山羊颈椎间盘纤维环成纤维细胞成骨潜能的体外观察

目的 诱导培养椎间盘纤维环成纤维细胞，探讨其成骨潜能，比较重组人骨形成蛋白2（recombinant human bone morphogenetic protein2，rhBMP-2）和肿瘤坏死因子α（tumor necrosis factor α，TNF—α）对其成骨的不同影响。方法利用体外细胞培养技术，建立实验山羊颈椎间盘纤维环成纤维细胞培养体系，根据培养液的不同，分为空白对照组、TNF-α组（加入50U／ml TNF-α）、rhBMP-2组（加入0．1μg／ml rhBMP-2）和TNF—α＋rhBMP-2组（加入50U／ml TNF-α＋0．1μg／ml rhBMP-2），培养3周观察纤维环成纤维细胞在条件培养液诱导下的成骨表型变化、碱性磷酸酶（alkaline phosphatase，ALP）和骨钙素（osteocalcin，OC）等成骨指标变化。结果 各实验组培养液对细胞增殖无明显影响。rhBMP-2组和TNF-α＋rhBMP-2组细胞趋化性生长明显，矿化结节出现，茜素红染色呈深红色钙阳性反应。各实验组ALP活力与空白对照组比较差异均有统计学意义（P〈0．05）。rhBMP-2组与TNF—α＋rhBMP-2组成纤维细胞的OC分泌量与空白对照组比较差异均有统计学意义（P〈0．05），TNF—α组与空白对照组比较差异无统计学意义（P〉0．05）。结论 体外培养的椎问盘纤维环成纤维细胞有成骨潜能，rhBMP-2对纤维环成纤维细胞有明确的诱导成骨作用。     

山羊颈椎间盘纤维环组织成骨潜能的体内观察

目的：观察山羊颈椎椎间融合器内填充松质骨、纤维环组织后在体内的组织学变化过程，以了解纤维环组织在骨融合过程中的成骨潜能。方法：实验山羊按常规颈椎前路减压、内同定术式施术，术中随机取C2～C6椎间隙中相邻的两个间隙．每个间隙各置入两枚钛合金颈椎窄心螺纹式柱状内同定器(CHTF)，分别填充单纯松质骨(A组)：松质骨 纤维环(B组)：纤维环(C组)及空白对照(D组)。术后应用X线片、颈椎CT扫描等影像学检查及组织切片观察植骨融合及局部组织反应情况。结果：X线片及CT示内置CHTF与椎体的骨一金属界面周围有骨组织生长．CHTF与椎体终板接触部位有成骨现象，骨桥形成。A组切片观察示新生软骨、骨小梁存在，原植入骨坏死：B组纤维组织有坏死．原骨小梁、纤维环周围新牛骨存在，新生软骨堆积；C组术后6周纤维组织内有纤维软骨存在．术后12周新生软骨存在；D组术后6周组织学观察无阳性染色结果，术后12周有少量新生软骨。结论：颈椎间盘纤维环组织有成骨潜能，成骨形式可能是成纤维细胞的软骨化骨。     

成纤维细胞成骨潜能的体外培养研究

本研究采用新西兰种家兔皮肤的成纤维细胞进行体外培养，通过活体相关显微缩时录像，体视显微镜摄影，ＨＥ染色以及组织化学染色等技术做系列观察。发现成纤维细胞在体外培养条件下，不仅能产生粘多糖，胶原等基质成分，而且还能提供钙盐沉积。培养中期，成纤维细胞培养液显示Ａ１Ｐ阳性反应；培养后期，培养液与成纤维细胞都可显示Ａ１Ｐ阳性反应。     

成纤维细胞成骨潜能的研究进展

在体内组织中，有两类生成骨细胞的前体细胞：成骨前体细胞(DOPC)和可诱导的成骨前体细胞(IOPC)。前者主要存在于骨髓的基质细胞中，具有干细胞的特点，能自身复制分化成一定类型的细胞，此种基质细胞可分化成为成骨细胞、成纤维细胞、网状细胞等，即基质细胞中只有一部分属于骨祖细胞，能自发地分化为成骨细胞，骨髓多能干细胞在     

