Haruhito Aoki

Membrane-associated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis

Prostaglandin E synthase (PGES) including isoenzymes of membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES) is the recently identified terminal enzyme of the arachidonic acid cascade. PGES converts prostaglandin (PG)H2 to PGE2 downstream of cyclooxygenase (COX). We investigated the expression of PGES isoenzyme in articular chondrocytes from patients with osteoarthritis (OA). Chondrocytes were treated with various cytokines and the expression of PGES isoenzyme mRNA was analyzed by the reverse transcription–polymerase chain reaction and Northern blotting, whereas Western blotting was performed for protein expression. The subcellular localization of mPGES-1 was determined by immunofluorescent microscopy. Conversion of arachidonic acid or PGH2 to PGE2 was measured by enzyme-linked immunosorbent assay. Finally, the expression of mPGES-1 protein in OA articular cartilage was assessed by immunohistochemistry. Expression of mPGES-1 mRNA in chondrocytes was significantly induced by interleukin (IL)-1β or tumor necrosis factor (TNF)-α, whereas other cytokines, such as IL-4, IL-6, IL-8, IL-10, and interferon-γ, had no effect. COX-2 was also induced under the same conditions, although its pattern of expression was different. Expression of cPGES, mPGES-2, and COX-1 mRNA was not affected by IL-1β or TNF-α. The subcellular localization of mPGES-1 and COX-2 almost overlapped in the perinuclear region. In comparison with 6-keto-PGF1α and thromboxane B2, the production of PGE2 was greater after chondrocytes were stimulated by IL-1β or TNF-α. Conversion of PGH2 to PGE2 (PGES activity) was significantly increased in the lysate from IL-1β-stimulated chondrocytes and it was inhibited by MK-886, which has an inhibitory effect on mPGES-1 activity. Chondrocytes in articular cartilage from patients with OA showed positive immunostaining for mPGES-1. These results suggest that mPGES-1 might be important in the pathogenesis of OA. It might also be a potential new target for therapeutic strategies that specifically modulate PGE2 synthesis in patients with OA.

The role of C-C chemokines and their receptors in osteoarthritis.

OBJECTIVE To evaluate the involvement of the chemokine/chemokine receptor system in cartilage degradation in osteoarthritis (OA). METHODS Expression of the 4 C-C chemokines monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, and their receptors CCR-2 and CCR-5, was assessed in 11 OA patients and 5 normal controls, by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunochemistry, and flow cytometry on untreated or interleukin-1beta (IL-1beta)- and/or tumor necrosis factor alpha (TNFalpha)-stimulated chondrocytes. The effects of these chemokines on the expression of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases were assayed by RT-PCR and ELISA. The effects on proteoglycan synthesis and release were also assayed, using 35S-sulfate incorporation and 35S-proteoglycan release. RESULTS The C-C chemokines and their receptors CCR-2 and CCR-5 were found to be expressed in normal and OA chondrocytes. However, regulation of chemokine expression by IL-1beta and TNFalpha differed between normal and OA chondrocytes. Intracellular staining revealed that approximately 20% of the chondrocytes contained CCR-2 and CCR-5 in the cytoplasm, whereas cell surface expression was detected less frequently. Interestingly, RANTES induced expression of its own receptor, CCR-5, suggesting an autocrine/paracrine pathway of the chemokine within the cartilage milieu. Finally, addition of MCP-1 or RANTES not only induced MMP-3 expression, but also inhibited proteoglycan synthesis and enhanced proteoglycan release from the chondrocytes. CONCLUSION The differential expression of chemokines and their receptors under the regulation of IL-1beta and TNFalpha suggests that the cytokine-triggered chemokine system may play a key role in the cartilage degradation of OA, possibly acting in an autocrine/paracrine manner.

