葛根素联合国产多孔钽对人成骨细胞COL-I、OC、OPN表达和细胞增殖的影响

目的研究探索葛根素联合国产多孔钽支架材料对人成骨细胞MG-63 COL-I、OC、OPN表达和细胞增殖的影响。方法实验分为:葛根素组:不同浓度的葛根素与成骨细胞共培养(CCK-8法筛选出葛根素作用最佳剂量浓度组);多孔钽组:国产多孔钽与成骨细胞共培养;葛根素多孔钽组:葛根素、国产多孔钽与成骨细胞共培养;空白组:成骨细胞培养。扫描电镜观察多孔钽及葛根素多孔钽-成骨细胞复合物表面形态;CCK-8法检测成骨细胞的生长增殖状态;实时荧光定量PCR法(RT-PCR)和免疫细胞化学染色法检测成骨细胞COL-I、OC、OPN mRNA和蛋白的表达。结果葛根素和多孔钽组较空白组均可促进成骨细胞的增殖(P0.05);葛根素多孔钽组较其它各组细胞的增殖活性最显著(P0.05)。RT-PCR法和免疫细胞化学染色法结果显示,葛根素和国产多孔钽对成骨细胞COL-I、OCmRNA和蛋白表达均有影响(P0.05),但对OPN mRNA和蛋白无显著影响(P0.05)。结论葛根素和国产多孔钽支架具有协同影响成骨细胞COL-I、OC、OPN mRNA和蛋白的表达,以及促进成骨细胞的增殖。     

不同程度低氧培养对多孔钽-人成骨细胞复合物细胞形态及COL-Ⅰ、OC蛋白表达的影响

[目的]探讨低氧培养条件对多孔钽-成骨细胞形态学、增殖能力以及COL-Ⅰ、OC蛋白表达的影响。[方法]选用MG63成骨细胞原代培养及传代。将第3代细胞以1×10~6/L~(-1)细胞浓度接种于多孔钽材料进行复合培养。采用三气培养箱,以20%氧体积分数作为常氧环境,氧体积分数2%作为缺氧环境,同时设1%及3%氧体积分数作为缺氧反应对比条件。实验分组:将多孔钽-成骨细胞复合物分为4组:常氧钽组(对照组),3%缺氧钽组、2%缺氧钽组及1%缺氧钽组(实验组)。CCK-8法检测多孔钽-成骨细胞复合培养第1、3、5、7、9、11 d各组成骨细胞生长和增殖状态;扫描电镜观察缺氧对多孔钽-成骨细胞复合物形态特征影响;透射电镜观察缺氧条件下成骨细胞超微结构改变;免疫细胞化学法检测缺氧各组成骨细胞COL-Ⅰ、OC蛋白的表达。[结果]CCK-8法检测显示缺氧条件下各实验组与对照组比较,细胞随培养时间的延长,成骨细胞生长及增殖速度明显放慢,除第1 d外,其余各时间点OD值差异均有统计学意义(P<0.05);各实验组组间比较除第3 d外,其余时间点各组间比较差异均存在统计学意义(P<0.05),扫描电镜显示在缺氧条件下多孔钽-成骨细胞对缺氧反应表现为体积变小,伪足少而细,只分泌极少量的细胞外基质覆盖于多孔钽表面。透射电镜显示:在缺氧条件下钽-成骨细胞复合培养与常氧培养的成骨细胞相比,线粒体、内质网扩张,空泡样变性,线粒体嵴断裂或消失。免疫细胞化学检测结果得出:缺氧条件下各实验组成骨细胞COL-Ⅰ、OC蛋白表达较对照组显著降低(P<0.05)。[结论]多孔钽-成骨细胞复合物在缺氧条件下成骨细胞增殖能力降低,细胞形态及超微结构改变,功能降低以及成骨蛋白COL-Ⅰ、OC表达降低。提示:监测骨缺损局部氧分压将有助于三维结构骨移植材料内部成骨细胞生长及骨形成量。     

