Innervation of pathologies in the lumbar vertebral end plate and intervertebral disc

Background contextMagnetic resonance imaging (MRI) has limited diagnostic value for chronic low back pain because of the unclear relationship between any anatomic abnormalities on MRI and pain reported by the patient. Assessing the innervation of end plate and disc pathologies—and determining the relationship between these pathologies and any abnormalities seen on MRI—could clarify the sources of back pain and help identify abnormalities with enhanced diagnostic value.PurposeTo quantify innervation in the vertebral end plate and intervertebral disc and to relate variation in innervation to the presence of pathologic features observed by histology and conventional MRI.Study design/settingA cross-sectional histology and imaging study of vertebral end plates and intervertebral discs harvested from human cadaver spines.MethodsWe collected 92 end plates and 46 intervertebral discs from seven cadaver spines (ages 51–67 years). Before dissection, the spines were scanned with MRI to grade for Modic changes and high-intensity zones (HIZ). Standard immunohistochemical techniques were used to localize the general nerve marker protein gene product 9.5. We quantified innervation in the following pathologies: fibrovascular end-plate marrow, fatty end-plate marrow, end-plate defects, and annular tears.ResultsNerves were present in the majority of end plates with fibrovascular marrow, fatty marrow, and defects. Nerve density was significantly higher in fibrovascular end-plate marrow than in normal end-plate marrow (p<.001). Of the end plates with fibrovascular and fatty marrow, less than 40% were Modic on MRI. Innervated marrow pathologies collocated with more than 75% of the end plate defects; hence, innervation was significantly higher in end plate defects than in normal end plates (p<.0001). In the disc, nerves were observed in only 35% of the annular tears; in particular, innervation in radial tears tended to be higher than in normal discs (p=.07). Of the discs with radial tears, less than 13% had HIZ on T2 MRI. Innervation was significantly less in radial tears than in fibrovascular end-plate marrow (p=.05) and end-plate defects (p=.02).ConclusionsThese findings indicate that vertebral end-plate pathologies are more innervated than intervertebral disc pathologies and that many innervated end-plate pathologies are not detectable on MRI. Taken together, these findings suggest that improved visualization of end-plate pathologies could enhance the diagnostic value of MRI for chronic low back pain.     

1991 Volvo Award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate: an immunolocalization study

Several types of collagen are known to exist in the intervertebral disc in addition to the fibrillar collagens, Types I and II. Although they constitute only a small percentage of the total collagen content, these minor collagens may have important functions. This study was designed to investigate the presence of Types I, II, III, IV, VI, and IX collagens in the intervertebral disc and cartilage end plate by immunohistochemistry, thereby establishing their location within the tissues. Types III and VI collagen have a pericellular distribution in animal and human tissue. No staining for Type IX collagen was present in normal human disc, but in rat and bovine intervertebral disc, it was also located pericellularly. These results show that cells of the intervertebral disc and cartilage end plate sit in fibrous capsules, forming chondrons similar to those described in articular cartilage. In pathologic tissue the amount and distribution of the collagen types, and the organization of the pericellular capsule, differ from that seen in control material.     

椎间盘髓核细胞组织块法原代培养

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

A phenotypic comparison of intervertebral disc and articular cartilage cells in the rat

The basic molecular characteristics of intervertebral disc cells are still poorly defined. This study compared the phenotypes of nucleus pulposus (NP), annulus fibrosus (AF) and articular cartilage (AC) cells using rat coccygeal discs and AC from both young and aged animals and a combination of microarray, real-time RT-PCR and immunohistochemistry. Microarray analysis identified 63 genes with at least a fivefold difference in fluorescence intensity between the NP and AF cells and 41 genes with a fivefold or greater difference comparing NP cells and articular chondrocytes. In young rats, the relative mRNA levels, assessed by real-time RT-PCR, of annexin A3, glypican 3 (gpc3), keratin 19 (k19) and pleiotrophin (ptn) were significantly higher in NP compared to AF and AC samples. Furthermore, vimentin (vim) mRNA was higher in NP versus AC, and expression levels of cartilage oligomeric matrix protein (comp) and matrix gla protein (mgp) were lower in NP versus AC. Higher NP levels of comp and mgp mRNA and higher AF levels of gpc3, k19, mgp and ptn mRNA were found in aged compared to young tissue. However, the large differences between NP and AC expression of gpc3 and k19 were obvious even in the aged animals. Furthermore, the differences in expression levels of gpc3 and k19 were also evident at the protein level, with intense immunostaining for both proteins in NP and non-existent immunoreaction in AF and AC. Future studies using different species are required to evaluate whether the expression of these molecules can be used to characterize NP cells and distinguish them from other chondrocyte-like cells.     

