Programmed cell death in intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is largely a process of destruction and failure of the extracellular matrix (ECM), and symptomatic IVD degeneration is thought to be one of the leading causes of morbidity or life quality deterioration in the elderly. To date, however, the mechanism of IVD degeneration is still not fully understood. Cellular loss from cell death in the process of IVD degeneration has long been confirmed and considered to contribute to ECM degradation, but the causes and the manners of IVD cell death remain unclear. Programmed cell death (PCD) is executed by an active cellular process and is extensively involved in many physiological and pathological processes, including embryonic development and human degenerative diseases. Thus, the relationship between PCD and IVD degeneration has become a new research focus of interest in recent years. By reviewing the available literature concentrated on PCD in IVD and discussing the methodology of detecting PCD in IVD cells, its inducing factors, the relationship of cell death to ECM degradation, and the potential therapy for IVD degeneration by modulation of PCD, we conclude that IVD cells undergo PCD via different signal transduction pathways in response to different stimuli, that PCD may play a role in the process of IVD degeneration, and that modulation of PCD might be a potential therapeutic strategy for IVD degeneration.     

Matrix-assisted cell transfer for intervertebral disc cell therapy

Cell therapy seems to be a promising way to reconstitute degenerated discs. We elucidate the basic aspects of intervertebral disc (IVD) cell therapy to estimate its potential in disc regeneration. Cell transfer efficiency and survival was quantified by luciferase expression after injection of recombinant cells into healthy, nucleotomized or mechanically degenerated rabbit IVDs in vitro, in situ or in vivo. A two-component fibrin matrix was adapted to allow injection of a fluid cell suspension that quickly polymerizes in IVDs. Thirty-five to fifty percent of matrix injected cells remained in the nucleus and transition zone in contrast to a rapid loss of medium-injected cells. Nucleotomy, which reduces intradiscal pressure, was crucial to the survival of the transferred cells over 3 days and nutritional enrichment of the fibrin matrix with potent biomolecules from serum significantly enhanced cell viability. In conclusion, advanced matrix substitutes are needed for efficient transfer and improved cell survival in the low-nutrient intradiscal environment to further improve disc cell therapy.     

不同月龄大鼠椎间盘退变与多效生长因子表达的关系

目的观察不同月龄大鼠椎间盘的形态学变化并检测椎间盘中多效生长因子（pleiotrophin,PTN）的表达,探讨PTN与椎间盘退变的关系。方法取Wistar大鼠50只,以1,3,6,12,18个月龄不同分为5组,每组10只。采用苏木精-伊红染色观察椎间盘的形态学变化。采用SABC免疫组织化学方法,检测椎间盘中PTN的表达情况;结果（1）随着月龄的增加,椎间盘组织结构紊乱的程度逐渐增加,髓核内基质降解、正中出现空腔,胶原纤维增生、粗大、排列紊乱、并可见纤维断裂或缺失。（2）随着大鼠月龄的增加（1-12月龄）,椎间盘细胞中PTN的表达有逐渐减低的趋势,但至18月龄,PTN表达又有所增加;6和12月龄组椎间盘细胞中PTN的表达显著低于1月龄组,而18月龄组PTN的表达显著高于12月龄组。同月龄组椎间盘细胞中,PTN在终板的表达高于髓核和纤维环,髓核和纤维环中PTN的表达未见明显差异。结论大鼠椎间盘结构随月龄增加发生退行性变,PTN参与了大鼠椎间盘的退变,并可能通过促进椎间盘组织中新生血管的形成,延缓椎间盘的退变。     

Advances in susceptibility genetics of intervertebral degenerative disc disease

The traditional view that the etiology of lumbar disc herniation is primarily due to age, gender, occupation, smoking and exposure to vehicular vibration dominated much of the last century. Recent research indicates that heredity may be largely responsible for the degeneration as well as herniation of intervertebral discs. Since 1998, genetic influences have been confirmed by the identification of several genes forms associated with disc degeneration. These researches are paving the way for a better understanding of the biologic mechanisms. Now, many researchers unanimously agree that lumbar disc herniation appears to be similar to other complex diseases, whose etiology has both environmental and hereditary influence, each with a part of contribution and relative risk. Then addressing the etiological of lumbar disc herniation, it is important to integrate heredity with the environment factors. For the purpose of this review, we have limited our discussion to several susceptibility genes associated with disc degeneration.     