Bcl2及Bak1重组表达腺病毒对MG63细胞的影响

目的研究Bcl2、Bak1重组表达腺病毒载体单独或共转染对MG63细胞活性及结缔组织生长因子(connective tissue growth factor,CTGF)、Runx2、骨桥蛋白(osteopontin,OPN)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的影响。方法收集对数期MG63细胞,分为4组:A组:空载腺病毒干预组;B组:Bcl2重组表达腺病毒转染组;C组:Bak1重组表达腺病毒转染组;D组:Bcl2、Bak1重组表达腺病毒共转染组。A组给予空载腺病毒转染,B、C组分别给予Bcl2重组表达腺病毒、Bak1重组表达腺病毒转染,D组给予Bcl2、Bak1重组表达腺病毒共转染,MTT法检测细胞活性,碱性磷酸酶(alkaline phosphates,ALP)活性检测试剂盒检测ALP活性,流式细胞仪检测钙离子浓度,Western Blot检测CTGF、Runx2、OPN、TNF-α的表达。结果与A组相比,B组细胞活性、ALP活性升高,钙离子浓度降低,CTGF、OPN、Runx2蛋白相对表达量均升高,TNF-α表达量则降低,差异有统计学意义(P0.05);C组细胞活性、ALP活性降低,钙离子浓度升高,各蛋白相对表达趋势与B组相反,差异有统计学意义(P0.05);D组细胞活性、ALP活性、钙离子浓度升高,CTGF、Runx2相对表达量升高,OPN、TNF-α相对表达量降低,但差异均无统计学意义(P0.05)。(2)B组、C组、D组各检测指标组间比较,差异均有统计学意义(P0.05)。结论 Bcl2重组表达腺病毒载体转染MG63细胞可增强细胞活性及钙化能力,促进骨形成。     

RNA干扰沉默CTGF表达对人成骨样MG63细胞I型胶原、ALP mRNA表达的影响

目的本研究将利用RNA干扰技术,阻断CTGF在人成骨样MG63细胞中的表达,观察CTGF降表达后对人成骨样MG63细胞Ⅰ型胶原、碱性磷酸酶 mRNA表达的影响。方法针对人CTGF mRNA440、875、910位点设计、合成3对21核苷酸siRNA（siRNA1、siRNA2、siRNA3）;在阳离子脂质体介导下转染人成骨样MG63细胞,以空白及非特异性siRNA作为对照,转染48h后收集细胞。采用Northern杂交研究CTGF mRNA表达水平的改变,半定量RT-PCR观察Ⅰ型胶原、ALP mRNA表达的改变,Western blotting观察CTGF蛋白表达的变化。MTT法测定RNA干扰后人成骨样MG63细胞活力的改变。结果空白对照组相比,转染siRNA1、siRNA3的MG63细胞CTGF mRNA和蛋白表达明显下调,转染siRNA2及非特异性siRNA的MG63细胞CTGF的表达无明显变化。转染siRNA1、siRNA3的MG63细胞Ⅰ型胶原、ALP mRNA表达明显下调,细胞活力明显降低。结论针对人CTGF mRNA设计、合成的siRNA可有效抑制人成骨样MG63细胞CTGF的转录和表达;CTGF表达下调可抑制MG63细胞表型标志物Ⅰ型胶原、ALP mRNA的表达,降低MG63细胞活力,这说明CTGF可能在维持骨代谢平衡中具有重要的作用。     

益肾胶囊对糖尿病肾病肾组织SOCS3及Col-Ⅰ、Ⅳ表达的影响

目的:观察益肾胶囊对糖尿病肾病大鼠肾组织SOCS-3、Ⅰ、Ⅳ型胶原表达的影响,探讨益肾胶囊延缓糖尿病肾病肾纤维化的机制。方法:36只健康雄性Wistar大鼠,通过尾静脉单次注射链脲佐菌素(60 mg/kg),制造糖尿病大鼠模型,后随机分为DN模型组、益肾胶囊组、氯沙坦钾组,各12只,益肾胶囊组每只灌胃益肾胶囊(625 mg.kg-1.d-1),氯沙坦组每只灌胃氯沙坦钾片(30 mg.kg-1.d-1),另取12只健康大鼠作为正常对照组。正常对照组及模型组每日给予等量蒸馏水。12周后测定各组大鼠体重、24 h尿蛋白定量、血肌酐、尿素氮;采用HE、Masson和PAS染色观察肾组织病理变化,采用免疫组化法检测肾组织中SOCS-3、Ⅰ、Ⅳ型胶原表达水平。结果:实验12周末,与正常组比较,模型组大鼠病理改变较明显,24 h尿蛋白定量、血肌酐、尿素氮明显上升(P<0.05),肾组织中SOCS-3、Ⅰ、Ⅳ型胶原表达明显上调。与模型组比较,治疗组大鼠病理改变减轻,24 h尿蛋白定量、血肌酐、尿素氮水平明显降低(P<0.05);肾组织中SOCS-3表达显著上调,Ⅰ、Ⅳ型胶原表达显著抑制(P<0.05)。结论:益肾胶囊可能通过上调SOCS-3表达,抑制Ⅰ、Ⅳ型胶原的表达,从而减轻了DN大鼠肾小球硬化和肾小管间质纤维化的程度,延缓糖尿病肾病的进展。     

