Protection by CD4 or CD8 T Cells against Pulmonary Mycobacterium bovis Bacillus Calmette-Guérin Infection

Mice deficient in CD8 T cells demonstrated levels of Th1 cytokines and granulomatous responses in the lungs very similar to those demonstrated by normal control mice and were fully capable of controlling pulmonary mycobacterial infection by Mycobacterium bovis BCG as assessed at day 37 postinfection. In comparison, mice deficient in CD4 T cells had similar levels of interleukin-12 (IL-12) and tumor necrosis factor alpha but lower levels of gamma interferon in the lungs and were still able to mount tissue granulomatous responses and control pulmonary mycobacterial infection. In contrast, IL-12^−/− mice with impaired CD4 and CD8 T-cell responses had a markedly weakened control of infection, whereas SCID mice deficient in all T cells succumbed to such pulmonary mycobacterial infections.     

Responses of MC3T3-E1 cells to three dental resin-based restorative materials

This study aimed to examine the influences of three dental resin-based restorative materials on cells associated with hard tissue regeneration using osteoblastic MC3T3-E1 cells. A Bis-GMA-based resin composite [Clearfil AP-X (APX)], an MMA-based resin cement [Superbond C&B (SB)], and a resin-modified glass-ionomer [Fuji Ionomer Type II LC (LC)] were tested. A zinc oxide eugenol cement [Super EBA (EBA)] was included in the study for comparison. MC3T3-E1 cells were cultured on set materials for 3, 7, 14, or 21 days. Cell attachment and proliferation were observed by scanning electron microscopy, and mitochondrial dehydrogenase and alkaline phosphatase (ALP) activities of the cells were evaluated. Cell cultures on polystyrene tissue culture dishes served as controls. On APX and SB, cells demonstrated attachment, spreading, and proliferation similar to the controls. In contract, cells adhered and proliferated poorly on LC and EBA. The mitochondrial function and ALP activity of the cells were significantly suppressed (p < 0.05, Scheffe's F test) throughout the experimental period when cultured on LC or EBA, although APX and SB exhibited less inhibition. The results indicate that APX and SB are less toxic to proliferation and differentiation of MC3T3-E1, suggesting that a smaller influence on cementogenesis on these materials can be expected.     

Bone Morphogenetic Protein-2 Counterregulates Interleukin-18 mRNA and Protein in MC3T3-E1 Mouse Osteoblastic Cells

Fibroblast growth factors-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) are two of the main factors that regulate differentiation of osteoblasts. Interleukin-18 (IL-18), originally cloned as an interferon γ-inducing factor, has been reported to inhibit maturation of osteoclasts by upregulation of osteoprotegerin secreted from osteoblasts. Little is known about the functional relationship between IL-18 and the two growth factors in osteoblast differentiation. To better understand this relationship, we analyzed the effect of BMP-2 and FGF-2 on the mRNA expression levels of IL-18, as well as IL-1α and IL-6, in MC3T3-E1 mouse osteoblastic cells. Following this, the effects of BMP-2 on the expression of IL-18 protein and caspase-1 protein were analyzed by immunofluorescence staining. Real-time PCR and immunofluorescence staining analysis showed that FGF-2 had no effect on the expression of IL-18 mRNA and protein, but while BMP-2 reduced IL-18 mRNA levels, increased immunostaining of both IL-18 protein and caspase-1 protein was detected in BMP-2-treated MC3T3-E1 cells. Although the significance and mechanisms of this counterregulation of IL-18 mRNA and protein were not determined in this study, the increase of IL-18 protein suggested that BMP-2 may induce an active form of IL-18.     

Spironolactone Attenuates Methylglyoxal-induced Cellular Dysfunction in MC3T3-E1 Osteoblastic Cells

BackgroundMethylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells.MethodsMC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured.ResultsPretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone.ConclusionSpironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone''s inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.     

