J A Hoyland

Nerve ingrowth into diseased intervertebral disc in chronic back pain

BACKGROUNDnIn the healthy back only the outer third of the annulus fibrosus of the intervertebral disc is innervated. Nerve ingrowth deeper into diseased intervertebral disc has been reported, but how common this feature is and whether it is associated with chronic pain are unknown. We examined nerve growth into the intervertebral disc in the pathogenesis of chronic low back pain.nnnMETHODSnWe collected 46 samples of intervertebral discs from 38 patients during spinal fusion for chronic back pain. 30 samples were from pain levels clinically established by discography and 16 samples were from adjacent vertebral levels with no pain. We obtained 34 control samples of intervertebral disc from previously healthy individuals with normal histology within 8 h of recorded death. We used standard immunohistochemical techniques to test for a general nerve marker, a nociceptive neurotransmitter (substance P), and a protein expressed during axonogenesis (growth-associated protein 43 [GAP43]).nnnFINDINGSnWe identified nerve fibres in the outer third of the annulus fibrosus in 48 (60%) of the 80 samples of intervertebral discs. Nerves were restricted to the outer or middle third of the annulus fibrosus in the 34 control samples. Among the patients with chronic low back pain, nerves extended into the inner third of the annulus fibrosus and into the nucleus pulposus in 21 (46%) and ten (22%) samples, respectively. Nerves usually accompanied blood vessels, but in 14 of the samples from back-pain patients, isolated nerve fibres were seen in the discal matrix. Both types of nerve fibres expressed substance P, but only non-vessel-associated fibres expressed GAP43. Deep nerve ingrowth into the inner third of the annulus fibrosus, the nucleus pulposus, or both was seen in four (25%) of 16 biopsy samples from non-pain levels and in 17 (57%) samples from pain levels. Of the 16 paired samples from both pain and non-pain levels, five pain-level samples and one non-pain-level sample showed deep nerve ingrowth.nnnINTERPRETATIONnOur finding of isolated nerve fibres that express substance P deep within diseased intervertebral discs and their association with pain suggests an important role for nerve growth into the intervertebral disc in the pathogenesis of chronic low back pain.

Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs

Aims: To investigate the phenotype of cells in normal and degenerate intervertebral discs by studying the expression of molecules characteristic of chondrocytes in situ. Methods: Human intervertebral discs taken at surgery were graded histologically, and classified on this basis as normal or degenerate. Eighteen of each type were selected, and in situ hybridisation was performed for the chondrocytic markers Sox9 and collagen II using 35S labelled cDNA probes. Aggrecan was located by immunohistochemistry, using the monoclonal antibody HAG7E1, and visualised with an avidin–biotin peroxidase system. Results: In the normal discs, strong signals for Sox9 and collagen II mRNA, and strong staining for the aggrecan protein were seen for the cells of the nucleus pulposus (NP), but reactions were weak or absent over the cells of the annulus fibrosus (AF). In degenerate discs, the Sox9 and collagen II mRNA signals remained visible over the cells of the NP and were again absent in the AF. Aggrecan staining was not visible in the NP cells, and was again absent in the AF. Conclusions: Cells of the normal NP showed expression of all three markers, clearly indicating a chondrocytic phenotype. In degeneration, there was evidence of a loss of aggrecan synthesis, which may contribute to the pathogenesis of disc degeneration. AF cells showed no evidence of a chondrocytic phenotype in either normal or degenerate discs.

Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis

Aims: Preliminary studies have suggested that there is an increase in adipocytic tissue in osteoporotic (OP) bone, supporting in vitro evidence for a switch in differentiation of stromal cells from the osteoblastic to the adipocytic lineage. To investigate this the variation of the ratio of adipose tissue to haemopoietic/stromal tissue in OP bone was measured. Methods: The ratio of adipocytic to haemopoietic/stromal tissue (A/H) was measured by semi-automated image analysis in iliac crest biopsies from 127 patients with osteoporosis (84 female patients, 48 male patients; mean age, 55 years; range, 5–80). Fourteen patients with normal histomorphometric data (nine women; five men; mean age, 48 years; range 21–70) acted as controls. Results: The ratio of A/H was higher in OP bone than in the normal controls (OP mean 43.06% v normal mean 22.4%; p < 0.001). Multiple regression analysis showed that 98.5% of the variability in the A/H ratio was the result of age and several measures of bone formation, including cancellous wall thickness, osteoid volume, cancellous thickness, cortical wall thickness, cancellous apposition rate, and bone formation rate, together with cancellous separation (each significant at p < 0.001). Those with the greatest effect on the A/H ratio (in decreasing order) were cancellous apposition rate, osteoid volume, and age. Conclusions: Cancellous apposition rate, osteoid volume, and age were associated with the increase in the proportion of adipose tissue present in OP bone. Of these, cancellous apposition rate reflects osteoblast activity, indicating that the increase in the volume of adipose tissue in osteoporosis is associated with reduced bone formation, supporting the postulated switch in differentiation of stromal cells from the osteoblastic to the adipocytic pathway in osteoporosis.

