力学刺激对大鼠成骨细胞Integrins α2、β1、β3表达和细胞增殖、合成功能的影响

探讨在成骨细胞中Integrins调节力学信号转导可能的分子机制和周期性双轴力学应变对成骨细胞增殖与合成功能的影响。将3月龄雌性SD大鼠颅顶骨分离的成骨细胞在含10％胎牛血清的F-12培养液中培养并接种在Flexercell type I dish中。当细胞生长至亚融合状态(Subconfluence)，给细胞施加力学刺激，频率为1Hz，力学刺激分别为400、1000、4000μ strain；作用时间分别为每天30min，2h，4h，和8h，共加载2d。以未受力学刺激的细胞为对照组，受力学刺激的细胞为实验组，并进行比较。采用流式细胞技术测定和分析Integrinsα2，β1，β3在成骨细胞膜上的表达量、细胞周期分布与细胞增殖变化；采用同位素标记方法检测成骨细胞骨钙素、Ⅰ型胶原C端前肽(PICP)和总蛋白的分泌量。结果表明：(1)在3月龄雌性SD大鼠成骨细胞膜表面有Integrins α2，β1，β3的表达，β1的表达最高。(2)周期性双轴力学应变对3月龄雌性SD大鼠成骨细胞膜表面Integrins α2，β1，β3的表达量有明显上调作用，但不同强度、不同作用时间的力学应变对成骨细胞膜表面Integrins α2，β1，β3表达量上调的作用亦不相同，而β3对力学刺激最敏感；在4000μ strain力学刺激下，成骨细胞膜表面Integrins α2，β1，β3表达量上调最为明显；(3)周期性双轴力学应变400、1000μ strain的力学刺激可提高成骨细胞的增殖与合成分泌功能；在4000μ strain力学刺激下，可明显增加成骨细胞的增殖功能但成骨细胞的合成分泌功能明显受抑制。在我们实验中，3月龄雌性SD大鼠成骨细胞对力学应变的这些反应提示：(1)Integrins α2，β1，β3表达量随着应变幅度值增大而增高。(2)Integrinsα2、β1、β3的表达量的变化与成骨细胞的增殖分化功能有密切的相关关系，在低应变幅度值，随Integrinsα2、β1、β3的上调，可增加成骨细胞的增殖与合成分泌功能；在高应变幅度值，随Integrinsα2、β1、β3的上调，可明显提高成骨细胞增殖功能，但明显抑制成骨细胞的合成功能。表明Integrinsα2、β1、β3在成骨细胞的力学信号转导和细胞增殖、功能活性方面具有重要的调节作用。     

缺氧大鼠成骨细胞增殖、分化及基因表达

背景：研究表明,低氧会引起骨折延迟愈合或不愈合,骨密度减低,使骨质疏松、骨折等疾病的发病率升高。成骨细胞是骨形成、生长和发育主要的功能细胞。 目的：观察缺氧对体外培养成骨细胞增殖、分化及基因表达的影响。 方法：选用新生Wistar大鼠颅盖骨,使用胰酶-胶原酶序贯消化法获取成骨细胞,进行体外传代培养及鉴定。在缺氧培养下应用MTT法测定成骨细胞增殖率,对硝基苯磷酸盐法测定成骨细胞碱性磷酸酶活性,反转录-聚合酶链反应法测定成骨细胞内骨钙素及Ⅰ型胶原的表达。 结果与结论：缺氧具有抑制成骨细胞增殖,降低碱性磷酸酶活性及下调大鼠成骨细胞中Ⅰ型胶原α1、骨钙素基因表达的作用,随缺氧时间的增加作用更加明显。提示缺氧可通过抑制成骨细胞增殖、分化成熟及下调Ⅰ型胶原α1、骨钙素基因表达而降低成骨能力,从而促进骨质疏松的发生。     

