Effects of Titanium Micro-Nanopermeable Structures on Osteogenic Differentiation

To evaluate the effects of different Ti surface micro-nanopermeable structures on osteoblast proliferation and differentiation and explore related mechanisms, hybrid technology of sandblast, acid etching, and hydrothermal (HT) was used to form the micro-nanopermeable surface of Ti. Scanning electron microscopy (SEM), surface profiler, and contact angle meter were utilized to assess the surface morphology, roughness, and hydrophilicity. MTT, SEM, alkaline phosphatase (ALP) activity assay, and real-time PCR were performed to investigate proliferation, adhesion and spreading, and differentiation of MC3T3-E1 cells grown on polished Ti (control), sandblast + acid etching- (SLA-) treated Ti, and SLA + HT-treated Ti. MAPK signal pathway activity was evaluated by Western blotting. The results showed that SLA + HT could result in not only formation of microscale groove containing submicroscale and nanoscale porous structures in Ti samples but also rough and hydrophilic surface. SLA + HT treatment has the best effects on cell adhesion and spreading. Significantly increased levels of ALP activity and osteogenic genes including Alp, Ocn, Opn, Runx2, and Bsp, as well as p38 but not ERK phosphorylation, were found in the SLA + HT group. In conclusion, sandblast, acid etching, and hydrothermal treatment on Ti regulates osteoblast differentiation, while activation of the MAPK p38 signaling pathway served as the mechanism.

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Synergistic stimulation of osteoblast differentiation of rat mesenchymal stem cells by leptin and 25(OH)D3 is mediated by inhibition of chaperone-mediated autophagy

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02623-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qiting He ◽

Ruixi Qin ◽

Julie Glowacki ◽

Shuanhu Zhou ◽

Jie Shi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Vitamin D ◽

Mesenchymal Stem Cells ◽

Osteoblast Differentiation ◽

Signal Pathway ◽

Calcium Deposition ◽

Qrt Pcr ◽

Alp Activity ◽

Chaperone Mediated Autophagy

Abstract Background Vitamin D is important for the mineralization of bones by stimulating osteoblast differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are a target of vitamin D action, and the metabolism of 25(OH)D3 to biologically active 1α,25(OH)2D3 in BMMSCs promotes osteoblastogenesis in an autocrine/paracrine manner. Our previous study with human BMMSCs showed that megalin is required for the 25(OH)D3-DBP complex to enter cells and for 25(OH)D3 to stimulate osteoblast differentiation in BMMSCs. Furthermore, we reported that leptin up-regulates megalin in those cells. Leptin is a known inhibitor of PI3K/AKT-dependent chaperone-mediated autophagy (CMA). In this study, we tested the hypothesis that leptin acts synergistically with 25(OH)D3 to promote osteoblastogenesis in rat BMMSCs by a mechanism that entails inhibition of PI3K/AKT-dependent CMA. Methods BMMSCs were isolated from rat bone marrow (4-week-old male SD rats); qRT-PCR and western immunoblots or immunofluorescence were used to evaluate the expression of megalin, ALP, COL1A1, RUNX2, OSX, OSP, and CMA in rBMMSCs. The osteoblast differentiation was evaluated by ALP activity, ALP staining, and calcium deposition. The viability of rBMMSCs was assessed with the CCK-8 kit. Biosynthesis of 1α,25(OH)2D3 was measured by a Rat 1α,25(OH)2D3 ELISA Kit. Results The combination of leptin and 25(OH)D3 treatment significantly enhanced osteoblast differentiation as shown by ALP activity, ALP staining, and calcium deposition, the expression of osteogenic genes ALP, COL1A1, RUNX2, OSX, and OSP by qRT-PCR and western immunoblots in rBMMSCs. Leptin enhanced the expression of megalin and synthesis of 1α,25(OH)2D3 in rBMMSCs. Our data showed that leptin inhibited CMA activity of rBMMSCs by activating PI3K/AKT signal pathway; the ability of leptin to enhance 25(OH)D3 promoted osteoblast differentiation of rBMMSCs was weakened by the PI3K/AKT signal pathway inhibitor. Conclusions Our data reveal the mechanism by which leptin and 25(OH)D3 promote osteoblast differentiation in rBMMSCs. Leptin promoted the expression of megalin by inhibiting CMA, increased the utilization of 25(OH)D3 by rBMMSCs, and enhanced the ability of 25(OH)D3 to induce osteoblast differentiation of rBMMSCs. PI3K/AKT is at least partially involved in the regulation of CMA. These data indicate the importance of megalin in BMMSCs for vitamin D’s role in skeletal health.