Gene expression of matrix metalloproteinases 1, 3, and 9 by chondrocytes in osteoarthritic human knee articular cartilage is zone and grade specific.

OBJECTIVES Matrix metalloproteinases (MMPs) are thought to be major mediators of cartilage destruction. Osteoarthritis (OA) is characterised by cartilage degradation. This study explores gene expression of three MMPs in articular chondrocytes during the histological development of the cartilage lesion of OA. METHODS Biopsy specimens of human normal and OA cartilage, classified into four grades on the basis of histology, were probed for MMPs 1, 3, and 9 using 35S-labelled cDNA probes. The signal was measured at four different depths (zones) using an automated image analyser and compared with signal from sections probed with λDNA. Rheumatoid synovium was used as a positive control for MMP gene expression. RESULTS Rheumatoid tissue contained mRNA for all three MMPs. Expression in chondrocytes varied with the depth of the chondrocyte in the cartilage and the histomorphological extent of the OA changes. There was no detectable mRNA signal for these three MMPs in normal cartilage. In general, in OA, MMP-1 gene expression was greatest in the superficial cartilage in established disease. By contrast mRNAs for MMP-3 and 9 were expressed deeper in the cartilage, MMP-9 early in disease and MMP-3 with a biphasic pattern in early and late stage disease, most pronounced in the latter. This was a consequence of differential expression in single cells and chondrocyte clusters in late disease. CONCLUSION The data indicate that expression of genes for MMPs 1, 3, and 9 is differentially regulated in human articular chondrocytes and, in individual cells, is related to the depth of the chondrocyte below the cartilage surface and the nature and extent of the cartilage lesion.

Degeneration of intervertebral discs: current understanding of cellular and molecular events, and implications for novel therapies.

Recent advances in surgical techniques have made it possible to study the changes that are known as degeneration of the intervertebral disc (IVD) in greater depth than ever before. This is an important area of study because degeneration of the lumbar IVDs is associated, perhaps causally, with low back pain: one of the most common and debilitating conditions in the western world. The term degeneration implies an inevitable progression that is characteristic of wear-and-tear-associated conditions. However, modern research on human tissue has shown that this is not the case. Here, disruption of the micro-anatomy that is described as degeneration is an active process, which is probably regulated by locally produced cytokines. This one observation opens up the possibility of inhibiting or even reversing the processes of degeneration. The immediate question is how can the slowly progressive changes of degeneration be treated, particularly as connective tissue repair is thought to be a slow and highly regulated process. In this review, the possibility of using gene therapy as a single-shot, long-lasting therapeutic strategy is discussed, together with an outline of developments in this area to date.

Herniated intervertebral disc-associated periradicular fibrosis and vascular abnormalities occur without inflammatory cell infiltration.

Study Design Prospective histologic comparison of perineural tissues from patients requiring decompression surgery for herniated intervertebral disc with those from cadaveric controls. Objectives To examine the significance of herniated intervertebral-disc-associated perineural vascular and fibrotic abnormalities with respect to back pain symptom generation. Summary of Background Data Previous cadaveric studies have demonstrated perineural vascular congestion, dilatation, and thrombosis and perineural and intraneural fibrosis occurring in association with herniated intervertebral disc. It was suggested that these neural abnormalities were the result of ischemia, due to venous outflow obstruction, and also represented a possible cause of ongoing back pain symptoms. Criticisms of such a conclusion arose, however, because the possibility could not be excluded that these abnormalities were the result of postmortem artifact. Methods Histologic and immunohistochemical comparison of discal and peridiscal tissues removed from 11 patients with radiographically proven herniated intervertebral disc requiring decompressive surgery and from 6 fresh cadavers without history of back pain in life. Results Histology and immunohistochemistry of perineural and extraneural tissues from patients revealed vascular congestion, neovascularization, and endothelial abnormalities including luminal platelet adhesion, in association with reductions in von Willebrand factor levels, together with perivascular and perineural fibrosis. Elevated fibrogenic cytokine concentrations were also detected in patients tissues. These changes occurred without evidence of inflammation and were absent in cadaveric control tissues. Conclusions The vascular abnormalities detected in patients may represent an important etiopathologic factor predisposing to intraneural and perineural fibrosis, and hence to chronic pain symptoms, after disc herniation. It seems important to preserve the perineural microcirculation following disc herniation

Age related histomorphometric changes in bone in normal British men and women.