表面矿化修饰煅烧骨/BMP2活性多肽复合MC3T3-E1细胞构建组织工程骨

探讨表面矿化修饰煅烧骨/BMP₂活性多肽复合材料、表面矿化修饰煅烧骨和单纯煅烧骨复合MC3T3-E1细胞构建的组织工程骨在体内的成骨能力。实验分3组,A组：表面矿化修饰煅烧骨/BMP₂活性多肽复合材料,B组：表面矿化修饰煅烧骨,C组：单纯煅烧骨。诱导培养MC3T3-E1细胞,将其分别接种于3组材料,倒置相差显微镜和环境扫描电镜分别观察细胞生长情况。将18只新西兰大白兔随机分成3组,每只大白兔作两侧骶棘肌肌袋模型,然后将3组材料分别植入大白兔骶棘肌内,分别于术后第4、8周各组处死大白兔3只,行组织学观察及新生骨面积测定。通过观察发现MC3T3-E1细胞在3组材料表面生长良好,表面矿化修饰煅烧骨/BMP₂活性多肽复合材料能促进MC3T3-E1细胞在其表面粘附与增殖并能较好地保持细胞的形态。术后第4、8周组织学观察A组材料新生骨明显多于B组,B组多于C组。术后4周和8周新生骨面积测定值A、B、C组分别为（19 712.524±3 782.126）μm²、（28 227.617±2 455.375）μm²,（11 648.507±1 047.221）μm²、（14 592.892±899.532）μm²,（7 986.655±903.487）μm²,（11 254.822±669.508）μm²。表面矿化修饰煅烧骨/BMP₂活性多肽复合材料是一种较理想的骨组织工程复合材料,有望应用于临床。     

Honokiol protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity

Objective  

Honokiol is a phenolic compound isolated from the bark of Magnolia officinalis, a plant widely used in traditional medicine. Antimycin A, which inhibits complex III of the electron transport system, has been used as a reactive oxygen species generator in biological systems. In the present study, we investigated the protective effects of honokiol on antimycin A-induced dysfunction in osteoblastic MC3T3-E1 cells.     

纳米壳聚糖对MC3T3-E1成骨细胞生长的影响

背景：已有体内急性毒理实验证实,壳聚糖纳米微囊的半数致死量高于2 000 mg/kg,但其具体致病机制目前尚不明确。 目的：分析纳米壳聚糖作为骨替代材料对MC3T3-E1成骨细胞生长及大鼠肝、肾等器官生理功能的影响。 方法：将MC3T3-E1成骨细胞分别在含0(对照)、10 mg/L、100 mg/L、1 g/L、10 g/L纳米壳聚糖的DMEM培养液中培养,检测各组细胞A值。透射电镜观察10 g/L纳米壳聚糖溶液培养MC3T3-E1成骨细胞24 h后的细胞形态变化。采用PBS制备10 g/L纳米壳聚糖悬浮液,分别以166.67,16.67 mg/kg经腹腔注射至SD大鼠体内4周,每周3次,正常对照组注射等量生理盐水,血清生化指标分析大鼠肝、肾功能,病理切片观察组织形态学改变、炎症细胞浸润情况。 结果与结论：与对照组比较,10 mg/L、100 mg/L、1 g/L、10 g/L的纳米壳聚糖溶液均抑制MC3T3-E1细胞的生长(P < 0.05)。透射电镜见团聚的壳聚糖存在于MC3T3-E1细胞浆中,细胞表面的伪足形成,细胞膜呈波浪状起伏,细胞核变性、碎裂及固缩。与正常对照组比较,注射纳米壳聚糖悬浮液两组大鼠血尿素氮、Na+水平均有明显升高(P < 0.05),高剂量组K+水平明显降低(P < 0.01);肝脏、肾脏均出现组织细胞凋亡现象,高剂量组凋亡更加明显。表明纳米壳聚糖可导致细胞凋亡,超过一定剂量可造成肾功能受损,对机体生理功能造成影响。     

