Characterization of degenerative human facet joints and facet joint capsular tissues.

OBJECTIVE: Lumbar facet joint degeneration (FJD) may be an important cause of low back pain (LBP) and sciatica. The goal of this study was to characterize cellular alterations of inflammatory factor expression and neovascularization in human degenerative facet joint capsular (FJC) tissue. These alterations in FJC tissues in pain stimulation were also assessed. DESIGN: FJs were obtained from consented patients undergoing spinal reconstruction surgery and cadaveric donors with no history of back pain. Histological analyses of the FJs were performed. Cytokine antibody array and quantitative real-time polymerase chain reaction (qPCR) were used to determine the production of inflammatory cytokines, and western blotting analyses (WB) were used to assay for cartilage-degrading enzymes and pain mediators. Ex vivo rat dorsal root ganglion (DRG) co-culture with human FJC tissues was also performed. RESULTS: Increased neovascularization, inflammatory cell infiltration, and pain-related axonal-promoting factors were observed in degenerative FJCs surgically obtained from symptomatic subjects. Increased VEGF, (NGF/TrkA), and sensory neuronal distribution were also detected in degenerative FJC tissues from subjects with LBP. qPCR and WB results demonstrated highly upregulated inflammatory cytokines, pain mediators, and cartilage-degrading enzymes in degenerative FJCs. Results from ex vivo co-culture of the DRG and FJC tissue demonstrated that degenerative FJCs increased the expression of inflammatory pain molecules in the sensory neurons. CONCLUSION: Degenerative FJCs possess greatly increased inflammatory and angiogenic features, suggesting that these factors play an important role in the progression of FJD and serve as a link between joint degeneration and neurological stimulation of afferent pain fibers.

Type IX collagen neo-deposition in degenerative discs of surgical patients whether genotyped plus or minus for COL9 risk alleles.

STUDY DESIGN: Immunohistochemical analysis of type IX collagen in disc tissue from spinal fusion patients. OBJECTIVE: To determine if collagen IX can be detected in adult disc tissue removed at spinal fusion surgery from patients either with or without degeneration-associated tryptophan single nucleotide polymorphisms (SNPs) and whether the distribution is associated either with severity of degeneration or incidence of a collagen IX SNP genotype. SUMMARY OF BACKGROUND DATA: Genetic factors are strongly associated with risk of development and/or progression of disc degeneration. Two SNPs that introduce tryptophan polymorphisms in COL9A2 and COL9A3 are independently linked to an increased risk of lumbar disc disease. Although tryptophan variants are associated with accelerated degeneration, it is not known if collagen IX can be detected in adult disc tissue. METHODS: We selected age-matched disc samples from five clinical groups: fracture with Trp(-) (six cases), herniation (six cases), degeneration (five cases), spondylolisthesis with Trp(-) (eight cases), and spondylolisthesis/herniation/fracture with Trp(+) (six cases of Trp3 allele and one case of Trp2 allele). Using hematoxylin and eosin staining and immunohistochemical staining (collagens IX and IIA), 78 sections from 32 patients were analyzed. Selected disc tissues were assayed biochemically for collagen IX. RESULTS: Focal deposition of collagen IX was observed in regions of adult human disc tissue from spines showing degenerative changes in patients whether or not they were positive for a tryptophan SNP. However, in nondegenerative control disc tissue from fracture cases, little or no collagen IX was detected. The latter finding was confirmed by direct biochemical analyses for collagen IX in pooled samples of normal adult human annulus fibrosus or nucleus pulposus. CONCLUSION: During growth and maturation of the disc, collagen IX is presumably removed completely during matrix remodeling so that the protein is absent from normal adult annulus and nucleus but can reappear at sites of degeneration presumably as part of a repair response to mechanical injury.

Diabetes mellitus as a risk factor for the development of lumbar spinal stenosis.

