It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.
Alkaline Phosphatase/metabolism* ; Animals ; Electromagnetic Fields ; Osteoblasts/metabolism* ; Osteogenesis/genetics* ; Rats ; TRPP Cation Channels/physiology*

In this paper, we review the results of previous studies and summarize the effects of various factors on the regulation of bone metabolism in traumatic bone infections. Infection-related bone destruction incorporates pathogens and iatrogenic factors in the process of bone resorption dominated by the skeletal and immune systems. The development of bone immunology has established a bridge of communication between the skeletal system and the immune system. Exploring the effects of pathogens, skeletal systems, immune systems, and antibacterials on bone repair in infectious conditions can help improve the treatment of these diseases.
Anti-Bacterial Agents/administration & dosage* ; Bone and Bones/metabolism* ; Cellular Microenvironment ; Humans ; Immune System/immunology* ; Lymphocyte Subsets/immunology* ; Osteitis/microbiology* ; Osteoblasts/physiology* ; Osteoclasts/physiology* ; Staphylococcal Infections

Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.
Animals ; Cell Differentiation ; physiology ; Core Binding Factor Alpha 1 Subunit ; Homeodomain Proteins ; metabolism ; Mesenchymal Stem Cells ; metabolism ; Mice ; Mice, Nude ; Osteoblasts ; metabolism ; Osteocalcin ; drug effects ; Osteogenesis ; physiology ; Transcription Factors ; metabolism ; Up-Regulation

In this study, we aim to investigat the effect of microgravity on osteoblast differentiation in osteoblast-like cells (MC3T3-E1). In addition, we explored the response mechanism of nuclear factor-kappa B (NF-κB) signaling pathway to "zero- " in MC3T3-E1 cells under the simulated microgravity conditions. MC3T3-E1 were cultured in conventional (CON) and simulated microgravity (SMG), respectively. Then, the expression of the related osteoblastic genes and the specific molecules in NF-κB signaling pathway were measured. The results showed that the mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin (OCN) and type Ⅰ collagen (CoL-Ⅰ) were dramatically decreased under the simulated microgravity. Meanwhile, the NF-κB inhibitor α (IκB-α) protein level was decreased and the expressions of phosphorylation of IκB-α (p-IκB-α), p65 and phosphorylation of p65 (p-p65) were significantly up-regulated in SMG group. In addition, the IL-6 content in SMG group was increased compared to CON. These results indicated that simulated microgravity could activate the NF-κB pathway to regulate MC3T3-E1 cells differentiation.
3T3 Cells ; Animals ; Cell Differentiation ; Mice ; NF-kappa B ; physiology ; Osteoblasts ; Signal Transduction ; Weightlessness Simulation

Nephronectin (NPNT) is a novel extracellular matrix protein and a new ligand of integrin α8β1. Recent studies showed that NPNT is highly expressed in kidney, lung, thyroid, etc, and it may play an important role in many pathological conditions. NPNT is involved in the process of kidney development and acute kidney injury, regulates proliferation and differentiation of osteoblast, and induces the vasculogenesis in vitro. NPNT may play a key role in pathological osteoporosis and therefore be a new therapeutic target of bone diseases. NPNT gene variants are not only associated with lung function, but also potentially implicated in chronic airway diseases development. Moreover, NPNT is also an important factor that mediates pathology of cardiac, epidermis, breast, liver and teeth diseases. In this paper, we reviewed some research progresses on the structure, distribution, physiological and pathophysiological functions of NPNT.
Cell Differentiation ; Cell Proliferation ; Extracellular Matrix Proteins ; physiology ; Humans ; Kidney ; physiology ; Osteoblasts ; cytology ; Osteoporosis