热损伤角质形成细胞培养上清对真皮成纤维细胞Ⅰ、Ⅲ型胶原及基质金属蛋白酶1表达的影响

目的探讨角质形成细胞(keratinocytes,KC)热损伤后对真皮成纤维细胞(fibroblasts,Fb)Ⅰ、Ⅲ型胶原及基质金属蛋白酶1(matrix metalloproteinase 1,MMP-1)表达的影响。方法分离培养人正常Fb及KC,分别建立KC、Fb热损伤模型;收集正常及热损伤12 h后细胞培养上清,并配制成浓度为50%的细胞条件培养液。根据培养液不同将第3～5代Fb分为3组,分别采用含50%热损伤KC培养上清(A组)、含50%正常KC培养上清(B组)的条件培养液及单纯DMEM(C组)培养,24 h后收集3组细胞;另于培养0、1、2、6、12、24、48 h分别收集A组细胞。采用含50%热损伤Fb培养上清的条件培养液培养Fb,于0、1、2、6、12、24、48 h收集细胞。采用实时荧光定量PCR检测各时间点KC热损伤条件培养上清对FbⅠ、Ⅲ型胶原及MMP-1 mRNA表达影响,以及Fb热损伤条件培养上清对Fb MMP-1mRNA表达影响。结果 KC热损伤条件培养上清培养24 h,A组Ⅰ、Ⅲ型胶原及MMP-1 mRNA相对表达量均显著高于B、C组,差异有统计学意义(P<0.05)。培养2、6、12、24、48 h,A组Ⅰ、Ⅲ型胶原及MMP-1 mRNA相对表达量均高于0 h(P<0.05),1 h与0 h差异无统计学意义(P>0.05);随培养时间延长相对表达量逐渐增高,2 h后各时间点间比较差异均有统计学意义(P<0.05)。Fb热损伤条件培养上清培养1、2、6、12、24、48 h,MMP-1 mRNA相对表达量与0 h比较,差异均有统计学意义(P<0.05);培养2 h后相对表达量逐渐降低,各时间点间差异均有统计学意义(P<0.05)。结论热损伤后KC培养上清对FbⅠ、Ⅲ型胶原及MMP-1的表达具有调控作用。     

胰岛素样生长因子结合蛋白相关蛋白1在活化肝星状细胞中的表达及意义

目的 研究胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor binding protein-related protein-1,IGFBP-rP1)在转化生长因子β1(tronstorming growth factor β1,TGFβ1)诱导的肝星状细胞中表达的变化及抗IGFBP-rP1抗体对TGFβ1诱导的肝星状细胞产生Ⅰ型胶原的影响.方法 大鼠肝星状细胞株(HSC-T6)体外培养,分别设空白对照组(加入等量PBS)、不同浓度的TGFβ1.处理组、不同浓度的抗IGFBP-rP1抗体处理组,处理因素作用24h,采用免疫组织化学染色、Western blot检测IGFBP-rP1在HSC-T6中的表达,ELISA检测Ⅰ型胶原的含量并进行IGFBP-rP1与Ⅰ型胶原的相关性分析.结果 TGFβ1各处理组IGFBP-rP1的表达均较空白对照组显著增强.抗IGFBP-rP1抗体可以拮抗TGFβ1诱导的HSC-T6产生的过多Ⅰ型胶原.IGFBP-rP1与Ⅰ型胶原呈正相关(r=0.833,P＜0.01).结论 IGFBP-rP1参与了肝纤维化的形成,且可能在肝纤维化的发生、发展中占有重要地位.     

手术创伤中腹膜组织Sp1、COL1A1和TIMP-1的表达

目的　探讨人手术创伤腹膜组织中核转录因子Sp1激活 ,COL1A1和TIMP 1表达变化与腹膜纤维化之间的关系。方法　采用凝胶电泳迁移率改变分析法 (EMSA)检测手术创伤后不同时间的腹膜组织核转录因子Sp1的表达水平 ,WesternBlot检测COL1A1和TIMP 1蛋白表达 ,Masson染色观察腹膜组织中胶原纤维的变化。结果　Sp1在手术创伤后 0 .5h被活化 ,随着手术时间延长Sp1活性逐渐增强 ,至创伤后 4h时达高峰 ,同时创伤腹膜组织中的COL1A1和TIMP 1蛋白表达水平逐渐升高 ,存在差异显著性 (P <0 .0 1)。在手术创伤期内随手术时间的延长腹膜组织中胶原纤维增加。结论　核转录因子Sp1活化导致Ⅰ型胶原合成增加 ,细胞外基质降解减少 ,从而启动腹膜纤维化进程。     

