循环牵张应力对人退变纤维环细胞增殖的影响

目的 探讨不同水平循环牵张应力对人退变椎间盘细胞增殖的影响.方法 取1例29岁手术切除的腰椎间盘突出患者的椎间盘,通过病理学方法评价其退变程度,酶消化法分离椎间盘纤维环细胞.选取P3代细胞,利用ElectroForce 3200力学试验仪搭载的BioDynamic生物反应舱系统,以3h为固定时间点对其进行不同水平的频率...     

Research development of tissue-engineering in intervertebral disc anulus fibrosus

Disc degeneration disease (DDD) is the major cause of lower back pain, resulting in significant serious social problems and economic burdens due to its high mortality rate and disability rate. Currently available clinic treatments are inefficient for solving spinal structure and function insufficiency caused by the DDD, especially for patients with degenerated or broken anulus fibrosus. Tissue engineering provides a promising and available strategy to regenerate a new anulus fibrosus with complete bio-functions. This review following primary introduces the current research progress and the potential development orientation of tissue engineering anulus fibrosus.     

A stimulation unit for the application of mechanical strain on tissue engineered anulus fibrosus cells: a new system to induce extracellular matrix synthesis by anulus fibrosus cells dependent on cyclic mechanical strain

A bioreactor system consisting of a multifunctional stimulation unit and common 6-well culture plate is introduced to activate extracellular matrix synthesis in intervertebral disc cells due to cyclic mechanical strain. The developed stimulation unit is sterilizable and reusable. It is viable for cultivation and mechanical stimulation of cartilage tissue and tissue engineered cell matrix constructs in combination with the common 6-well culture plate. The custom made device allows long-term cultivations in batch- or continuous operation mode. Manual handling and thereby the risk of contamination is reduced. Sampling, changing the medium, and addition of supplements are easily performed from the connected conditioning vessel. This bioreactor system enables stimulation of different samples independently during one run. For the work presented here anulus fibrosus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs. Over a period of 14 days the constructs were subjected to 10% compression under cyclic mechanical pressure with a frequency of 0.1 Hz. Afterwards the constructs were biochemically examined for hydroxyproline and sulphated glycosaminoglycanes. These proven constituents of extracellular matrix were found to be released depending on the applied compressive strain.     

纤维环损伤诱导兔椎间盘退变模型

目的:探讨纤维环刺伤法诱导腰椎间盘退变建立动物模型的可行性.方法:普通级新西兰大白兔(雄性,体质量约3.5 kg,1岁龄).分为正常对照组及模型组,以腰4～5椎问盘为正常对照组,以每只兔的腰2～3椎间盘为模型组.用16号穿刺针于腰2～3椎间盘纤维环前外侧刺入,分别于术前及术后4、8、16周对模型组及正常对照组椎问盘进行磁共振(MRI)检查,动物处死取材后行组织学及免疫组织化学观察.结果:术后4～16周,模型组椎间盘髓核MRI T<,2>W<,1>信号呈逐渐减弱趋势,组织学观察髓核脊索细胞逐渐减少,免疫组织化学观察到髓核中Ⅰ型胶原表达较正常对照组逐渐增多,而Ⅱ型胶原表达较对照组逐渐减少.结论:本实验可为椎间盘退变的进一步研究提供动物模型.     