恒定磁场中大鼠成骨细胞的增殖和功能活性

背景：磁场对成骨细胞生物学的影响存在一定的分歧。 目的：探讨不同强度恒定磁场作用不同时间后成骨细胞增殖和功能活性的改变。 方法：用体外培养的SD大鼠成骨细胞第3~5代分别在0,5,22,86,135 mT恒定磁场下培养,观察8,12,24 h后细胞增殖和凋亡变化及细胞上清液中骨钙素及Ⅰ型前胶原C端前肽的表达。 结果与结论：磁场作用8,12 h后,5,22,86,135 mT磁场培养的细胞增殖指数均高于对照组(P < 0.05),作用24 h时,仅5 mT组高于对照组(P < 0.05)。而0,5,22,86,135 mT恒定磁场下培养8,12,24 h的凋亡百分数差异无显著性意义。磁场作用8 h后,22,86,135 mT组骨钙素分泌量均高于对照组(P < 0.05);作用24 h后,135 mT组骨钙素分泌量则明显低于对照组(P < 0.05);而磁场作用8,12 h后,22,86 mT组Ⅰ型前胶原C端前肽分泌量明显高于对照组(P < 0.05),作用24 h后,135 mT组Ⅰ型前胶原C端前肽分泌量则明显低于对照组(P < 0.05)。表明低强度磁场、短时间作用可促进成骨细胞的增殖及成骨物质的分泌,而高强度磁场或磁场暴露时间过长则抑制成骨细胞增殖及成骨物质的分泌。     

力学刺激对MG-63成骨样细胞增殖分化的影响

为探讨力学刺激对成骨细胞增殖分化的影响,应用根据四点弯曲原理设计的加力装置对MG-63成骨样细胞进行力学刺激,观察不同大小和作用时间的机械应变对成骨细胞增殖分化的影响。本研究中蛋白表达、碱性磷酸酶(ALP)活性测定分别采用Western blot、α-磷酸奈酚法,采用茜素红染色观察矿化结节的形成。结果发现:⑴与对照组相比,加力组增殖细胞核抗原(PCNA)蛋白表达、ALP活性均明显增加,但这种作用并不随着力刺激大小的增加而增加。⑵在适宜的力刺激(2 000μstrain)下,随着加力时间的增加,PCNA、Ⅰ型胶原(COLⅠ)蛋白表达及ALP活性逐渐增加,并且适宜的力刺激也促进了矿化结节的形成。该结果提示适宜的力学刺激能够促进成骨细胞的增殖分化。     

rhBMP-7对人真皮成纤维细胞增殖和碱性磷酸酶活性的影响

碱性磷酸酶(ALPase)的活性是成骨细胞分化成熟的重要标志之一,其活性的高低可反映细胞钙化的能力和向成骨细胞分化的趋势[1]。重组人骨形成蛋白(rhBMP)可刺激成骨细胞、类成骨细胞及已分化的骨细胞,促进其增殖、胶原合成以及细胞中碱性磷酸酶和骨钙素的表达升高[2]。本实验中主     

去卵巢骨质疏松大鼠骨髓间充质干细胞体外成脂肪分化能力的研究

探讨不同月龄、去卵巢对大鼠骨髓间充质干细胞(MSCs)向脂肪细胞分化能力的影响。选用健康3月龄和6月龄Spraque-Dawley(SD)雌性大鼠行双侧卵巢切除术,建立骨质疏松症的动物模型;使用密度梯度离心法分别获取各组骨髓间充质干细胞(MSCs),体外培养传至3代,加入成脂肪诱导液进行诱导,油红O染色观察细胞内脂滴;并用RT-PCR法检测成脂肪分化标志物脂蛋白脂酶(LPL)mRNA表达水平。实验分为8组:1)3月龄正常大鼠骨髓间充质干细胞组(MSCs3control);2)3月龄去卵巢骨质疏松大鼠骨髓间充质干细胞组(MSCs3ovx);3)6月龄正常大鼠骨髓间充质干细胞组(MSCs6control);4)6月龄去卵巢骨质疏松大鼠骨髓间充质干细胞组(MSCs6ovx);5)3月龄正常大鼠骨髓间充质干细胞成脂肪诱导组(MSCs ADI3control);6)3月龄去卵巢骨质疏松大鼠骨髓间充质干细胞成脂肪诱导组(MSCs ADI3ovx);7)6月龄正常大鼠骨髓间充质干细胞成脂肪诱导组(MSCs ADI6control);8)6月龄去卵巢骨质疏松大鼠骨髓间充质干细胞成脂肪诱导组(MSCs ADI6ovx)。结果显示:(1)年老组大鼠MSCs成脂肪细胞分化能力大于年幼组大鼠;(2)成脂肪诱导后,成脂肪诱导各组MSCs胞浆内均有脂滴形成,MSCs ADI3ovx和MSCs ADI6ovx明显多于相应对照组。(3)成脂诱导后,与相应的对照组相比,MSCs ADI3ovx和MSCs ADI6ovx LPL mRNA表达水平明显增高。研究结果表明,年老组和去卵巢骨质疏松大鼠(MSCs)向脂肪细胞分化能力皆增强。     

