The proliferation and differentiation of osteoblasts in co-culture with human umbilical vein endothelial cells: An improved analysis using fluorescence-activated cell sorting

The interaction of osteoblasts and endothelial cells plays a pivotal role in osteogenesis. This interaction has been extensively studied using their direct co-culture in vitro. However, co-culture experiments require clear discrimination between the two different cell types in the mixture, but this was rarely achieved. This study is the first to use fluorescence-activated cell sorting (FACS) for the separation and quantitative analysis of the proliferation and differentiation of MG-63 cells grown in direct co-culture with human umbilical vein endothelial cells (HUVECs). The cells of the MG-63 cell line have properties consistent with the characteristics of normal osteoblasts. We labeled HUVECs with fluorescent antibody against CD31 and used FACS to measure the proportions of each cell type and to separate them based on their different fluorescence intensities. The rate of proliferation of the MG-63 cells was estimated based on a count of the total viable cells and the proportion of MG-63 cells in the mixture. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), collagen type 1 (Coll-1) and osteocalcin (OC) in the MG-63 cells were measured via real-time PCR after the separation via FACS. We found that HUVECs stimulated the proliferation of the MG-63 cells after 72 h of co-culture, and inhibited it after 120 h of co-culture. The mRNA expression levels of ALP and Coll-1 significantly increased, whereas that of OC significantly decreased in MG-63 after co-culture with HUVECs. Using FACS for the quantitative analysis of the proliferation and differentiation of osteoblasts directly interacting with endothelial cells could have merit for further co-culture research.     

In vitro effects of low frequency electromagnetic fields on osteoblast proliferation and maturation in an inflammatory environment

An in vitro model was set up to investigate the effects of low frequency pulsed electromagnetic fields (PEMF) and its induced electric fields on osteoblast cells under inflammatory conditions. Osteoblasts (7F2) were seeded on top of chitosan scaffolds and co-cultured with macrophage cells (RAW 264.7) growing on the bottom of culture wells, stimulated by lipopolysaccharide to release reactive oxygen species including nitric oxide (NO). The co-culture was exposed to PEMF (magnitude of the magnetic field = 1.5 mT; induced electric voltage = 2.5 mV; frequency = 75 Hz; pulse duration = 1.3 ms) for 9 h. The osteoblasts were examined for their proliferation, viability, alkaline phosphatase (ALP) activity, and genetic expressions of type I collagen (COL I) and osteocalcin (OC), immediately and 7 days after PEMF exposure (days 0 and 7). Macrophage cell viability and NO concentration in the medium were monitored before and after PEMF exposure. The PEMF-exposed co-culture released a significantly higher amount of NO (65 μM) compared to control (17 μM) on day 7. Despite the high level of NO in the medium that was reported to be cytotoxic, PEMF-exposed osteoblasts had enhanced cell proliferation (23%), viability (36%), and COL I mRNA expression (3.4-fold) compared to the controls. The osteoblasts subjected to the PEMF had 41% less ALP activity than the control, which was associated with the active cell proliferation and COL I expression. The expression of OC mRNA was not seen in either the PEMF or control group, indicating cells had not entered the mineralization stage by day 7.     

The influence of RAMP1 overexpression on CGRP‐induced osteogenic differentiation in MG‐63 cells in vitro: An experimental study

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor‐like receptor (CRLR) in MG‐63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG‐63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG‐63 cells. Real‐time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post‐induction of differentiation. The CGRP‐induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP‐mediated differentiation of MG‐63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP‐induced MG‐63 differentiation. J. Cell. Biochem. 114: 314–322, 2013. © 2012 Wiley Periodicals, Inc.     

牙源性间充质干细胞对成骨前体细胞成骨分化的影响

目的:探讨牙源性间充质干细胞对成骨前体细胞成骨分化的影响.方法:将小鼠成骨前体细胞MC3T3-El分为两组,观察组为牙源性间充质干细胞与MC3T3-E1细胞共培养,对照组为单一MC3T3-E1细胞培养.采用CCK-8法检测细胞增殖水平,采用酶联免疫法检测碱性磷酸酶(Alkaline phosphatase,ALP)活性...     

Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study

The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.     

Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. In conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.     

Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.     

Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation

Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic cell differentiation and osteogenesis. It is unknown whether BMPs may act on human osteoblastic cells by increasing immature cell growth and/or differentiation. We investigated the short- and long-term effects of recombinant human (rh)BMP-2 on cell growth and osteoblast phenotype in a new model of human neonatal pre-osteoblastic calvaria cells (HNC). In short-term culture, rhBMP-2 (20-100 ng/ml) inhibited DNA synthesis and increased alkaline phosphatase (ALP) activity without affecting osteocalcin (OC) production. When cultured for 3 weeks in the presence of ascorbic acid and inorganic phosphate to induce cell differentiation, HNC cells initially proliferated, type 1 collagen mRNA and protein levels rose, and then decreased, whereas OC mRNA and protein levels, and calcium accumulation into the extracellular matrix increased at 2 to 3 weeks. A transient treatment with rhBMP-2 (50 ng/ml) for 1 to 7 days which affected immature HNC cells, decreased cell growth, increased ALP activity and mRNA, and induced cells to express ALP, osteopontin, and OC at 7 days, as shown by immunocytochemistry. At 2 to 3 weeks, matrix mineralization was markedly increased despite cessation of treatment, and although OC and Col 1 mRNA and protein levels were not changed. A continuous treatment with rhBMP-2 for 3 weeks which affected immature and mature cells reduced cell growth, increased ALP activity and mRNA at 1 week and increased OC mRNA and protein levels and calcium content in the matrix at 3 weeks, indicating complete osteoblast differentiation. These results indicate that the differentiating effects of BMP-2 on human neonatal calvaria are dependent on duration of exposure. Although long-term exposure led to complete differentiation of OC-synthesizing osteoblasts, the primary effect of rhBMP-2 was to promote osteoblast marker expression in immature cells, which was sufficient to induce optimal matrix mineralization independently of cell growth and type 1 collagen expression.     

高浓度地塞米松通过下调LMP-1表达抑制大鼠成骨细胞的分化

为探讨地塞米松（dexamethasone,DEX)抑制成骨细胞分化的机制,观察了不同浓度DEX对体外培养大鼠成骨细胞的碱性磷酸酶活性,骨钙素（osteocalcin,OC)合成,I型胶原蛋白表达的影响。并用RT-PCR方法检测了成骨细胞中LIM矿化蛋白1mRNA的表达量,结果显示：低浓度（10^-9mol/L）的DEX能增强碱性磷酸酶的活性、OC的分泌和I型胶原蛋白的表达;而高浓度（10^-7mol/L)的DEX对它们则起抑制作用,并下调成骨细胞正调节因子LMP-1mRNA的表达,上述结果表明,低浓度的DEX促进成骨细胞的分化;高浓度的DEX则抑制成骨细胞的分化,其抑制作用可能是通过下调LMP-1mRNA的表达而实现的。     

Reciprocal control of osteoblast/chondroblast and osteoblast/adipocyte differentiation of multipotential clonal human marrow stromal F/STRO-1(+) cells

The regulation of human bone marrow stromal precursor cell differentiation toward the chondrocyte, osteoblast or adipocyte lineages is not known. In this study, we assessed the lineage-specific differentiation and conversion of immortalized clonal F/STRO-1(+) A human fetal bone marrow stromal cells under the control of dexamethasone (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F/STRO-1(+) A cells expressed markers mRNAs or proteins of the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline phosphatase (ALP), type 1 collagen], of the chondrocyte lineage (aggrecan, types 2, 9 and 10 collagen), and of the adipocyte lineage (PPARgamma2, C/EBPalpha, aP2, G3PDH, lipoprotein lipase, leptin). Treatment with Dex increased CBFA1, OC and ALP mRNA and protein levels. Exposure to LA enhanced expression of adipocytic genes and cytoplasmic triglycerides accumulation, and suppressed the Dex-induced stimulation of osteoblast marker genes. Indo/Ins stimulated the synthesis of aggrecan and type 2 collagen and increased types 9 and 10 collagen mRNA levels, and suppressed both basal and Dex-promoted expression of osteoblast markers. Conversely, stimulation of osteoblastogenesis by Dex suppressed both basal and Indo/Ins-stimulated chondrocyte genes. Thus, the clonal human fetal bone marrow stromal F/STRO-1(+) A cell line is a lineage-unrestricted common progenitor that expresses tripotential adipocyte, osteoblast or chondrocyte characteristics. Our data also show that differentiation towards one pathway in response to Dex, Indo/Ins and LA restricts expression of other lineage-specific genes, and provide evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of clonal F/STRO-1(+) human bone marrow stromal cells.     

