Spinal biomechanics and aging are major determinants of the proteoglycan metabolism of intervertebral disc cells

STUDY DESIGN: The proteoglycan metabolism of ovine disc nucleus pulposus and anulus fibrosus cells was investigated in relation to age, spinal level, and intrinsic spinal biomechanical properties. OBJECTIVE: To evaluate the hypothesis that with aging loss of proteoglycans from the lumbosacral disc exceeds that from upper lumbar discs because of its proximity to a rigid segment, the sacrum. SUMMARY OF BACKGROUND DATA: The proteoglycan and associated water of the disc decreases with aging. METHODS: Proteoglycans were extracted directly from the disc tissues using 4 M GuHCl and examined by composite agarose polyacrylamide gel electrophoresis. Disc cells were cultured in alginate beads, and their metabolic activity was assessed by 3H-thymidine incorporation into DNA and by bioreduction of a cell proliferation dye. Newly synthesized proteoglycans were radiolabeled with 35S, and their molecular weight distributions and ability to aggregate with hyaluronan were determined by Sephacryl S1000 gel chromatography. Resident proteoglycans extracted from disc tissues with 4 M GuHCl were similarly evaluated. A group of adult animals also were studied biomechanically to evaluate the range of spinal motion (L4/L5 to L7/S1). RESULTS: In contrast to the neonatal proteoglycan samples, the biosynthesis of proteoglycans by nucleus pulposus cells of adult discs increased progressively toward the sacrum. This correlated with increased metabolic activity. Analysis of the resident proteoglycans by composite agarose polyacrylamide gel electrophoresis indicated that although the aggrecan-1 population was present almost exclusively in the neonatal group, it was the aggrecan-2 population that predominated in the adult discs, and it became progressively more heterogeneous with aging and proximity of the disc to the sacrum. CONCLUSIONS: The proteoglycans of the lumbosacral disc of adult animals turned over faster than proteoglycans of adjacent lumbar discs. The reduced proteoglycan content and ability to aggregate, particularly in the nucleus pulposus of lumbosacral discs, indicated that proteoglycan catabolism exceeded the rate of biosynthesis. These events in the lumbosacral disc are thought to be determined mechanically by its proximity to the sacrum.     

Degeneration and the chemical composition of the human lumbar intervertebral disc

Fifteen lumbar spines were collected postmortem. The intervertebral discs were assigned morphological grades and were analyzed for water, collagen, and proteoglycan. In order of increasing degeneration, five grade 0, four grade 1, 45 grade 2, 12 grade 3A, and nine grade 3B discs were identified. The proteoglycan concentration fell progressively with increasing grade, although the concentrations of each component overlapped extensively among the grades. Grade 2 discs showed no consistent differences from adjacent grade 3A or 3B discs in the same spine. All discs in seven of the eight spines with grade 3A or 3B discs and all discs in three spines with no grade 3A or 3B discs had proteoglycan concentrations below 52 mg/g fresh weight. Four of five spines with at least one disc of proteoglycan concentration above this value contained no grade 3A or 3B discs. These observations support the hypothesis that low proteoglycan concentrations in all the discs of a spine precede degeneration.     

Swelling pressure of the lumbar intervertebral discs: influence of age, spinal level, composition, and degeneration

The fluid content of the intervertebral disc is important in determining its mechanical response and also its transport and biologic properties. Fluid content depends on the proteoglycan content of the tissue and on the relationship of the external load to the disc's swelling pressure. The influence of proteoglycan content and external load on the hydration of nuclei from 32 human lumbar discs was measured. Swelling pressure of the same specimens was measured by equilibrium dialysis. The influence of age (14-91 years) and spinal level was noted. Proteoglycan content of the discs fell with age, and for all spines tested, proteoglycan content was lowest in the L5-S1 disc; no systematic change in collagen content was found. The hydration of the discs, as received, also fell with increase in age; in each complete lumbar spine tested, the L1-L2 and the L5-S1 discs had the lowest hydration at postmortem examination. As the stress applied to the discs was increased, hydration decreased. Although a stress of 0.10-0.23 MPa maintained the disc slices at their postmortem hydration, under a stress of about 0.6-0.8 MPa, most discs lost 40-60% of their initial fluid. The relationship between change in hydration and swelling pressure was found to depend on the composition of the disc rather than on age or degree of degeneration; the relationship between equilibrium hydration and swelling pressure could be predicted satisfactorily for a disc of known collagen and proteoglycan content.     