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miR-34s inhibit osteoblast proliferation and differentiation in the mouse by targeting SATB2

The Journal of Cell Biology ◽

10.1083/jcb.201201057 ◽

2012 ◽

Vol 197 (4) ◽

pp. 509-521 ◽

Cited By ~ 164

Author(s):

Jianwen Wei ◽

Yu Shi ◽

Lihua Zheng ◽

Bin Zhou ◽

Hiroyuki Inose ◽

...

Keyword(s):

Osteoblast Differentiation ◽

Matrix Protein ◽

Molecular Mechanisms ◽

Loss Of Function ◽

Osteoblast Proliferation ◽

Nuclear Matrix Protein ◽

Critical Determinant ◽

Proliferation And Differentiation ◽

Bone Mass Accrual

A screen of microRNAs preferentially expressed in osteoblasts identified members of the miR-34 family as regulators of osteoblast proliferation and/or differentiation. Osteoblast-specific gain- and loss-of-function experiments performed in vivo revealed that miR-34b and -c affected skeletogenesis during embryonic development, as well as bone mass accrual after birth, through two complementary cellular and molecular mechanisms. First, they inhibited osteoblast proliferation by suppressing Cyclin D1, CDK4, and CDK6 accumulation. Second, they inhibited terminal differentiation of osteoblasts, at least in part through the inhibition of SATB2, a nuclear matrix protein that is a critical determinant of osteoblast differentiation. Genetic evidence obtained in the mouse confirmed the importance of SATB2 regulation by miR-34b/c. These results are the first to identify a family of microRNAs involved in bone formation in vivo and to identify a specific genetic pathway by which these microRNAs regulate osteoblast differentiation.

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Collagen Hydrolysate Gly-Pro-Hyp on Osteoblastic Proliferation and Differentiation of MC3T3-E1 Cells

Journal of Clinical and Nursing Research ◽

10.26689/jcnr.v1i3.158 ◽

2017 ◽

Vol 1 (3) ◽

Author(s):

Geng Min

Keyword(s):

Alkaline Phosphatase ◽

Transcription Factor ◽

Bone Formation ◽

Osteoblast Differentiation ◽

Control Medium ◽

Collagen Hydrolysate ◽

Alp Activity ◽

Proliferation And Differentiation ◽

Related Transcription Factor ◽

Set Up

Objectives: Bone formation and bone resorption continuouslyoccur in bone tissue to prevent the accumulation of old bone, thisbeing called bone remodeling. Osteoblasts especially play a crucialrole in bone formation through the differentiation and proliferation.Therefore, in this study, we investigated the effects of collagenhydrolysate Glycine-Prolyl-Hydroxyproline (Gly-Pro-Hyp) onosteoblastic proliferation and differentiation in MC3T3-E1 cells.Methods: Four groups including control, Gly-Pro-Hyp 20 Î¼M,100 Î¼M, 500 Î¼M groups were set up in this study. Cells werecultured with blank control medium or Gly-Pro-Hyp of thedifferent dosages for 24 h. CCK 8 assay was analyzed cellproliferation. Assay of Alkaline phosphatase (ALP)activity wasanalyzed osteoblast differentiation. The expression levels of ALP,Col 1, Runx 2 and Osterix in MC3T3-E1 cells were measured byWestern blot. Results: The results indicated the treatment of Gly-Pro-Hyp promoted the proliferation of MC3T3-E1 cells andimproved ALP activity. In addition, cells treated with Gly-Pro-Hypsignificantly upregulated protein expression of ALP, type 1collagen, runt-related transcription factor 2 and osterix.Conclusion: The results demonstrate that Gly-Pro-Hyp promotedifferentiation inducement and proliferation of MC3T3-E1 cells,therefore may help to elucidate the transcriptional mechanism ofbone formation and possibly lead to the development of bone-forming drugs.

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Adenosine Regulates Bone Metabolism Via A1, A2A and A2B Receptors in Bone Marrow Cells From Normal and Patients with Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.4977.4977 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4977-4977

Author(s):