AIMS--To define the iliac crest histomorphometry of static variables in 234 individuals aged 16-100 years (91 men, 143 women) and of dynamic variables in 84 individuals aged 19-94 years (33 men, 51 women) from the North West of England. METHODS--Iliac crest biopsy specimens were sectioned, undecalcified, and examined using image analysis. RESULTS--The decrease in the quantity of cortical and trabecular bone and the connectivity of trabecular bone was more pronounced in women than men. This was associated with a reduction in bone formation and increased bone resorption which was greater in women at both the tissue and cellular level. Some of these histomorphometric differences first became evident at the natural menopause, and therefore provide clues as to the cause of the high prevalence of osteoporosis in postmenopausal women. CONCLUSIONS--These results show an age and sex dependent variation both in static and dynamic parameters, which differ, in some respects, from other studies and confirm the need for large regional studies to provide a database of normal morphometric results for a specific population.

In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage

OBJECTIVES Chondrocytic matrix metalloproteinases (MMPs) are believed to be important in osteoarthritic cartilage degradation. The cartilage lesion of osteoarthritis (OA) is focal and often progressive. During its development chondrocytes differentially up and down regulate production of mRNA for individual MMPs. This observation has potential implications for understanding the disease processes that lead to progressive cartilage loss in OA and designing appropriate targeted treatment. The complex regulation of MMP mediated effects means there is a pressing need to establish whether visualisation of MMP mRNA or protein equates to enzyme activity. The technique of in situ zymography (ISZ) offers a way of examining diseased human tissue for in vivo production of an excess of degrading enzyme over inhibitor. The primary objective of this study was to assess, and if positive follow, collagen II degrading activity in cartilage during development of the OA lesion. A secondary objective was to assess whether there was any correlation between sites of collagen II degrading activity and expression of the collagenase (MMP-13), recently implicated in type II collagen degredation in this lesion. METHODS Biopsied human normal and osteoarthritic cartilage, showing various degrees of damage, was examined by in situ zymography, with and without enzyme inhibitors, to establish sites of type II collagenase activity. Paired samples were probed for MMP-13 mRNA using 35S-labelled oligonucleotide probes. Comparative analyses were performed. RESULTS In situ zymography showed collagen II degrading activity over chondrocytes only in osteoarthritic cartilage. Distribution and amount varied with the extent of cartilage damage and position of chondrocytes, being greatest in deep cartilage and in cartilage lesions where fissuring was occurring. The enzyme causing the degradation behaved as a matrix metalloproteinase. MMP-13 mRNA expression codistributed with the type II collagenase activity. CONCLUSION In OA, chondrocytes can degrade type II collagen. The type II collagen degrading activity varies in site and amount as the cartilage lesion progresses and throughout codistributes with MMP-13 mRNA expression.

Mature osteoblasts in human non-union fractures express collagen type III

AIMS: High levels of collagen type III are biochemically detectable in biopsies of non-uniting fractures, and in the serum of patients suffering from this condition. The aim of this study was to determine whether the expression of collagen type III was limited to fibrous tissue in non-unions, or whether some was present in bone. METHODS: Biopsies from normally healing human fractures and non-unions were examined using in situ hybridisation and immunohistochemistry. RESULTS: The mesenchymal cell population, which includes fibroblast and osteoblast precursors, expressed mRNA for collagen type III. However, mature osteoblasts on the surface of woven bone varied profoundly between normally healing fractures (in which they were negative or occasionally weakly positive) and non-unions (in which they were strongly positive). Areas of woven bone that had osteoblasts positive for collagen type III mRNA also immunostained positively for the protein. CONCLUSIONS: This study shows that non-union fracture callus osteoblasts on the surfaces of woven bone exhibit an unusual phenotype: they express collagen type III, a molecule characteristic of an earlier stage of osteoblast differentiation, which is not expressed by osteoblasts on woven bone surfaces of bone that develops normally. This finding may be useful in developing an early clinical test for impending non-union.

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype.

Histological examination of long bones from 1‐day‐old bcl‐2 knockout and age‐matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35‐day‐old bcl‐2 knockout mice that are growth retarded or ‘dwarfed’, the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age‐matched wild‐type controls. The most marked histological effects of bcl‐2 ablation were on osteoblasts and bone. 35‐day‐old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl‐2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo‐woven appearance. The results suggest an important role for Bcl‐2 in controlling osteoblast phenotype and bone deposition in vivo.