Development and reliability of a standard rating system for outcome measurement of foot and ankle disorders I: development of standard rating system

BackgroundThe aim of this study was to report the five scales comprising the rating system that the Japanese Society for Surgery of the Foot (JSSF) devised (JSSF standard rating system) and the newly offered interpretations and criteria for determinations of each assessment item.MethodsWe produced the new scales for the JSSF standard system by modifying the clinical rating systems established by the American Orthopaedic Foot and Ankle Society (AOFAS scales) and the Japanese Orthopaedic Association’s foot rating scale (JOA scale). We also provided interpretations of each assessment item and the criteria of determinations in the new standard system.ResultsWe improved the ambiguous expressions and content in the conventional standard rating systems so they would be easily understood by Japanese people. The result was five scales in total. Four were designed for use specifically for ankle-hindfoot, midfoot, hallux metatarsophalangeal-interphalangeal, and lesser metatarsophalangeal-ineterphalangeal sites; and the fifth was for the foot and ankle with rheumatoid arthritis. Furthermore, we described interpretations and criteria for determinations with regard to evaluation items in each scale.ConclusionsConventionally, the AOFAS scales or the JOA scale have been separately applied depending on the sites or disorders concerned, but it was often difficult to decide on scores during practical evaluations because of differing expressions in different languages and also because of ambiguity in the interpretation of each evaluation item and in scoring standards as well. JSSF improved these scales and added definite interpretations of evaluation items as well as criteria for the rating (to be reported here in part I). Because these steps were expected to improve the reliability of outcomes assessed by each scale, we examined the reliability in scores of the newly developed scales, which are reported in part II (in this issue).

Development and reliability of a standard rating system for outcome measurement of foot and ankle disorders II: interclinician and intraclinician reliability and validity of the newly established standard rating scales and Japanese Orthopaedic Association rating scale

BackgroundThis study evaluated the validity and inter- and intraclinician reliability of (1) the Japanese Society of Surgery of the Foot (JSSF) standard rating system for four sites [ankle-hindfoot (AH), midfoot (MF), hallux (HL), and lesser toe (LT)] and the rheumatoid arthritis (RA) foot and ankle scale and (2) the Japanese Orthopaedic Association’s foot rating scale (JOA scale).MethodsClinicians from the same institute independently evaluated participating patients from their institute by two evaluations at a 1- to 4-week interval. Statistical evaluation was as follows. (1) The intraclass correlation coefficient (ICC) was calculated from data collected from at least two examinations of each patient by at least two evaluating clinicians (Data A). (2) Total scores for the two evaluations were determined from the distribution of differences in data between the two evaluations (Data B); each item was evaluated by determining Cohen’s coefficient of agreement. (3) The relation between patient satisfaction and total score was investigated only for patients who underwent surgery (Data C). Spearman’s rank correlation coefficient was obtained.ResultsParticipants were 65 clinicians and 610 patients, including those with disorders of the AH (313), MF (47), HL (153), and LT (50) and those with RA (47). From Data A, the ICC was high for AH and HL by JSSF scales and for AH, MF, and LT by the JOA scale. From Data B, the coefficient showed high validity for both scales for AH, with almost no difference between the two scales; the validity for HL was higher with the JOA scale than with the JSSF scale. From Data C, correlations were significant between patient satisfaction and outcome for AH and HL by the JSSF scales and for AH, HL, and LT by the JOA scale.ConclusionsThe validity of both scales was high. Clinical evaluation of the therapeutic results using these scales would be highly reliable.

A 15-year prospective epidemiological account of acute traumatic injuries during official professional soccer league matches in Japan.