Wenjie He ◽

Jeesun Park ◽

Amitabha Mazumder ◽

Bruce Cronstein

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Resorption ◽

Receptor Agonist ◽

Osteoblast Differentiation ◽

Osteoclast Differentiation ◽

Activity Assay ◽

Alizarin Red ◽

Alp Activity ◽

A2a Receptor

Abstract Abstract 4977 Background: Multiple myeloma is characterized by osteolytic bone lesions, wherein coupled bone remodeling is disrupted with increased osteoclast activation and decreased osteoblast differentiation. We have previously demonstrated that adenosine, acting via A2A receptors, diminishes human and murine osteoclast formation and others have reported that adenosine, acting at A2Breceptors, promotes osteoblast differentiation in murine osteoblast precursors and cell lines. In this study, we examined the effect of adenosine on osteoblast and osteoclast differentiation derived from multiple myeloma (MM) patients. Methods: Human bone marrow was collected from multiple myeloma patients. Bone marrow stromal cells (BMSCs) and bone marrow derived mononuclear (BMMs) cells were isolated and osteoblasts and osteoclastswere cultured, respectively. Adenosine A1 receptor agonist CHA and antagonist Rolofylline, A2A receptor agonist CGS and antagonist ZM, and A2b receptor agonist BAY and antagonist MRS 1754, A3receptor agonist IB-MECA and antagonist MRS 1191; and dipyridamole, a nucleoside transport inhibitor, were added to the culture media. Alkaline phosphatase (ALP) activity assay was used to quantitate the osteoblast differentiation. In vitro osteoblast calcification was determined by alizarin red staining. TRAP+ staining was used to examine the osteoclast differentiation and bone resorption assay was used to study the osteoclast activity. Results: We found that A1R blockade by rolofyllineand A2aR ligation by CGS21680 inhibited differentiation of both normal and MM BMMs into TRAP+ multinucleated cells (IC50= 1nM for A1R, IC50= 10μM for A2AR;p<0. 001, n=3 for both). The inhibition of osteoclast differentiation by Rolofylline was also seen in bone resorption assay (Pit formation assay). The A2A receptor antagonist completely reversed the effects of CGS21680 on osteoclast differentiation. Moreover, enhanced adenosine accumulation in the presence of dipyridamole (0. 5μM) and A2BR activation promoted the differentiation of BMSCs from myeloma patients into osteoblasts shown byArlizarin red staining and ALP activity assay (by 1. 8 ± 0. 41 and 1. 57 ± 0. 26 fold, respectively, p<0. 05, compared with osteogenic media only, n=3 for both). Conclusions: These results indicate that adenosine A2 receptors may be useful targets for the treatment and prevention of MM-induced bone disease. Disclosures: No relevant conflicts of interest to declare.

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Isolation and identification of peptides from Pinctada martensii with osteogenic activity

Acta Alimentaria ◽

10.1556/066.2021.00109 ◽

2021 ◽

Keyword(s):

Mass Spectrometry ◽

Alkaline Phosphatase ◽

Liquid Chromatography Mass Spectrometry ◽

Osteoblast Proliferation ◽

Isolation And Identification ◽

Osteogenic Activity ◽

Alp Activity ◽

Pinctada Martensii ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation

Abstract Marine organisms have attracted considerable attention in recent years. In this study, peptides with osteogenic activity from Pinctada martensii were isolated and identified. Additionally, the effects of the hydrolysates on MC3T3-E1 cell proliferation and differentiation were evaluated using the MTT and alkaline phosphatase (ALP) assays, respectively. First, trypsin, pancreatin, and neutral protease were used to hydrolyse the intact shellfish. The hydrolysates with the greatest effects on osteoblast proliferation and ALP activity were separated and purified. Second, fraction WP2 was isolated and purified using a Sephadex G-25 column. WP2, which had the highest osteogenic activity, increased cell growth by 48.57 ± 0.05% and ALP activity by 6.27 ± 0.07 mU. Finally, four novel peptides were identified in WP2 (FDNEGKGKLPEEY, IVLDSGDGVTH, IVLDSGDGVSH, and SSENSDLQRQ) by Orbitrap Fusion Lumos Tribrid orbital liquid chromatography-mass spectrometry. Our findings revealed that P. martensii contains peptides with potential osteogenic activity.

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Irisin promotes osteoblast proliferation and differentiation via activating the MAP kinase signaling pathways

Scientific Reports ◽

10.1038/srep18732 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 80

Author(s):

Xiaoyong Qiao ◽

Ying Nie ◽

Yaxian Ma ◽

Yan Chen ◽

Ran Cheng ◽

...

Keyword(s):

Map Kinase ◽

P38 Mapk ◽

Calcium Deposition ◽

Kinase Signaling ◽

Osteoblast Proliferation ◽

Skeletal Health ◽

Alp Activity ◽

Proliferation And Differentiation ◽

Map Kinase Signaling

Abstract Physical exercise is able to improve skeletal health. However, the mechanisms are poorly known. Irisin, a novel exercise-induced myokine, secreted by skeletal muscle in response to exercise, have been shown to mediate beneficial effects of exercise in many disorders. In the current study, we demonstrated that irisin promotes osteoblast proliferation and increases the expression of osteoblastic transcription regulators, such as Runt-related transcription factor-2, osterix/sp7; and osteoblast differentiation markers, including alkaline phosphatase, collagen type 1 alpha-1, osteocalcin and osteopontin in vitro. Irisin also increase ALP activity and calcium deposition in cultured osteoblast. These osteogenic effects were mediated by activating the p38 mitogen-activated protein kinase (p-p38 MAPK) and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK by SB203580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runx2 expression and ALP activity. Together our observation suggest that irisin directly targets osteoblast, promoting osteoblast proliferation and differentiation via activating P38/ERK MAP kinase signaling cascades in vitro. Whether irisin can be utilized as the therapeutic agents for osteopenia and osteoporosis is worth to be further pursued

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SPAK-p38 MAPK signal pathway modulates claudin-18 and barrier function of alveolar epithelium after hyperoxic exposure

BMC Pulmonary Medicine ◽

10.1186/s12890-021-01408-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chih-Hao Shen ◽

Jr-Yu Lin ◽

Cheng-Yo Lu ◽

Sung-Sen Yang ◽

Chung-Kan Peng ◽

...

