vivo micro-computed tomography (µCT) and histology revealed that stathmin deficiency results in a significant reduction of bone mass in adult mice concurrent with decreased osteoblast and increased osteoclast numbers in bone tissues. Phenotypic analyses of primary calvarial cells and bone marrow cells showed that stathmin deficiency inhibited osteoblast differentiation and induced osteoclast formation. In vitro overexpression studies showed that increased stathmin levels enhanced osteogenic differentiation of preosteoblast MC3T3-E1 cells and mouse bone marrow-derived cells and attenuated osteoclast formation from osteoclast precursor Raw264.7 cells and bone marrow cells. Results of immunofluorescent studies indicated that overexpression of stathmin disrupted radial microtubule filaments, whereas deficiency of stathmin stabilized the microtubule network structure in these bone cells. In addition, microtubule-targeting drugs that inhibit microtubule assembly and induce osteoblast differentiation lost these effects in the absence of stathmin. Collectively, these results suggest that stathmin, which alters microtubule dynamics, plays an essential role in maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone. \