Background: Few prospective epidemiological studies on soccer match injuries have collected continuous data using subjects from the same group. Purpose: To investigate long-term injury-induced changes during official matches in the professional Japanese soccer league. Study Design: Descriptive epidemiological study. Methods: Acute injuries during official matches among top-division Japanese professional soccer leaguers were prospectively collected from 1993 to 2007. Injuries preventing player participation for 7 days or more were defined as a reportable injury. Interseasonal variations of injury rate (IR: injuries/1000/player hours) and injury pattern (type, location, circumstances, severity, injury time, positional role, and relationship to weather) were analyzed. Results: Throughout the study period, 2947 injuries from 3984 matches occurred. Mean annual IR was 21.77/1000 player hours, and annual variance showed gradual decrement throughout the study period. The proportion of injury type and location were not significantly changed. Sprain and contusion as injury type and thigh and ankle joint as location were the most common in every season. Contact-related injuries comprised 73.3% on average and were observed to occur more frequently during the last 15 minutes and extra time of match play. The proportion of foul play–related injuries showed a clear declining trend. The proportion of severe injury showed a sporadic increase from 2001 to 2004. The second, fifth, and sixth 15-minute match segments showed a higher IR. Goalkeepers had a lower IR versus other field players. Matches on rainy days resulted in a lower IR than did those held under other weather conditions. Conclusion: Long-term surveillance and statistical feedback of injury characteristics to organization members were considered effective in improving safer play awareness among players and for referees to reduce injury incidence, particularly foul play–related injuries.

Expression of the anaphylatoxin receptor C5aR (CD88) by human articular chondrocytes

Abstract. Although the complement system is implicated in the inflammatory process in arthritic diseases, a direct interaction between chondrocytes and complement has not been demonstrated. In this study, we investigated expression of the C5a receptor (C5aR) on chondrocytes of cartilage from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and bone fracture as normal controls by reverse transcriptase polymerase chain reaction (RT-PCR), flow cytometry, and immunohistochemistry. The RT-PCR detected mRNA for C5aR in most or all of the tested samples (73% in OA, 100% in RA, 89% in normal). The FACS analysis revealed different expression ratios between individuals varying from 0.7% to 77.1%; however, expression ratios of C5aR were significantly higher in RA than in controls (26.0% in RA, 9.0% in OA, 6.9% in normal). The expression of C5aR was upregulated significantly by addition of IL-1β in RA and normal samples but not in OA. In addition, the C5aR-positive chondrocytes were confirmed by immunohistochemistry. In conclusion, expression of C5aR and the effect of IL-1β on the expression were different between RA and OA. The C5aR may contribute to chondrocyte metabolism and the pathogenesis of arthritis differently between in RA and OA.

Incidence of injury among adolescent soccer players: a comparative study of artificial and natural grass turfs

Objective:To investigate the incidence of acute injuries and soccer-related chronic pain from long-term training and during matches in adolescent players using natural grass turfs (NT) and artificial turfs (AT). Design:Case-controlled prospective study. Setting:Institutional-level Fédération Internationale de Football Association Medical Centre of Excellence. Participants:Youth soccer players (12-17 years of age) from 6 teams, with a predominant tendency to train on either NT or AT, were included. Of 332 players enrolled in this study, 301 remained to completion. Interventions:Medically diagnosed acute injuries and chronic pain were recorded daily by team health care staff throughout 2005, and reports were provided monthly to the authors. Assessment of Risk Factors:Noninvasive prospective study. Independent Variables:Age and turf type. Main Outcome Measures:Acute injuries per 1000 player hours on each surface and chronic complaints per 1000 player hours were evaluated according to frequency of surface used ≥80% of the time. Incidence rate ratio (IRR) of acute injuries and chronic complaints during play on NT and AT was calculated. Results:There was no significant difference in the incidence of acute injuries between the 2 surfaces during training and competition. However, the AT group showed a significantly higher incidence of low back pain during training (IRR, 1.62; 95% confidence interval, 1.06-2.48). Early adolescence and prolonged training hours were factors associated with an increased incidence of chronic pain in the AT group. Conclusion:Adolescent players routinely training on AT for prolonged periods should be carefully monitored, even on AT conforming to new standards.

Transplantation of myocyte precursors derived from embryonic stem cells transfected with IGFII gene in a mouse model of muscle injury.