BACKGROUND: Diabetes mellitus is a multi-organ disorder affecting many types of connective tissues, including bone and cartilage. Certain skeletal changes are more prevalent in diabetic patients than in non-diabetic individuals. A possible association of diabetes mellitus and lumbar spinal stenosis has been raised. OBJECTIVES: To compare the prevalence of diabetes mellitus in patients with spinal stenosis, degenerative disk disease or osteoporotic vertebral fractures. METHODS: A cross-sectional analysis was performed of 395 consecutive patients diagnosed with spinal stenosis, degenerative disk disease or osteoporotic vertebral fractures. All the patients were examined by one senior author in the outpatient orthopedic clinic of a large general hospital between June 2004 and January 2006 and diagnosed as having lumbar spinal stenosis (n=225), degenerative disk disease (n=124), or osteoporotic vertebral fractures (n=46). RESULTS: The prevalence of diabetes mellitus in the three groups (spinal stenosis, osteoporotic fracture, degenerative disk disease) was 28%, 6.5% and 12.1%, respectively, revealing a significantly higher prevalence in the spinal stenosis group compared with the others (P=0.001). The higher prevalence of diabetes in the stenotic patients was unrelated to the presence of degenerative spondylolisthesis. CONCLUSIONS: There is an association between diabetes and lumbar spinal stenosis. Diabetes mellitus may be a predisposing factor for the development of lumbar spinal stenosis.

Fas ligand expression on human nucleus pulposus cells decreases with disc degeneration processes.

BACKGROUND: The intervertebral disc has been reported to be an immunologically privileged environment, possibly mediated by Fas ligand (FasL) expression. On the other hand, recent studies have shown the infiltration of host immune cells into the degenerated disc, which may indicate the failure of the immune-privilege feature of the disc with degeneration. However, the relationship between FasL expression and disc degeneration is still unclear. Therefore, the purpose of this study was to clarify the relationship between FasL expression and disc degeneration. METHODS: Ten human degenerated disc specimens were obtained from spondylolisthesis patients and ten nondegenerated discs from idiopathic scoliosis patients during surgical procedures. Immunohistochemical staining was performed to determine the presence of FasL in cross-sections of those discs. Parts of the disc tissues were used to examine FasL expression quantitatively with Western blot analysis. To examine whether the change in FasL expression was influenced by aging, an animal study comparing the discs from young and old rats were performed using magnetic resonance imaging (MRI) and real-time polymerase chain reaction (PCR) assessment. RESULTS: Nucleus pulposus cells showed strong positive staining for FasL in all specimens examined. Quantitative examination demonstrated a significant decrease in FasL expression in the degenerated group compared with the nondegenerated group (average 67.6%, P<0.05). MRI showed no significant differences in the grade of disc degeneration between young and old rats, and also no significant difference in FasL mRNA in real-time PCR assay. CONCLUSIONS: The current results indicate that FasL and its potential mechanism of immunological privilege could influence the protection of the intervertebral disc against degeneration.

Calcium pyrophosphate crystal deposition in the ligamentum flavum of degenerated lumbar spine: histopathological and immunohistological findings.

We investigated the histological and immunohistochemical features of degenerative changes in the ligamentum flavum of the lumbar spine with calcium crystal deposition. We investigated degenerative changes in 270 ligamentum flavum specimens harvested from 198 patients who underwent decompressive surgeries for lumbar spinal canal stenosis. En bloc sections of the ligamentum flavum were examined histologically. We also examined immunoreactivity for transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF), CD34, and CD68; immunoblot analysis for VEGF; and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end-labeling (TUNEL) method. The ligamentum flavum showed fragmented and disorganized elastic fiber bundles with increased collagen fibrils in the matrix. Calcium deposition, which was identified as calcium pyrophosphate dihydrate crystals, was evident in 72 of 198 patients and in 99 of 270 samples, and was associated with appearance of hypertrophic chondrocytes and new blood vessel formation. Areas of calcium deposits were surrounded by abundant hypertrophic chondrocytes (with marked immunoreactivity to TGF-beta and VEGF) and a significant number of TUNEL-positive chondrocytes. Calcium crystal deposition in the lumbar ligamentum flavum progresses with reduction in elastic fibers and accumulation of collagen fibrils in the matrix as well as expansion of chondrometaplastic areas.

Changes in immune and glial markers in the CSF of patients with Complex Regional Pain Syndrome.