家兔皮肤成纤维细胞体外成骨潜能的研究

作者将家兔皮肤的中厚皮片制备成小皮块后，作体外培养，在倒置相差显微镜下观察，并作扫描电镜检查，结果显示成纤维细胞自皮块游出，不断增多，开始汇合，细胞呈现鳞片形状，具树枝样分叉，并形成多层结构，成纤维细胞开始分泌众多微小颗粒，堆集在细胞的表面及周围，以后在细胞表面出现细针结晶。结晶及颗粒结合在一起，不断增大，成为结节，成纤维细胞以辐射状排列在结节的四周。相邻的结节联在一起，形成小梁状的组织，用新生骨     

成纤维细胞生长因子增强骨形态发生蛋白在小鼠体内骨诱导作用

在啮类动物体内植入骨形态发生蛋白后，引起细胞内一系列生化和形态学变化，在植入物内和其周转形成新骨。这过程受多因素影响。本实验将重组人成纤维细胞生长因子与部分纯化的ＢＭＰ悬液混合后注入小鼠肌肉，观察成骨反应。单独注射牛骨ＢＭＰ组和ｂＦＧＦ组为对照。     

骨形态发生蛋白和碱性成纤维细胞生长因子激发纤维环细胞成骨的潜能

目的研究联合使用重组人骨形态发生蛋白（recombinant human bone morphogenetic protein-2,rhBMP-2）和碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）椎间盘纤维环细胞成骨潜能的激发作用。方法向体外培养的纤维环细胞中分别及联合加入rhBMP-2和bFGF,观察纤维环细胞的表型表达特点。结果联合使用rh-BMP-2和bFGF能够明显促进椎间盘细胞增殖,提高细胞内碱性磷酸酶活力,增加I型胶原分泌,提高钙盐沉积程度,提高骨钙素的表达水平。结论联用rhBMP-2和bFGF能够诱导纤维环细胞向成骨细胞方向分化,分泌钙盐并形成钙结节。     

重建端粒酶活性的正常人成纤维细胞的成骨潜能研究

目的　通过重建端粒酶活性延长人成纤维细胞寿命 ,并对其成骨潜能进行研究 ,为解决组织工程骨修复种子细胞老化问题提供实验依据。方法　将人端粒酶催化亚基 (h TERT)基因用电穿孔法导入正常人原代成纤维细胞 ,用 TRAP- PCR检测细胞端粒酶活性 ,用β-半乳糖苷酶活性测定评价细胞衰老情况。在此基础上用骨形成蛋白(BMP- 2 )和肿瘤坏死因子 -α(TNF-α)联合诱导已重建端粒酶活性的成纤维细胞在体外培养条件下成骨 ,用四环素活体标记和茜素红染色显示钙盐结节形成。结果　转染 h TERT的人成纤维细胞能稳定表达端粒酶活性 ,培养超过 5 0代后细胞仍保持β-半乳糖苷酶阴性。经 BMP- 2和 TNF-α诱导后 ,转染后传 80代的人成纤维细胞仍可形成四环素标记和茜素红染色均为阳性的钙盐结节。结论　重建端粒酶活性、寿命延长的人成纤维细胞仍然维持了其本身所具有的成骨潜能     

体外诱导成纤维细胞成骨活性表达的研究

目的探讨成纤维细胞(fibroblast,FB)体外成骨表达的诱导因素,为其能否成为骨组织工程的种子细胞提供理论依据.方法分离和纯化新西兰兔肉芽组织FB,按1×105/ml分别接种于含纤维结合蛋白(fibronectin,FN)10、20、40、60、80μg/ml条件培养液中(实验1～5组),对照组为不含FN的无血清培养液.于培养14 d后,行细胞组织学观察及钙化结节形成率、趋骨性四环素荧光标记、3H-TdR标记、骨钙素测定及3H脯氨酸标记,检测FB成骨表型表达的标志.结果组织学观察实验组发现FB由梭形逐渐趋向多突形和圆形,细胞核偏向一侧,细胞重叠成多层结构;其表面有钙盐堆积,逐渐呈云雾状物质,经不断融合和扩大形成不透光的结节.干预培养14 d钙化结节形成率:对照组15.35%±3.45%,实验1～5组分别为53.73%±9.49%、75.21%±9.80%、98.34%±15.20%、61.83%±10.04%、45.11%±8.70%,实验组与对照组比较,差异有统计学意义(P＜0.05);实验3组与其它各实验组比较,差异有统计学意义(P＜0.05).趋骨性四环素特异性标记为新生骨组织;FB增殖活性,实验3、4、5组7 d时,实验2、3、4组14 d时与对照组比较,差异有统计学意义(P＜0.05).实验1～5组FB分泌骨钙素,实验2～5组3H-脯氨酸掺入量高于对照组,7、14d时与对照组比较差异有统计学意义(P＜0.05).结论适量的FN可以促进FB增殖、胶原蛋白的合成及骨钙素分泌,FN可以诱导FB成骨表达,适宜浓度为40～60μg/ml.     