Background. Reconstruction of skeletal muscle tissue is hampered by the lack of availability of functional substitution of the tissue. Methods. Embryonic stem (ES) cells were transfected with the insulin-like growth factor (IGF) II gene and were selected with G418. The resultant cell clones were analyzed regarding their myogenic differentiation in vitro and in vivo. Results. The cells expressed early and late myogenic differentiation markers, including myoD, myogenin, and dystrophin in vitro. They had phosphorylated Akt within the cells, suggesting their activation by the secreted IGFII. Transplantation of the cells to injured anterior tibial muscle of mice significantly improved their motor functions compared to injured mice transplanted with undifferentiated ES cells and injured mice given vehicle alone. The transfected cells adapted to the injured muscle, formed myofibers positive for dystrophin and negative for MyoD and myogenin. Trichrome staining and toluidine blue staining support myofiber formation in vivo. The enzymatic activity of acetylcholine esterase suggested the functional activity of the regenerated motor units. The evoked electromyogram of anterior tibial muscle transplanted with the transfected cells showed significantly higher potentials compared to that transplanted with undifferentiated ES cells and that injected with phosphate-buffered saline (control injury). Electron microscopic examination confirmed the myofiber formation in the cells in vivo. Conclusions. Transfection of IGFII gene into ES cells may be applicable for transplantation therapy of muscle damage due to injury and myopathies.

An experimental study on initial fixation strength in transpedicular screwing augmented with calcium phosphate cement.

Study Design. An experimental study. Objective. To clarify the optimal insertion timing of transpedicular screws when the initial fixation strength reaches in maximum as calcium phosphate cement (CPC) hardens, in cases augmented by CPC to the vertebrae. Summary of Background Data. CPC goes easily into the bone trabeculae and excels in the bone compatibility. However, it is still unknown as for differences of fixation effects by CPC hardening time at actual insertion of the pedicle screw. Methods. Fifty-seven vertebrae obtained from 11 human cadavers. The CPC and titanium pedicle screws were used. Experimental groups were decided as follows. (1) Control group (without CPC). (2) CPC group (augmented with CPC); the mixed CPC infused into the screw hole, afterwards the pedicle screw inserted at a set time (passage time from the initiation of powder and liquid agent mixing). The CPC group was further divided into 3 subgroups, with respect to insertion time of the pedicle screws: 2, 5, and 10 minute subgroups. Maximum pull-out strength was compared, and cross sectioned specimens of the 5 and 10 minute groups were prepared and observed. Results. CPC group showed a pull-out strength of about 177% that of the control group. For inserting timing of the pedicle screw and pull-out strength, no apparent statistically significant difference was found between each subgroups, although the 10-minute group showed the lowest. Cross sectional observations revealed that the CPC diffused deeper into the bone trabeculae in the 5-minute group than in the 10 minutes. Conclusion. CPC augmentation enabled an average 77% increase of the maximum pull-out strength compared to the control group. The study of screw insertion timing augmented with CPC was indicative of the fact that an increase in the initial fixation of the pedicle screw can be achieved when the screw is inserted before initiation of CPC hardening.

Functional Overloading Facilitates the Regeneration of Injured Soleus Muscles in Mice

The effect of functional overloading on the regenerating process of injured skeletal muscle was investigated in 10-week-old male mice (C57BL/6J). Functional overloading on soleus of both hindlimbs was performed by cutting the distal tendons of plantaris and gastrocnemius muscles for 2 weeks before cardiotoxin (CTX) injection as the preconditioning and also during 10 weeks of recovery. To activate the necrosis-regeneration cycle, 0.1 ml of 10-microM CTX was injected into soleus muscle. The mean values of absolute muscle weight and the percentage of Pax7-positive nuclei in soleus were increased by the preconditioning. These values, as well as total muscle protein content, in the group with CTX injection plus overloading were larger than in the group with CTX injection alone. Fibers with central nucleus were noted in the group with CTX injection with or without overloading. The rate of disappearance of fibers having central nucleus during recovery was stimulated by overloading. Histological analyses revealed that the regeneration of injured soleus muscle with overloading proceeded more rapidly than the muscle without overloading. These results, in combination with previous lines of evidence, strongly suggest that functional overloading may facilitate the regeneration of injured skeletal muscles.