周期性牵张椎间盘纤维环细胞肌动蛋白骨架的重排

BACKGROUND: When the intervertebral disc is under stress, the hydraulic pressure generated inside the nucleus pulposus makes the annulus fibrosus extend outward and expand, and the annulus collagen fibers are stretched so that the extracellular matrix of annulus fibrosus cells is also under the pressure. In the intervertebral disc, aggrecan is the main component of proteoglycans, matrix metalloproteinase-2 is a major enzyme for extracellular matrix degradation, and tissue inhibitor of metalloproteinase is a multifunctional specific inhibition factor for matrix metalloproteinase activity. There is a mutual regulation between the latter two to keep the homeostasis between them. OBJECTIVE: To investigate the mechanism of cyclic tensile strain in the metabolism of intervertebral disc annulus matrix. METHODS: Rat anulus fibrosus cells were subjected to 2% or 10% cyclic tensile strain at 1.0 Hz for 2 and 12 hours using Flexcell4000 tension system. Then cells were collected and cultured in conditioned medium for gene and protein detection. Real-time quantitative PCR was used to detect mRNA expression of aggrecan, matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2. Gelatin zymography was used to detect matrix metalloproteinases-2 activity. RESULTS AND CONCLUSION: The use of 2% cyclic tensile strain had no obvious effect on the stress fiber of actin cytoskeleton, whereas actin cytoskeleton was depolymerized in response to 10% cyclic tensile strain. The 2% cyclic tensile strain raised the expression of Aggrecan at 12 hours; whereas raised the matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2 at 2 hours, both of which were in homeostasis; matrix metalloproteinases-2 activity had no significant changes. 10% cyclic tensile strain had no effect on the mRNA expression of Aggrecan. No matter stretching 2 or 12 hours, the matrix metalloproteinases-2 was up-regulated, and the tissue inhibitor of metalloproteinase-2 was down-regulated, both of which were not in balance. Moreover, the matrix metalloproteinases-2 activity was not significantly changed. These findings indicate that the mRNA expressions of Aggrecan, matrix metalloproteinases-2 and tissue inhibitor of metalloproteinase-2 alter in response to cyclic tensile strain in rat anulus fibrosus cells, and the tensile strain induces different mechano-responses in the actin cytoskeleton.      

Regional variations in the density and arrangement of elastic fibres in the anulus fibrosus of the human lumbar disc

Elastic fibres are critical components of the extracellular matrix in dynamic biological structures that undergo extension and recoil. Their presence has been demonstrated in the anulus fibrosus of the human lumbar intervertebral disc; however, a detailed regional analysis of their density and arrangement has not been undertaken, limiting our understanding of their structural and functional roles. In this investigation we have quantitatively described regional variations in elastic fibre density in the anulus fibrosus of the human L3-L4 intervertebral disc using histochemistry and light microscopy. Additionally, a multiplanar comparison of patterns of elastic fibre distribution in the intralamellar and interlamellar zones was undertaken. Novel imaging techniques were developed to facilitate the visualization of elastic fibres otherwise masked by dense surrounding matrix. Elastic fibre density was found to be significantly higher in the lamellae of the posterolateral region of the anulus than the anterolateral, and significantly higher in the outer regions than the inner, suggesting that elastic fibre density in each region of the anulus is commensurate with the magnitude of the tensile deformations experienced in bending and torsion. Elastic fibre arrangments in intralamellar and interlamellar zones were shown to be architecturally distinct, suggesting that they perform multiple functional roles within the anulus matrix structural hierarchy.     

The superficial anulus fibrosus ligament. An incipient description of a separate ligament between the lumbar anterior longitudinal ligament and the intervertebral disc

A previously unknown ligament, the superficial anulus fibrosus ligament (SAFL), situated on the ventral part of the L5 intervertebral disc (ID) was observed and described from autopsy material. Twenty-eight cadaveric specimens from 12 black and 16 white persons aged 17-30 years were studied during routine forensic autopsies. The anterior longitudinal ligament was separated from the ID and the ventral part of the SAFL was visualized. The SAFL samples were removed, measured and studied with both conventional histology and examination by transmission electron microscopy. The SAFL was a completely separate ligament at the level of the L5-S1 ID situated between the anterior longitudinal ligament and the anulus fibrosus of the ID. The fibers of the ligament were vertically oriented. A difference in distance between the L5-S1 vertebral bodies ventrally was noted in the two groups studied (18.7 +/- 1.2 mm in the black vs. 15.2 +/- 1.0 mm in the white, p < 0.001), indicating a difference in the ventral thickness of the intervertebral disc. Also, there was a difference in the length (black: 17.7 +/- 1.6 mm vs. white: 14.1 +/- 1.1), thickness (black: 3.3 +/- 0.3 mm vs. white: 2.1 +/- 1.9), and the cross-sectional area (black: 58.2 +/- 6.7 mm(2) vs. white: 26.5 +/- 2.7 mm(2), p < 0.001) of the SAFL. Conventional light microscopy revealed no obvious differences. However, transmission electron microscopy revealed notably larger collagen fibril diameter in black than white subjects. In conclusion, the interbody distances were greater in the black group, indicating a greater intervertebral disc thickness, compared to that of the white. Furthermore, the SAFL was significantly longer and thicker in the black than in the white group. Albeit unsubstantiated, these race-specific macroscopic findings may have implications for understanding the etiology of various low back stress problems.     