Role of Sandhika: A Polyherbal Formulation on MC3T3-E1 Osteoblast-like Cells

Sandhika is a polyherbal formulation, (water soluble fraction of Commiphora mukul, Boswellia serrata, Semecarpus anacardium and Strychnos nux vomica), which has been in clinical use in India for last 20 years. Its modified formulation BHUx has shown specific inhibition of cyclooxygenase (COX)-2 and lipoxygenase (LOX)-15 and has prevented diet-induced atherosclerosis in rabbits. In order to explore the possibility of the use of Sandhika for the management of osteoporosis, we have examined its influence on MC3T3-E1 osteoblast-like cells in presence of lipopolysaccharide (1 μg/ml) in terms of calcium nodule formation and alkaline phosphatase activity. MC3T3-E1 osteoblast-like cells (80% confluence in 6-well plates) were treated with water extract of Sandhika, for 10 days, in the concentration range of 0.5 to 16 mg/ml final concentration, in presence of LPS. Media was changed on every third day and culture supernatant was collected after every change to assess the alkaline phosphatase activity and on the tenth day, cells were washed and stained with “Alizarin S” for visualization of calcium nodules by using Meta Morph software (Universal Imaging, Downingtown, PA). The results showed significant enhancement in calcium nodule formation in the dose dependent manner up to 2 mg/ml, followed by gradual decrease at higher concentrations. This change was accompanied with the increase in the alkaline phosphatase activity in these plates, indicating a potential anabolic effect of this polyherbal formulation on osteoblast-like cells under inflammatory conditions induced by LPS.     

Antigenic Definition of Plasma Membrane Proteins of Bacillus Calmette-Guérin: Predominant Activation of Human T Cells by Low-Molecular-Mass Integral Proteins

Mycobacterial plasma membrane proteins, in particular the detergent-soluble or 'integral' ones, comprise a class of mostly unexplored antigens capable of inducing potent activation of human T cells. Plasma membrane isolated from culture-grown Bacillus Calmette-Guérin (BCG; Indian vaccine; Danish strain) was subjected to a Triton X-114-based biphasic extraction procedure for isolation of peripheral (water-soluble) and integral proteins (PMP and IMP). A distinction between the two protein pools was evident from results of SDS-PAGE and immunoblotting using antisera raised in rabbits. An enzyme-linked immunosorbant assay with a panel of WHO-IMMYC monoclonal antibodies against various mycobacterial antigens revealed that three well-known antigens, 19 kDa, 33/36 kDa (proline rich) and 38 kDa (PstS homologue), were part of the IMP pool; and another such antigen, 14/16 kDa alpha-crystallin homologue, partly constituted the PMP pool. Apparently, antigenically distinct species of the immunomodulatory moiety lipoarabinomannan partitioned in aqueous and detergent phases. Human T-cell proliferation assays in donors comprising tuberculoid leprosy and pulmonary tuberculosis patients and healthy BCG vaccinees showed significantly greater potency of IMP over PMP and this immunodominance appeared to be directed towards CD4+ cells. IMP of < 56 kDa were resolved by 'continuous elution SDS-PAGE' into 15 fractions which, after extraction of SDS, were used in T-cell proliferation assays for the identification of immunodominant constituents. Proteins falling within three low-molecular-mass zones (all < 35 kDa) performed better than the rest, particularly a approximately 22 kDa fraction, which strongly stimulated T cells from all five donors. Partial overlap between IMP and secreted proteins, as noticed in this study, could provide clues to immunodominance of the latter. The apparent uniqueness and a high T-cell activating potency make mycobacterial IMP attractive candidates for designing future vaccines or immunotherapeutic agents.     

Mycobacterium bovis Bacillus Calmette Guérin infection prevents apoptosis of resting human monocytes

Apoptosis plays an essential role in the development and homeostasis of multi-cellular organisms. Some infectious agents interfere with this programmed cell death to their own benefit. Here, we show that infection of resting human monocytes with Mycobacterium bovis Bacillus Calmette Guérin (BCG) increases monocyte viability by preventing them from undergoing apoptosis. Heat-killed BCG also prevented apoptosis, indicating that replication of BCG is not required to prevent cell death. Analysis of BCG-infected monocytes revealed an up-regulation of the A1 mRNA, whereas the bcl-2 mRNA was not up-regulated. Interestingly, preinfection with BCG renders the cells resistant to interleukin (IL)-10-induced apoptosis which may be one of the mechanisms mycobacteria use to modulate immune responses. BCG infection was also accompanied by an impairment of the capacity of monocytes to secrete IL-10 and by an induction of the capacity to secrete tumor necrosis factor-α, two cytokines known to induce and prevent human monocyte apoptosis, respectively. Since it has been reported that apoptosis is involved in killing of intracellular mycobacteria, the prevention of apoptosis may represent a strategy for mycobacterial survival in the infected host.     

周期性机械牵拉通过上调Runx2表达促进MC3T3-E1细胞迁移

目的 探究周期性牵拉对小鼠胚胎成骨细胞MC3T3-E1迁移功能的影响及其相关机制。方法 使用应变加载系统对体外培养的MC3T3-E1细胞施加15%幅度的牵拉,模拟细胞体内受力情况。使用划痕愈合实验检测MC3T3-E1细胞的迁移功能,使用蛋白免疫印迹法检测Runx2表达情况,使用RNA干扰技术特异性降低Runx2表达量。结果 幅度15%、频率1.25 Hz、持续24 h的周期性机械牵拉可以促进MC3T3-E1细胞的迁移,升高细胞内Runx2表达水平。在静态培养条件下,干扰Runx2能抑制MC3T3-E1细胞的迁移。干扰MC3T3-E1细胞中Runx2表达可以部分降低机械牵拉对细胞迁移的促进效应。结论 周期性牵拉可以促进MC3T3-E1细胞的迁移,在此过程中Runx2可能发挥重要作用。研究结果为寻找促进骨折愈合的创新临床治疗方法提供实验依据。     

Contribution of α/β and γ/δ T lymphocytes to immunity against Mycobacterium bovis Bacillus Calmette Guérin: studies with T cell receptor-deficient mutant mice

Mutant mice with defined T cell deficiencies were infected with Mycobacterium bovis bacillus Calmette Guérin (BCG) and the relative contribution of α/β T cells and γ/δ T cells to the host immune response was assessed. Recombinase activating gene (RAG-1)^?/? mutants as well as T cell receptor (TcR) β^?/?, but not TcR-δ^?/?, mutants succumbed to M. bovis BCG infection and failed to develop granulomatous lesions. Antigen-induced IFN-γ production by spleen cells in vitro was abrogated in RAG-1^?/? mutants and markedly diminished in TcR-β^?/? and TcR-δ^?/? mice. Reconstitution experiments suggest that both α/β and γ/δ T cells are essential for antigen-specific IFN-γ secretion. Our data formally prove the crucial role of α/β T cells and reveal accessory functions of γ/δ T cells in optimum immunity against M. bovis BCG.     

Protective effect of quercitrin against hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells

The protective effect of quercitrin on the response of osteoblastic MC3T3-E1 cells to oxidative stress was evaluated. Osteoblasts were incubated with H₂O₂ and/or quercitrin, and markers of osteoblast function and oxidative damage were examined. Quercitrin treatment reversed the cytotoxic effect of H₂O₂ significantly (P < 0.05). This effect was blocked by ICI182780 and LY294002, suggesting that quercitrin's effect might be involved in estrogen action and results from PI3K mediated signaling pathway. Pretreatment of quercitrin increased collagen content, alkaline phosphatase (ALP) activity, and calcium deposition of osteoblasts compared with H₂O₂ treated cells and these effects were blocked by ERKs and p38 mitogen-activated protein kinases (MAPKs) inhibitors such as PD98059 and SB203580, respectively. These suggest that quercitrin-induced protective effect against osteoblast dysfunction by oxidative stress is associated with increased activation of ERKs and p38 MAPK. Pretreatment with quercitrin also reduced the increase in bone-resorbing factor, receptor activator of nuclear factor-kB ligand (RANKL) and oxidative damage markers (malondialdehyde, protein carbonyl, and nitrotyrosine) induced by H₂O₂. These results suggest that quercitrin may be protective against H₂O₂-induced dysfunction in osteoblasts.     