退变椎间盘聚集蛋白聚糖中硫酸软骨素链变化的实验研究

目的：研究内破裂及突出椎间盘细胞合成的聚集蛋白聚糖中硫酸软骨素链（CS）在长度及数量分布上的变化.方法：将正常椎间盘、内破裂（IDD）及突出椎间盘（LIDP）髓核或纤维环的组织20mg培养于24孔板中,用放射性同位素35S-硫酸盐和3H-丝氨酸标记新合成的蛋白聚糖分子.将聚集蛋白聚糖单体从培养的组织片中提取,用四氢硼酸钠或木瓜蛋白酶消化后,凝胶包谱分析硫酸软骨链的变化特征.结果：IDD椎间盘组织内细胞合成的聚集蛋白聚糖内CS链的数量明显减少,但长度保持相对正常;突出椎间盘组织中CS的数量和长度有明显下降和缩短,CS链数量的减少较IDD组织中更为严重.结论：IDD组织合成CS的减少主要是由于生成的CS链数量减少所致,而数量的进一步减少以及CS链长度的缩短是LIDP组织中CS合成量下降的主要原因.     

Proteoglycan core protein in human urine and its possible role on calcium oxalate urolithiasis

BACKGROUND: There are only a few papers reporting on the role of proteoglycan core protein in calcium oxalate stone formation. The present study was carried out to investigate the role of core protein of proteoglycan in human urine on calcium oxalate (CaOx) crystallization. METHODS: Proteoglycans were collected from whole human urine. The covalently bound glycosaminoglycans (GAG) of proteoglycans were then digested by GAG lyase. The inhibitory activity on CaOx crystal growth in vitro was measured before and after enzyme digestion of proteoglycans. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the core protein of proteoglycans and the analysis of amino acid sequence were performed. RESULTS: The core protein showed significant inhibitory activity on CaOx crystal growth, which scarcely changed when compared with that of proteoglycans before enzyme digestion. The SDS-PAGE revealed that the core protein was a single unit with a molecular weight of 26 kDa and amino acid sequencing demonstrated high homology to interalpha-trypsin inhibitor (ITI) light chain (bikunin) with Kunitz inhibitor domain as a core protein. CONCLUSIONS: The results suggested that human urine contains proteoglycans and a major part of them is ITI light chain (bikunin). The Kunitz inhibitor domain, a core protein of bikunin, has significant inhibitory activity on CaOx crystallization without GAG bound covalently to the core protein.     

不同年龄及退变腰椎间盘中蛋白多糖酶活性测定

目的 测定不同年龄以及退变腰椎间盘中的蛋白多糖酶活性,探讨蛋白多糖酶与年龄、退变之间的关系.方法 对20个腰椎间盘的蛋白多糖酶活性进行测定,其中8个腰椎间盘来自胎儿,6个是退变的成人腰椎间盘,6个是正常的成人腰椎间盘.用含有10mmol/L CaCl2的0.05mol/L Tris/HC对腰椎间盘中的蛋白多糖酶进行提取,用1mmol/L APMA将其激活,与取自牛肩胛软骨的蛋白多糖单体底物进行反应,以酶标仪来测定蛋白多糖酶的活性.结果 胎儿腰椎间盘、退变成人腰椎间盘以及正常成人腰椎间盘的蛋白多糖酶活性的吸光度值分别是1.963±0.487、1.719±0.337、0.870±0.133.胎儿腰椎间盘与正常成人腰椎间盘的吸光度值差异有统计学意义(P<0.01),退变成人椎间盘的吸光度值也高于正常成人椎间盘(P<0.01). 结论腰椎间盘中的蛋白多糖酶活性与年龄、退变相关.     

腰椎间盘基质降解酶的研究

椎间盘基质大分子的变化可使其生物力学性能丧失,这种变化涉及到能使基质中的胶原和蛋白多糖发生改变的细胞外酶。作者以氚－Ⅰ型胶原为底物,对４１例手术治疗的腰椎间盘突出者的椎间盘组织及３４个正常尸检腰椎间盘组织作了胶原酶活力的测定,纤维环与髓核分别测定。用ＰＡＧＥ法以变性胶原为底物,光密度扫描峰值面积自动积分法对６个正常与１６个退变椎间盘作了中性蛋白酶相对含量的初步研究。结果显示,正常纤维环与髓核的胶原酶活力相似,仅含极微量的中性蛋白酶,退变椎间盘胶原酶与中性蛋白酶活性明显增强,尤其是退变髓核。破裂型椎间盘突出者的髓核胶原酶活力高于凸起型。提示基质降解酶在腰椎间盘退变过程中起重要作用,退变程度的差异是临床不同突出类型的生化基础。     

