兔腰椎间盘成分抗原性差异的研究

目的 初步了解兔椎间盘不同成分的免疫源性差别,进一步提示人体内不同椎间盘细胞的抗原性差别.方法 根据生物基因相似性原理及在椎间盘不同部位髓核成分含量存在差异的基础上,采用手术切取实验动物不同区域椎间盘组织并埋植于术野中邻近的椎旁肌内,从而建立髓核糖蛋白埋植组(n=20)和纤维环胶原蛋白埋植组(n=20),并且建立埋植自体肌肉的空白对照组(n=20).分别于第1、2、4、6、8周分批处死动物取标本进行HE染色,观察新生血管化和淋巴细胞浸润;进行免疫组织化学染色,观察CD4、CD8 T淋巴细胞阳性率.结果 HE染色显示糖蛋白埋植组标本在术后1周可见淋巴细胞浸润,4周可见典型新生血管化,并且持续至第8周;胶原蛋白埋植组标本在术后4周才可见少量淋巴细胞浸润,不典型新生血管化;肌肉埋植组在各个时间点均未见典型淋巴细胞浸润和新生血管化形成.各组标本总新生血管化率差异有统计学意义.各时间点所取标本进行免疫组织化学染色,计数CD4+和CD8+T淋巴细胞,发现糖蛋白埋植组CD4+和CD8+淋巴细胞计数均高于胶原蛋白埋植组和肌肉埋植组,胶原蛋白埋植组CD4+和CD8+淋巴细胞计数高于肌肉埋植组,差异有统计学意义.结论 兔腰椎间盘成分中髓核糖蛋白相比纤维环胶原蛋白更易诱导T淋巴细胞分化为CD4+和CD8+细胞,进而推论糖蛋白的自身免疫源性强于胶原蛋白,而胶原蛋白强于肌肉组织,具有弱抗原性.     

兔腰椎间盘成分抗原性差异的研究

目的 初步了解兔椎间盘不同成分的免疫源性差别,进一步提示人体内不同椎间盘细胞的抗原性差别.方法 根据生物基因相似性原理及在椎间盘不同部位髓核成分含量存在差异的基础上,采用手术切取实验动物不同区域椎间盘组织并埋植于术野中邻近的椎旁肌内,从而建立髓核糖蛋白埋植组(n=20)和纤维环胶原蛋白埋植组(n=20),并且建立埋植自体肌肉的空白对照组(n=20).分别于第1、2、4、6、8周分批处死动物取标本进行HE染色,观察新生血管化和淋巴细胞浸润;进行免疫组织化学染色,观察CD4、CD8 T淋巴细胞阳性率.结果 HE染色显示糖蛋白埋植组标本在术后1周可见淋巴细胞浸润,4周可见典型新生血管化,并且持续至第8周;胶原蛋白埋植组标本在术后4周才可见少量淋巴细胞浸润,不典型新生血管化;肌肉埋植组在各个时间点均未见典型淋巴细胞浸润和新生血管化形成.各组标本总新生血管化率差异有统计学意义.各时间点所取标本进行免疫组织化学染色,计数CD4+和CD8+T淋巴细胞,发现糖蛋白埋植组CD4+和CD8+淋巴细胞计数均高于胶原蛋白埋植组和肌肉埋植组,胶原蛋白埋植组CD4+和CD8+淋巴细胞计数高于肌肉埋植组,差异有统计学意义.结论 兔腰椎间盘成分中髓核糖蛋白相比纤维环胶原蛋白更易诱导T淋巴细胞分化为CD4+和CD8+细胞,进而推论糖蛋白的自身免疫源性强于胶原蛋白,而胶原蛋白强于肌肉组织,具有弱抗原性.     

Cyclic mechanical stretch stress increases the growth rate and collagen synthesis of nucleus pulposus cells in vitro