mTORC1 信号促进前成骨细胞MC3T3-E1 的分化成熟

目的:研究mTORC1 信号对前成骨细胞MC3T3-E1 向成骨细胞分化成熟的调控作用。方法:通过向MC3T3-E1 转染pcDNA3.1-Raptor,对mTORC1 信号相关蛋白Raptor 进行过表达。向MC3T3-E1 转染Raptor siRNA,对mTORC1 信号蛋白Raptor 进行基因沉默。通过Real-time PCR 方法测定Raptor 的基因表达,通过蛋白免疫印迹法测定Raptor 蛋白水平,并通过茜素红染色检测成骨矿化情况,以测定成骨分化程度。通过Real-time PCR 检测成骨分化指标的基因表达。结果:与对照组相比,Raptor 过表达组的Raptor mRNA 和蛋白水平明显增加;茜素红染色结果显示Raptor 过表达组染色更深,说明成骨矿化程度更高;荧光定量PCR 结果显示,Raptor 过表达组的成骨分化标记基因以及成骨转录因子的表达量均高于对照组。与对照组相比,Raptor siRNA 组的Raptor mRNA 和蛋白水平明显降低;茜素红染色结果显示Raptor siRNA 组染色更浅,说明成骨矿化程度更低;荧光定量PCR 结果显示,Raptor siRNA 组的成骨分化标记基因以及成骨转录因子的表达量均低于对照组。结论:mTORC1 信号促进前成骨细胞MC3T3-E1 向成骨细胞分化成熟。     

骨形态发生蛋白2诱导C2C12和MC3T3-E1的成骨分化与自噬

背景：一系列研究表明自噬与分化有密切联系;骨形态发生蛋白2是诱导C2C12、MC3T3-E1成骨分化经典途径,是研究成骨分化过程的理想模型。 目的：观察自噬与骨形态发生蛋白2诱导细胞株C2C12、MC3T3-E1成骨分化的关系。 方法：Real-Time PCR检测MC3T3-E1与C2C12在骨形态发生蛋白2(100 μg/L)诱导培养3 d后成骨与自噬相关指标变化。碱性磷酸酶染色检测不同浓度3-甲基腺嘌呤(0,1,5,10 mmol/L)对骨形态发生蛋白2(100 μg/L)诱导培养7 d MC3T3-E1与C2C12成骨指标碱性磷酸酶变化,Western Blot检测C2C12和MC3T3-E1在骨形态发生蛋白2(100 μg/L)诱导不同时间点(0,12,24,48,72,96 h)LC3-Ⅰ/Ⅱ蛋白表达水平。 结果与结论：在骨形态发生蛋白2诱导细胞株C2C12、MC3T3-E1成骨分化过程中,诱导自噬相关mRNA与蛋白水平均有增高趋势,且自噬相关蛋白LC3水平增高与时间相关。同时,抑制自噬成骨分化过程中碱性磷酸酶表达水平降低。因此,自噬与骨形态发生蛋白2诱导细胞株C2C12、MC3T3-E1成骨分化过程有密切关系。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

High glucose promotes apoptosis and autophagy of MC3T3-E1 osteoblasts

IntroductionDiabetes and osteoporosis are common metabolic diseases. Abnormal high glucose can lead to the apoptosis of osteoblasts. Autophagy is a highly conserved cellular process that degrades proteins or organelles. In the present study, we comparatively analyzed the effects of high glucose and glucose fluctuation on apoptosis and autophagy of MC3T3-E1 osteoblasts.Material and methodsMC3T3-E1 cells were respectively treated with different concentrations of D-glucose: 5.5 mM for the control group, 25 mM for the high glucose group and 5.5/25 mM for the glucose fluctuation group.ResultsHigh glucose and glucose fluctuation decreased MC3T3-E1 proliferation and activated autophagy. Also, high glucose and glucose fluctuation might induce the production of reactive oxygen species, decline the mitochondrial membrane potential and trigger apoptosis. The differences in the glucose fluctuation treatment group were more significant. Moreover, N-acetylcysteine, an antioxidant reagent, dramatically eliminated the intracellular reactive oxygen species induced by high glucose and glucose fluctuation, and significantly inhibited the autophagy and apoptosis in MC3T3-E1 osteoblasts. Furthermore, treatment with chloroquine, an inhibitor of autophagy, significantly increased the apoptosis of MC3T3-E1 osteoblasts.ConclusionsHigh glucose, especially high glucose fluctuation, inhibits proliferation and promotes apoptosis and autophagy of MC3T3-E1 osteoblasts. This may occur through inducing oxidative stress and mitochondrial damage in the osteoblasts.     