基因物质p63、β1整合素对正常人不同部位皮肤表皮干细胞的表达和意义

目的　探讨基因物质 p63、β1整合素对正常人不同部位皮肤表皮干细胞的表达情况及意义。方法　在 5例正常人尸体上各取 2 1个不同部位的全层皮肤组织 ,采用免疫组织化学SP法 ,用基因物质 p63、β1整合素作为第一抗体标记表皮干细胞 ,并通过图像分析 ,研究表皮干细胞在正常人不同部位皮肤中的表达情况。结果　 (1)头皮 p63的PU值 [阳性单位 (% ) ] (4 2 .17± 3 .10 )最大 ,与面皮、腋部、上臂内侧、前臂内侧、腰背部、手背、足背及足底差异有显著性 (P <0 .0 5 ) ;足背p63的PU值(2 1.19± 6.89)最小 ,与身体其他部位差异均有显著性 (P <0 .0 5 )。头皮CD2 9的PU值 (2 9.13± 4.99)最大 ,与身体其他部位差异均有显著性 (P <0 .0 5 ) ;足底CD2 9的PU值 (11.89±1.5 2 )最小 ,阴囊与面皮、腰背、上臂内外侧、手掌、手背、腹部及足底差异有显著性 (P <0 .0 5 )。(2 )正常人不同部位多毛组与汗毛组 p63、CD2 9的PU值差异有显著性(P <0 .0 0 0 1)。(3 )躯干组与四肢组p63、CD2 9的PU值差异无显著性 (P >0 .0 5 )。(4 )屈侧与伸侧p63、CD2 9的PU值差异无显著性 (P >0 .0 5 )。结论　 (1) p63、CD2 9对正常人不同部位皮肤表皮干细胞的表达不同 ,头皮、阴阜、阴囊皮肤组织中的阳性表达较高 ,而其他部位皮肤组织中     

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.     

Immunoelectron microscopy of osteonectin and type I collagen in osteoblasts and bone matrix

Summary The pathway of production, secretion, and extracellular deposition of type I collagen and osteonectin was studied by immunoelectron microscopy using respective polyclonal antibodies. Protein A gold and immunogold methods yielded to similar results in human callus tissue used as a model of bone formation. The intracellular distribution of osteonectin in active osteoblasts is found as a faint immunolabeling of vesicular Golgi fields and some lamellae of rough endoplasmic reticulum. A more intensive labeling occurs in opaque cytoplasmic vesicles pointing to the process of secretion as some of the vesicles are connected with the basal cell membrane. Our type I collagen antibody did not label the respective intracellular compartments. Extracellulary, the type I collagen antibody showed a continuous labeling from the immature subcellular osteoid to the mineralized bone. Osteonectin antibodies were bound first to the deeper layer of osteoid maturation with intensity increasing below the mineralization front. Osteonectin is thought to be associated with mineralization of bone matrix.     

染料木黄酮对成骨细胞C-fos、ALP及骨钙蛋白基因表达的影响

目的研究不同浓度的染料木黄酮（genistein，GEN）对体外培养大鼠成骨细胞C-fos、碱性磷酸酶（alkaline phosphatase，ALP）及骨钙蛋白（osteocalcin）基因表达的影响。方法采用新生大鼠颅骨分离培养成骨细胞，加入不同浓度的染料木黄酮（10^-8mol·L^-1、10^-7mol·L^-1、10^-6mol·L^-1、10^-5mol·L^-1），以妊马雌酮（商品名：倍美力）（10^-8mol·L^-1、10^-7mol·L^-1、10^-6mol·L^-1、10^-5mol·L^-1）作为阳性对照组，提取总RNA，RT-PCR半定量分析其表达。结果染料木黄酮对成骨细胞C-fos、ALP及骨钙蛋白基因水平的影响呈正相关性，并与其剂量成正相关性，但这些作用均低于雌激素水平（P〈0．05）。结论染料木黄酮能诱导成骨细胞C-fos、ALP及骨钙蛋白基因的表达，并与其浓度有关，作用与雌激素相似。     

