Epigenetic DNA de-methylation at specific CpG promoter sites is associated with abnormal synthesis of matrix-degrading enzymes in human osteoarthritis (Arthritis Rheum 52:3110–24), but the mechanisms that trigger or cause loss of DNA methylation are not known. Since inflammatory cytokines are known to induce abnormal gene expression in cultured chondrocytes, we wanted to know whether this induction also involved loss of DNA methylation. If so, the abnormal gene expression would be permanent and transmitted to daughter cells rather than a simple up-regulation. To test this hypothesis, we selected IL-1b as the abnormally expressed gene. Healthy chondrocytes, harvested from human femoral head cartilage following a fracture, were divided into five groups: non-culture; control culture; culture with the de-methylating agent 5-aza-deoxycyti-dine (5-aza-dC); culture with the inflammatory cytokine IL-1b; or with TNF-a/oncostatin M. Total RNA and genomic DNA were extracted at confluency, relative mRNA expression of IL-1b was quantified by Syb-rGreen-based real-time PCR, and a method for quantifying the percent of cells with DNA methylation at a specific CpG site was developed (Epigenetics 2: 86–95).

The methylation status of 16 CpG sites in the promoter of IL-1b was determined by the bisulfite modification method. The two CpG sites important for the epigenetic regulation of IL-1b were at -247bp and -290bp, the latter was selected to quantify DNA methylation. 5-aza-dC halved DNA methylation, which resulted in 4–8 fold increases in IL-1b expression; showing that DNA de-methylation per se increases gene expression. However, far greater effects were seen with the inflammatory cytokines. IL-1b increased its own expression 50–100 fold, whereas TNF-a/OSM increased IL-1b expression 500–1000 fold. DNA methylation varied inversely, IL-1b reducing methylation to ~15% and TNF-a/OSM abolishing DNA methylation almost completely.

This is the first demonstration that inflammatory cytokines have the capacity to cause loss of DNA methylation. We also confirmed previous work that IL-1b induces its own expression in healthy chondrocytes, thus setting up a dangerous positive feed-back mechanism. If true in vivo, both the auto-induction and the heritable expression of IL-1b by a growing number of chondrocytes could explain the unrelenting progression of osteoarthritis.

Osteoarthritis (OA) is characterized by progressive erosion of articular cartilage due to degradation of the cartilage matrix. The major enzymes involved are the matrix metalloproteases and aggrecanases, which are either derived from the synovium or synthesized by chondrocytes as OA progresses. This abnormal enzyme synthesis is part of a phenotypic change from normal to ‘degradative’ chondrocytes. If this change could be prevented, then disease progression might be slowed.

In early OA, degradative chondrocytes are only present in the superficial zone, but with increasing severity of OA, more chondrocytes become degradative cells so that, in high-grade OA, these cells are also located in the deep zone. We hypothesized the existence of a ‘factor X’, which diffuses from the superficial to the deep zone and induces cells to change phenotype and express the pro-teases. We further hypothesize that this factor is released by degradative chondrocytes. To test the hypothesis, we co-cultured explants of human superficial-zone OA cartilage (which contains degradative cells and thus factor X) with explants of deep-zone cartilage from fracture neck of femur patients (#NOF), which contains mostly normal chondrocytes that do not express the proteases. We investigated MMP expression by real time RT-PCR and protein synthesis by immunohistochemistry.

Before culture, MMP-2, -3, -9, or -13 were expressed in the superficial-zone OA cartilage, but not in deep-zone #NOF cartilage, as expected. After 4 weeks with separate culture of superficial zones and deep zones, no MMPs was expressed in deep zone chondrocytes, suggesting that culture per se did not induce expression of these enzymes. Neither did culture abolish expression in the superficial zone, as confirmed by RT-PCR and immunohistochemistry. However, when superficial-zone cartilage was co-cultured with deep-zone cartilage, MMP-3 expression were induced in deep- zone chon-drocytes, suggesting that a diffusible factor X, derived from degradative chondrocytes, had induced normal articular chondrocytes to express MMP-3. These experiments provide evidence for the existence of a factor that, when diffusing through the cartilage matrix, has the potential to induce normal non-enzyme expressing cells to become degradative chondrocytes.

In the pubertal growth plate, sex hormones play important roles for the regulation of the proliferation, differentiation, maturation and programmed death of chondrocytes. Many studies have been reported on the regulation of oestrogen in long bone growth, however, some of the mechanisms have remained unclarified to date including its role for cell kinetics in the growth plate chondrocytes. The aim of this study was to clarify the effect of the deficiency of oestrogen on growth plate chondrocytes.

We obtained the growth plates of femoral head from the normal and ovariectomized Japanese white rabbits at 10, 15, 20 and 25 weeks. Ovariectomy was performed at 8 weeks. The cell kinetics of chondrocytes as defined by the numbers of proliferating and programmed dying cells was investigated using immunohistological methods.

The lengths of the femur were almost same both in the ovariectomised and normal rabbits. The height of the growth plate was larger in the former. The total number of chondrocytes in the ovariectomised rabbits was less than that of normal rabbits of the same age. Immunostaining of proliferating cell nucleous antigen (PCNA) showed a decrease number of proliferating chondrocytes and that of caspase-3 indicated a little increased number of apoptotic chondrocytes.

Oestrogen regulates endochondral bone formation through several pathways. It directly binds oestrogen receptor alpha and beta, and the former accelerates longitudinal bone growth whereas the latter represses it. Another pathway is through the GH-IGF-I axis: it closely interacts with GH and IGF-I for the control of longitudinal bone growth. In addition, there might be other mediators including transforming growth factor-beta, other IGFs and still unknown paracrine or auto-crine factors as IHH PTHrP. Our study suggests that in the rabbit growth plate during puberty, oestrogen mainly acts through the GH-IGF-I axis since its defi-ciency declined the proliferating ability of chondrocytes, which led the decrease of the number of chondrocytes.

Idiopathic osteoarthritis (OA) is a complex, late-onset disease whose causes are still unknown. In spite of tremendous efforts, the search for the genes pre-disposing towards osteoarthritis has so far met with little success. We hypothesize that epigenetic changes play a major role in the pathology of OA. Epigenetics refers to stable, heritable, but potentially reversible modifications of gene expression that do not involve mutations in the DNA sequence, for example DNA methylation or histone modification. Epigenetic changes are gene and cell-type specific, may arise sporadically with increasing age or be provoked by environmental factors. To investigate whether epigenetic changes are significant factors in OA, we examined the DNA methylation status of the promoter regions of three genes that are expressed by OA, but not by normal, articular chondrocytes, namely MMP-3 (stromelysin-1), MMP-9 (gelatinase B) and MMP-13 (collagenase3). We hypothesized that these genes are silenced in normal chondrocytes by methylation of the cytosines of CpG dinucleotides in the respective promoter regions, but that abnormal expression is associated with a de-methylation, leading to eunsilencing f of gene expression. Cartilage was obtained from the femoral heads of 16 OA and 10 femoral neck fracture (#NOF) patients, which served as controls due to the inverse relationship between osteoporosis and OA. The cartilage was milled in a freezer mill with liquid nitrogen, DNA was extracted with a Qiagen kit, digested with methylation sensitive restriction enzymes, followed by PCR amplification. These enzymes will cut at their specific cleavage sites only if the CpGs is not methylated and thus allow us to determine methylation status of specific CpG sites.

Results. Less than 5% of the chondrocytes in superficial layer from #NOF cartilage expressed degradative enzymes, whereas all cloned chondrocytes from advanced-stage OA cartilage were immunopositive. The overall % of CpG demethylation in the promoters of control patients (whose chondrocytes did not express the enzymes) was 20.1%, whereas 48.6% of CpG sites were demethylated in degradative chondrocytes of OA patients (p< 0.001). For MMP-13, the increase in demethylation between control and OA was from 4 ^..20%; for MMP-9 from 47 ^..81% and for MMP-3 from 30 ^..57%. However, not all available CpG sites were equally demethylated. Some sites were uniformly methylated in both OA and controls, others were demethylated even in controls. However, there was at least one crucial site for each degradative enzyme, where the differences in the degree of methylation were greatest and statistically different. These sites were at −110 for MMP-13; −36 for MMP-9; −635 for MMP-3. There was no relation between the % demethylation and the patient fs age and no apparent difference between males and females.

Conclusions: We have demonstrated an association between abnormal gene expression of MMP-3, MMP-9 and MMP-13 and promoter DNA demethylation. This epigenetic dysregulation of genes appeared to be clonally inherited by daughter cells and may be typical for osteoarthritis and other complex, late-onset diseases.