Keyword(s):

P38 Mapk ◽

Knockout Mice ◽

Barrier Function ◽

Alveolar Epithelium ◽

Signal Pathway ◽

Control Group ◽

Barrier Dysfunction ◽

Hyperoxic Exposure ◽

Monolayer Permeability ◽

Mapk Signal Pathway

Abstract Background Hyperoxia downregulates the tight junction (TJ) proteins of the alveolar epithelium and leads to barrier dysfunction. Previous study has showed that STE20/SPS1-related proline/alanine-rich kinase (SPAK) interferes with the intestinal barrier function in mice. The aim of the present study is to explore the association between SPAK and barrier function in the alveolar epithelium after hyperoxic exposure. Methods Hyperoxic acute lung injury (HALI) was induced by exposing mice to > 99% oxygen for 64 h. The mice were randomly allotted into four groups comprising two control groups and two hyperoxic groups with and without SPAK knockout. Mouse alveolar MLE-12 cells were cultured in control and hyperoxic conditions with or without SPAK knockdown. Transepithelial electric resistance and transwell monolayer permeability were measured for each group. In-cell western assay was used to screen the possible mechanism of p-SPAK being induced by hyperoxia. Results Compared with the control group, SPAK knockout mice had a lower protein level in the bronchoalveolar lavage fluid in HALI, which was correlated with a lower extent of TJ disruption according to transmission electron microscopy. Hyperoxia down-regulated claudin-18 in the alveolar epithelium, which was alleviated in SPAK knockout mice. In MLE-12 cells, hyperoxia up-regulated phosphorylated-SPAK by reactive oxygen species (ROS), which was inhibited by indomethacin. Compared with the control group, SPAK knockdown MLE-12 cells had higher transepithelial electrical resistance and lower transwell monolayer permeability after hyperoxic exposure. The expression of claudin-18 was suppressed by hyperoxia, and down-regulation of SPAK restored the expression of claudin-18. The process of SPAK suppressing the expression of claudin-18 and impairing the barrier function was mediated by p38 mitogen-activated protein kinase (MAPK). Conclusions Hyperoxia up-regulates the SPAK-p38 MAPK signal pathway by ROS, which disrupts the TJ of the alveolar epithelium by suppressing the expression of claudin-18. The down-regulation of SPAK attenuates this process and protects the alveolar epithelium against the barrier dysfunction induced by hyperoxia.

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Thyroid-stimulating hormone decreases the risk of osteoporosis by regulating osteoblast proliferation and differentiation

BMC Endocrine Disorders ◽

10.1186/s12902-021-00715-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Tuo Deng ◽

Wenwen Zhang ◽

Yanling Zhang ◽

Mengqi Zhang ◽

Zhikun Huan ◽

...

Keyword(s):

Thyroid Function ◽

Thyroid Stimulating Hormone ◽

Mrna Levels ◽

Dependent Manner ◽

Osteoblast Proliferation ◽

Normal Thyroid ◽

Normal Thyroid Function ◽

Gene And Protein Expression ◽

Proliferation And Differentiation ◽

Stimulating Hormone

Abstract Background As the incidence of secretory osteoporosis has increased, bone loss, osteoporosis and their relationships with thyroid-stimulating hormone (TSH) have received increased attention. In this study, the role of TSH in bone metabolism and its possible underlying mechanisms were investigated. Methods We analyzed the serum levels of free triiodothyronine (FT3), free thyroxine (FT4), and TSH and the bone mineral density (BMD) levels of 114 men with normal thyroid function. In addition, osteoblasts from rat calvarial samples were treated with different doses of TSH for different lengths of time. The related gene and protein expression levels were investigated. Results A comparison of the BMD between the high-level and low-level serum TSH groups showed that the TSH serum concentration was positively correlated with BMD. TSH at concentrations of 10 mU/mL and 100 mU/mL significantly increased the mRNA levels of ALP, COI1 and Runx2 compared with those of the control (P < 0.05, P < 0.01). Bone morphogenetic protein (BMP)2 activity was enhanced with both increased TSH concentration and increased time. The protein levels of Runx2 and osterix were increased in a dose-dependent manner. Conclusions The circulating concentrations of TSH and BMD were positively correlated with normal thyroid function in males. TSH promoted osteoblast proliferation and differentiation in rat primary osteoblasts.

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