STUDY DESIGN: A rabbit model designed to investigate the effects of applied cyclic tensile stress on the cell division rate and the collagen synthesis in the rabbit nucleus pulposus cells in vitro. OBJECTIVE: To evaluate the effects of mechanical stress on nucleus pulposus cells, thus adding to the understanding of the adaptation of the intervertebral disc to mechanical stress. SUMMARY OF BACKGROUND DATA: Intervertebral disc cells in vivo are exposed to a multitude of physical forces during physical motion. Although it is known that in intervertebral disc disease, a common pathway of disc degeneration is mechanical stress on the nucleus pulposus or the anulus fibrosus or both, the underlying mechanism has been less well defined. METHODS: Nucleus pulposus cells were isolated from 4-week-old Japanese white rabbits. These cells were subjected to the mechanical cyclic stretch stress using a computerized, pressure-operated instrument that physically deformed the cells. The DNA synthesis rate, collagen synthesis rate, and cell cycle progression were measured. RESULTS: Cyclic tensile stretch increased the DNA synthesis rate in nucleus pulposus cells and in the population of cells in the S phase of the cell cycle during 1 to 2 days of subjugation to stress. Cyclic tensile stretch also increased collagenous protein synthesis in nucleus pulposus cells during 1 to 4 days of stress. CONCLUSIONS: Mechanical stress on nucleus pulposus cells promotes the proliferation of cells and alters the properties of intervertebral disc cells. This study may reflect the adaptation of the intervertebral disc to increased motion and stress.     

Viscoelastic properties of intervertebral disc cells. Identification of two biomechanically distinct cell populations

STUDY DESIGN: A combined experimental and theoretical biomechanical study to quantify the mechanical properties of living cells of the porcine intervertebral disc. OBJECTIVES: To quantify zonal variations in the mechanical properties and morphology of cells isolated from the intervertebral disc. SUMMARY OF BACKGROUND DATA: Cellular response to mechanical stimuli is influenced by the mechanical properties of cells and of the extracellular matrix. Significant zonal variations in intervertebral disc matrix properties have been reported. No information is currently available on the corresponding regional variations in the mechanical properties of intervertebral disc cells, despite evidence of significant differences in cellular phenotype and biologic response to loading. METHODS: The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of cells isolated from the anulus fibrosus, transition zone, and nucleus pulposus. RESULTS: Intervertebral disc cells exhibited viscoelastic solid behaviors. Highly significant differences were observed in the morphology, cytoskeletal arrangement, and biomechanical properties of the nucleus pulposus cells as compared with anulus fibrosus or transition zone cells. Cells of the nucleus pulposus were approximately three times stiffer and significantly more viscous than cells of the anulus fibrosus or transition zone. CONCLUSIONS: The findings of this study provide new evidence for the existence of two biomechanically distinct cell populations in the intervertebral disc. These differences in mechanical behavior may be related to observed differences in the cytoskeletal architecture between these cells, and may further play an important role in the development, maintenance, and degeneration of the intervertebral disc.     

Age-related changes in fibromodulin and lumican in human intervertebral discs.

STUDY DESIGN: An analysis of proteoglycans of the intervertebral disc using immunoblotting of tissue extracts. OBJECTIVES: To investigate the changes in structure and abundance of fibromodulin and lumican in human intervertebral discs during aging and degeneration. SUMMARY OF BACKGROUND DATA: Fibromodulin and lumican are keratan sulfate proteoglycan constituents of the disc's extracellular matrix, whose interaction with collagen fibrils may contribute to the mechanical properties of the tissue. Changes in their abundance and/or structure that occur with aging and degeneration therefore may have an impact on disc function. METHODS: Lumbar intervertebral discs were obtained from individuals of different ages, and extracts of anulus fibrosus and nucleus pulposus were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for fibromodulin and lumican. RESULTS: The major changes in abundance observed with age were a decrease in fibromodulin in the adult nucleus pulposus and an increase in lumican in anulus fibrosus during early juvenile development. In addition, fibromodulin in the anulus fibrosus exhibited a structural change with increasing age, characterized by a shift toward the predominance of its glycoprotein form lacking keratan sulfate. Fibromodulin was more abundant in the anulus fibrosus than in nucleus pulposus at all ages, whereas lumican was much more abundant in nucleus pulposus than in anulus fibrosus in the young juvenile; in the adult, however, lumican was present in comparable levels in both tissues. With increasing degrees of degeneration, fibromodulin exhibited an increase in abundance. CONCLUSIONS: Growth, aging, and degeneration of the intervertebral disc are associated with changes in the abundance and structure of fibromodulin and lumican, which presumably influence the functional properties of the tissue.     

Cell culture of the intervertebral disc of rats: factors influencing culture, proteoglycan, collagen, and deoxyribonucleic acid synthesis.

To establish cell culture of the nucleus pulposus and anulus fibrosus of rat intervertebral disc, the effects of culture conditions on the growth of cells and the synthesis of DNA, proteoglycan, and collagen were studied. For cell culture of the nucleus pulposus, the use of 3-week-old rats and a medium adjusted to pH 7.0 was optimal. There was almost no difference in growth between cells in Ham's F12 medium and those in Dulbecco's Modified Eagle Medium. In cells isolated from the anulus fibrosus, a medium adjusted to pH 7.0-7.6 was preferable, but irrespective of rat age. Culture cells of the nucleus pulposus were composed of large cells with vacuoles and small polygonal cells. These cells had a slight growth activity and a fair capability of proteoglycan and collagen synthesis. Culture cells of the anulus fibrosus were composed of polygonal and spindle-shape cells, and the growth was more vigorous with the potentials for proteoglycan and collagen synthesis than the nucleus cells.     

The influence of human intervertebral disc tissue on the metabolism of osteoblast-like cells

Extensive studies have been performed to evaluate different factors that may affect on spinal interbody fusion, but the role of intervertebral disc tissue in the fusion process remains unclear. To study the influence of intervertebral disc tissue on osteoblast metabolism, we harvested disc tissue from patients who had undergone spinal surgery. The nucleus pulposus and annulus fibrosus were separately co-cultured with osteoblast-like cells SaOS-2 by means of culture inserts or organ culture. We assayed alkaline phosphoatase activity, 3H-thymidine incorporation into the DNA, and production of collagen type I, IL-1beta, IL-6, IL-10, and TNF-alpha. Exposure of the nucleus pulposus (NP) to osteoblast-like cells revealed stimulation of alkaline phosphatase production, 3H-thymidine incorporation and collagen type I production. Exposure of the annulus fibrosus (AF) stimulated 3H-thymidine incorporation and collagen type I production, but did not affect ALP activity. IL-6 was detected after application of NP and AF. Interleukin IL-10, IL-1beta and TNF-alpha were all below detection levels after application of disc tissue. Our findings show that frozen disc tissue stimulates the metabolism of osteoblast-like cells in vitro.     

Phenotypic characteristics of rabbit intervertebral disc cells. Comparison with cartilage cells from the same animals.

STUDY DESIGN: Intervertebral disc cells were extracted from the surrounding matrix, and their metabolic activities and phenotypes were studied. OBJECTIVES: To compare the metabolic activities and phenotypes of cell populations extracted from the intervertebral discs of young rabbits with those of articular and growth plate chondrocytes from the same animals. SUMMARY OF BACKGROUND DATA: The phenotype of intervertebral disc cells has been poorly studied and still is debated. METHODS: The intervertebral discs as well as articular and vertebral growth plate cartilage of rabbits were digested enzymatically. The morphology of freshly isolated cells was examined. Their contents of collagen II and X mRNAs were determined by Northern blot analysis, and their sulfation activity by 35S-sulfate incorporation as chondrocytic markers. Cells were cultured at high density or low density and grown in primary culture. The stability of their phenotype was monitored by evaluating the collagen I and II mRNA ratio. The proteoglycans newly synthesized by the cells also were quantified, and their elution profile analyzed on Sepharose 2B columns. RESULTS: The anulus fibrosus cells were morphologically undistinguishable from articular chondrocytes. The nucleus pulposus contained mainly large vacuolated cells and a few smaller cells. All freshly extracted cells expressed different levels of collagen II mRNA. Anulus fibrosus and nucleus pulposus cells contained, respectively, 22% and 8% of collagen II mRNA compared with that found in articular or growth plate chondrocytes from the same animal. Only growth plate chondrocytes expressed collagen X. When anulus fibrosus cells were incubated for 48 hours at high density, they had collagen II mRNA contents similar to those of articular and growth plate chondrocytes, but synthesized five to six times fewer sulfated proteoglycans. When seeded at low density, anulus fibrosus cells divided more slowly than articular chondrocytes and incorporated four times fewer 35S-sulfate into proteoglycans. Their collagen II mRNA content was 2.75-fold lower than that of chondrocytes, and the procollagen alpha 1II/alpha 1I mRNA ratio was 3.1 for anulus fibrosus cells and 7 for chondrocytes. No collagen X mRNA was detected. When incubated for 48 hours at high density, the nucleus pulposus giant cells had four times less collagen II mRNA content than cartilage cells but synthesized the same amounts of sulfated proteoglycans. They did not divide during 21 days in culture and still contained collagen II mRNA but no collagen X mRNA. CONCLUSIONS: Findings showed that intervertebral disc cells all express cartilage-specific matrix proteins with quantitative differences, depending on their anatomic situation. It is suggested that anulus fibrosus cells are chondrocytic cells at a different stage of differentiation than articular and growth plate chondrocytes. The phenotype of nucleus pulposus cells still is unclear. They could be chondrocytic or notochordal. A definitive answer to this important question requires differentiating markers of notochordal cells.     