Sex hormones play important roles in the regulation of the proliferation, maturation and death of chondrocytes in the epiphyseal growth plate. We have investigated the effects of male castration on the cell kinetics of chondrocytes as defined by the numbers of proliferating and dying cells. The growth plates of normal rabbits and animals castrated at eight weeks of age were obtained at 10, 15, 20 and 25 weeks of age.

Our study suggested that castration led to an increase in apoptosis and a decrease in the proliferation of chondrocytes in the growth plate. In addition, the number of chondrocytes in the castrated rabbits was less than that of normal animals of the same age.

The efficacy of traction before an attempted closed reduction for patients with developmental dislocation of the hip remains controversial. We treated 55 children (62 dislocations of the hip) by preliminary, prolonged traction for a mean of eight weeks. All were followed up for at least two years in order to observe the development of any avascular changes within the femoral head. Of the 55 children, 27 (31 dislocations) were followed up until they were over six years of age. Fifty-seven of the 62 hips (92%) showed a successful closed reduction. Only one had radiological evidence of avascular necrosis of the femoral head. Of the 31 hips which were followed up to over six years of age, 15 (48%) showed residual subluxation. Our method of prolonged preliminary traction leads to a high rate of successful closed reduction, a low incidence of avascular necrosis and a reduced need for secondary operations.

Endochondral ossification involves a well ordered sequence of cellular events. Chondrocytes change their morphology and functions and are ultimately removed by the process of apoptosis. A variety of apoptotic-related signals have been characterised. These include Fas receptor (FasR)/Fas ligand (FasL), p53 and Bcl family. However, there is little known regarding the activity of these signals in the process of fracture healing. The purpose of this study was to investigate mRNA expression of apoptotic signals using RNase protection assay (RPA) and immunohistochemistry in endochondral bone formation.

BALB/C mice aged 8 to 10 weeks were used for this study. First, a transverse fracture was made in the right tibia. Mice were euthanised at 1, 2 and 3 weeks postfracture. The calluses were harvested and studied for the expression of caspase-8, a key enzyme of apoptosis, and apoptosis inducers: tumour necrosis factor-alpha (TNF-α) and its receptor p55, FasL and Fas receptor (FasR), and TNF-related apoptosis-inducing ligand (TRAIL). Four mice at each timepoint were used for immunostaining of fracture callus. Sections were incubated with primary antibody then labelled by avi-din-biotin complex method. Another four to ten tibiae were used for RPA. Fracture callus were harvested and snap frozen in liquid nitrogen. RNA was isolated by TRI reagent and BCP, and mRNAs expression of apoptotic signals were detected.

At each timepoint, mRNA of caspase-8, TNF-α, p55, FasL,FasR and TRAIL were detected by RPA. Immunostainings clearly showed that those apoptotic-related proteins were expressed by callus chondrocytes. Cartilaginous callus is replaced by woven bone in endochondral ossification. In this process, chondrocytes should be removed by the process of apoptosis in which death factors are elaborated directly in both an autocrine and paracrine manner.

Purpose: There are a wide variety of operative procedures for lumbar spinal canal stenosis. Bilateral fenestration, preserving the continuity of the lamina and spinous processes, has widely been employed in our department and its affiliated hospitals. The following questions are raised: Are decompressive effects of fenestration and spinal stability maintained without spinal fusion or instrumentation? In order to answer the questions, we compared the rates of revision after fenestration with those after laminectomy alone and decompressive surgery with spinal fusion.

Materials and methods: The registry of spinal surgeries of our university and affiliated hospitals from 1988 to 1997 was consulted.

During the first 5-years period 1159 patients underwent decompressive surgery. 908 of them had spondylosis and 251 had degenerative spondylolisthesis (DO) as a contributing factors of neural compression. Fenestration was done in 740 (81%) of patients with spondylosis and in 176 (70%) of patients with DO.

Results: Regarding the whole series 31 out of 1159 patients had a revision. The revision rate was 2.7%. 15 out of 908 patients (1.7%) with spondylosis and 16 out of 251 patients (6.4%) with DO underwent revisions. 11 out of 740 (1.5%) with spondylosis and 11 out of 176 (6.3%) with DO underwent revisions after fenestration. No significant differences were found among the revision rate of fenestration, laminectomy and decompressive surgery with spinal fusion.

Conclusion: The decompressive effect of fenestration was maintained long enough, even for degenerative spondylolisthesis. As a first operation spinal fusion is not necessarily indicated for lumbar canal stenosis.