Compressive Force Induces Osteoclast Differentiation via Prostaglandin E2 Production in MC3T3-E1 Cells

In orthodontic tooth movement, prostaglandin E₂ (PGE₂) released from osteoblasts can alter the normal process of bone remodeling. We previously showed that compressive force (CF) controls bone formation by stimulating the production of PGE₂ and Ep2 and/or Ep4 receptors in osteoblasts. The present study was undertaken to examine the effect of CF on the production of PGE₂, cyclooxygenase-2 (COX-2), macrophage colony-stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) using osteoblastic MC3T3-E1 cells and to examine the indirect effect of CF on osteoclast differentiation using RAW264.7 cells as osteoclast precursors. MC3T3-E1 cells were cultured with or without continuous CF (1.0 or 3.0 g/cm²) for 24 hr, and PGE₂ production was determined using ELISA. The expression of COX-2, M-CSF, RANKL, and OPG genes and proteins was determined using real-time PCR and ELISA, respectively. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of RAW 264.7 cells cultured for 10 days with conditioned medium from CF-treated MC3T3-E1 cells and soluble RANKL. As CF increased, PGE₂ production and the expression of COX-2, M-CSF, and RANKL increased, whereas OPG expression decreased. The number of TRAP-positive cells increased as CF increased. Celecoxib, a specific inhibitor of COX-2, blocked the stimulatory effect of CF on TRAP staining and the production of PGE₂, M-CSF, RANKL, and OPG. These results suggest that CF induces osteoclast differentiation by increasing M-CSF production and decreasing OPG production via PGE₂ in osteoblasts.     

Hydroxyapatite accelerates differentiation and suppresses growth of MC3T3-E1 osteoblasts

Hydroxyapatite describes both the natural mineral phase of bone as well as the widely used calcium-phosphate implant substitute. Given that hydroxyapatite is a major component of the in vivo surface with which osteoblasts interact, it is surprising that most studies examining the regulation of osteoblast growth and differentiation utilize plastic surfaces. Here we demonstrate that the phenotype of mouse MC3T3-E1 osteoblasts is significantly altered on hydroxyapatite compared with plastic surfaces. Specifically, alkaline phosphatase activity and messenger RNA levels, markers of early stages of osteoblast differentiation, are increased in osteoblasts cultured on hydroxyapatite. The precocious appearance of alkaline phosphatase activity on the hydroxyapatite surface suggests that osteoblast differentiation is activated earlier compared with plastic surfaces. Osteocalcin expression, a marker of late-stage differentiation, is also increased on hydroxyapatite and further demonstrates enhanced differentiation. Cell counts indicate that fewer osteoblasts are present on hydroxyapatite versus plastic surfaces 24 h after plating. Measurement of osteoblast attachment, apoptosis, and necrosis indicated no differences between surfaces. In contrast, the number of bromodeoxyuridine-incorporating cells was significantly decreased on hydroxyapatite compared with plastic surfaces. Taken together, our findings indicate that hydroxyapatite enhances osteoblast differentiation while also suppressing growth.     

Bone morphogenetic protein-2 counterregulates interleukin-18 mRNA and protein in MC3T3-E1 mouse osteoblastic cells

Fibroblast growth factors-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) are two of the main factors that regulate differentiation of osteoblasts. Interleukin-18 (IL-18), originally cloned as an interferon gamma-inducing factor, has been reported to inhibit maturation of osteoclasts by upregulation of osteoprotegerin secreted from osteoblasts. Little is known about the functional relationship between IL-18 and the two growth factors in osteoblast differentiation. To better understand this relationship, we analyzed the effect of BMP-2 and FGF-2 on the mRNA expression levels of IL-18, as well as IL-1alpha and IL-6, in MC3T3-E1 mouse osteoblastic cells. Following this, the effects of BMP-2 on the expression of IL-18 protein and caspase-1 protein were analyzed by immunofluorescence staining. Real-time PCR and immunofluorescence staining analysis showed that FGF-2 had no effect on the expression of IL-18 mRNA and protein, but while BMP-2 reduced IL-18 mRNA levels, increased immunostaining of both IL-18 protein and caspase-1 protein was detected in BMP-2-treated MC3T3-E1 cells. Although the significance and mechanisms of this counterregulation of IL-18 mRNA and protein were not determined in this study, the increase of IL-18 protein suggested that BMP-2 may induce an active form of IL-18.