重组人胰岛素样生长因子I对大鼠成骨细胞增殖、凋亡及I型胶原蛋白合成的影响

目的观察胰岛素样生长因子I(IGF-I)对体外培养的大鼠成骨细胞的增殖、凋亡及I型胶原蛋白合成的影响,探讨IGF-I对骨代谢影响的可能机制。方法不同浓度rhIGF-I刺激体外培养的大鼠成骨细胞,采用噻唑蓝(MTT)法测定细胞增殖能力;肿瘤坏死因子α(TNF-α)单独或与rhIGF-I共同刺激成骨细胞,流式细胞仪检测细胞周期和凋亡率;rhIGF-I刺激成骨细胞,免疫细胞化学结合计算机图像分析系统检测I型胶原蛋白的表达。结果一定浓度IGF-I能明显增加成骨细胞数量(P<0·05),在0·1~100ng/ml这种作用与IGF-I的浓度呈正相关;TNF-α在0·1~100ng/ml浓度范围内呈剂量依赖性地促进成骨细胞凋亡(P<0·05),并使S期细胞减少(P<0·05),而IGF-I能抑制TNF-α对成骨细胞的促凋亡作用(P<0·05);经IGF-I的刺激,成骨细胞I型胶原蛋白的表达明显高于对照组(P<0·05)。结论IGF-I对大鼠成骨细胞有明显的促增殖作用,在0·1~100ng/ml之间呈浓度依赖性,IGF-I能抑制TNF-α诱导的大鼠成骨细胞凋亡,IGF-I能促进大鼠成骨细胞I型胶原蛋白的合成。     

Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures

IntroductionOsteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization.We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event.MethodsIn this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined.Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis.Results64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p < 0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~ 0; p = 0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p = 0.029). ucOC levels were higher in the fracture group although not significantly (p = 0.058). No differences were found regarding total OC (p = 0.602), apoE (p = 0.467) and Vitamin K (p = 0.371).In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (β = 0.607; p = 0.013) and stiffness (β = 0.693; p = 0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (β = − 0.411; p = 0.030) and stiffness (β = − 0.487; p = 0.009).ConclusionWe demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.     

Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures

IntroductionOsteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization.We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event.MethodsIn this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined.Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis.Results64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p < 0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~ 0; p = 0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p = 0.029). ucOC levels were higher in the fracture group although not significantly (p = 0.058). No differences were found regarding total OC (p = 0.602), apoE (p = 0.467) and Vitamin K (p = 0.371).In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (β = 0.607; p = 0.013) and stiffness (β = 0.693; p = 0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (β = ? 0.411; p = 0.030) and stiffness (β = ? 0.487; p = 0.009).ConclusionWe demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.     

Influence of vitamin D and retinoids on the gammacarboxylation of osteocalcin in human osteosarcoma MG63 cells

Osteocalcin (OC) is a bone matrix protein, synthesized by osteoblasts, which contains three residues of gammacarboxyglutamic acid (GLA). A fraction of circulating OC, which is not fully carboxylated and does not bind to hydroxyapatite, is called undercarboxylated OC (ucOC). In elderly institutionalized women, we have shown an increase of circulating ucOC level which may result not only from vitamin K deficiency but also from vitamin D deficiency (Szulc et al., J Clin Invest 91: 1769; 1993). This intriguing finding prompted us to study the effect of vitamin D on the secretion of ucOC by osteoblastic cells in vitro in the presence of warfarin, an inhibitor of gammacarboxylation of GLA-containing proteins. The potential influence of retinoic acid (RA) was also studied, because its mechanism of action involves pathways that are similar to vitamin D. In the presence of warfarin (0.05 μg/mL), 1,25(OH)₂D (10⁻⁸-10⁻⁶ mol/L) decreased dose dependently ucOC secretion by human osteosarcoma MG63 cells (from 3.87 ± 0.96 to 2.12 ± 0.13 ng/10⁶ cells). When expressed as a fraction of total OC, secretion ucOC decreased from 47.4 ± 1.4% to 24.8 ± 3.2% in the MG63 cells. The secretion of total OC was stimulated by RA and by Ro 13-7410, which is a specific ligand of retinoic acid receptor (RAR), but not by 9-cis retinoic acid (9-cisRA), which is a physiologic ligand of retinoid X receptor (RXR). RA and Ro 13-7410 decreased ucOC secretion and ucOC% in warfarin-treated MG63 cells (RA: from 50.4 ± 13.3% to 13.5 ± 2.8%; Ro 13-7410: from 28.4 ± 8.2% to 11.3 ± 8.4%). 9-cisRA had no effect on OC gammacarboxylation. These results show that vitamin D, RA, and Ro 13-7410, but not 9-cisRA, may modify the gammacarboxylation of OC in human MG63 cells.