Ⅰ型胶原/聚己内酯/凹凸棒石复合支架材料体外诱导成骨的研究

目的:研究Ⅰ型胶原(ColⅠ)/聚己内酯(PCL)/凹凸棒石(ATP)复合支架材料的生物相容性及体外骨诱导性。方法:采用溶液浇铸-粒子滤沥法制备三种不同ATP含量(0% wt、10% wt、30% wt)的ColⅠ/PCL/ATP复合支架材料;将D1细胞与三种支架材料共培养,扫描电镜、鬼笔环肽和H&E染色、CCK-8法评价支架材料的生物相容性;D1细胞复合三种支架材料培养7天、14天、21天后RT-qPCR检测其成骨相关基因(Runx-2、Osterix、ALP、Col I、OPN、OC)的相对表达量,分别评价比较三种支架材料的成骨诱导效应。结果:SEM、鬼笔环肽和H&E染色显示D1细胞在三种支架材料表面均呈现良好的黏附;CCK-8结果显示,细胞在ATP含量30% wt的支架材料上增殖率显著高于其他两组,RT-qPCR检测结果显示,与0% wt、10% wt ATP相比,30% wt ATP组的Runx-2相对表达量在7天时显著升高, 14天、21天降低;ALP相对表达量在14天时显著升高,21天时显著降低;Osterix、Col I、OPN、OC的相对表达量随时间和ATP剂量的增加显著上调(P<0.05)。结论:ColⅠ/PCL/ATP复合支架材料具有良好的生物相容性及骨诱导性,有望成为一种理想的骨组织工程支架材料。     

葛根素对体外培养的人老年女性骨质疏松症成骨细胞分化的初步研究

目的:观察葛根素对体外培养老年女性骨质疏松症骨折患者体外分离培养的成骨细胞分化的影响。方法:采集老年女性骨质疏松症股骨颈骨折行人工股骨头置换术中取下的股骨颈松质骨,采用骨组织块法原代培养成骨细胞。观察不同浓度(0、0.01、0.1、1μmol/L)葛根素对成骨细胞表达碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OC)的影响,并比较各组成骨细胞钙化能力的大小。结果:0.01~1.00μmol/L的葛根素随着浓度增加,成骨细胞表达ALP、OC活性呈增强趋势,各浓度组两两比较差异均有统计学意义(P0.05);随葛根素浓度增高,成骨细胞钙化结节数量也明显增加,组间比较差异有统计学意义(P0.05)。结论:葛根素呈浓度依赖性促进成骨细胞表达ALP、OC并能提高成骨细胞的钙化能力。     

Three-dimensional type I collagen co-culture systems for the study of cell-cell interactions and treatment response in bone metastases

The available monolayer culture systems for the study of bone metastases constitute a suboptimal simulation of the in vivo pathophysiology of bone metastases, and therefore, do not provide sufficient information to assess the morphologic evidence of bone reaction to cancer cells, the nature of cell-specific mediators of osteolysis and osteoplasia and the response to treatment. Therefore, we have developed a three-dimensional (3-D) type I collagen gel system that allows co-culture of human osteoblasts (MG-63) with cancer cells, such as MCF-7, MDA-MB-231 or ZR-75 breast cancer cells, PC-3 prostate cancer, KLE endometrial cancer cells and Calu-1 lung cancer cells. We used type I collagen purified from rat tail tendons and the 3-D system was prepared by mixing MG-63 cells with type I collagen in 24-well plates. The 3-D system was inoculated with cancer cells and processed with standard cell culture procedures. After 1 week of culture, the matrix gel was fixed with formalin and embedded in paraffin. Serial sections were stained with trichrome Masson stain and modified Masson-Goldner stain, as well as analyzed by in situ hybridization, immunohistochemistry and the TUNEL technique for semi-quantitative detection of apoptotic cell death, assessing the response to adriamycin therapy. The inoculation of PC-3 cells in this collagen matrix produced a blastic reaction, documented by an increased number of MG-63 cells and increased density of type I collagen. The human KLE cells and inoculation of cell-free media produced no reaction, while ZR-75, MCF-7 and Calu-1 cells produced local degradation of the collagen matrix. In situ hybridization revealed the expression of Insulin-like growth factor 1 (IGF-1) and urokinase-type plasminogen activator (uPA) mRNA, while immunohistochemistry detected differential expression of uPA and cathepsin D. Adriamycin induced apoptotic cell death in prostate cancer cells and estrogen receptor negative (ER-) MDA-MB-231 breast cancer cells, while adriamycin did not induce apoptosis but cytostasis in ER+ MCF-7 cells. The adriamycin-induced apoptosis was inhibited by co-culture with osteoblast-like cells (MG-63). We conclude that this 3-D culture system is a useful in vitro model allowing the analysis of local mediators of osteolytic and osteoblastic reactions to bone metastases and treatment response.     