Diagnostic indices for the determination of involved nerve root in cervical radiculopathy have been described by Yoss (1957), Murphey (1973) and Hoppenfeld (1976). However, there has been criticism that their indices are inappropriate for the diagnosis, because involved nerve root can not necessarily be determined using them. Difficulties in diagnosis have been attributed to the variable patterns of symptoms and signs caused by nerve root compression.

Purpose: To develop the new diagnostic indices for determination of involved nerve root in cervical radiculopathy.

Methods: Forty-five cases operated on through posterior foraminotomy were reviewed. The sites of neck pain(s) (in nape, in suprascapular, superior angle of scapula, interscapular, or scapular regions), and arm pain (anterior, lateral, posterior or medial) in anatomical position were preoperatively recorded. The finger(s) with subjective paraesthesia or objective sensory change, and the finger(s) of the most severe involvement were recorded. Affected muscle(s) (deltoid, biceps, wrist extensor, wrist flexor, triceps, finger extensor, or intrinsic), and the muscle(s) of the weakest were recorded. All of 45 cases were decompressed unilaterally at only 1 level, and showed improvements just after operation. Involved nerve roots and number of their cases were as follows: C5, 7; C6, 12; C7, 13; C8, 13.

Results: Pain in the suprascapular region frequently (82%) indicated C5 or C6 radiculopathy. Interscapular or scapular pain always (100%) indicated C7 or C8 radiculopathy. Lateral, posterior, or medial arm pain frequently indicated C6, C7 or C8 radiculopathy, respectively. Involved nerve roots and number of patterns of finger paraesthesia [or sensory change] were as follows: C5, 0 [0]; C6, 4 [5]; C7, 8 [10]; C8, 4 [5]. However, when the most severe involvement was that of the thumb, the index or long finger, or the little finger, the indication was C6, C7 or C8 radiculopathy, respectively. Although patterns in affection of muscles were also variable, when the weakest muscle was deltoid, biceps or wrist extensor, wrist flexor or triceps, or intrinsic, the indication was C5, C6, C7 or C8 radiculopathy, respectively.

Conclusion: The sites of the neck and arm pain are important for the diagnosis of the involved nerve root. Not the fingers with paraesthesia but the fingers with the most severe involvement lead to the diagnosis.

Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper hypertrophic zone at five weeks, whereas after ten weeks it was found mostly in the lower hypertrophic zone. The proliferating chondrocytes tended to show nuclear staining and the hypertrophic cells cytoplasmic staining, although the terminal hypertrophic chondrocytes sometimes expressed the protein in their nuclei. In the younger rabbits, c-Myc co-localised with proliferating cell nuclear antigen, whereas in the hypertrophic zone of older rabbits, it was present in some chondrocytes the nuclei of which also contained DNA breaks.

Our study suggests that, in the rabbit growth plate, c-Myc is associated with different cellular processes, depending on the age and the developmental stage of the chondrocytes.

Chondrocytes of the growth plate are generally assumed to undergo apoptosis, but the mechanisms which induce this cell death are not known. The Fas receptor is a mediator of the apoptotic signal in some systems. We studied its expression in situ in growth plates of rabbits aged from five to 20 weeks. In addition, we investigated the immunolocalisation in the growth plates of the bone proteins, osteonectin and osteocalcin, and the changes in their expression with age.

The Fas-positive chondrocytes were found mostly in the hypertrophic zone, as were the osteonectin-positive and osteocalcin-positive cells. The percentage of Fas-positive cells increased with age whereas little change was found in the number of osteonectin-positive and osteocalcin-positive chondrocytes.

Many of the Fas-positive chondrocytes were also TUNEL-positive. This strongly suggests that apoptosis in the growth plate is mediated through the Fas system.

Double immunostaining for osteocalcin and Fas showed that not all hypertrophic chondrocytes were of the same cell type. Some chondrocytes stained for osteocalcin only, others for Fas only, while some were positive for both.

The growth plates of the femoral head of Japanese white rabbits aged 5, 10, 15 and 20 weeks were stained for apoptotic and proliferating chondrocytes using the TUNEL and PCNA antibody staining techniques. Both TUNEL- and PCNA-positive chondrocytes were detected in all of the specimens. The positive ratios of both stainings were calculated for the whole plate and for the resting, proliferating and hypertrophic zones. The highest ratios in both stainings occurred in the hypertrophic zone in all age groups. With growth, the TUNEL-positive ratio increased whereas the proliferating ratio decreased.

We suggest that the increase in chondrocytic death by apoptosis and the decrease in cell proliferation potential led to closure of the growth plate.