Application of methods for viral clearance in stem cell production

Regenerative medicine therapies will allow in the future the transplant of cells of human origin in some diseases that until now have been incurable. The assurance of the safety and quality, especially from a microbiological point of view, is very important for these therapeutic products. Depending on the starting material, there are several sources of pathogen presence, mainly human viruses. Also, the use of feeders of animal origin as layers in which the stem cells can grow may permit the transmission of animal pathogens to these cells. However, cell sources are limited due to the low availability of spare in vitro fecundation human embryos and the low rate of success in the derivation of human stem cell lines. Thus, in several cases, it will be necessary to evaluate the possibility of removing or inactivating these microorganisms. In this paper, we summarize the main methods of viral clearance and we have provided an overview of the main features taking into account in the viral clearance techniques.     

Reduced nucleus pulposus glycosaminoglycan content alters intervertebral disc dynamic viscoelastic mechanics

The intervertebral disc functions over a range of dynamic loading regimes including axial loads applied across a spectrum of frequencies at varying compressive loads. Biochemical changes occurring in early degeneration, including reduced nucleus pulposus glycosaminoglycan content, may alter disc mechanical behavior and thus may contribute to the progression of degeneration. The objective of this study was to determine disc dynamic viscoelastic properties under several equilibrium loads and loading frequencies, and further, to determine how reduced nucleus glycosaminoglycan content alters dynamic mechanics. We hypothesized that (1) dynamic stiffness would be elevated with increasing equilibrium load and increasing frequency, (2) the disc would behave more elastically at higher frequencies, and finally, (3) dynamic stiffness would be reduced at low equilibrium loads under all frequencies due to nucleus glycosaminoglycan loss. We mechanically tested control and chondroitinase ABC injected rat lumbar motion segments at several equilibrium loads using oscillatory loading at frequencies ranging from 0.05 to 5 Hz. The rat lumbar disc behaved non-linearly with higher dynamic stiffness at elevated compressive loads irrespective of frequency. Phase angle was not affected by equilibrium load, although it decreased as frequency was increased. Reduced glycosaminoglycan decreased dynamic stiffness at low loads but not at high equilibrium loads and led to increased phase angle at all loads and frequencies. The findings of this study demonstrate the effect of equilibrium load and loading frequencies on dynamic disc mechanics and indicate possible mechanical mechanisms through which disc degeneration can progress.     

Is the sheep a suitable model to study the mechanical alterations of disc degeneration in humans? A probabilistic finite element model study

Intervertebral disc degeneration is one major source of low back pain, which because of its complex multifactorial nature renders the treatment challenging and thus necessitates extensive research. Experimental animal models have proven valuable in improving our understanding of degenerative processes and potentially promising therapies. Currently, the sheep is the most frequently used large animal in vivo model in intervertebral disc research. However, despite its undoubted value for investigations of the complex biological and cellular aspects, to date, it is unclear whether the sheep is also suited to study the mechanical aspects of disc degeneration in humans.A parametric finite element (FE) model of the L4–5 spinal motion segment was developed. Using this model, the geometry and the material properties of both the human and the ovine spinal segment as well as different appearances of disc degeneration can be depicted. Under pure and combined loads, it was investigated whether degenerative changes to both the human and the ovine model equivalent caused the same mechanical response.Different patterns of degeneration resulted in large variations in the ranges of motion, intradiscal pressure, ligament and facet loads. In the human, but not in the ovine model, all these results differed significantly between different degrees of degeneration.This FE model study highlighted possible differences in the mechanical response to disc degeneration between human and ovine intervertebral discs and indicates the necessity of further, more detailed, investigations.     