家兔皮肤成纤维细胞的成骨潜能（组织化学及放射自显影研究）

家兔皮肤的中厚皮片，经制备成小皮块以后作体外培养，成纤维细胞自皮块游出，不断增多，以后开始汇合，形成多层结构，成纤维细胞分泌众多颗粒，堆积在细胞的表面，逐步形成去雾状物质，云雾状物质增大，成为圆形或卵圆形结节。相邻的结节可被条状小梁联在一起，用新生骨细胞的特异标记物－四环素及茜素红，作组织化学观察，结节及小梁均呈阳性反应，说明成纤维细胞所形成的结节及小梁均为骨组织，将体外培养的成纤维细胞用＾Ｈ－Ｔ     

颈椎关节突关节矢状角不对称对颈椎间盘纤维环应力影响的三维有限元研究

目的:利用有限元技术探究颈椎关节突关节矢状角不对称对颈椎间盘纤维环和关节突关节的应力影响.方法:选择一名26岁健康女性志愿者的颈椎CT扫描数据构建颈椎有限元模型.首先,构建C3-C7的颈椎正常模型,该模型通过先前发表的研究数据进行了验证.其次,提取C5-C6的数据,并改变C5-C6关节突关节矢状角的角度以重新构建实验模...     

脱细胞纤维环基质对兔纤维环源干细胞分化的影响

目的观察脱细胞纤维环基质(DAFM)对纤维环源干细胞(AFSC)分化行为的影响,为新型纤维环组织工程支架材料的开发提供依据。方法将从新西兰大白兔获得的AFSC接种于由猪纤维环组织制备的DAFM膜和无DAFM膜培养皿,分别进行成脂、成骨、成软骨分化诱导,考察DAFM对AFSC分化行为的影响。结果 AFSC在DAFM膜上生长良好。DAFM膜上AFSC成脂、成软骨诱导分化水平低于无DAFM膜者,而成骨诱导分化水平显著高于无DAFM膜者;DAFM膜上的成脂、成骨及成软骨诱导比值(诱导组基因表达量/对照组基因表达量)均高于无DAFM膜者。结论猪DAFM有利于促进兔AFSC成骨分化,抑制其成脂及成软骨分化,较好地维持AFSC的差异性分化潜能。     

Both expression of cytokines and posterior annulus fibrosus rupture are essential for pain behavior changes induced by degenerative intervertebral disc: An experimental study in rats

The aim of this study was to analyze the relationship between intervertebral disc degeneration and low back pain (LBP). Rat L4/5 disc degeneration model was established by annular puncture using a 0.4 mm needle anteriorly or posteriorly. In both anterior and posterior puncture models, magnetic resonance imaging (MRI) and histological analyses revealed marked disc degeneration 2 weeks after puncture. Cytokine expression was up‐regulated in different level in nucleus pulposus (NP) from 3 days after puncture. Pain behavioral tests indicated that the anterior disc puncture did not induce pain behavior changes, whereas the posterior disc puncture resulted in mechanical allodynia from 1 day to 21 days after injury. Besides, cytokine expression was significantly increased in dorsal root ganglion (DRG) at 1 and 2 weeks after posterior puncture, but not after the anterior puncture. These findings indicate the NP of the degenerative disc expresses different levels of inflammatory cytokines, and posterior disc puncture produced mechanical allodynia. The expression phase of cytokines in the NP was accordance with mechanical hyperalgesia in the posterior disc puncture model. Both expression of cytokines and posterior annulus fibrosus (AF) rupture in degenerative intervertebral disc are essential for pain behavior changes. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:262–272, 2014.     