Optimizing nonviral-mediated transfection of human intervertebral disc chondrocytes

BACKGROUND CONTEXT: The use of viral vectors for transfection of human disc chondrocytes has been well documented. However, because of immunological and cell toxicity concerns, nonviral reagents may provide gene delivery to intervertebral disc (IVD) chondrocytes without these associated obstacles. Several studies have been done using nonviral delivery systems with varying degrees of success. PURPOSE: The purpose of the study was to determine the efficiency, toxicity, and optimal conditions for gene delivery into human degenerative IVD cells via nonviral reagents in vitro. STUDY DESIGN/SETTING: In vitro viral and nonviral gene transfer. PATIENT SAMPLE: Human disc chondrocytes from 21 patients undergoing discectomy for trauma, disc herniation, and fusion for scoliosis or degenerative low back pain. OUTCOME MEASURES: Cell cytotoxicity and transfection efficiency as determined by microscopy, luciferase assay, and flow cytometry. METHODS: Seventeen lipid-based nonviral reagents coupled to DNA plasmids coding for luciferase were transfected into cultured chondrocytes. Cells were transfected with varying ratios of DNA plasmid to reagent, harvested at 48 hours and analyzed for transfection rates and cell viability. Transfections with adenoviral constructs were comparisons. The three most efficient reagents were then coupled to green fluorescent protein and the experiments repeated. The most efficient reagent after these experiments (LT1) was tested in standard chondrocyte-maintenance medium and a minimal medium mixture devoid of antibiotics, buffers, and amino acids. Finally, LT1 in minimal medium with various hyaluronidase treatments was tested. The most effective reagents and relative toxicity as measured by flow cytometry were analyzed using repeated measures analysis of variance. RESULTS: LT1 was most efficient and least toxic of nonviral reagents tested. LT1 had a mean percent survival of 78.1% versus 26.6% for TKO, 15.8% for T-Jurkat, and 70.8% in controls. Transfection was 1.5%. LT1 in minimal medium was significantly better than other reagents for both cell viability and transfection percentages. Minimal medium increased transfection with other reagents, yet cell viability with TKO and T-Jurkat was poor. Hyaluronidase had no effect on the viability of controls and decreased viability from 74.9% to an overall mean of 62.6% for all treatments. Transfection percentages increased from 1.8% without treatment to 15.2% with 40 units and 10.4% with four units of hyaluronidase given 24 hours before transfection and left in throughout the experiment. When treated at the time of transfection, efficiency was not significantly different to samples without hyaluronidase added. Additionally, hyaluronidase added 24 hours before transfection and washed out at the time of transfection significantly increased transfection percentages. CONCLUSIONS: LT1 was the most efficient reagent in terms of transfection ability and cell toxicity compared with other reagents. Treatments in minimal medium yielded significant increases in transfection and no significant difference in toxicity as compared with controls. Hyaluronidase treatments improve transfection significantly but also increase toxicity. These results suggest that the nonviral reagent LT1 can be used to transfect IVD chondrocytes in vitro and may help facilitate gene transfection of IVD chondrocytes in vivo.     