实验性脊柱内固定后相应区域椎间盘超微结构观察

目的　观察脊柱内固定后相应区域椎间盘的超微结构变化。　方法　日本大耳白兔2 4只,随机分成实验组和对照组,每组12只。实验组骨膜下游离T1 0 ～L3棘突和关节突,克氏针制成“L”形,将钢丝横行穿过T1 1、1 2 ,L1、2 的关节突关节,并与置于T1 1 ～L3棘突两旁的克氏针系紧,对相应区域的脊柱行内固定术。对照组未行手术,仅喂养至实验完成。术后6个月,对两组动物摄X线片观察1次,随后处死动物。取两组动物的L1 椎间盘组织(髓核、纤维环内侧及纤维环外侧)行透射电镜观察,对两组T1 2 、L2 椎间盘组织分别行水平面和矢状面透射电镜及扫描电镜观察。　结果　X线片显示,实验组与对照组椎体及椎间隙差别不明显;透射电镜与扫描电镜观察,实验组椎间盘的髓核、纤维环内层细胞的结构改变较纤维环外层早;对照组的髓核、纤维环内层细胞的结构改变与纤维环外层差别不明显。在退变的椎间盘基质中,蛋白多糖颗粒和特殊结构明显减少。髓核与纤维环基质内有蛋白多糖颗粒和一种特殊结构,而特殊结构在髓核与纤维环内层的形态不一致。　结论　脊柱内固定术后6个月,实验组在异常应力环境下发生椎间盘退变。髓核、纤维环内层基质内的特殊结构分布有特殊规律,与蛋白多糖颗粒在椎间盘退变中的生物学行为密切相关。     

Intradiscal solid phase displacement as a determinant of the centripetal fluid shift in the loaded intervertebral disc

STUDY DESIGN: The movement of cross sections of the monofilament nylon threads inserted into the axially loaded intervertebral disc was traced with magnetic resonance imaging (MRI). This technique allowed the observation of the sequential solid phase displacement of the loaded intervertebral disc. OBJECTIVES: To clarify sequential solid phase displacement of the axially loaded intervertebral disc to elucidate the cause of centripetal fluid shift within a disc. SUMMARY OF BACKGROUND DATA: We already have reported that there is a centripetal fluid shift within the axially loaded intervertebral disc during the early phase of loading. We assumed that there should be an elaborate intradiscal matrix displacement that generates a pressure gradient within the disc to cause a centripetal fluid shift. METHODS: Thirteen freshly obtained bovine caudal intervertebral discs were prepared. Three to five monofilament nylon threads were inserted into each disc in the anterior-posterior direction to trace the intradiscal solid phase displacement on the midcoronal MR images. Sequential displacement of the disc matrix was recorded during a 294 N axial loading. RESULTS: Relatively large centrifugal expansion at the inner layer of the anulus fibrosus compared with less centrifugal expansion of the outer anulus fibrosus was observed in accord with gradual creep of the disc thickness. CONCLUSIONS: The uneven displacement of the intradiscal solid phase observed in the present study expels the fluid phase from the inner anulus fibrosus, thus resulting in accumulation of fluid phase in the nucleus pulposus. The present study suggests the presence of a mechanism that retains water within the normal intervertebral disc, in spite of an external load, because it forms a water-abundant nucleus pulposus, which is surrounded by an anulus fibrosus with decreased water permeability caused by fluid loss. A more detailed analysis is required to clarify topographic volumetric changes within the disc.     