不同强度50 Hz脉冲电磁场促进大鼠颅骨成骨细胞矿化成熟最佳参数的筛选

为研究不同强度脉冲电磁场(pulse electromagnetic fields,PEMFs)对大鼠颅骨成骨细胞（rat skull osteoblasts,OB）增殖及成熟矿化的影响,将大鼠颅骨成骨细胞随机分为 7 组. 检测大鼠颅骨成骨细胞的增殖,细胞内碱性磷酸酶（ALP）活性变化,细胞沉积钙盐的情况,组织化学染色以及成骨细胞内标志性分子表达量的改变.结果显示,0.6 mT组促细胞增殖作用最强（P <0.01）;0.6 mT、1.8 mT、3.0 mT和3.6 mT均能提高ALP活性,其中0.6 mT ALP活性最高（P＜0.01）;在磁场处理4 ～12 d时细胞沉积钙盐逐渐增加,6种强度的脉冲电磁场均能促进钙盐沉积,尤以0.6 mT水平最高; ALP 染色、茜素红染色0.6 mT 组均显著高于对照组（P<0.01）;0.6 mT组 Bmp-2和Collagen-1 mRNA 的表达明显（P<0.01）高于对照组,磁场处理组Rankl mRNA 的表达均比对照组低. 0.6 mT 50 Hz 脉冲电磁场是促进成骨细胞增殖和矿化成熟的最佳参数,这为采用脉冲电磁场治疗骨质疏松症提供了治疗参数的基础支持.     

Tunicamycin negatively regulates BMP2-induced osteoblast differentiation through CREBH expression in MC3T3E1 cells

Tunicamycin, an endoplasmic reticulum (ER) stress inducer, specifically inhibits N-glycosylation. The cyclic AMP (cAMP) response element-binding protein H (CREBH) was previously shown to be regulated by UPR-dependent proteolytic cleavage in the liver. On the other hand, the role of CREBH in other tissues is unknown. In the present study, tunicamycin increased the level of CREBH activation (cleavage) as well as mRNA expression in osteoblast cells. Adenoviral (Ad) overexpression of CREBH suppressed BMP2-induced expression of alkaline phosphatase (ALP) and osteocalcin (OC). Interestingly, the BMP2-induced OASIS (structurally similar to CREBH, a positive regulator of osteoblast differentiation) expression was also inhibited by CREBH overexpression. In addition, inhibition of CREBH expression using siRNA reversed the tunicamycin-suppressed ALP and OC expression. These results suggest that CREBH inhibited osteoblast differentiation via suppressing BMP2-induced ALP, OC and OASIS expression in mouse calvarial derived osteoblasts.     

Glucosamine sulfate promotes osteoblastic differentiation of MG-63 cells via anti-inflammatory effect

Glucosamine sulfate (SGlc) has been known to be effective in controlling osteoarthritis (OA) symptoms in several clinical studies. However, the mechanisms of this positive effect of SGlc in human OA still remain elusive. Therefore, first, the effects of SGlc on the differentiation of osteoblast-like MG-63 cells were investigated. Our results demonstrate that SGlc can increase ALP activity, collagen synthesis, osteocalcin secretion, and mineralization in osteoblastic cells in vitro. Furthermore, it was observed that SGlc exhibited anti-inflammatory effect on production of TNF-alpha, IL-1beta, and PGE(2) in macrophage, RAW264.7 cells. In conclusion, these results suggest that SGlc can promote cell differentiation in cultured MG-63 osteoblast cells via anti-inflammatory effect.