Micromechanics of annulus-end plate integration in the intervertebral disc

Background contextThe intervertebral disc plays a major functional role in the spinal column, providing jointed flexibility and force transmission. The end plate acts as an important structural transition between the hard vertebral tissues and the compliant disc tissues and is therefore a region of potentially high stress concentration. The effectiveness of anchorage of the tough annulus fibers in the end plate will have a major influence on the overall strength of the motion segment. Failure of the end plate region is known to be associated with disc herniation.PurposeThe aim of this study was to investigate the mechanism of anchorage of the annular fibers in the end plate.Study designA microstructural analysis of the annulus–end plate region was carried out using motion segments obtained from the lumbar spines of mature ovine animals.MethodsMotion segments were fixed and then decalcified. Samples incorporating the posterior annulus–end plate were then removed and cryosectioned along the plane of one of the lamellar fiber directions to obtain oblique interlamellar sections. These sections were imaged in their fully hydrated state using differential interference contrast optical microscopy.ResultsThe annular fiber bundles on entering the end plate are shown to subdivide into subbundles to form a three-dimensional multileaf morphology with each leaf separated by cartilaginous end plate matrix. This branched morphology increases the interface area between bundle and matrix in proportion to the number of subbundles formed.ConclusionsGiven both the limited thickness of the end plate and the intrinsic strength of the interface bond between bundle and end plate matrix, the branched morphology is consistent with a mechanism of optimal shear stress transfer wherein a greater strength of annular fiber anchorage can be achieved over a relatively short insertion distance.     

Triamcinolone decreases bupivacaine toxicity to intervertebral disc cell in vitro

Background contextLocal anesthetics combined with corticosteroids are commonly used for management of back pain in interventional spinal procedures. Several recent studies suggest cytotoxicity of bupivacaine, whereas others report protective and cytotoxic effects of corticosteroids on chondrocytes and intervertebral disc cells. Considering the frequent use of these agents in spinal interventions, it is meaningful to know how they affect intervertebral disc cells.PurposeThis study was conducted to assess the effects of bupivacaine and triamcinolone, both alone and in combination, on viability of intervertebral disc cells in vitro.Study designControlled laboratory study.MethodsNucleus pulposus cells were isolated from human disc specimens from patients undergoing surgery because of disc herniation or degenerative disc disease. They were grown in three-dimensional alginate beads for 1 week to maintain their differentiated phenotypes and to allow for matrix formation before analysis. After 1 week of culture, the cells were exposed to bupivacaine (0.1%, 0.25%, 0.5%, and 1%) or bupivacaine (0.1%, 0.25%, 0.5%, and 1%) with 1 mg of triamcinolone for 1, 3, or 6 hours. Cell viability was measured using trypan blue exclusion assay and flow cytometry. Live cell/dead cell fluorescent imaging was assessed using confocal microscopy.ResultsTrypan blue exclusion assays demonstrated dose- and time-dependent cytotoxic effects of bupivacaine on human nucleus pulposus cells. Similar but reduced cytotoxicity was observed after exposure to the combination of bupivacaine and 1 mg of triamcinolone. Flow cytometry showed a dose-dependent cytotoxic effect of bupivacaine on nucleus pulposus cells after 3 hours of exposure. The reduced cytotoxicity of bupivacaine combined with 1 mg of triamcinolone was also confirmed in flow cytometry. Confocal images showed that the increase in dead cells correlated with the concentration of bupivacaine. Nevertheless, fewer cells died after exposure to several different concentrations of bupivacaine combined with 1 mg of triamcinolone than did after exposure to bupivacaine alone.ConclusionsThe combination of bupivacaine and triamcinolone induced dose- and time-dependent cytotoxicity on human intervertebral disc cells in vitro, but the cytotoxicity was much weaker than that of bupivacaine alone. This study shows a potential protective influence of triamcinolone on intervertebral disc cells.     

大鼠不同部位软骨细胞的形态及表型特征比较研究

目的:比较研究大鼠椎间盘软骨终板和膝关节软骨的细胞表型特征的相关性.方法:大鼠的软骨终板和关节软骨细胞分别予以消化培养.进行光镜、电镜观察其形态.使用免疫组化技术分别检测不同部位细胞的Ⅱ型胶原表达.结果:大鼠椎间盘软骨终板和关节软骨细胞形状相似,并且均表达Ⅱ型胶原.结论:本研究提示软骨终板表达软骨细胞的特征性胶原,与关节软骨细胞相似.     

PDGF‐BB inhibits intervertebral disc cell apoptosis in vitro

Degeneration of the intervertebral disc (IVD) results in deterioration of the spinal motion segment and can lead to debilitating back pain. Given the established mitotic and anti‐apoptotic effects of recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB) in a variety of cell types we postulated that rhPDGF‐BB might delay disc cell degeneration through inhibition of apoptosis. To address this hypothesis, we treated human IVD cells isolated from five independent patients with rhPDGF‐BB in monolayer and 3D pellet cultures. The anti‐apoptotic potential, cell proliferative capacity, morphology/pellet differentiation, and gene expression of PDGF‐treated IVD cells were evaluated via flow cytometry/immunohistochemistry, MTT assays, histology, and quantitative RT‐PCR, respectively. We found that rhPDGF‐BB treatment significantly inhibited cell apoptosis, increased cell proliferation and matrix production, and maintained mRNA expression of critical extracellular matrix genes. This study suggests two possible mechanisms for the anti‐degenerative effects of rhPDGF‐BB on human IVD cells. First, PDGF treatment strongly inhibited IVD cell apoptosis in 3D cultures. Second, rhPDGF‐BB acts as an anabolic agent, promoting maintenance of IVD cell phenotype in 3D culture, based on the molecular and protein expression analysis. We speculate that rhPDGF‐BB may be used as a biologic treatment to target early degenerative IVD disease in the future. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1181–1188, 2014.     

PDGF,bFGF and IGF-I stimulate the proliferation of intervertebral disc cells in vitro via the activation of the ERK and Akt signaling pathways

Intervertebral disc (IVD) degeneration is frequently characterized by increased cell proliferation, probably as a tissue regenerative response. Although many growth factors and their receptors have been shown to be expressed normally in the disc, and generally to be over-expressed during degeneration, not all of them have been thoroughly studied concerning their effects on IVD cell proliferation. In the present report, three potent mitogens, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) are examined regarding their capacity to induce proliferation in vitro of bovine coccygeal nucleus pulposus (NP) and annulus fibrosus (AF) cells, as well as to activate major intracellular signal transduction pathways. PDGF, bFGF and IGF-I were found to induce DNA synthesis in quiescent IVD cells in a dose-dependent manner. Maximum stimulation was induced by PDGF, while stimulation by all three factors simultaneously exceeded only slightly that caused by PDGF alone. All three growth factors were shown to phosphorylate immediately extracellular-signal regulated kinases (ERKs), while the stimulation by bFGF especially resulted in sustained ERK phosphorylation. Furthermore, all three growth factors induced phosphorylation of Akt in both Thr308 and Ser473 residues immediately after stimulation, although bFGF-induced phosphorylation was much weaker than that provoked by PDGF and IGF-I. In addition, the MEK inhibitor PD98059 and the PI 3-K inhibitor wortmannin were shown to block growth factor-induced ERK- and Akt-phosphorylation, respectively, in IVD cells. Inhibition of the MEK/ERK or the PI 3-K/Akt pathways provoked a significant decline of the proliferative effects of PDGF, bFGF or IGF-I on IVD cell cultures, while the simultaneous inhibition of both signaling pathways abolished completely the mitogenicity of these growth factors. The above effects of the three growth factors were reproduced similarly in both NP and AF cell cultures. Overall, the above results indicate that PDGF, bFGF and IGF-I stimulate the proliferation of IVD cells via the ERK and Akt signaling pathways.     

Effect of cell density on the rate of glycosaminoglycan accumulation by disc and cartilage cells in vitro

Aggrecan concentration falls markedly during cartilage and disc degeneration with unfortunate biomechanical and physiological consequences. There is thus now an increasing interest in developing biological methods for its replacement. One approach is to stimulate aggrecan and hence glycosaminoglycan (GAG) production by resident cells through growth factor or genetic engineering. Another approach is to implant autologous cells into the cartilage or disc to enhance GAG production. In both instances GAG accumulation depends both on the number of active cells in the cartilage or disc and the rate of aggrecan synthesis per cell. Here we examine how cell density influences the rate of glycosaminoglycan accumulation in a three‐dimensional cell culture system. In the results, at cell densities found in vivo (standard condition) in the articular cartilage and the disc nucleus viz. 4 million cells/ml and at 21% oxygen the concentration of GAG in the bead reached 520.9±62.4 μg/ml and 649.0±40.1 μg/ml, respectively, in 5 days. These concentrations could be increased to 2‐4‐fold by raising cell density to 25 million cells/ml. However, rates of GAG production per cell decreased by 50‐60%. These results showed that rate of accumulation of glycosaminoglycan in this culture system in vitro is slow and is limited both by the rate of production of GAG per cell and by the cell density which can be maintained in a viable state. Although GAG production can be increased somewhat by use of higher cell densities, the consequent fall in concentration of oxygen and other nutrients in the center of three‐dimensional constructs slows metabolism and leads to apoptosis and cell death, which again limits the rate that the cells can produce matrix. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:493–503, 2008     

Disproportion of end plates and the lumbar intervertebral disc herniation

Background contextIt is suggested that the shape of the vertebral end plates may play a role in the development of abnormalities in the intervertebral disc. On midsagittal magnetic resonance images of the spine in patients with lumbar intervertebral disc herniation, a notable disproportion frequently exists between the end plates of two vertebrae to which the disc is attached. There is apparently no study in the literature examining possible association of this disproportion with development of disc herniation.PurposeTo determine whether a disproportion between two neighboring vertebral end plates is associated with the presence of disc herniation at the same level.Study designCase-control study.Patient sampleTwo hundred fifty patients with primary lumbar disc herniation in the case group and 250 age- and sex-matched normal individuals in the control group.Outcome measuresOn midsagittal sections, the difference of anteroposterior diameter of upper and lower end plates neighboring a herniated (in the case group) or normal (in the control group) intervertebral disc was calculated and expressed as “difference of end plates” or “DEP.”MethodsSubjects with previous spinal surgery, spondylolisthesis, or a significant vertebral deformity were excluded. For the main outcome variable, DEP was calculated at the level with herniated intervertebral disc in the case group, and the mean value was compared with mean DEP at the same level in the controls.ResultsMean DEP was significantly higher in the case group at both L4–L5 (2.45±0.28 vs. 2.08±0.27 mm, p=.02) and L5–S1 (3.32±0.18 vs. 2.51±0.13 mm, p<.001) levels. Similar differences were only marginally insignificant at L2–L3 (1.96±0.14 mm in the cases vs. 1.33±0.15 mm in the controls, p=.07) and L3–L4 (2.17±0.11 mm in the cases vs. 1.55±0.09 mm in the controls, p=.06) levels, with no significant difference at L1–L2 level (1.81±0.10 mm in the cases vs. 1.28±0.09 mm in the controls, p=.12). Each 1 mm increase of DEP at L4–L5 and L5–S1 levels was associated with 53% and 56% elevation in disc herniation risk at the corresponding levels, respectively.ConclusionsDifference of end plate is a significant and probably independent risk factor for lumbar disc herniation.     

腰椎间盘突出上移症

1　病例介绍病例 1,女 ,4 8岁 ,农民。因腰扭伤疼痛向右下肢放射痛3年来院就诊。体格检查 :腰生理弯曲变直 ,Ｌ3 ,4、Ｌ4,5右旁叩痛阳性 ,右直腿抬高试验 2 0°阳性 ,右足下垂 ,右膝反射消失 ,右侧股四头肌肌力Ⅲ级 ,右大腿外及右小腿前内侧和右小腿外及右足背皮肤感觉麻木。ＣＴ检查示 :Ｌ4,5右偏型椎间盘突出 ,Ｌ3 ,4未见椎间盘突出。腰椎管造影Ｘ线片示 :Ｌ4,5椎间隙严重变窄 ,平齐第 4腰椎右侧椎管内组织及腰 4和腰 5右侧神经根明显压迫影像 ,造影剂充盈缺损 (图 1)。诊断为Ｌ4,5右偏型椎间盘突出上移伴高位神经压迫。施行Ｌ4,5后路右侧…     

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.