Part 1: dual-tuned proton/sodium magnetic resonance imaging of the lumbar spine in a rabbit model

STUDY DESIGN.: Development of a dual-tuned proton/sodium radiofrequency (RF) coil for magnetic resonance imaging (MRI) of the rabbit spine and quantification of sodium concentration in intervertebral discs. OBJECTIVE.: To develop the dual-tuned proton/sodium MRI of rabbit lumbar spine to investigate proteoglycan matrix content and intervertebral disc degeneration (IDD). SUMMARY OF BACKGROUND DATA.: IDD is a common chronic condition that may lead to back pain, limited activity, and disability. Early-stage IDD involves the loss of proteoglycan matrix and water content in the disc. Sodium MRI is a promising noninvasive technique for quantitative measurement of proteoglycan changes associated with IDD. The combined structural (proton) and biochemical (sodium) MRI facilitates the investigation of morphological and molecular changes associated with degeneration of discs. METHODS.: Multichannel dual-tuned proton/sodium transceiver RF coil of the rabbit spine was developed and optimized at 3T human scanner-8 channels allocated for the sodium coil and 4 channels for the proton coil. High-resolution anatomy proton images of the discs were acquired using turbo spin echo and dual echo steady state sequence. Sodium concentration of the discs was quantified from sodium magnetic resonance (MR) images that were calibrated for signal attenuation because of RF field inhomogeneity, sodium MR relaxation times, and disc thickness. Twelve rabbits (～1-yr old, female, 5.2 ± 0.4 kg) were used for measuring disc sodium concentration. RESULTS.: High-resolution in vivo proton and sodium MR images of rabbit discs (≤2-mm thickness) were successfully obtained using an in-house dual-tuned proton/sodium RF coil at 3T. The total acquisition time for each set of images was approximately 40 minutes. Sodium concentration of normal rabbit lumbar discs was measured at 269.7 ± 6.3 mM, and this measurement was highly reproducible, with 5.3% of coefficient of variation. CONCLUSION.: Sodium concentrations of rabbit lumbar discs were reliably measured using our newly developed dual-tuned multichannel proton/sodium RF coil at 3T.     

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.     

P物质阳性神经纤维在退变腰椎间盘的分布及意义

目的研究P物质（substance P,SP）阳性神经纤维在腰椎间盘中的出现及分布,以探讨其在椎间盘退变中的潜在作用。方法收集16例患者的21个病变腰椎间盘和来自于新鲜尸体的12个正常对照椎间盘,行组织学检查和SP免疫组织化学染色检查。结果SP阳性神经纤维偶见于正常椎间盘,在病变椎间盘内可见较多的SP免疫反应阳性神经纤维分布,阳性神经纤维数量与腰椎间盘退变程度呈正相关。结论腰椎间盘退变程度和SP阳性神经纤维数量有明显的相关性,SP可能作为神经炎性介质加速了腰椎间盘的退变。     

Does long-term compressive loading on the intervertebral disc cause degeneration?

STUDY DESIGN: Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs of dogs for up to 53 weeks. OBJECTIVE: To test the hypothesis that compressive forces applied to the intervertebral disc for a long period of time cause disc degeneration in vivo in a dog model. SUMMARY OF BACKGROUND DATA: It is a commonly held belief that high forces applied to the intervertebral disc, and to joints in general, play a role in causing degeneration. METHODS: Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs (L3/L4) of 12 dogs. After up to a year, the dogs were killed, and their lumbar spines were removed and radiographed. The L3/L4 disc and the controls (T13/L1 and L4/L5) were excised and examined for visible signs of degeneration. The discs then were assessed using immunohistochemical analysis and enzyme-linked immunosorbent assay. Disc chondrocytes also were assayed for apoptosis. RESULTS: No obvious signs of degeneration in the discs (L3/L4) that had been under compression for up to a year could be observed. There was no disc bulging, anular fissures, or disc space narrowing. Some changes were observed at the microscopic level, although no thickening of the endplate was apparent. The enzyme-linked immunosorbent assay analysis provided significant data for all three regions of the disc (nucleus, inner anulus, and outer anulus). When comparing the compressed disc (L3/L4) with either of the control discs (T13/L1 and L4/L5), in the compressed disc: 1) the nucleus contained less proteoglycan and more collagen I and II; 2) the inner anulus contained less proteoglycan and collagen I; and 3) the outer anulus contained more proteoglycan and less collagen I. The collagen II differences for the inner and outer anulus were not significant. CONCLUSION: Compression applied to the lumbar intervertebral discs of dogs for up to a year does not produce degeneration in any visible form. It does produce microscopic changes and numerical changes, however, in the amounts of proteoglycan and collagen in the nucleus, inner anulus, and outer anulus. The present results add no credence to the commonly held belief that high compressive forces play a causative role in disc degeneration.     

