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.     

Water, fixed charge density, protein contents, and lysine incorporation into protein in chymopapain-digested intervertebral disc of rabbit

Chymopapain (Discase) was injected at a dose of 0.125 nanokatal unit into the intervertebral discs of rabbits, and sequential changes in the metabolism of water, proteoglycan, collagen, and noncollagenous protein were investigated separately in the nucleus pulposus, anterior, and posterior anulus fibrosus. One week after chymopapain injection, the water and proteoglycan content was lower in all of the fractionated tissues of the anterior and posterior anulus and nucleus pulposus of the discs than in the control discs. In the anterior and posterior anulus, the proteoglycan content recovered after 12 weeks, but there was no recovery in the nucleus pulposus. The collagen content continued to increase up to the 12th week in the nucleus pulposus, while the noncollagenous protein content decreased in all tissue fractions after 1 week. In the anterior and posterior anulus, the content of noncollagenous protein recovered after 3 to 6 weeks, but there was no recovery in the nucleus pulposus. The lysine incorporation in collagen and noncollagenous protein was inhibited in all tissue fractions after 12 weeks, suggesting a decrease in synthetic activity. The intradiscal pressure calculated from proteoglycan hydration at 1 to 6 weeks after chymopapain injection showed a marked decrease to 0.8 to 0.9 atm, but it recovered to 1.6 atm after 12 weeks.     

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.     

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.     

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.     

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.     

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.     

Effect of extracellular matrix protein on the rate of proteoglycan synthesis in rabbit intervertebral disc cells

OBJECTIVE: The extracellular matrix (ECM) is very important for fundamental cellular processes. However, the effects of ECM proteins on intervertebral disc (IVD) cell proliferation and metabolism have not been clarified. To verify the effects of ECM proteins on DNA and proteoglycan (PG) synthesis of IVD cells, PG synthesis rate was measured in IVD cells cultured in monolayer with or without ECM protein. METHODS: Nucleus pulposus (NP) cells and anulus fibrosus (AF) cells isolated from adolescent rabbits were cultured in monolayer with or without ECM protein and at different concentrations of ECM protein for 4-6 days. [S]Sulfate incorporation into PG in the cell-associated matrix (CM) formed around cells and the further-removed matrix (FRM) in labeling medium was measured and standardized to DNA content. CONCLUSIONS: NP cells in type I or type II collagen-coated plates significantly increased the rate of PG synthesis in both the CM and the FRM compared with those in uncoated plates and in fibronectin-coated plates; however, AF cells with ECM proteins did not increase the rate significantly. The rate of PG synthesis of nucleus cells was contra-dose dependent on both type I and type II collagen.     

Sensitivity of anulus fibrosus cells to interleukin 1 beta. Comparison with articular chondrocytes

STUDY DESIGN: Anulus fibrosus cells from rabbits were grown in primary culture 1) to study their ability to produce prostaglandin E2 and Type II phospholipase A2, and to express stromelysin-1 messenger ribonucleic acid; and 2) to study the effect of interleukin 1 beta on this production and on proteoglycan aggregation. OBJECTIVES: To investigate the potency of anulus fibrosus cells to respond to interleukin 1 beta by producing degradative and inflammatory agents as compared with the potency of articular chondrocytes in the same animal. SUMMARY OF BACKGROUND DATA: Interleukin 1 beta has been implicated in the degradation of intervertebral discs. The way anulus fibrosus cells differ from articular chondrocytes in their responses to interleukin 1 beta remains to be established. METHODS: Anulus fibrosus cells and articular chondrocytes were obtained from young rabbits, grown in primary culture, and incubated with interleukin 1 beta. The newly synthesized proteoglycan was measured by labeling with [35S]-sulfate. Proteoglycan aggregation was analyzed by the elution profile on Sepharose 2B columns. The contents of collagen Type II and stromelysin-1 messenger ribonucleic acid were assessed by Northern blot analysis. The Type II phospholipase A2 activity was measured using a fluorometric substrate. Prostaglandin E2 production was evaluated by radioimmunoassay. RESULTS: Anulus fibrosus cells had 2.5-fold less Type II collagen messenger ribonucleic acid than articular chondrocytes, and interleukin 1 beta had no significant effect on this. Anulus fibrosus cells synthesized and secreted four-fold less proteoglycan than articular chondrocytes. Interleukin 1 beta reduced the anulus fibrosus content of total [35S]-sulfated proteoglycan by 35% (P < 0.01), and that of articular cells by 41% and decreased proteoglycan aggregation. Interleukin 1 beta induced the production of stromelysin-1 messenger ribonucleic acid in both cell types. The stromelysin-1 messenger ribonucleic acid content of anulus fibrosus cells was one half that of articular cells. Interleukin 1 beta increased the production of prostaglandin E2 and caused a dose-dependent secretion of Type II phospholipase A2 activity in both cell types. Its effect was 2.5-fold lower in anulus fibrosus cells than in articular chondrocytes. CONCLUSION: Anulus fibrosus cells can be stimulated by interleukin 1 beta to produce factors implicated in local degradative and inflammatory processes. This production is associated with decreased proteoglycan aggregation. Anulus fibrosus cells respond slightly less well to interleukin 1 beta in vitro than do articular 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。     

微载体立体培养腰椎间盘细胞蛋白多糖表达的研究

目的研究腰椎间盘细胞在微载体培养与单层培养中细胞表达蛋白多糖含量的差别。方法椎间盘疾病手术病例的术中切除组织采用酶消化法分别进行微载体三维细胞培养和单层细胞培养;取胎儿椎间盘组织,显微镜下区分髓核细胞和纤维环细胞,分别进行培养,同成入组对照。利用^35S放射标记渗入放免定量测定的方法进行蛋白多糖含量的检测。结果①椎间盘细胞胞内的蛋白多糖含量（cpm）,细胞单层培养组为101．909±11．439,微载体立体培养组为136．607±10．792,P〈0．05;②椎间盘细胞表达的蛋白多糖含量（cpm）,细胞单层培养组为105．119±13．040,微载体立体培养组为174．231±17．676,P〈0．05;③各组椎间盘细胞表达的蛋白多糖含量均高于细胞内的含量;④胎儿腰椎间盘细胞蛋白多糖的含量及表达量均高于成人退变椎间盘细胞,胎儿髓核细胞蛋白多糖的表达量高于纤维环细胞的表达量。结论椎间盘细胞的微载体三维立体培养相对单层培养具有较高细胞蛋白多糖的表达量,是一种较好的细胞培养方式。     

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.     

单纯Ⅱ型胶原酶消化法分离、培养人退变椎间盘髓核细胞的形态学观察

目的:探讨单纯Ⅱ型胶原酶消化法体外培养扩增人退变椎间盘髓核细胞的可行性。方法:收集20例人退变椎间盘髓核,单纯Ⅱ型胶原酶消化分离出髓核细胞并连续培养传代,倒置相差显微镜和HE染色观察细胞形态学变化,甲苯胺蓝染色检测髓核细胞内聚集蛋白聚糖的表达,免疫细胞化学法行Ⅱ型胶原染色,观察髓核细胞的类软骨表型表达情况。结果:单纯Ⅱ型胶原酶消化法可较好的分离培养人退变椎间盘髓核细胞,20例人退变椎间盘髓核,培养成功16例;原代髓核细胞平均7d贴壁,呈类圆形或多角形,P1代髓核细胞平均12h贴壁,呈大梭形或多角形,两代细胞融合95%所需时间分别为30d和7d,差异有统计学意义(P0.01);聚集蛋白聚糖和Ⅱ型胶原主要表达于原代和P1代髓核细胞浆内,被甲苯胺蓝染成天蓝色,免疫细胞化学染色主要表现为黄褐色沉淀,两代间聚集蛋白聚糖和Ⅱ型胶原的表达无统计学差异(P0.05)。结论:单纯Ⅱ型胶原酶消化法可简化髓核细胞分离步骤,提高培养效率;传一代后髓核细胞增殖速率提高,但仍维持类软骨表型,表达聚集蛋白聚糖和Ⅱ型胶原。     

Complications following chemonucleolysis with collagenase

Lumbar disc tissue from eight patients previously submitted to unsuccessful chemonucleolysis with collagenase was studied by light and scanning electron microscopy (SEM). Similar material from eight patients subjected to primary disc surgery served as control. The control discs revealed the characteristic signs of degeneration of collagen tissue, microcystic areas, and giant chondromas. However, the anulus fibrosus and the end-plates remained intact. Following chemonucleolysis with collagenase, "digestion" of the nucleus pulposus, the anulus fibrosus, and extensive damage to the end-plates, bone, ligaments, and epidural fat were seen.     

Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion,and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs

The present study was undertaken to investigate the effect of insulin-like growth factor-1 on proteoglycan synthesis and the autocrine/paracrine mechanisms involving insulin-like growth factor-1 in the bovine coccygeal intervertebral disc. Insulin-like growth factor-1 stimulated proteoglycan synthesis in cultured cells of the nucleus pulposus of bovine intervertebral discs in a dose-dependent manner, and the effect was inhibited by an anti-insulin-like growth factor-1 monoclonal antibody. In situ hybridization histochemistry revealed the expression of insulin-like growth factor-1 mRNA in the cultured cells, and its production in these cells was demonstrated by radioimmunoassay. Insulin-like growth factor-1 receptor in the cultured cells was also demonstrated immunohistochemically. Scatchard analysis using an [¹²⁵I]insulin-like growth factor-1 binding assay showed that the cells cultured in monolayer had a single type of insulin-like growth factor-1 receptor, whose affinity and number were estimated to be 7.38 × 10⁸/M and 9.27 × 10⁴/cell, respectively. These results suggest that insulin-like growth factor-1 stimulates proteoglycan synthesis in cells of the nucleus pulposus and that these cells in culture have an insulin-like growth factor-1 autocrine/paracrine mechanism. The expressions of insulin-like growth factor-1 mRNA and insulin-like growth factor-1 receptor in disc tissue were greater in cells of the nucleus pulposus of fetal bovine intervertebral discs than in those of the adult discs. These findings suggest that the action of autocrine/paracrine insulin-like growth factor-1 is more active in cells of the young nucleus pulposus than in cells of mature subjects.     

Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study.

Reinsertion of autogenous nucleus pulposus, an innovative method to delay further disc degeneration, has been proved with an experimental animal model. This study examined whether coculture of nucleus pulposus cells with annulus fibrosus cells (a) activates annulus fibrosus cells and (b) retards disc degeneration when reinserted into the disc in a rabbit model of disc degeneration. Coculture of the two cell types stimulated proliferation of each, as indicated by increased DNA synthesis measured by increases in DNA polymerase alpha expression and uptake of 5-bromo-2'deoxy-uridine assessed by an enzyme-linked immunosorbent assay. In a model of disc degeneration in rabbits, reinsertion of activated nucleus pulposus cells delayed the formation of clusters of chondrocyte-like cells, the destruction of disc architecture, and the elaboration of type-II collagen as measured immunohistochemically compared with no treatment. The direct reinsertion of activated nucleus pulposus cells into the disc offers a promising line of investigation for delaying intervertebral disc degeneration, although these results obtained with notochordal cells may not necessarily apply when mature central nucleus pulposus cells are used.     

The effect of hydrostatic pressure on intervertebral disc metabolism.

STUDY DESIGN: By the use of pressure vessels, hydrostatic pressure was applied to intervertebral disc cells cultured in an alginate. OBJECTIVE: To test the hypothesis that hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells. SUMMARY OF BACKGROUND DATA: The influence of compression (both hydrostatic and mechanical) on chondrocyte metabolism was examined in a number of earlier studies. However, in most of these studies, articular cartilage, not intervertebral disc, was used, and in none of these was hydrostatic pressure applied to intervertebral disc cells cultured in alginate. METHODS: Fresh cells were harvested from the lumbar intervertebral discs of dogs. Before their suspension in an alginate gel system, the cells were plated and expanded until they reached confluence. Then, by use of the alginate gel system, the cells were exposed (for up to 9 days) to specific values of hydrostatic pressure inside two stainless steel pressure vessels. One vessel was kept at 1 MPa and the other at atmospheric pressure. The effects of 1 MPa were compared against atmospheric pressure by measuring the incorporation of [3H]-proline and [35S]-sulfate into collagen and proteoglycans, respectively, for the anulus cells and nucleus cells separately, and by determining whether this incorporation was reflected by changes in the levels of mRNA for aggrecan and Types I and II collagen. RESULTS: Comparisons with atmospheric pressure yielded the following findings: 1) In the incorporation studies, the nucleus and anulus cells exhibited a differential response to a hydrostatic pressure of 1 MPa. Collagen and proteoglycan syntheses were stimulated in the nucleus cells and inhibited in the anulus cells. 2) There was no significant increase in cell proliferation, as measured by DNA content, at 1 MPa for either the anulus or nucleus cells. 3) The mRNA levels of collagen (Col 1A1 and Col 2A1) and aggrecan increased at 1 MPa in both the nucleus and anulus cells. CONCLUSIONS: Hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells.