The effect of static in vivo bending on the murine intervertebral disc.

BACKGROUND CONTEXT: Intervertebral disc cell function in vitro has been linked to features of the local environment that can be related to deformation of the extracellular matrix. Epidemiologic data suggest that certain regimens of spinal loading accelerate disc degeneration in vivo. Yet, the direct association between disc cell function, spinal loading and ultimately tissue degeneration is poorly characterized. PURPOSE: To examine the relationships between tensile and compressive matrix strains, cell activity and annular degradation. STUDY DESIGN/SETTING: An in vivo study of the biologic, morphologic and biomechanical consequences of static bending applied to the murine intervertebral disc. SUBJECT SAMPLE: Twenty-five skeletally mature Swiss Webster mice (12-week-old males) were used in this study. OUTCOME MEASURES: Bending neutral zone, bending stiffness, yield point in bending, number of apoptotic cells, annular matrix organization, cell shape, aggrecan gene expression, and collagen II gene expression.METHODS: Mouse tail discs were loaded for 1 week in vivo with an external device that applied bending stresses. Mid-sagittal sections of the discs were analyzed for cell death, collagen II and aggrecan gene expression, and tissue organization. Biomechanical testing was also performed to measure the bending stiffness and strength. RESULTS: Forceful disc bending induced increased cell death, decreased aggrecan gene expression and decreased tissue organization preferentially on the concave side. By contrast, collagen II gene expression was symmetrically reduced. Asymmetric loading did not alter bending mechanical behavior of the discs. CONCLUSIONS: In this model, annular cell death was related to excessive matrix compression (as opposed to tension). Collagen II gene expression was most negatively influenced by the static nature of the loading (immobilization), rather than the specific state of stress (tension or compression).     

Mechanical evaluation of a canine intervertebral disc spacer: In situ and In vivo studies

An elastomeric intervertebral disc spacer with hydroxyapatite ingrowth surfaces was implanted in a canine model. We studied (a) the mechanical behavior of motion segments at time 0 and at 3, 6, and 12 months and (b) the effect of the interface between the spacer and vertebral bone on implant stability and bone ingrowth. A polymeric spacer was designed with compressive and torsional properties similar to those of the isolated canine lumbar disc. Implantation of the spacer in canine cadaver motion segments permitted in situ biomechanical evaluation at time 0. An in vivo study permitted continuous neurological monitoring of animals, with evaluation of mechanical behavior, stability, and ingrowth at 3, 6, and 12 months. Mechanical testing of cadaver motion segments with the spacer in situ resulted in decreased compressive and torsional stiffnesses, averaging 25 and 42%, respectively. This decrease was due to a combination of the surgical insult to the annulus and decortication of adjacent vertebral endplates. In the in vivo study, all 12 animals tolerated the surgery well and none had permanent neurological impairment. The measured parameters indicated that behavior of the spacer-motion segment composite appeared to return to normal within 3-6 months. However, despite use of a porous hydroxyapatite on the implant surface, there was no significant bone ingrowth. Instead, a layer of dense fibrous connective tissue was formed at the spacer-vertebral bone interface. Early migration of five of the 12 spacers resulted in eccentric loading patterns with consistent reactive osteophyte formation.     

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。     

Accumulation and localization of macrophage phenotypes with human intervertebral disc degeneration

Background Context

Chronic inflammation is an important component of intervertebral disc (IVD) degeneration, but there is limited knowledge about the identity and source of inflammatory cells involved with the degenerative processes. Macrophages can exhibit multiple phenotypes and are known inflammatory regulators in many tissues, but their phenotypes have not been characterized in IVD degeneration.

血必净对椎间盘突出组织中巨噬细胞表达的影响

[目的]研究中药血必净注射液对椎间盘突出组织中巨噬细胞CD68表达的影响。[方法]以猪为实验动物，手术制作椎间盘突出的动物模型，用血必净试验治疗，检测椎间盘突出组织中CD68表达情况。[结果]模型组CD68阳性率为11／16，低剂量组CD68阳性率为7／16，高剂量组CD68阳性率为6／16，2组与模型组比较P〈0．05。[结论]椎间盘突出组织中的巨噬细胞是引起腰腿痛的重要病理生理基础之一，血必净对其有明显的下调作用，为血必净治疗椎间盘突出症提供了一定的基础理论依据。     

Lumbar intervertebral disc and ligament deformations measured in vivo

The physiologic deformations of the anterior and posterior margins of the annulus fibrosus and the interspinous ligaments were defined in flexion and extension by accurate measurements taken from lateral roentgenograms of the lumbar spines of 11 normal males. The anterior and posterior disc heights were shown to compress and extend up to 35% and 60%, respectively, while the interspinous distance extended up to 369%. In relation to the known mechanical characteristics in vitro, these deformations implied that the soft-tissue elements were lax or in compression during part of the range of motion. Significantly, the interspinous ligament could be active only in the extremes of flexion. These data provide basic information for further studies of the function of the soft parts of the intervertebral joints.     

椎间盘细胞培养及调控

现代科学对椎问盘退变疾病虽然做了大量的研究，但关于体外培养的椎问盘细胞的研究及认识却相对较少。由于细胞与组织培养技术具有较好的重复性、费用低、结果分析较简单，无伦理问题等特点，使其在椎问盘退变研究中引起重视并不断得到应用。     

Leptin expression by annulus cells in the human intervertebral disc.

BACKGROUND CONTEXT: It is now known that leptin acts not only as a metabolic signal related to energy homeostasis but also as an endocrine hormone regulating traditional endocrine systems and neuroendocrine function in various cells. It participates in bone remodeling and acts as a growth factor stimulating proliferation. Expression of leptin and the presence of leptin receptors have not been explored in disc tissue. PURPOSE: To determine (1) whether leptin is produced by cells in the human annulus in vivo, (2) whether annulus cells have leptin receptors in vivo and in vitro, and (3) whether measurable amounts of leptin are produced during three-dimensional culture of human annulus cells. STUDY DESIGN/SETTING: Studies were approved by the human subjects Institutional Review Board. Surgical and donor disc tissue was obtained and assessed by using immunocytochemistry of paraffin-embedded disc tissue. Annulus cells were also cultured from disc specimens and conditioned media assessed for the production of leptin during three-dimensional culture. PATIENT SAMPLE: Disc tissue was examined from 7 young subjects and 29 adult subjects. OUTCOME MEASURES: Immunodetection of leptin and leptin receptors in cells of the human annulus; conditioned media was analyzed for production of leptin in vitro by human disc cells. METHODS: Human annulus tissue and cultured cells were examined by using immunohistochemical methods to identify the presence of leptin and leptin receptors. Human disc cells were assayed for leptin production in three-dimensional culture. RESULTS: Immunocytochemistry showed the presence of intracellular leptin and the presence of leptin receptors in some (but not all) annulus cells in the human disc. Production of leptin by annulus cells was further confirmed by assays of conditioned media from three-dimensional annulus cell culture. CONCLUSIONS: These novel studies identify the presence of a heretofore unrecognized cytokine/hormone and its receptor in human annulus cells. Because of the mitogenic role of leptin in other tissues, the present work points to the importance of future studies to explore whether leptin has a mitogenic function in maintaining disc cell numbers.     

Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer: implications for the clinical management of intervertebral disc disorders

STUDY DESIGN: Human intervertebral disc cells were cultured in monolayer and treated with adenovirus-containing marker genes to determine the susceptibility of the cells to adenovirus-mediated gene transfer. OBJECTIVES: To test the efficacy of the adenovirus-mediated gene transfer technique for transferring exogenous genes to human intervertebral disc cells in vitro. SUMMARY OF BACKGROUND DATA: Upregulated proteoglycan synthesis after direct in vivo adenovirus-mediated transfer of growth factor genes to the rabbit intervertebral disc has previously been reported. Before contemplating extending this approach to the treatment of human disc disease, it is necessary to demonstrate that human intervertebral disc cells are indeed susceptible to adenovirus-mediated gene transduction. METHODS: Human intervertebral disc cells were isolated from disc tissue obtained from 15 patients during surgical disc procedures. The cells were cultured in monolayer and treated with saline containing five different doses of adenovirus carrying the lacZ gene (Ad/CMV-lacZ), saline containing adenovirus carrying the luciferase gene (Ad/CMV-luciferase), or saline alone. Transgene expression was analyzed by 5-bromo-4-chloro-3-indolyl-beta-galactosidase (X-Gal) staining and luciferase assay. RESULTS: Adenovirus efficiently transferred lacZ and luciferase marker genes to cells from degenerated discs as well as to cells from nondegenerated discs. A minimum dose of 150 MOI Ad/CMV-lacZ was found to be sufficient to achieve transduction of approximately 100% of disc cells-regardless of patient age, sex, surgical indication, disc level, and degeneration grade. No statistically significant difference in the luciferase activities could be detected in disc cell cultures from degenerated and nondegenerated discs treated with Ad/CMV-luciferase. CONCLUSIONS: In vitro transducibility of human intervertebral disc cells by adenovirus is relatively insensitive to disc degeneration grade. Because the rate-limiting step for successful gene therapy is the ability to transfer genes efficiently to the target tissue, the achievement of efficient gene transfer to human intervertebral disc cells(using a direct, adenovirus-mediated approach) is an important and necessary step in the development of gene therapy strategies for the management of human intervertebral disc disorders.     

辛伐他汀对兔髓核细胞的影响

目的 观察辛伐他汀对兔髓核细胞Ⅱ型胶原(ColⅡ)及聚集蛋白聚糖(Agg)表达的影响.方法 取兔髓核细胞进行原代培养,传至第3代行ColⅡ免疫组织化学鉴定后随机分为5组,以不同浓度辛伐他汀处理:A组:空白对照组;B、C、D、E组分别为0.1、0.2、0.4、0.8 μmol/L辛伐他汀组.运用半定量逆转录-聚合酶链反应(RT-PCR)检测ColⅡ、Agg含量的变化,并行细胞活力检测.结果 辛伐他汀浓度超过0.2 μmol/L时ColⅡ及Agg的表达增加,0.4 μmoL/L时达到高峰(P＜0.05),0.8 μmol/L时ColⅡ及Agg表达下降.0.8 μmol/L处理组影响细胞活力,0.1～0.4 μmol/L范围时细胞活性无明显影响(P＜0.05).结论 辛伐他汀可促进兔髓核细胞ColⅡ及Agg的表达,改善椎间盘退变进程;在＜0.4 μmol/L的较低浓度内对细胞活力无明显影响.     

In vivo intervertebral disc remodeling: Kinetics of mRNA expression in response to a single loading event

Kinetics of mRNA expression following a single loading event was measured using an in vivo rat tail model. Animals were instrumented and loaded in compression for 1.5 h at 1 MPa and 1 Hz. Real‐time RT‐PCR was used to measure mRNA levels 0, 8, 24 and 72 h after mechanical stimulation for genes associated with matrix proteins (aggrecan, collagen‐I, collagen‐II), proteases (MMP‐2, MMP‐3, MMP‐13, ADAMTS‐4), and their inhibitors (TIMP‐1, TIMP‐3) in anulus fibrosus and nucleus pulposus regions. Baseline mRNA levels were of greatest abundance for matrix proteins and lowest for proteases. The mRNA levels reached maximum levels 24 h following mechanical stimulation for the majority of genes evaluated, but some had maximum levels 8 and 72 h following loading. The mRNA levels returned to baseline levels for all genes in the nucleus 72 h following loading, but the majority of genes in the anulus remained upregulated. Results support a coordinated strategy of relative mRNA expression that varied over time beginning with inhibition of tissue breakdown, followed by synthesis of aggrecan and matrix degrading enzymes, and eventually collagen metabolism days following loading. Consequently, optimal time for tissue harvest for mRNA measurements depends on genes of interest. Results suggest attempts at anabolic remodeling must be given adequate time for metabolic processes and protein synthesis to occur, and that changes in TIMP and MMP levels may have greater potency in affecting structural protein abundance than direct changes in the structural protein messages. Results have important implications for disc remodeling and tissue engineering. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:579–588, 2008     

椎间盘髓核细胞组织块法原代培养

椎间盘退变为引起颈肩痛、腰腿痛的主要原因,其确切的发生机理迄今仍不十分清楚,所以对椎间盘髓核细胞的研究越来越深入。椎间盘髓核细胞原代培养在研究髓核细胞生物学、转基因治疗以及组织工程等方面日渐成为一种不可缺少的技术。传统的酶消化法髓核细胞原代培养存在操作烦琐、污染率高以及胰蛋白酶和胶原酶的应用导致细胞活性降低等缺点。因此,有必要对其进行改进。