OBJECTIVE: To investigate the effects of the Arg-Gly-Asp polypeptedes (RGD) peptides-modified porous tantalum surface on osteoblasts morphology and expressions of osteogenesis factors, and to evaluate RGD peptides promotes junctura ossium of tantalum-bone interface in vivo. METHODS: RGD peptides of different concentrations (1 g/L, 5 g/L, and 10 g/L) were loaded to porous tantalum slices with a diameter of 10 mm and a thickness of 3 mm by physical absorption. The 3rd generation of MG63 cells were co-cultured with tantalum and divided into 4 groups: Ta-cells (control) group, 1 g/L cells/Ta/RGD group, 5 g/L cells/Ta/RGD group, and 10 g/L cells/Ta/RGD group. Porous tantalum compo-sites and osteoblasts-tantalum interface were observed by scanning electron microscopy. The adhesion rate of osteoblasts was detected and immunocytochemistry was used to detect the expressions of filamentous actin (F-actin), osteocalcin (OC) and fibronectin (FN). RESULTS: The scanning electron microscope (SEM) revealed that osteoblasts distributed on the surface of porous tantalum and secreted extracellular matrix on outside and inner of micro-pores. The osteoblasts adhesion rate on porous tantalum modified with RGD was higher than that in the unmodified porous tantalum at the end of 24, 48, and 72 hours. The best adhesion effect was got in 5 g/L cells/Ta/RGD group at hour 48 [(68.07±3.80) vs. (23.40±4.39), P<0.05]. The results of immunocytochemistry showed that the expressions intensity of F-actin, OC and FN in osteoblasts on porous tantalum modified groups with RGD were stronger than that in the unmodified groups, and the expressions of 5 g/L cells/Ta/RGD group were significantly higher than those in the 10 g/L group and 1 g/L group [OC: (18.08±0.08) vs. (15.14±0.19), P<0.05; (18.08±0.08) vs. (14.04±0.61), P<0.05. FN: (24.60±0.98) vs. (15.90±0.53), P<0.05; (24.60±0.98) vs. (15.30±0.42), P<0.05. F-actin: (29.20±1.31) vs. (24.50±1.51), P<0.05; (29.20±1.31) vs. (16.92±0.40), P<0.05]. Correspondingly F-actin in osteoblasts was showed in longitudinal arrangement, and the expressions intensity was stronger than those OC and FN. CONCLUSION The RGD peptides is beneficial to enhance adhesion of osteoblast, spreading and reorganization of cytoskeleton on porous tantalum surface and improve the interface morphology, further promoting osteoblasts-tantalum conjunctive interface osseointegration.
Cell Adhesion ; Oligopeptides ; Osteoblasts/physiology* ; Osteogenesis ; Tantalum

OBJECTIVE: This study aimed to investigate the effect of calcium ion (Ca²⁺) on the migration and osteogenic differentiation of human osteoblasts and explore the proper concentration and correlation mechanism. METHODS: A series of Ca²⁺ solutions with different concentrations was prepared. Osteoblast migration was assessed by Transwell assay, and proliferation was studied via the CCK-8 colorimetric assay. The mRNA expression of osteogenic genes was examined via reverse transcription-polymerase chain reaction (RT-PCR), and the mineralized nodule was examined by alizarin red-S method. After calcium sensitive receptor (CaSR) antagonism, Ca²⁺-induced migration and osteogenic differentiation were analyzed. RESULTS: In the migration experiment, 2, 4, and 6 mmol·L⁻¹ Ca²⁺ could promoted osteoblast migration at three timepoints (8, 16, and 24 h), whereas 10 mmol·L⁻¹ Ca²⁺ considerably inhibited migration at 8 h. The Ca²⁺ concentration range of 2-10 mmol·L⁻¹ could promote proliferation, osteogenic differentiation, and mineralization of human osteoblasts. Moreover, mineralization was predominantly induced by 8 and 10 mmol·L⁻¹ Ca²⁺. CaSR antagonism could reduce Ca²⁺-induced migration and osteogenic differentiation of human osteoblasts. CONCLUSIONS Low Ca²⁺ concentration favored osteoblast migration, whereas high Ca²⁺ concentration favored osteogenic differentiation. The Ca²⁺ concentrations of 4 and 6 mmol·L⁻¹ could substantially induce osteoblast migration and osteogenic differentiation, and the Ca²⁺-CaSR pathway participated in signal transduction.
Calcium ; physiology ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Humans ; Osteoblasts ; Osteogenesis ; physiology ; Signal Transduction

