丹皮酚对过氧亚硝基阴离子致大鼠成骨细胞分化抑制的影响

探讨丹皮酚（Pae）拮抗过氧亚硝基阴离子（ONOO^-）对体外培养大鼠成骨细胞分化的影响。用改良的组织块法分离培养新生大鼠颅骨成骨细胞,采用淬灭流动反应方法体外制备ONOO^-,以不同终浓度加入成骨细胞培养体系,在作用不同时间后,用对硝基苯二钠动力学（PNPP）法检测细胞内碱性磷酸酶（ALP）的活性,用Lowry法测定蛋白含量,并以不同终浓度Pae消除ONOO^-（1000μmoL/L）对成骨细胞分化的影响。结果显示,不同浓度的ONOO^-（50-1000μmoL/L）均能抑制碱性磷酸酶的活性,影响分化;高浓度的丹皮酚（10^-3-10^-6mol/L）能消除ONOO^-（1000μmoL/L）对碱性磷酸酶活性的抑制,拮抗ONOO^-抑制成骨细胞分化的作用。     

Real-Time Vital Mineralization Detection and Quantification during In Vitro Osteoblast Differentiation

Background

In vitro studies of osteoblasts traditionally use Alizarin Red as a golden standard for the detection and quantification of mineralization, which is a marker of osteoblast differentiation. However, this method presents a number of drawbacks, including the need to fix cells, which prevents additional measurements. Years ago, Calcein Green was proposed as an alternative to Alizarin Red, with the advantage to be directly detectable in live cells. However, the protocol was still time-consuming, and it never managed to replace Alizarin Red. Now, with more efficient imaging systems, we present a protocol using Calcein Green which provides significant advantages.

Results

The osteoblast mineralization was efficiently detected and accurately quantified in real time at any desired time point across the entire differentiation period, with a minimum time expenditure.

Conclusions

The combination of Calcein Green and the real-time imaging station IncuCyte ZOOM can efficiently replace the Alizarin Red method, and allows very accurate and time-saving assessment of the level and the dynamics of matrix mineralization.

     

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.     

Low-Dose X-Ray Irradiation Promotes Osteoblast Proliferation,Differentiation and Fracture Healing

Great controversy exists regarding the biologic responses of osteoblasts to X-ray irradiation, and the mechanisms are poorly understood. In this study, the biological effects of low-dose radiation on stimulating osteoblast proliferation, differentiation and fracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5 Gy) X-ray irradiation induced the cell viability and proliferation of MC3T3-E1 cells. However, high-dose (5 Gy) X-ray irradiation inhibited the viability and proliferation of osteoblasts. In addition, dynamic variations in osteoblast differentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterix and osteocalcin, were observed after both low-dose and high-dose irradiation by Western blot analysis. Second, fracture healing was evaluated via histology and gene expression after single-dose X-ray irradiation, and low-dose X-ray irradiation accelerates fracture healing of closed femoral fractures in rats. In low-dose X-ray irradiated fractures, an increase in proliferating cell nuclear antigen (PCNA)-positive cells, cartilage formation and fracture calluses was observed. In addition, we observed more rapid completion of endochondral and intramembranous ossification, which was accompanied by altered expression of genes involved in bone remodeling and fracture callus mineralization. Although the expression level of several osteoblast differentiation genes was increased in the fracture calluses of high-dose irradiated rats, the callus formation and fracture union were delayed compared with the control and low-dose irradiated fractures. These results reveal beneficial effects of low-dose irradiation, including the stimulation of osteoblast proliferation, differentiation and fracture healing, and highlight its potential translational application in novel therapies against bone-related diseases.     

minTBP-1/IGF-1融合蛋白钛涂层对高糖高脂环境下成骨细胞分化活性的影响

目的:评价自行设计的minTBP-1与IGF-1融合蛋白涂层修饰的纯钛表面对高糖高脂环境下成骨相关基因及蛋白表达的影响。方法:借助互联网Prot Param工具设计并筛选出亲水性较高的minTBP-1与IGF-1融合蛋白,用于修饰钛表面,分别将正常成骨细胞和高糖高脂环境下培养的成骨细胞接种于其表面,以钛表面无涂层的正常成骨细胞为对照组,通过碱性磷酸酶(ALP)、茜素红S染色及定量,比较成骨细胞的生物学特性;1周、2周时,Real-time PCR检测成骨细胞ALP、Ι型胶原、Runx2和OCN基因表达水平的变化;1周、2周和3周时,Western Blot分别检测成骨细胞ALP、OCN蛋白的表达量。结果:高糖高脂环境下培养的成骨细胞扁平、呈不规则多边形,其ALP的表达、钙化结节形成均低于正常成骨细胞(P0.05)。高糖高脂环境下,在目标融合蛋白修饰的钛表面接种成骨细胞1周时,ALP基因及蛋白均有明显表达,与正常成骨细胞相比无统计学差异(P0.05);2周时Ι型胶原、Runx2以及OCN的基因明显高表达,第3周时OCN蛋白显著表达,与正常成骨细胞相比无统计学差异(P0.05)。结论:minTBP-1与IGF-1融合蛋白涂层修饰的纯钛表面有利于改善高糖高脂环境下成骨细胞的生物学活性、促进了成骨相关基因及蛋白的表达,有望为2型糖尿病条件下钛-骨结合的种植体表面改性提供新策略。     

Rac Regulates Integrin-Mediated Spreading and Increased Adhesion of T Lymphocytes

Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte–ECM adhesion include (i) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (ii) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras were previously shown to affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. In this study, we demonstrated that expression of an activated mutant of Rac triggered dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect was not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading was accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells was observed. We demonstrate that expression of RacV12 did not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, our results indicate that Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. Furthermore, we show that the Rac-mediated signaling pathway leading to spreading of T lymphocytes did not require activation of c-Jun kinase, serum response factor, or pp70^{S6 kinase} but appeared to involve a phospholipid kinase.     