Matrix replenishment by intervertebral disc cells after chemonucleolysis in vitro with chondroitinase ABC and chymopapain

BACKGROUND CONTEXT: One of the advantages of chemonucleolysis for the treatment of a herniated intervertebral disc is the potential for the disc to self-repair. It has been suggested that the enzymes used for chemonucleolysis differentially affect the potential of the disc cells to promote repair. PURPOSE: To test the ability of nucleus pulposus and anulus fibrosus cells to repair the extracellular matrix degraded in vitro by either chondroitinase ABC or chymopapain. STUDY DESIGN: An alginate cell culture system was used to monitor the progress of matrix repair after chemonucleolysis in vitro. METHODS: Rabbit nucleus pulposus or anulus fibrosus cells precultured for 10 days in alginate gel were briefly exposed to low concentrations of chondroitinase ABC or chymopapain and then returned to normal culture conditions for up to 4 weeks. At each time point, the contents of DNA and matrix macromolecules and proteoglycan synthesis were measured. RESULTS: The DNA content of enzyme-treated alginate beads during the following 4 weeks of culture was higher in the chondroitinase ABC group than in the chymopapain group (NP, p<.01, and AF, p<.05). The content of proteoglycan in beads containing nucleus pulposus and anulus fibrosus cells in the chondroitinase ABC group was higher than that in the chymopapain group (NP and AF, p<.001). The rate of proteoglycan synthesis and the content of collagen did not, however, differ between those two groups. CONCLUSIONS: Intervertebral disc cells exposed to chondroitinase ABC reestablish a matrix richer in proteoglycan than cells exposed to chymopapain. This may be because of differences in the substrate spectrum of each enzyme. Although these results cannot be translated directly to the in vivo situation, they suggest the possibility that cells in discs subjected to chondroitinase ABC-induced chemonucleolysis retain a greater ability to replenish their extracellular matrix with proteoglycans than cells in discs exposed to chymopapain.     

Mechanobiology of the intervertebral disc and relevance to disc degeneration

Mechanical loading of the intervertebral disc may contribute to disc degeneration by initiating degeneration or by regulating cell-mediated remodeling events that occur in response to the mechanical stimuli of daily activity. This article is a review of the current knowledge of the role of mechanical stimuli in regulating intervertebral disc cellular responses to loading and the cellular changes that occur with degeneration. Intervertebral disc cells exhibit diverse biologic responses to mechanical stimuli, depending on the loading type, magnitude, duration, and anatomic zone of cell origin. The innermost cells respond to low-to-moderate magnitudes of static compression, osmotic pressure, or hydrostatic pressure with increases in anabolic cell responses. Higher magnitudes of loading may give rise to catabolic responses marked by elevated protease gene or protein expression or activity. The key regulators of these mechanobiologic responses for intervertebral disc cells will be the micromechanical stimuli experienced at the cellular level, which are predicted to differ from that measured for the extracellular matrix. Large hydrostatic pressures, but little volume change, are predicted to occur for cells of the nucleus pulposus during compression, while the highly oriented cells of the anulus fibrosus may experience deformations in tension or compression during matrix deformations. In general, the pattern of biologic response to applied loads suggests that the cells of the nucleus pulposus and inner portion of the anulus fibrosus experience comparable micromechanical stimuli in situ and may respond more similarly than cells of the outer portion of the anulus fibrosus. Changes in these features with degeneration are critically understudied, particularly degeneration-associated changes in cell-level mechanical stimuli and the associated mechanobiology. Little is known of the mechanisms that regulate cellular responses to intervertebral mechanobiology, nor is much known with regard to the precise mechanical stimuli experienced by cells during loading. Mechanical factors appear to regulate responses of the intervertebral disc cells through mechanisms involving intracellular Ca(2+) transients and cytoskeletal remodeling that may regulate downstream effects such as gene expression and posttranslational biosynthesis. Future studies should address the broader biologic responses to mechanical stimuli in intervertebral disc mechanobiology, the involved signaling mechanisms, and the apparently important interactions among mechanical factors, genetic factors, cytokines, and inflammatory mediators that may be critical in the regulation of intervertebral disc degeneration.     

Temporo-spatial distribution of blood vessels in human lumbar intervertebral discs