Lumbar disc degeneration: correlation with age, sex, and spine level in 600 autopsy specimens

Using data from 16 published reports, the authors correlated macroscopic disc degeneration grades with age, sex, and spine level in 600 lumbar intervertebral discs from 273 cadavers (ages: 0-96 years). Male discs were more degenerated than female discs at most ages; significantly so in the second, fifth, sixth, and seventh decades. On average, L4-L5 and L3-L4 level discs showed more degeneration than discs at other lumbar levels. These macroscopic findings corroborate radiographic data from epidemiologic studies. The calculations suggest that higher mechanical stress, perhaps combined with longer nutritional pathways, may be responsible for the earlier degeneration of male discs.     

The effect of spinal fusion on the collagen and proteoglycans of the canine intervertebral disc

The effects on disc composition of 16 months of lumbar spinal arthrodesis in the greyhound (a nonchondrodystrophoid breed) have been investigated using age-matched controls. The guanidinium chloride-extractable proteoglycans of the disc's nucleus pulposus and annulus fibrosus were compared using equilibrium density gradient ultracentrifugation under associative conditions. No differences in proteoglycan sedimentation behavior were detected between discs from experimental or control animals. Analysis of individual discs for collagen, total nitrogen, noncollagenous protein, uronic acid, or total hexosamine content also failed to demonstrate statistically significant differences between the experimental and control groups.     

Lumbar motion-sparing surgery

Surgical treatment of the painful lumbar spine segment is a challenging endeavor. The ideal surgical intervention has a predictably good clinical effect, a low incidence of complications, and long-term durability. The gold standard for surgical intervention is a lumbar spine fusion, but the long-term durability of lumbar fusions is compromised with the development of adjacent level degeneration. Due to these concerns, motion-sparing surgical implants, including artificial discs, dynamic stabilization techniques, and nucleus pulposus replacements, have been developed to simultaneously treat the painful motion segment while at the same time preserving sufficient motion to decrease the chances of adjacent level degeneration.     

Levels of keratan sulfate-bearing fragments rise predictably following chemonucleolysis of dog intervertebral discs with chymopapain

Chymopapain (1 mg) was injected into each of four-lumbar intervertebral discs of adult mongrel dogs. As expected, at 2 weeks, all injected discs exhibited marked loss of height (mean: 50% of original height) indicative of severe proteoglycan depletion. The appearance of keratan sulfate-bearing fragments in plasma was monitored by an ELISA-inhibition assay which uses a monoclonal antibody (1/20/5-D-4) specific for an epitope present only in the longest keratan sulfate chains. Levels of plasma keratan sulfate rose within 30 minutes and reached a maximum between 24 and 72 hours later. Levels then declined progressively but were still elevated at 2 weeks postinjection. Keratan sulfate-bearing fragments in plasma were purified by ion exchange chromatography on DEAE-Sephacryl and fractionated by sieve chromatography on Sepharose CL-6B. These plasma keratan sulfate-bearing fragments were found to be similar in size to keratan sulfate-bearing fragments generated by chymopapain digestion of dog nucleus pulposus proteoglycans, but slightly larger than single keratan sulfate chains obtained by alkaline borohydride treatment of dog nucleus pulposus proteoglycans. The results of this study show that measurements of blood levels of keratan sulfate could prove useful in monitoring effective degradation of disc proteoglycans in chemonucleolysis in man and help discriminate between ineffective enzyme placement, and alternative mechanisms of treatment failure.     

Proteoglycan alterations during developing experimental osteoarthritis in a novel hip joint model

Degenerative hip joint disease was induced in dogs by extra-articular surgery that created a condition that mimics hip dysplasia. Decreased acetabular coverage of the femoral head gave altered mechanical load, with ensuing cartilage degeneration. For comparison, degenerative knee joint disease was induced in other dogs by transection of the anterior cruciate ligament of the knee. The femoral head articular cartilage showed macroscopic signs of degeneration within a month. No macroscopical changes of synovitis were present. Chemical analysis of cartilage samples showed loss of proteoglycans. Guanidine hydrochloride extracts of the cartilage contained proteoglycan fragments that could be separated by equilibrium density gradient centrifugation in cesium chloride. The data indicate that proteoglycans are fragmented by proteolytic cleavage and lost from the cartilage. The proteoglycans remaining in the tissue are smaller and have lost the ability to aggregate with hyaluronic acid. Similarly, in experimental knee joint osteoarthritis, the proteoglycan content of the cartilage decreased. The structural changes of those proteoglycans remaining were of a different nature, with no changes in proteoglycan size or aggregation properties, possibly indicating that both degradation and repair took place in the knee articular cartilage and/or that fragments were rapidly lost from the tissue. This may follow from different surgical procedures, only the one used for the hip joint being extra-articular, or from the different anatomy and physiology of the hip joint and the knee joint.     