Differential Roles of Akt, Rac, and Ral in R-Ras-Mediated Cellular Transformation, Adhesion, and Survival

Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.     

3D水凝胶压应力模型建立及压应力对成骨细胞的影响

目的:通过建立3D水凝胶细胞模型,采用不同强度、频率、时间对MC3T3-E1细胞进行机械加压干预,进而探讨促进成骨细胞生长的适宜压应力方案。方法:设计不同强度、频率、时间的压应力梯度方案对成骨细胞MC3T3-E1进行干预。加压干预结束后即刻收集细胞,对样本中的ATF4、ALP、Runx2、Osteocalcin、RANKL和RANK mRNA进行定量检测。结果:经统计学分析后发现不同强度和频率对ALP(P0.05)、Runx2(P0.01)交互作用显著。此外,4h加压Runx2的表达量比12h加压的高(P0.05);4h加压RANKL的表达量比12h加压的低(P0.05)。结论:确定了压力强度和频率后,对3D细胞水凝胶模型施加一段时间的压应力发现,以1%强度、0.5Hz频率、4h的压应力干预方案能够促进成骨细胞系的生长。     

N-Cadherin Negatively Regulates Osteoblast Proliferation and Survival by Antagonizing Wnt,ERK and PI3K/Akt Signalling

Background

Osteoblasts are bone forming cells that play an essential role in osteogenesis. The elucidation of the mechanisms that control osteoblast number is of major interest for the treatment of skeletal disorders characterized by abnormal bone formation. Canonical Wnt signalling plays an important role in the control of osteoblast proliferation, differentiation and survival. Recent studies indicate that the cell-cell adhesion molecule N-cadherin interacts with the Wnt co-receptors LRP5/6 to regulate osteoblast differentiation and bone accrual. The role of N-cadherin in the control of osteoblast proliferation and survival remains unknown.

Methods and Principal Findings

Using murine MC3T3-E1 osteoblastic cells and N-cadherin transgenic mice, we demonstrate that N-cadherin overexpression inhibits cell proliferation in vitro and in vivo. The negative effect of N-cadherin on cell proliferation results from decreased Wnt, ERK and PI3K/Akt signalling and is restored by N-cadherin neutralizing antibody that antagonizes N-cadherin-LRP5 interaction. Inhibition of Wnt signalling using DKK1 or Sfrp1 abolishes the ability of N-cadherin blockade to restore ERK and PI3K signalling and cell proliferation, indicating that the altered cell growth in N-cadherin overexpressing cells is in part secondary to alterations in Wnt signalling. Consistently, we found that N-cadherin overexpression inhibits the expression of Wnt3a ligand and its downstream targets c-myc and cyclin D1, an effect that is partially reversed by N-cadherin blockade. We also show that N-cadherin overexpression decreases osteoblast survival in vitro and in vivo. This negative effect on cell survival results from inhibition of PI3K/Akt signalling and increased Bax/Bcl-2, a mechanism that is rescued by Wnt3a.

Conclusion

The data show that N-cadherin negatively controls osteoblast proliferation and survival via inhibition of autocrine/paracrine Wnt3a ligand expression and attenuation of Wnt, ERK and PI3K/Akt signalling, which provides novel mechanisms by which N-cadherin regulates osteoblast number.     

Effect of Hydrofluoric Acid Etching Time on Titanium Topography,Chemistry, Wettability,and Cell Adhesion

Titanium implant surface etching has proven an effective method to enhance cell attachment. Despite the frequent use of hydrofluoric (HF) acid, many questions remain unresolved, including the optimal etching time and its effect on surface and biological properties. The objective of this study was to investigate the effect of HF acid etching time on Ti topography, surface chemistry, wettability, and cell adhesion. These data are useful to design improved acid treatment and obtain an improved cell response. The surface topography, chemistry, dynamic wetting, and cell adhesiveness of polished Ti surfaces were evaluated after treatment with HF acid solution for 0, 2; 3, 5, 7, or 10 min, revealing a time-dependent effect of HF acid on their topography, chemistry, and wetting. Roughness and wetting increased with longer etching time except at 10 min, when roughness increased but wetness decreased. Skewness became negative after etching and kurtosis tended to 3 with longer etching time. Highest cell adhesion was achieved after 5–7 min of etching time. Wetting and cell adhesion were reduced on the highly rough surfaces obtained after 10-min etching time.     

The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation

The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content) and differentiation (alkaline phosphatase (ALP), collagen and osteocalcin secretion and calcium deposition) were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day) treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB₂ and TRPV1, but not CB₁ in HOBs. Anandamide-induced changes in HOB differentiation were CB₁ and CB₂-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.