白细胞介素-17对体外培养髓核细胞增殖和代谢的影响

目的:探究白细胞介素-17(interleukin-17,IL-17)对体外培养髓核细胞增殖和细胞代谢的影响。方法:髓核细胞取自经核磁共振影像确认需手术的退变椎间盘组织,建立体外培养体系。用2、5、10、15、20 ng/mL IL-17刺激髓核细胞72 h后,MTS法检测细胞增殖情况。用适当浓度IL-17刺激细胞48 h或96 h后,采用实时定量-PCR和免疫印迹方法检测基质和组织代谢相关基因的mRNA和蛋白表达。结果:IL-17刺激可以抑制体外培养髓核细胞的增殖,且15 ng/mL浓度的抑制作用最强。15 ng/mL IL-17刺激髓核细胞后,聚集蛋白聚糖(aggrecan,ACAN)和I型胶原(type I collagen,COL1A1)mRNA表达水平显著下降(P0.05),基质金属蛋白酶(matrix metalloproteinase-3,MMP3)、金属蛋白酶3组织抑制剂(tissue inhibitor of metalloproteinase-3,TIMP3)的mRNA表达水平显著上升(P0.05)。COL2A1 mRNA的表达下降,MMP13、含Ⅰ型血小板结合蛋白基序的结聚蛋白样金属蛋白酶(a disintegrin like and metalloproteinase with thrombospondin typeⅠ motifs-4,ADAMTS4)、ADAMTS5、TIMP1 mRNA的表达上升,但差异均不显著(P0.05)。IL-17刺激48 h时,COL1A1的蛋白水平明显下降(P=0.010),而ADAMTS5的蛋白水平显著上升(P=0.005)。但刺激96h时,COL1A1的蛋白表达下降,ADAMTS5的蛋白表达上升,但无显著差异(P0.05);COL2A1的蛋白表达水平显著下降(P=0.037)。结论:IL-17可抑制体外培养髓核细胞的增殖及代谢,在椎间盘的退变过程中可能发挥了重要的促进作用。     

干细胞治疗脊髓损伤研究进展

脊髓损伤后的常规治疗手段是在有效时间内进行手术缓减外力压迫,防止脊髓神经进一步受损。细胞替代治疗理论上可治愈脊髓损伤,不同类型细胞可从各角度产生治疗作用,包括损伤后的脊髓轴突再生、神经元再建和轴突髓鞘化等,进而促进功能恢复。对近年来干细胞治疗脊髓损伤研究中的最新结果进行了概述,以期为干细胞治疗脊髓损伤的研究提供参考。     

Human L3L4 intervertebral disc mean 3D shape,modes of variation,and their relationship to degeneration

Intervertebral disc mechanics are affected by both disc shape and disc degeneration, which in turn each affect the other; disc mechanics additionally have a role in the etiology of disc degeneration. Finite element analysis (FEA) is a favored tool to investigate these relationships, but limited data for intervertebral disc 3D shape has forced the use of simplified or single-subject geometries, with the effect of inter-individual shape variation investigated only in specialized studies. Similarly, most data on disc shape variation with degeneration is based on 2D mid-sagittal images, which incompletely define 3D shape changes. Therefore, the objective of this study was to quantify inter-individual disc shape variation in 3D, classify this variation into independently-occurring modes using a statistical shape model, and identify correlations between disc shape and degeneration. Three-dimensional disc shapes were obtained from MRI of 13 human male cadaver L3L4 discs. An average disc shape and four major modes of shape variation (representing 90% of the variance) were identified. The first mode represented disc axial area and was significantly correlated to degeneration (R²=0.44), indicating larger axial area in degenerate discs. Disc height variation occurred in three distinct modes, each also involving non-height variation. The statistical shape model provides an average L3L4 disc shape for FEA that is fully defined in 3D, and makes it convenient to generate a set of shapes with which to represent aggregate inter-individual variation. Degeneration grade-specific shapes can also be generated. To facilitate application, the model is included in this paper?s supplemental content.     

