Takao Kubota

Synovial fluid cytokines and proteinases as markers of temporomandibular joint disease

PURPOSE In this article, biochemical markers in the synovial fluid (SF) for detecting intraarticular inflammation and early cartilage degradation of the temporomandibular joint (TMJ) disease were examined. PATIENTS AND METHODS SF was obtained from 25 TMJs in 22 patients with internal derangement (ID) or osteoarthritis (TMJ-OA), 15 asymptomatic TMJs in 11 normal volunteers, and 10 osteoarthritic knee joints (KNEE-OA). Cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA), and the proteinase activities were detected by enzymography. RESULTS SF from TMJs with ID and OA showed higher (P < .05) levels (330.1 +/- 347.7 pg/100 microg SF protein) of IL-1beta than the asymptomatic control TMJs (76.7 +/- 95.3 pg/100 microg of SF protein). SF from TMJs with OA contained significantly (P < .05) higher levels of IL-1beta (531.8 +/- 379.6 pg/100 microg of SF protein) and IL-6 (979 +/- 552 pg/100 microg SF protein) than those with ID (IL-1beta: 216.7 +/- 280.1 pg, IL-6: 293 +/- 434 pg). Two matrix metalloproteinases (MMPs) with gelatinolytic activities at 92 kDa and 72 kDa were consistently detected in both the TMJ-SF (either normal or disease) and SF from KNEE-OA. Also detected were weak bands with molecular weight of 83 and 66 kDa. These bands were clearly shown, particularly in knee joints with advanced stages of OA. Western blot analysis delineated that these were active forms of MMP-9 (83 kDa) and MMP-2 (66 kDa). The same bands were also detected in TMJs with OA that showed high levels of IL-1beta and IL-6. CONCLUSION These findings suggest that concomitant increases in the levels of cytokines (IL-1 and IL-6) and active forms of MMPs could be potential catabolic markers for cartilage degradation in the TMJ.

Interleukin 1β and stromelysin (MMP3) activity of synovial fluid as possible markers of osteoarthritis in the temporomandibular joint

PURPOSE This study investigated the early signs of synovitis and cartilage degradation by means of synovial fluid analysis in temporomandibular joints (TMJs) with internal derangement (closed lock) or osteoarthritis (OA). PATIENTS AND METHODS Synovial fluid was obtained from 25 TMJs in 22 patients diagnosed with closed lock and from 15 asymptomatic TMJs of 12 normal controls. IL-1 beta concentrations were measured using enzyme-linked immunosorbent assay (ELISA), and proteinase activity was detected by means of gelatin enzymography. RESULTS Nine of the 25 TMJs with closed lock (CL group) exhibited osteolytic changes on the surface of the condyle. TMJs in the normal control group did not show any bony changes. Mean IL-1 beta concentration in the synovial fluid (SF) protein in the CL group was 330.1 +/- 347.7 pg per 100 micrograms protein, which was significantly higher than in the normal control (76.7 +/- 95.3 pg/100 micrograms SF-protein). Synovial fluid from the TMJs with osteolytic changes contained higher levels of IL-1 beta (531.8 +/- 379.6 pg/100 micrograms SF-protein) than those without bony changes (216.7 +/- 280.1 pg/100 micrograms SF-protein). Matrix metalloproteinase (MMP) activity with a molecular weight of 50 kd (stromelysin or MMP3) was detected in a highly augmented form in two synovial fluid samples of seven closed lock patients. CONCLUSION The results suggest that IL-1 beta levels in synovial fluid of the TMJ have a positive correlation with OA change. The MMP3 activity detected was greatly increased in patients with cartilage degradation. These findings suggest that both changes may be important markers of early bone deterioration in TMJs that are undetectable by radiograph imaging.

Multiple forms of SppI (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: Regulation by TGF-β

Metabolic labeling has revealed that rat bone cell populations in culture synthesize several forms of the secreted phosphoprotein, SppI. Most cell populations produced two major [32PO4]-labeled forms that behaved anomolously on SDS-PAGE migrating at 60 kDa and 56 kDa on 10% gels and 55 kDa and 44 kDa on 15% gels. Minor forms of intermediate sizes were also resolved. In normal bone cells the 60 kDa form was predominant and was the only form produced by the clonal bone cell line, RCA 11, whereas the 56 kDa a form predominated in the transformed bone cell line, ROS 17/2.8. In all populations [35S]-methionine-labeling revealed SppIs at approximately 60 kDa but no 56 kDa form. Each form of SppI was specifically cleaved by thrombin which generated fragments of approximately 28 kDa. Transforming growth factor beta 1 increased SppI mRNA levels 3 to 6-fold within 24 h in the normal bone cells, but no increase occurred in the ROS 17/2.8 cells. The elevated expression of SppI was reflected in a selective increase in the synthesis of the [32PO4]-and [35S]-methionine-labeled 60 kDa SppIs.

Influence of an intermittent compressive force on matrix protein expression by ROS 17/2.8 cells, with selective stimulation of osteopontin

The purpose of this study was to determine the response of bone cells to physical stress. Intermittent compressive force (ICF) was applied to 13 kPa to subconfluent ROS 17/2.8 cells at 18 cycles/min. After 48 h of this application, the cells were labelled with [35S]-methionine or [32PO4]. Application of ICF over this time did not alter the synthesis of type I collagen, fibronectin or bone SPARC (osteonectin) compared to that of control cells. However, the activity of alkaline phosphatase was increased 1.5-fold, and the synthesis of a 32PO4-labelled, 75-kDa phosphoprotein, recognized as osteopontin by immunoprecipitation with specific antibodies, was increased 1.4-fold. Also, an increase in osteopontin mRNA starting within 12h of ICF application was observed. The selective increase in osteopontin expression associated with ICF may be important in the remodelling of bone tissues during growth and development and in response to functional forces.

EXTRACELLULAR PROCESSING OF BONE AND DENTIN PROTEINS IN MATRIX MINERALIZATION

There are two steps in the process of matrix-mediated bone and dentin mineralization. First, as in other soft tissues, osteoblasts/odontoblasts synthesize collagenous matrices and second, mineral deposits in these matrices at a location distant from the cells that synthesized the matrices. We suggest a sequence of events that lead the matrix to mineralization: the phosphoproteins of bone and dentin are posttranslationally processed by limited proteolysis, then they are extracellularly processed into a more phosphorylated species that, we believe, facilitates mineralization. Our in situ phosphorylation experiments done with [gamma-32P] GTP suggest the existence of extracellular phosphorylation by a casein kinase II (CKII)-like enzyme, the enzyme known to phosphorylate most of the phosphate residues in dentin phosphophoryn and bone sialoproteins (osteopontin and BSP II).