Immunohistochemical localization of proteoglycans in interstitial elements of human bladder cancer

The immunohistochemical localization of glycosaminoglycan side chains and core protein of proteoglycan was observed, using monoclonal antibodies, on 8 specimens of non-tumor bladder tissues and 26 specimens of bladder tumors obtained from total cystectomy or transurethral resection of bladder tumor (TUR-Bt). In non-tumor tissues of human bladder examined, the surface of urothelial epithelium consisted of heparan sulfate as revealed with antibody HepSS-1, and submucosal interstitial elements consisted mainly of small proteoglycan having chondroitin 4-sulfate side chains as revealed with antibodies 6B6 and 9A2, respectively. On the other hand, in bladder tumors examined, the interstitial fibrous elements, the so-called "specific stroma" in cancer cell nests, consisted mainly of large proteoglycans having chondroitin 4-sulfate or 6-sulfate side chains as revealed with antibodies 2B1, 9A2 and 3B3, respectively. Antibodies 2B1 and 3B3 are considered to be useful to demonstrate the involvement by invasive growth of bladder tumor cells.     

椎间盘退变时水通道蛋白1表达变化研究

目的阐明水通道蛋白1在椎间盘退变时表达变化特点。方法采用腰椎失稳的方法建立大鼠腰椎间盘退变的动物模型,影像学观察模型建立的可靠性,收集大鼠椎间盘组织,部分用于提取RNA,采用RT-PCR的方法检测水通道蛋白1mRNA表达的变化,部分标本用western blot进行AQP1蛋白的检测。结果腰椎失稳可以建立大鼠腰椎间盘退变模型,AQP1mRNA的表达随椎间盘退变的进程进行性下降,其蛋白表达也呈现进行性下降的趋势,术后3月时同术前相比相差显著(P<0.05),术后6月和9月则下降更为明显(P<0.01);而在手术对照组而言,于术后6月时也出现AQP1基因和蛋白表达水平的下降(P<0.05)。结论腰椎间盘退变时,AQP1在基因和蛋白的表达水平下降,且与退变的时间长短相关,提示AQP1参予了椎间盘退变的发生发展,AQP1表达的下降可能是引起椎间盘退变的原因之一。     

Immunodetection and partial cDNA sequence of the proteoglycan, superficial zone protein, synthesized by cells lining synovial joints.

We have previously described a large proteoglycan named superficial zone protein that was isolated and purified from culture medium of superficial slices of bovine articular cartilage. Monoclonal antibodies were raised against superficial zone protein and used as probes in Western blot analyses for immunohistochemical studies both to determine precisely which cells within the joint synthesize the proteoglycan and to isolate a cDNA fragment from a bovine chondrocyte lambdagt11 library that encodes part of the proteoglycan. The cDNA fragment that was obtained with use of monoclonal antibody 6-A-1 encodes the 3' end of the sequence for superficial zone protein. On Western blots, monoclonal antibody 3-A-4 recognized an epitope on native, but not reduced, superficial zone protein, whereas monoclonal antibody 6-A-1 reacted with both native and denatured antigen. The proteoglycan was immunolocalized with monoclonal antibody 3-A-4 in chondrocytes predominantly within the superficial zone of fetal and adult articular cartilage and in some cells of the synovial lining. However, the proteoglycan was not detected in chondrocytes deep in articular cartilage, in nasal septal cartilage, or in synovial stromal cells. The only matrix staining positively for superficial zone protein was at the articular surface bordering the synovial cavity in adult, but not fetal, joints. Isolated chondrocytes and synovial cells showed intracellular binding of monoclonal antibody 3-A-4, and flow-cytometric analysis with the antibody gave the following percentages of immunopositive cells: 37.4, 52.5, 3.4, and 7.5 from chondrocytes from the full-thickness, superficial, and deep zones and from synovial cells, respectively. Thus, both chondrocytes and synovial cells bordering the joint cavity synthesize superficial zone protein and substantiate its usefulness as a phenotypic marker of particular cellular species lining the articular cavity.