不同接种方法构建组织工程化椎间盘纤维环细胞支架复合体的对比研究

目的 在以脱矿脱细胞骨基质环为支架以纤维环细胞为种子细胞构建组织工程化椎间盘纤维环细胞支架复合体过程中,探索最佳的构建复合体方法.方法 分别使用纤维蛋白凝胶接种技术和传统的直接接种技术一静置法构建技术构建组织工程化椎间盘纤维环细胞支架复合体.对构建产物进行倒置显微镜观察、扫描电镜观察和细胞计数,比较两方法的效果.结果 纤维蛋白凝胶接种技术构建的细胞支架复合体.细胞粘附更多、增殖更迅速.结论 纤维蛋白凝胶接种技术在以脱矿脱细胞骨基质环为支架以纤维环细胞为种子细胞构建组织工程化椎间盘纤维环细胞支架复合体过程中,比静置法效果更好.     

经皮穿刺纤维环建立兔腰椎间盘退变模型

目的探讨使用自制穿刺针经皮穿刺纤维环制备兔腰椎间盘退变模型的可行性。方法将18只新西兰大白兔分为实验组、假手术组及空白对照组。实验组及假手术组使用自制穿刺针穿刺L_(3~4)、L_(4~5)和L_(5~6)椎间盘位置,实验组穿刺椎间盘深度为5 mm,假手术组钝性穿刺但不损伤椎间盘,空白对照组不作处理。术后3、6、9周每组取2只兔麻醉后行腰椎MRI检查,处死行大体观察并取椎间盘行HE染色及髓核蛋白多糖含量测定。结果术后3周开始实验组MRI信号强度、髓核蛋白多糖含量与其他两组相比明显下降(P0.05),其后呈逐渐下降趋势,大体观察及HE染色显示实验组髓核及纤维环呈逐渐退变趋势。结论自制穿刺针经皮穿刺纤维环法能成功建立兔腰椎间盘退变模型,具有操作简单、损伤小、动物存活率高等优点。     

Mechanical properties of the extra‐fibrillar matrix of human annulus fibrosus are location and age dependent

The mechanical behavior of the annulus fibrosus (AF) of the intervertebral disc can be modeled as a mixture of fibers, extra‐fibrillar matrix (EFM), ions, and fluid. However, the properties of the EFM have not been measured directly. We measured mechanical properties of the human EFM at several locations, determined the effect of age and degeneration, and evaluated whether changes in EFM properties correspond to AF compositional changes. EFM mechanical properties were measured using a method that combines osmotic loading and confined compression. AF samples were dissected from several locations, and mechanical properties were correlated with age, degeneration, and composition. EFM modulus was found to range between 10 and 50 kPa, increasing nonlinearly with compression magnitude and being highest in the AF outer‐anterior region. EFM properties were not correlated with composition or degeneration. However, the EFM modulus, its relative contribution to tissue modulus, and model parameters were correlated with age. These measurements will result in more accurate predictions of deformations in the intervertebral disc. Additionally, parameters such as permeability and diffusivity used for biotransport analysis of glucose and other solutes depend on EFM deformation. Consequently, the accuracy of biotransport simulations will be greatly improved. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1725–1732, 2013     

Quantifying the contributions of structure to annulus fibrosus mechanical function using a nonlinear, anisotropic, hyperelastic model.

The annulus fibrosus of the intervertebral disc is comprised of concentric lamella of oriented collagen fibers embedded in a hydrated proteoglycan matrix with smaller amounts of minor collagens, elastin, and small proteoglycans. Its structure and composition enable the disc to withstand complex loads and result in inhomogeneous, anisotropic, and nonlinear mechanical behaviors. The specific contributions of the annulus fibrosus constituent structures to mechanical function remain unclear. Therefore, the objective of this study was to use a structurally motivated, anisotropic, nonlinear strain energy model of annulus fibrosus to determine the relative contributions of its structural components to tissue mechanical behavior. A nonlinear, orthotropic hyperelastic model was developed for the annulus fibrosus. Terms to describe fibers, matrix, and interactions between annulus fibrosus structures (shear and normal to the fiber directions) were explicitly included. The contributions of these structures were analyzed by including or removing terms and determining the effect on the fit to multidimensional experimental data. Correlation between experimental and model-predicted stress, a Bland-Altman analysis of bias and standard deviation of residuals, and the contribution of structural terms to overall tissue stress were calculated. Both shear and normal interaction terms were necessary to accurately model multidimensional behavior. Inclusion of shear interactions more accurately described annulus fibrosus nonlinearity. Fiber stretch and shear interactions dominated contributions to circumferential direction stress, while normal and shear interactions dominated axial stress. The results suggest that interactions between fibers and matrix, perhaps facilitated by crosslinks, elastin, or minor collagens, augment traditional (i.e., fiber-uncrimping) models of nonlinearity.