While there is consensus in the literature that blood vessels are confined to the outer anulus fibrosus of normal adult intervertebral disc, debate continues whether there is a vascular in-growths into inner parts of the intervertebral disc during degeneration. We therefore tested the hypothesis that vascular in-growth is not a distinct feature of disc degeneration. The specific endothelial cell marker CD 31 (PECAM) was used to immunohistochemically investigate 42 paraffin-embedded complete mid-sagittal human intervertebral disc sections of various ages (0–86 years) and varying extent of histomorphological degeneration. Additionally, 20 surgical disc samples from individuals (26–69 years) were included in this study. In discs of fetal to infantile age, blood vessels perforated the cartilaginous end plate and extended into the inner and outer anulus fibrosus, but not into the nucleus pulposus. In adolescents and adults, no blood vessels were seen except for the outer zone of the anulus fibrosus adjacent to the insertion to ligaments. The cartilaginous end plate remained free of vessels, except for areas with circumscribed destruction of the end plate. In advanced disc degeneration, no vessels were observed except for those few cases with complete, scar-like disc destruction. However, some rim lesions and occasionally major clefts were surrounded by a small network of capillary blood vessels extending into deeper zones of the anulus fibrosus. A subsequent morphometric analysis, revealed slightly “deeper” blood vessel extension in juvenile/adolescent discs when compared to young, mature and senile adult individuals with significantly “deeper” extension in the posterior than anterior anulus. The analysis of the surgical specimens showed that only sparse capillary blood vessels which did not extend into the nucleus pulposus even in major disc disruption. Our results show that vascular invasion deeper than the periphery was not observed during disc degeneration, which supports the hypothesis that vascular in-growth is not a distinct feature of disc degeneration. This study was supported by a grant from the AO/ASIF Foundation Switzerland (00-B72) and a grant from the AO Spine (SRN 02/103).     

退变颈椎间盘中IL-17表达与分布的研究

目的 观察退变颈椎间盘中自细胞介素-17(IL-17)的表达与分布,并探讨其与颈椎间盘退变发生发展的关系.方法 实时荧光相对定量PCR(RQ-PCR)检测30例退变颈椎间盘及10例正常对照椎间盘中IL-1β、IL-17、肿瘤坏死因子-α(TNF-α)和孤核受体(retinoid-related orphan re-ce...     

Effects of anulus fibrosus and experimentally degenerated nucleus pulposus on nerve root conduction velocity: relevance of previous experimental investigations using normal nucleus pulposus

STUDY DESIGN: Nerve conduction velocity was measured in the pig cauda equina after local application of anulus fibrosus or in vitro/postmortem degenerated nucleus pulposus from the same pig. OBJECTIVES: To analyze the effects of anulus fibrosus and degenerated nucleus pulposus on nerve conduction velocity. SUMMARY OF BACKGROUND DATA: Previous studies on nucleus pulposus-induced effects on nerve roots have used normal, nondegenerated nucleus pulposus. Because both anulus fibrosus and degenerated nucleus pulposus are commonly seen in the clinical situation of disc herniation, the value of the previous work could be questioned. METHODS: Anulus fibrosus and nucleus pulposus were harvested using a retroperitoneal approach. The nucleus pulposus was degenerated artificially either by addition of sodium lactate with HCl added to form a pH of either 6.0 or 3.5 (in vitro degeneration), or by storing the nucleus pulposus at 4 C until a pH of 6.0 (postmortem degeneration) was reached. After epidural application, the nerve conduction velocity was determined at 7 days (anulus fibrosus) or 3 days (degenerated nucleus pulposus). RESULTS: Application of anulus fibrosus did not induce any reduction of nerve conduction velocity. In vitro and postmortem degenerated nucleus pulposus induced a reduction of nerve conduction velocity similar to that of normal nucleus pulposus. CONCLUSIONS: Although only nerve function and not pain was assessed, it seems likely that previous experiments using normal nucleus pulposus may be relevant for evaluating the pathophysiologic mechanisms behind the nucleus pulposus-induced nerve root injury, also in a clinical perspective.     

The effect of various nucleotomy techniques on biomechanical properties of the intervertebral disk]

AIM OF THE STUDY: The purpose of the present investigation was to determine the significance of the technique of nucleotomy for the biomechanical properties of the intervertebral disc. METHODS: From 29 human cadavers the lumbar-vertebral segment L2/3 was taken. The laminae were removed and the segment was tested by an mechanical testing device. Measurements were taken under physiological loads in flexion/extension, lateral bending and rotation direction. There after nucleotomy was simulated by four different procedures: perforation of the anulus with a diameter of 3 mm, rectangular excision (1 x 1 cm) of the anulus, discotomy with 3 g and 6 g nucleus pulposus. In each step the measurement was repeated. RESULTS: The size of the defect in the anulus fibrosus did not have significant influence on the stiffness of the disc. There was a significant relation between quantity of removed disc material and flexibility of the motion segment. CONCLUSION: The quantity of removed nucleus pulposus plays a crucial role in the segmental mobility whereas the size of the approach in the anulus fibrosus, f.e. endoscopic with a hole diameter of 3 mm or conventionally with a rectangular defect of 1 x 1 cm, did not influence mobility.     