Ruptures of the anulus fibrosus of cervical intervertebral discs studied by cryomicrotomy and magnetic resonance

Degeneration of the anulus fibrosus in the cervical region has not been studied as systematically as in the lumbar intervertebral discs. We used cryomicrotomy and magnetic resonance (MR) to study the cervical discs in 18 cadavers. Three types of anular tears were identified: (1) a concentric tear due to delamination of the anulus, (2) a radial tear in which all layers of the anulus were disrupted, and (3) a transverse tear of the peripheral anulus. The latter two tear types could be demonstrated by MR. On the basis of their frequency the concentric and transverse tears are not likely symptomatic. Radial tears, which some investigators have suggested are symptomatic, may be studied clinically with MR.     

组织工程椎间盘纤维环支架的研究进展*

背景：组织工程为椎间盘退行性病变治疗提供了新的治疗方法。 目的：综述了组织工程椎间盘纤维环支架的种类及构建纤维环支架的不同方法。 方法：应用计算机检索CNKI和PubMed数据库中1988/2011关于组织工程纤维环支架的文章,在标题和摘要中以“组织工程;椎间盘;支架;纤维环;构建;综述”或“tissue engineering, intervertebral disc, scaffold, annulus fibrosus, construction”为检索词进行检索。 结果与结论：纤维环支架是椎间盘组织工程构建的关键,其相关研究尚处于初期阶段。纤维环支架材料有两大类：蚕丝、藻酸盐等天然生物材料;聚氨酯、聚羟基乙酸和聚乳酸及纳米纤维等人工合成材料;支架材料种类繁多,各有利弊。目前用于纤维环支架的构建的方法有致孔剂法、湿纺技术及静电纺丝法。构建的纤维环支架结构与椎间盘纤维环自然结构还有一定差距,目前的方法制作单纤维层比较容易实现,复层纤维环的构建还存在技术的问题,单纤维层的抗拉强度仍然达不到自然结构的标准。因此优化纤维环支架构建方法、固定纤维环支架等方面还需要需进一步的实验研究。      

Effect of strain and strain rate on fatigue-accelerated biodegradation of polyurethane

A diaphragm-type film specimen was used to study in vitro degradation of poly(etherurethane urea) (PEUU) under conditions of dynamic loading. This geometry allowed both uniaxial and biaxial loading in a single experiment. During testing, the film was exposed to a H(2)O(2)/CoCl(2) solution that simulated in vivo oxidation of PEUU. The combination of dynamic loading and biaxial tensile strain accelerated oxidative degradation. The effects of biaxial strain magnitude and strain rate were examined separately by increasing the frequency of fatigue loading from 0 to 1 Hz with constant maximum biaxial strain and by changing the maximum biaxial strain while maintaining constant strain rate. In the ranges of biaxial strain energy (0.17 to 0.55 MPa) and strain rate (0 to 46% s(-1)) tested, the rate of degradation increased with increasing strain rate whereas strain magnitude had essentially no effect on degradation rate. Although loading conditions affected the rate of oxidative degradation, ATR-FTIR analysis suggested that in all cases the mechanism of degradation did not change. Chemical degradation produced a brittle crosslinked surface layer marked by dimpling and pitting, as observed with scanning electron microscopy. Pits served as stress concentrators and initiated environmental stress cracks under dynamic loading but not under static (creep) loading. Small pits were sufficient to initiate cracks at higher strain rates whereas only large pits initiated cracks at lower strain rates. Consequently, a higher strain rate produced more profuse cracking.     

Effect of alternative crosslinking methods on the low strain rate viscoelastic properties of bovine pericardial bioprosthetic material

Early failures of bovine pericardial heart valves have been due to leaflet perforation/tearing and calcification. Since glutaraldehyde fixation has been shown to produce marked changes in leaflet mechanics and has been linked to the development of calcification, alternative crosslinking techniques have been suggested as means to overcome these obstacles. We have examined the low strain rate viscoelastic behavior of bovine pericardium: (1) fresh; (2) chemically treated with glutaraldehyde, cyanimide, or polyglycidyl ether; or (3) physically treated by freeze-drying or heat-drying. Shrinkage temperature tests were conducted to assess intrahelical crosslinking. Polyglycidyl ether and glutaraldehyde both produced substantial crosslinking, with the shrinkage temperature rising above 80 degrees C. Mechanical changes were nearly equivalent, both showing decreased stress relaxation and increased extensibility consistent with intrahelical crosslinking and shrinkage during fixation. Cyanimide, known to crosslink pure collagen materials, showed no evidence of crosslinking intact tissue. Heat-drying, also effective in pure collagen preparations, produced an increase in UTS and tissue modulus, but otherwise left the tissue unchanged. Freeze-drying had no mechanical effect, and therefore provides an attractive means for the storage of connective tissues for later mechanical testing.     

纤维环穿刺法与髓核抽吸法建立兔椎间盘退变模型的比较

目的:比较采用纤维环穿刺法和髓核抽吸法建立兔椎间盘退变动物模型的不同。方法:新西兰大白兔经腹膜外入路暴露腰3/4、腰4/5、腰5/6椎间隙,纤维环穿刺组采取针刺纤维环,髓核抽吸组抽吸髓核8周后核磁共振观察椎间盘变化,生化分析椎间盘中蛋白多糖含量,免疫组织化学检测Ⅱ型胶原表达。结果:术后8周与对照组相比,采用两种方法的椎间隙高度与T2信号手术前后变化比值,以及Ⅱ型胶原和蛋白多糖含量,差异显著。其中髓核抽吸组的Ⅱ型胶原和蛋白多糖含量明显低于纤维环穿刺组。结论:髓核抽吸法建立兔退变椎间盘模型退变程度大于纤维环穿刺法。     

Anisotropic tensile viscoelastic properties of vascular graft materials tested at low strain rates

Mechanical matching of vascular grafts and host vessels has been suggested to be important in determining graft patency rates. In this context, we have examined the anisotropic viscoelastic properties of natural vessels and some synthetic replacements using low strain rate tensile testing of circumferential and longitudinal strips. The canine iliac artery and iliac vein were compared with 6 mm diameter woven and knitted Dacron® grafts, expanded polytetrafluoroethylene (PTFE) grafts, and helically constructed prototype Polyurethane grafts at wrap angles of 45, 60 and 75°. A thick-walled pressure vessel analysis was used to approximate physiological stress levels, and SEM was used to correlate anisotropic properties with graft wall structure.     

经皮纤维环穿刺与经肌间隙纤维环刀刺建立兔椎间盘退变模型

背景：有研究表明椎间盘退变模型的建立可为椎间盘退变治疗提供实验载体,但目前尚缺乏公认的最佳实验动物模型。 目的：比较经皮纤维环穿刺法和经肌间隙纤维环刀刺法建立兔椎间盘退变模型的差异。 方法：将新西兰大白兔分别采用经皮纤维环穿刺法和经肌间隙纤维环刀刺法建立兔椎间盘退变模型,穿刺后4,8,16周通过磁共振和组织病理学检查观察腰椎间盘髓核变性及组织病理情况。 结果与结论：穿刺后4周,两组兔椎间盘髓核内T2加权像信号降低、变暗,椎间隙高度下降,但经皮纤维环穿刺组T2信号强度评分较经肌间隙纤维环刀刺组低(P < 0.05);穿刺后8周,两组T2信号强度评分均增高,经皮纤维环穿刺组T2信号强度评分较经肌间隙纤维环刀刺组低(P < 0.05);穿刺后16周,两组兔椎间盘髓核内T2信号强度评分达最高且差异无显著性意义,两组椎间隙均明显变窄,椎间盘均亮度变黑,两组差异不明显;经皮纤维环穿刺组手术时间少于经肌间隙纤维环刀刺组(P < 0.05),经肌间隙纤维环刀刺组感染率为5.6%,而经皮纤维环穿刺组无感染。结果证实,经皮纤维环穿刺法建立兔椎间盘退变模型建模时间短,感染率低,效果优于经肌间隙纤维环刀刺法造模。中国组织工程研究杂志出版内容重点：人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程全文链接：     

经皮纤维环穿刺法建立兔椎间盘退行性变动物模型

背景：合适的椎间盘退变动物模型是椎间盘组织工程研究的必要条件,但目前尚缺乏公认的模型制备方法。 目的：采用C形臂辅助下经皮纤维环穿刺法制备兔椎间盘退变动物模型,并评估其可行性。 方法：选定新西兰大白兔L2/3和L3/4椎间盘作为穿刺干预椎间盘,L1/2和L5/6椎间盘作为空白对照组,采用C形臂辅助下经皮穿刺法干预椎间盘。于术后2,4,8,12周各选择2只兔麻醉后拍摄兔腰椎核磁共振影像,处死动物采集椎间盘,进行椎间盘髓核蛋白多糖测定。核磁共振检查观察椎间盘退行性改变,二甲基亚甲蓝染色分光光度法测定髓核中蛋白多糖含量变化。 结果与结论：术后4周穿刺干预椎间盘髓核区域核磁共振信号强度及髓核内蛋白多糖含量同空白对照组相比均下降(P < 0.05),其后两者呈现逐渐下降趋势。结果证实,C形臂X射线机辅助下经皮纤维环穿刺法可用于椎间盘退变动物模型的制备。     