Complex Regional Pain Syndrome is a severe chronic pain condition characterized by sensory, autonomic, motor and dystrophic signs and symptoms. The pain in CRPS is continuous, it worsens over time, and it is usually disproportionate to the severity and duration of the inciting event. This study compares cerebrospinal fluid (CSF) levels of pro- and anti-inflammatory cytokines, chemokines and several biochemical factors (glial fibrillary acidic protein (GFAP), the nitric oxide metabolites (nitrate plus nitrite), the excitatory amino acid neurotransmitter glutamate, calcium, total protein and glucose) in patients afflicted with CRPS to levels found in patients suffering with other non-painful or painful conditions. The aim of the study is to determine the degree of involvement of glial cells and immune system mediators in the pathophysiology of CRPS. There was no elevation or reduction of a CSF marker that was specific for CRPS patients. However, there were several patterns of markers that could be helpful in both elucidating the mechanisms involved in the disease process and supporting the diagnosis of CRPS. The most common pattern was found in 50% (11 out of 22) of the CRPS patients and consisted of; elevated IL-6, low levels of IL-4 or IL-10, increased GFAP or MCP1 and increases in at least two of the following markers NO metabolites, calcium or glutamate. The results from this and other similar studies may aid in elucidating the mechanisms involved in the pathophysiology of CRPS. A better understanding of these mechanisms may lead to novel treatments for this very severe, life-altering illness.

Vertebral rounding deformity in pediatric spondylolisthesis occurs due to deficient of endochondral ossification of the growth plate: radiological, histological and immunohistochemical analysis of a rat spondylolisthesis model.

STUDY DESIGN: A study using rat spondylolisthesis models. OBJECTIVE: To clarify pathomechanism of vertebral rounding deformity in pediatric spondylolisthesis. SUMMARY OF BACKGROUND DATA: For high-grade slippage, rounding of sacrum surface associated with L5 spondylolisthesis is reported to be the most responsible risk factor. However, the exact pathomechanism of the rounding deformity is yet to be clarified. METHODS: Spondylolisthesis rat model (4-week-old) was used. Radiographs were taken weekly for 5 weeks after the surgery. The lumbar spines were harvested for histology. Hematoxylin and eosin, alcian blue staining, and tartrate-resistant acid phosphatase staining were used. Immunohistochemically, the growth plate cartilage was studied for type II and X collagen. A modified bone histomorphometric analysis was also performed. RESULTS: Radiographs showed slippage 1 week after surgery. Rounding deformity was obvious 2 weeks after surgery. The rounding deformity progressed with time. Three weeks after surgery, the specific columns of growth plate were unclear at the anterior corner, which corresponded to the rounding surface observed on radiographs. Instead, a huge mass of cartilage was observed at that site. Tartrate-resistant acid phosphatase-positive cells were observed in the vicinity of the growth plate except in relation with the anterior corner. The growth plate and cartilage mass at the anterior corner stained positive for type II collagen. Chondrocytes in the hypertrophied layer stained positively for type X collagen; however, staining was faint at the anterior corner. The results suggested that the chondrocytes at the anterior did not form, morphologically and functionally, the normal growth plate. From histomorphometrical analysis, the normal posterior growth plate made endochondral bone growth in 510 +/- 20 microm for a week, whereas the anterior corner in 200 +/- 15 microm. CONCLUSION: Deficient endochondral ossification of the growth plate in the anterior upper corner of the vertebra could be the pathomechanism of the rounding deformity of the sacrum.

Expression of estrogen receptor of the facet joints in degenerative spondylolisthesis.

STUDY DESIGN: Immunohistochemical study was done by harvesting articular cartilage of the facet joints during the decompressive surgery for spinal stenosis. OBJECTIVES: To observe the expression of estrogen receptor on the articular cartilage of the facet joints in degenerative spondylolisthesis (DS) SUMMARY OF BACKGROUND DATA: Few attempts have been made to evaluate the effect of sex-hormone, although DS is more common in females than in males. METHODS: After harvesting the articular cartilage of the facet joints in 17 DS and in 15 spinal stenosis (SS) patients, the expression of estrogen receptor and the severity of facet arthritis were observed by H-E and immunohistochemical staining, respectively. Measurements of both staining were made by using a semiquantitative analysis. RESULTS: The significantly increased expression of estrogen receptor correlated with the severity of facet arthritis (r = 0.78, P < 0.05). There was a significantly increased expression of estrogen receptor of the facet joint in DS compared with SS (P < 0.01). The histologic-histochemical grading of cartilage lesion in DS was 12.4 (SEM, 0.6), which was significantly higher than in SS (P < 0.05). CONCLUSIONS: These findings suggest that the higher expression of estrogen receptor might aggravate degenerative change of the facet articular cartilage and might also be considered one of the causative factors for DS in postmenopausal women.

Immunohistochemical analysis of the extracellular matrix in the posterior capsule of the zygapophysial joints in patients with degenerative L4-5 motion segment instability.

OBJECT: Although the hypertrophied shape of the zygapophysial joints in degenerative instability of the lumbar spine is well known, its underlying pathophysiological mechanism is unclear. The authors sought to provide evidence that there is increased fibrocartilaginous metaplasia in the posterior joint capsule resulting from greater mechanical loading; the authors suggest that these capsular changes are central to understanding the altered joint shape. METHODS: The LA-5 posterior articular complex was removed in 14 patients undergoing fusion for degenerative instability. After methanol-assisted fixation, cryosections were immunolabeled for a wide range of extracellular matrix molecules. These were collagens (Types I, II, III, V, and VI), glycosaminoglycans (chondroitin 4 and 6 sulfates; dermatan- and keratan-sulfate), and proteoglycans (versican, tenascin, aggrecan, and its associated link protein). The grade of degeneration of the articular complexes was assessed radiologically and histologically. CONCLUSIONS: The results of this study provide molecular evidence for an altered loading history on the joint capsule. The pronounced loss of intervertebral disc height that occurred in all patients with severe degeneration of the lumbar motion segment promotes an increased range of axial rotation that places the posterior capsule under greater mechanical load. Compared with normal joints studied previously, the posterior capsules involved in these degenerative joint complexes were hypertrophied and fibrocartilaginous throughout. Cartilaginous metaplasia was especially pronounced at the attachment sites (entheses) where the fibrocartilage now extended beyond the original level of the joint space, and capped the osseous spurs arising from these attachment sites.

Ultrastructural changes in paravertebral muscles associated with degenerative spondylolisthesis.

STUDY DESIGN: The paravertebral muscle of 30 patients with spondylolisthesis and 30 control patients were investigated histologically. OBJECTIVE: To propose myopathologic paravertebral muscle changes in cases of degenerative lumbar spondylolisthesis. SUMMARY OF BACKGROUND DATA: The stability of the vertebral column is based on both active and passive systems. The passive system is composed of the vertebrae, the intervertebral discs, and the ligaments. Surrounding muscles and tendons constitute the active system. The autochthonous back muscles take over support functions if the passive system is ineffective. In some cases, muscles are overstrained for a long period, ultimately leading to muscular changes. This study was performed to determine the histopathologic correlates of this permanent strain. METHODS: Between July 1998 and July 1999, paravertebral muscle biopsies were performed for 30 patients with monosegmental degenerative spondylolisthesis undergoing posterior lumbar interbody fusion. The tissue samples were submitted to histologic analysis including immune and enzyme histochemistry and electron microscopy. In addition, the muscle fibers were submitted to morphometry. RESULTS: Severe pathologic alterations were found. The findings showed that 22 patients (73.3%) had ragged red fibers with evident ultrastructural mitochondrial anomalies. The cristae appeared irregular in 12 patients (40%) Type 1 paracrystalline inclusions were detected in five samples (16.6%) and dense bodies in eight (26.6%). Fibers with ubiquitin-positive inclusions were detected by immunohistochemistry in 13 patients (43.3%). As shown by the electron microscope, these corresponded to granulofilamentous inclusions and polyglucosan bodies. The samples were submitted to genetical analysis because biochemical studies showed reduced activity of the respiratory chain enzymes. Normal mitochondrial deoxyribonucleic acids of unchanged length were detected. CONCLUSIONS: Apart from nonspecific myopathic changes such as those observed in rimmed vacuoles and rods, increased numbers of polyglucosan bodies were detected. This increase in polyglucosan bodies currently has not been described in patients with otherwise normal muscles.

Biochemical changes of intervertebral discs in patients with spondylolisthesis or with tears of the posterior annulus fibrosus.

Surgically removed discs from patients with either spondylolisthesis or tears of the posterior annulus fibrosus were analysed for water, proteoglycan, and collagen content and compared with post-mortem control material. Discs from patients with spondylolisthesis had a reduced proteoglycan content in all section sampled and less collagen in the outer annular layers. In contrast discs containing tears in the posterior annulus were unaltered biochemically, although extended studies on 2 patients indicated that there may be localised biochemical changes in the region of the tear itself. Collagen types I, II, and III and proteoglycan distributions were studies qualitatively by immunofluorescence. Collagen types I and III appeared to be reduced in discs from patients with spondylolisthesis, but again little change was found in patients with tears in the posterior annulus fibrosus.