The influence of human intervertebral disc tissue on the metabolism of osteoblast-like cells

Extensive studies have been performed to evaluate different factors that may affect on spinal interbody fusion, but the role of intervertebral disc tissue in the fusion process remains unclear. To study the influence of intervertebral disc tissue on osteoblast metabolism, we harvested disc tissue from patients who had undergone spinal surgery. The nucleus pulposus and annulus fibrosus were separately co-cultured with osteoblast-like cells SaOS-2 by means of culture inserts or organ culture. We assayed alkaline phosphoatase activity, 3 H-thymidine incorporation into the DNA, and production of collagen type I, IL-1^6;, IL-6, IL10, and TNF-^5;. Exposure of the nucleus pulposus (NP) to osteoblastlike cells revealed stimulation of alkaline phosphatase production, 3 H-thymidine incorporation and collagen type I production. Exposure of the annulus fibrosus (AF) stimulated 3 H-thymidine incorporation and collagen type I production, but did not affect ALP activity. IL-6 was detected after application of NP and AF. Interleukin IL-10, IL-1^6; and TNF-^5; were all below detection levels after application of disc tissue. Our findings show that frozen disc tissue stimulates the metabolism of osteoblast-like cells in vitro.     

Histology of intervertebral disc protrusion: an experimental study using an aged rat model

STUDY DESIGN: In vitro experimental intervertebral disc ruptures of aged rats were examined histologically. OBJECTIVES: To clarify the mechanism of intervertebral disc herniations by microscopic investigation of ruptured discs. SUMMARY OF BACKGROUND DATA: Clinically, disc herniations have been classified into two types: extrusion and protrusion. However, the pathogenesis of protrusion type herniations has not yet been demonstrated by any studies. To clarify this issue, it is essential to establish an appropriate model producing disc herniations, and to examine the sequential changes in the structure of herniated discs. METHODS: Lumbar discs of 2-year-old rats were examined histologically and compared with human lumbar discs. To examine structural changes in discs subjected to repetitive motion stress, 400 repetitions of a sequence of flexion (30 degrees ) and axial rotation (6 degrees ) were applied in vitro to the lumbar discs of the animals. RESULTS: The microstructure of normal lumbar discs in aged rats was similar in many ways to the human lumbar discs in a 20- to 40-year-old adult. Of 10 discs subjected to repetitive stress, 4 were ruptured at the junction between the posterior anulus fibrosus and the sacral cartilage endplate. One had an extruded nucleus pulposus, and three had a protruded anulus fibrosus, which displayed disorganized structure containing widened and flaccid lamellae. CONCLUSIONS: The results from this study indicate that disc protrusion can be caused by disorganization of the ruptured annular lamellae, not by focal compression of the nucleus pulposus.     

Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses

To study the relationships between the changes due to aging in lumbar intervertebral discs and the development of protrusion or prolapse, we carried out histological studies on operative specimens of thirty-one discs, of which twenty-two had been protruded and nine, prolapsed. The specimens were obtained during twenty-nine operations for herniation of a lumbar intervertebral disc in patients who were sixty years old or older. Changes in the anulus fibrosus were more extensive in the nine prolapsed discs than in the twenty-two protruded discs. Of the nine prolapsed discs, myxomatous degeneration, fibrosis, and swollen anular fibers were found in all nine, and cysts were seen in five. Of the twenty-two protruded discs, only five showed myxomatous degeneration; ten, fibrosis; one, a cyst; and sixteen, swollen fibers. For comparison, we also studied specimens that had been obtained at operation from twenty-one other patients, twenty to fifty-nine years old, who had a prolapsed disc. The anulus showed myxomatous degeneration in all twenty-one specimens, cysts in eight, and fibrosis in ten. In addition, we examined 368 autopsy specimens from people who had been between twenty-five and eighty-five years old at the time of death. In many of the subjects who had died in the sixth decade of life or later, we found that the orientation of the inner fiber bundles of the anulus fibrosus was reversed, so that they bulged inward. The reversal appeared to be the result of myxomatous degeneration of the middle fibers of the anulus, atrophy of the nucleus, and narrowing of the disc space. These histological findings suggest explanations for the predominance of protrusions of the nucleus pulposus in patients who are less than sixty years old and of prolapse of the anulus fibrosus in the few patients who are more than sixty years old who have herniation of an intervertebral disc.