动态力学应变对人骨膜细胞的生物学作用

探讨动态力学应变对体外分离培养的人骨膜细胞的生物学效应。使用Flexercell应力系统，将频率为1Hz、振幅为5％变形、正弦波状力学应变作用于体外培养的人骨膜细胞，在不同时间段检测细胞总蛋白含量、DNA合成量、ALP活性及骨钙素分泌量，并与空白对照组相比较，观察动态力学应变对人骨膜细胞生长、分化指标的影响。结果表明：所施加的动态力学应变对骨膜细胞的生长增殖无明显影响，但显著促进骨膜细胞向成骨细胞的分化。动态力学应变能够促进人骨膜细胞向成骨细胞分化，这可能是其对骨的生物学作用的机制之一。     

力学应变对大鼠骨髓间充质干细胞增殖和成骨分化能力的影响

探讨周期性双轴力学应变对大鼠骨髓间充质干细胞(M esenchym a l stem ce lls,M SC s)增殖和成骨分化能力的影响。选用9月龄健康SD雌性大鼠,分离股骨、胫骨提取骨髓,采用密度梯度离心法分离M SC s。体外培养M SC s传至第3代,以1×105细胞浓度接种于双轴力学应变系统,选取4 000μstra in,频率为1hz的力学应变对M SC s加载。每天加载3次,每次2 h,间隔2 h。观察力学应变作用后1 d、3 d,M SC s增殖和成骨分化能力的变化,并与相应未加力学应变对照组比较。结果表明:(1)力学应变可增高M SC s的碱性磷酸酶(ALP)和骨桥蛋白(OPN)表达量;力学应变作用3 d后M SC s的ALP和OPN表达量明显高于力学应变作用1 d。I型胶原(COL I)仅在力学应变作用3 d增高;骨钙素(OCN)在各组无明显变化。(2)力学应变可促进M SC s增殖,但力学应变作用1 d和3 d对M SC s增殖的作用无明显差异。上述结果提示:力学应变可以促进M SC s的增殖和成骨分化能力。     

磁性纳米多孔复合支架材料的细胞相容性研究

目的研究磁性纳米多孔复合(n-HA/PLLA/Fe2O3)材料的细胞相容性,探讨细胞在材料表面黏附、增殖、表达等生物学行为,为其医学应用提供实验依据。方法将大鼠成骨细胞与磁性纳米多孔复合材料共培养,采用CCK-8法检测细胞增殖、扫描电镜观察细胞在材料上的黏附、RT-PCR检测I型胶原和骨钙素基因的表达。结果 CCK-8检测显示实验组磁性纳米多孔复合材料上细胞的增殖与空白对照组没有差异性(0.05);扫描电镜观察到细胞在磁性纳米多孔复合材料的表面和孔隙内大量黏附、增殖和生长,随着共培养时间的增加,材料表面的细胞数量明显增多;RT-PCR显示随着共培养时间的增加,I型胶原基因的表达增强(0.05),骨钙素的表达无明显差异(0.05)。结论磁性纳米多孔复合支架材料适于成骨细胞的黏附、生长和分化,具有良好的细胞相容性。     

动态力学信号对人骨髓基质细胞、骨膜细胞生长和成骨表达的作用

目的 探讨动态力学信号对体外分离培养的人骨髓基质细胞、骨膜细胞生长与分化特征的生物学效应。方法 使用Flexcell应力系统，将频率为1Hz、振幅为5％变形、正弦波状力学信号作用于体外分离培养的正常人骨髓基质细胞和骨膜细胞，在不同时间段检测其对细胞DNA、总蛋白合成、碱性磷酸酶（ALP）表达和骨钙素分泌量的影响。结果 动态力学刺激对人骨髓基质细胞、骨膜细胞蛋白与DNA合成无明显作用。接受力学刺激信号后骨膜细胞受维生素D3刺激后分泌骨钙素显著增加，而骨髓基质细胞则显著下降。结论 动态力学信号能够促进人骨膜细胞向成骨细胞分化，这可能是其对骨的生物学作用的机制之一。     

周期性张应变作用下成骨细胞凋亡的体外研究

目的 研究机械周期性张应变对体外培养的成骨细胞凋亡的作用及其与细胞增殖和细胞分化之间的关系。方法 酶消化法分离培养新生SD大鼠颅顶骨成骨细胞,P2-P4代成骨细胞培养2d和4d后,用无血清培养法诱导成骨细胞凋亡,同时使用Flexcell 4000TM加力系统分别对细胞施加72h变形率为6%和13.6%的等轴循环牵张力。根据总培养时间分为5d和7d两组。运用流式细胞技术对成骨细胞凋亡水平进行检测,同时作细胞记数及碱性磷酸酶（ALP）蛋白含量检测。结果 与不加力的对照组相比,6%牵张力下5d组成骨细胞凋亡率下降了45%,成骨细胞数量增加了34%。在13.6%牵张力下7d组成骨细胞凋亡率增加了192%,细胞数量下降了64%。ALP活性在两种力值大小的牵张力刺激下均有下降。结论 周期性张应变对成骨细胞的凋亡有影响,不同力值大小的牵张力可通过促进或抑制细胞凋亡的方式调控成骨细胞的活性。     

Expression of vascular endothelial growth factor in normal, osteoarthritic and osteoporotic osteoblasts

To evaluate vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis in primary human osteoblast cultures from healthy, osteoporotic and osteoarthritic subjects. Normal primary human osteoblast cultures were obtained from healthy subjects undergoing surgery for the reduction in traumatic fractures. Pathological osteoblasts were obtained from patients undergoing to total hip replacement for osteoporotic hip fracture or advanced osteoarthritis. VEGF mRNA expression and protein synthesis were evaluated in cultured cells, by semiquantitative real-time PCR and ELISA, respectively, both under basal conditions than after vitamin D₃ stimulation. Osteoarthritic osteoblasts showed a significantly higher VEGF expression compared to the normal and OP osteoblasts, both under basal conditions than in the presence of vitamin D₃, whereas no difference was found between osteoporotic and normal osteoblast. Vitamin D₃ significantly enhanced VEGF expression in normal and pathological osteoblasts. This preliminary study supports the hypothesis that VEGF is involved in the pathogenic mechanisms underlying the bone alterations typical of osteoarthritis and confirms the crucial role of vitamin D₃ supplementation in metabolic bone diseases.     

不同质量浓度异烟肼对体外培养乳鼠成骨细胞的毒性作用

文题释义： 异烟肼：对结核杆菌有抑制和杀灭作用,其生物膜穿透性好,由于疗效佳、毒性小、价廉、口服方便,故被列为首选抗结核药。该药品常需和其他抗结核病药联合应用,以增强疗效和克服耐药菌。 成骨细胞：主要由内外骨膜和骨髓基质内的间充质始祖细胞分化而来,能特异性分泌多种生物活性物质,调节并影响骨的形成和重建过程。 背景：异烟肼作为抗结核的一线药物,对细胞内外结核菌都具有极强的杀菌效果,但目前还没有研究报道异烟肼对成骨细胞的毒性作用。 目的：探讨不同质量浓度异烟肼对新生SD大鼠成骨细胞增殖、分化的影响。 方法：将不同质量浓度(0,10,20,30,40,50,60,100 mg/L)异烟肼分别加入新生SD大鼠第3代成骨细胞中,采用CCK-8法、碱性磷酸酶活性试剂盒和免疫荧光染色法检测异烟肼对成骨细胞增殖、分化的影响。 结果与结论：与对照组比较,异烟肼质量浓度在30 mg/L时,成骨细胞增殖开始受到抑制;随着异烟肼质量浓度增高,碱性磷酸酶活性、Ⅰ型胶原表达明显降低。结果表明,异烟肼质量浓度过高对成骨细胞增殖和分化具有抑制作用。 ORCID: 0000-0002-8210-9063(陈强) 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     

Bone histology in young adult osteoporosis.

Bone histology was quantitated in 10 osteoporotic patients aged between 17 and 51 years and in six healthy subjects aged between 23 and 43 years. The osteoporosis was of varying aetiology and was clinically stable. All patients were given tetracycline before biopsy and double tetracycline labelling was used in seven patients. Bone forming and resorbing surfaces were defined by the presence of osteoblasts and osteoclasts, respectively, which were identified by histochemical techniques. The associations between bone forming and resorbing surfaces were similar in patients and controls, though the range of values was wider in the patients than in the controls. Mineral apposition rate was normal in the osteoporotic patients, but there was a reduction in mineralising (tetracycline) surface, whether related to osteoid surface or to osteoblast surface. This did not indicate osteomalacia as the directly and indirectly measured mineralisation lag times were normal. The osteoid seams were thinner in osteoporotic patients than in controls. The data suggest that osteoclast and osteoblast numbers were normal in this group of osteoporotic patients but that the metabolic activity of osteoblasts was impaired.     

Anti-osteoactivin antibody inhibits osteoblast differentiation and function in vitro

Osteoactivin (OA) is a novel protein identified by mRNA differential display using bone from osteopetrotic versus normal rats. Bioinformatic analysis showed that OA cDNA has an open reading frame of 1716 bp encoding a protein of 572 aa, the first 21 aa constitute a signal peptide. OA sequence analysis also demonstrated 13 putative N-glycosylation sites suggestive of a heavily glycosylated protein. In this study, we localized OA protein in primary osteoblast culture by immunofluorescent staining and Western blot analysis. Primary osteoblast cultures pass through three stages: proliferation from day 1 to 7, matrix formation from day 7 to 14, and matrix mineralization from day 14 to 21. OA protein was detected at all stages examined, with maximal expression at 3 weeks when osteoblasts are terminally differentiated. Using the Chariot transfection reagent as a vehicle to deliver anti-OA antibody into the cells, we demonstrated that anti-OA antibody significantly inhibited osteoblast differentiation markers, including alkaline phosphatase activity, nodule formation, osteocalcin production, and calcium deposition, without affecting cell proliferation or viability. These data suggest that OA is an osteoblast-related protein that plays an important role in the regulation of osteoblast differentiation and function.     

Bone tissue engineering on patterned collagen films: an in vitro study

This study aimed at guiding osteoblast cells from rat bone marrow on chemically modified and patterned collagen films to study the influence of patterns on cell guidance. The films were stabilized using different treatment methods including crosslinking with carbodiimide (EDC) and glutaraldehyde, dehydrothermal treatment (DHT), and deposition of calcium phosphate on the collagen membrane. Mesenchymal osteoprogenitor cells were differentiated into osteoblasts and cultured for 7 and 14 days on micropatterned (groove width: 27 microm, groove depth: 12 microm, ridge width: 2 microm) and macropatterned (groove width: 250 microm, groove depth: 250 microm, ridge width: 100 microm) collagen films to study the influence of pattern dimensions on osteoblast alignment and orientation. Fibrinogen was added to the patterned surfaces as a chemical cue to induce osteoblast adhesion. Cell proliferation on collagen films was determined using MTS assay. Deposition of calcium phosphate on the surface of the film increased surface hydrophilicity and roughness and allowed a good cell proliferation. Combined DHT and EDC treatment provided an intermediate wettability, and also promoted cell proliferation. Glutaraldehyde crosslinking was found to lead to the lowest cell proliferation but fibrinogen adsorption on glutaraldehyde treated film surfaces increased the cell proliferation significantly. Macropatterns were first tested for alignment and only microscopy images were enough to see that there is no specific alignment. As a result of this, micropatterned samples with the topography that affect cell alignment and guidance were used. Osteoblast phenotype expression (ALP activity) was observed to be highest in calcium phosphate deposited samples, emphasizing the effect of mineralization on osteoblast differentiation. In general ALP activity per cell was found to decrease from day 7 to day 14 of incubation. SEM and fluorescence microscopy revealed good osteoblast alignment and orientation along the axis of the patterns when micropatterned films were used. This study shows that it is possible to prepare cell carriers suitable for tissue engineering through choice of appropriate surface topography and surface chemistry. Presence of chemical cues and micropatterns on the surface enhance cell orientation and bone formation.