Effect of orientation and targeted extracellular matrix degradation on the shear mechanical properties of the annulus fibrosus

The intervertebral disc experiences combinations of compression, torsion, and bending that subject the disc substructures, particularly the annulus fibrosus (AF), to multidirectional loads and deformations. Combined tensile and shear loading is a particularly important loading paradigm, as compressive loads place the AF in circumferential hoop tension, and spine torsion or bending induces AF shear. Yet the anisotropy of AF mechanical properties in shear, as well as important structure-function mechanisms governing this response, are not well-understood. The objective of this study, therefore, was to investigate the effects of tissue orientation and enzymatic degradation of glycosaminoglycan (GAG) and elastin on AF shear mechanical properties. Significant anisotropy was found: the circumferential shear modulus, Gθz, was an order of magnitude greater than the radial shear modulus, Grθ. In the circumferential direction, prestrain significantly increased the shear modulus, suggesting an important role for collagen fiber stretch in shear properties for this orientation. While not significant and highly variable, ChABC treatment to remove GAG increased the circumferential shear modulus compared to PBS control (p=0.15). Together with the established literature for tensile loading of fiber-reinforced GAG-rich tissues, the trends for changes in shear modulus with ChABC treatment reflect complex, structure-function relationships between GAG and collagen that potentially occur over several hierarchical scales. Elastase digestion did not significantly affect shear modulus with respect to PBS control; further contributing to the notion that circumferential shear modulus is dominated by collagen fiber stretch. The results of this study highlight the complexity of the structure-function relationships that govern the mechanical response of the AF in radial and circumferential shear, and provide new and more accurate data for the validation of material models and tissue-engineered disc replacements.     

Regional variations in the cellular matrix of the annulus fibrosus of the intervertebral disc

The three-dimensional architecture of cells in the annulus fibrosus was studied by a systematic, histological examination using antibodies to cytoskeletal components, in conjunction with confocal microscopy. Variations in cell shape, arrangement of cellular processes and cytoskeletal architecture were found both within and between the defined zones of the outer and inner annulus. The morphology of three, novel annulus fibrosus cells is described: extended cordlike cells that form an interconnected network at the periphery of the disc; cells with extensive, sinuous processes in the inner region of the annulus fibrosus; and cells with broad, branching processes specific to the interlamellar septae of the outer annulus. The complex, yet seemingly deliberate arrangement of various cell shapes and their processes suggests multiple functional roles. Regional variations in the organization of the actin and vimentin cytoskeletal networks is reported across all regions of the annulus. Most notable is the continuous, strand arrangement of the actin label at the disc's periphery in contrast to its punctate appearance in all other regions. The gap junction protein connexin 43 was found within cells from all regions of the annulus, including those which did not form physical connections with surrounding cells. These observations of the cellular matrix in the healthy intervertebral disc should contribute to a better understanding of site-specific changes in tissue architecture, biochemistry and mechanical properties during degeneration, injury and healing.     

间歇牵拉应变对大鼠骨髓间充质干细胞增殖与成骨分化的影响

目的探讨体外间歇拉伸应变对大鼠骨髓间充质干细胞(rat bone mesenchymal stem cell,rBMSC)的增殖与成骨分化效应。方法应用Flexcell-4 000应力系统,将间歇拉伸应变(振幅10%,频率0.5 Hz,每天作用2次,4 h/次)作用于体外分离培养的正常rBMSC,加力1、3、5、7 d后检测应力对细胞形态改变、细胞增殖及骨形成核心结合因子Cbfα1、碱性磷酸酶(ALP)和I型胶原等成骨基因和Cbfα1蛋白的影响。结果 rBMSC在间歇牵拉应力作用1 d后即出现增殖速度减缓,一直延续到加力第7 d,ALP和I型胶原等成骨基因的表达自加力3 d后升高了3～6倍(P<0.05),并且Cbfα1的基因和蛋白表达在力学刺激下都出现上调。结论力学刺激在rBMSC的增殖与分化中起重要作用,适宜的间歇拉伸应变可以减缓BMSC的增殖,促进其成骨分化。