Tooth eruption is a series of complicated physiological processes occurring once the crown is formed completely, as well as when the tooth moves toward the occasion plane. As such, the tooth moves through the alveolar bone and the oral mucosa until it finally reaches its functional position. Most studies indicate that the process of tooth eruption involves the alveolar bone, dental follicles, osteoclasts, osteoblasts, and multiple cytokines. Dental follicles regulate both resorption and formation of the alveolar bone, which is required for tooth eruption. Furthermore, root formation with periodontal ligament facilitates continuous tooth eruption. However, the exact mechanism underlying tooth eruption remains unclear. Hence, this review describes the recent research progress on the cellular and molecular mechanisms of tooth eruption.
Dental Sac ; Humans ; Osteoblasts ; Osteoclasts ; Periodontal Ligament ; Tooth ; Tooth Eruption ; physiology ; Tooth Root

OBJECTIVEPrevious studies have clarified that calcitonin gene-related peptide (CGRP) can promote the biologi- cal activity of osteoblasts. To further reveal the role of CGRP in bone repair, we studied its influence on osteogenic differentia- tion of mouse bone marrow stromal cells (BMSCs) and initially explored the effect of the Hippo signaling pathway with this process.

METHODSBMSCs were induced to osteogenic differentiate osteoblasts by different concentrations of CGRP for a screening of the optimal concentration. CGRP was added in BMSCs, then the activity of alkaline phosphatase (ALP) and the number of mineralized nodules were examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days, respectively. The protein expression of p-Mst1/2 was measured by Western blot. Verteporfin was used to block the downstream Yap signaling. The mRNA expression of collagen type I (Col I) and runt-related transcription factor 2 (Runx2) were detected by reverse transcription-polymerase chain reaction.

RESULTSCompared to the blank group, different concentrations of CGRP (10⁻⁹, 10⁻⁸, 10⁻⁷ mol · L⁻¹), especially 10⁻⁸ mol · L⁻¹, significantly increased the ALP activity of BMSCs (P < 0.05). Alizarin red staining also showed more mineralized nodules in 10⁻⁸ mol · L⁻¹ group. The expression of p-Mst1/2 increased in the CGRP group (P < 0.05). Verteporfin treatment effectively decreased the mRNA expression of Runx2 and Col I (P < 0.05).

CONCLUSIONThe Hippo signaling pathway plays a role in CGRP-induced osteogenic differentiation in mouse BMSCs.

Alkaline Phosphatase ; Animals ; Calcitonin ; genetics ; metabolism ; Calcitonin Gene-Related Peptide ; metabolism ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; Core Binding Factor Alpha 1 Subunit ; Mesenchymal Stromal Cells ; physiology ; Mice ; Osteoblasts ; Osteogenesis ; physiology ; Signal Transduction

OBJECTIVEThis study investigated the effects of hydroxyapatite (HA)/chitosan (CS)-transforming growth factor-β1 (TGF-β1) composite coatings on titanium surfaces, as well as on the attachment and proliferation of osteoblasts.

METHODSHA/CS-TGF-β1 composite coatings were prepared on titanium surfaces by physical, chemical, and biological modifications. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FTIR), and other methods were employed to analyze the chemical composition and surface topography of the composite coatings. CCK-8 and immunofluorescence assays were used to analyze the effects of the coatings on the attachment and proliferation of osteoblasts.

RESULTSHA/CS-TGF-β1 composite coatings were successfully prepared. Their contact angle was almost zero. These composite coatings were applied in vitro, with a drug released early and a burst release effect. The growth of osteoblasts was not inhibited on it and it had obvious promoting effect on the adhesion and early proliferation of osteoblasts.

CONCLUSIONThe composite coatings significantly promote the adhesion and early proliferation of osteoblasts in vitro. This finding shows that the proposed method demonstrates a good prospective application in surface modification of titanium.

Cell Proliferation ; Chitosan ; chemistry ; Dental Bonding ; methods ; Durapatite ; chemistry ; Microscopy, Electron, Scanning ; Osteoblasts ; physiology ; Prospective Studies ; Spectroscopy, Fourier Transform Infrared ; Surface Properties ; Titanium ; chemistry ; Transforming Growth Factor beta ; chemistry ; Transforming Growth Factors ; X-Ray Diffraction