人骨形态蛋白-2对椎间盘细胞蛋白多糖和Ⅱ型胶原的影响

目的研究人骨形态蛋白(human bone morphogenetic protein-2,hBMP-2)对体外培养人腰椎间盘髓核细胞Ⅱ型胶原和aggrecan的影响。方法人退变髓核细胞体外分离培养,通过免疫组织化学鉴定椎间盘细胞。利用ELISA法检测对照组和不同剂量hBMP-2(10ng/ml和100ng/ml)组Ⅱ型胶原和aggrecan的表达水平。用RT-PCR检测细胞Ⅱ型胶原和aggrecan的mRNA表达水平。结果加入hBMP-2后Ⅱ型胶原和aggrecan表达增加,并存在剂量依赖关系(P<0.01)。在第6天RT-PCR结果显示hBMP-2组aggrecan和Ⅱ型胶原mRNA的表达高于对照组,并且随剂量增高aggrecan和Ⅱ型胶原mRNA的表达量逐渐增加。结论hBMP-2可促进体外培养人腰椎间盘细胞合成代谢,Ⅱ型胶原和aggrecan表达量增加,并存在剂量依赖关系,提示hBMP-2可能对退变椎间盘具有修复功能。     

Modeling the role of IGF-1 on extracellular matrix biosynthesis and cellularity in intervertebral disc

The insulin-like growth factor-1 (IGF-1) is a well-known anabolic agent for intervertebral disc (IVD), promoting both proteoglycan (PG) biosynthesis and cell proliferation. Accordingly, it is believed that IGF-1 may play a central role in IVD homeostasis. Furthermore, the exogenous administration of IGF-1 has been proposed as a possible therapeutic strategy for disc degeneration. The objectives of this study were to develop a new computational framework for describing the mechanisms regulating IGF-mediated homeostasis in IVD, and to apply this numerical tool for investigating the effectiveness of exogenous administration of IGF-1 for curing disc degeneration. A diffusive–reactive model was developed for describing competitive binding of IGF-1 to its binding proteins and cell surface receptors, with the latter reaction initiating the intracellular signaling mechanism leading to PG production and cell proliferation. Because PG production increases cell metabolic rate, and cell proliferation increases nutritional demand, nutrients transport and metabolism were also included into the model, and co-regulated, together with IGF-1, IVD cellularity. The sustainability and the effectiveness of IGF-mediated anabolism were investigated for conditions of pathologically insufficient nutrient supply, and for the case of exogenous administration of IGF-1 to degenerated IVD. Results showed that pathological nutrients deprivation, by decreasing cellularity, caused a reduction of PG biosynthesis. Also, exogenous administration of IGF-1 was only beneficial in well-nourished regions of IVD, and exacerbated cell mortality in malnourished regions. These findings remark the central role of nutrition in IVD health, and suggest that adequate nutritional supply is paramount for achieving a successful IGF-based therapy for disc degeneration.     

Multipotent Adult Progenitor Cells (MAPC) contribute to hepatocarcinoma neovasculature

The use of stem cells as a vehicle of therapeutic genes is an attractive approach for the development of new antitumoral strategies based on gene therapy. The aim of our study was to assess the potential of bone marrow-derived Multipotent Adult Progenitor Cells (rMAPCs) to differentiate in vitro and in vivo into endothelial cells and to be recruited to areas of tumor vasculogenesis. In vitro, rMAPCs obtained from Buffalo rats differentiated into cells expressing endothelial markers and demonstrated functional endothelial capacity. Intravenous injection of undifferentiated rMAPC transduced with a lentivirus expressing GFP in an orthotopic rat model of hepatocellular carcinoma, resulted in tumor recruitment of the injected cells and in vivo differentiation into endothelial cells in the tumor area with contribution to vasculogenesis. In summary, our results suggest that rMAPCs can be efficiently recruited by vascularized tumors and differentiate to endothelium and thus may represent a useful vehicle for delivery of therapeutic genes to sites of active tumor neovascularization.     

Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways

The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD.