N. Okumura

A novel exogenous concentration-gradient collagen scaffold augments full-thickness articular cartilage repair

OBJECTIVES A collagen scaffold has been long used in order to enhance the regeneration of articular cartilage. In the present study, we investigate the effectiveness of a concentration-gradient (CG) collagen that is designed to recruit efficiently the mesenchymal stem cells (MSCs) to the central region of the full-thickness cartilage defects via haptotaxis. METHODS The present study used Cellmatrix (0.3% type I collagen; Nitta gelatin, Osaka, Japan) as the collagen material. We prepared 33%CG collagen gel and 50%CG collagen gel. No gradient collagen gel served as negative control. Full-thickness cartilage defects were created at the patella groove of the rabbit knee, to which the three different collagen gels were transplanted. Bromodeoxyuridine (BrdU) positive, proliferating cells were enumerated and localized, whereas the histological grading score for cartilage regeneration was counted. The expression of type I and type II collagens was evaluated by immunohistochemistry. We also confirmed that the MSCs migrate toward the collagen substrate of higher concentration in a stringently in vitro haptotactic manner. RESULTS Enumeration of the BrdU-positive cells demonstrated that 33%CG collagen gel recruited a significantly larger number of proliferating cells to the central region of the cartilage defect. The histological grading score for the regenerated cartilage treated with 33%CG collagen gel was superior to the other groups. CONCLUSIONS CG collagen scaffold recruits effectively the MSCs to the center of full-thickness cartilage defect and enhances regeneration of the full-thickness cartilage defect.

Regulatory role of tyrosine phosphorylation in the swelling-activated chloride current in isolated rabbit articular chondrocytes

Articular chondrocytes are exposed in vivo to the continually changing osmotic environment and thus require volume regulatory mechanisms. The present study was designed to investigate (i) the functional role of the swelling‐activated Cl− current (ICl,swell) in the regulatory volume decrease (RVD) and (ii) the regulatory role of tyrosine phosphorylation in ICl,swell, in isolated rabbit articular chondrocytes. Whole‐cell membrane currents were recorded from chondrocytes in isosmotic, hyposmotic and hyperosmotic external solutions under conditions where Na+, K+ and Ca2+ currents were minimized. The cell surface area was also measured using microscope images from a separate set of chondrocytes and was used as an index of cell volume. The isolated chondrocytes exhibited a RVD during sustained exposure to hyposmotic solution, which was mostly inhibited by the ICl,swell blocker 4‐(2‐butyl‐6,7‐dichloro‐2‐cyclopentyl‐indan‐1‐on‐5‐yl)oxobutyric acid (DCPIB) at 20 μm. Exposure to a hyposmotic solution activated ICl,swell, which was also largely inhibited by 20 μm DCPIB. ICl,swell in rabbit articular chondrocytes had a relative taurine permeability (Ptau/PCl) of 0.21. Activation of ICl,swell was significantly reduced by the protein tyrosine kinase (PTK) inhibitor genistein (30 μm) but was only weakly affected by its inactive analogue daidzein (30 μm). Intracellular application of protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate (250 and 500 μm) resulted in a gradual activation of a Cl− current even in isosmotic solutions. This Cl− current was almost completely inhibited by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS, 500 μm) and was also largely suppressed by exposure to hyperosmotic solution, thus indicating a close similarity to ICl,swell. Pretreatment of chondrocytes with genistein significantly prevented the activation of the Cl− current by sodium orthovanadate, suggesting that the basal activity of endogenous PTK is required for the activation of this Cl− current. Our results provide evidence to indicate that activation of ICl,swell is involved in RVD in isolated rabbit articular chondrocytes and is facilitated by tyrosine phosphorylation.

17β‐Oestradiol inhibits doxorubicin‐induced apoptosis via block of the volume‐sensitive Cl‐ current in rabbit articular chondrocytes

BACKGROUND AND PURPOSE Chondrocyte apoptosis contributes to disruption of cartilage integrity in osteoarthritis. Recent evidence suggested that the volume‐sensitive organic osmolyte/anion channel [volume‐sensitive (outwardly rectifying) Cl‐ current (ICl,vol)] plays a functional role in the development of cell shrinkage associated with apoptosis (apoptotic volume decrease) in several cell types. In this study, we investigated the cellular effects of 17β‐oestradiol on doxorubicin‐induced apoptotic responses in rabbit articular chondrocytes.

Spatiotemporal control of proliferation and differentiation of bone marrow–derived mesenchymal stem cells recruited using collagen hydrogel for repair of articular cartilage defects

Articular cartilage has a poor healing capacity, and cartilage regeneration is not always warranted to achieve healing. On the other hand, collagen scaffolds have been shown to support regeneration of articular cartilage defects in animal models, whereas bone morphogenetic protein-2 (BMP-2) is known to cause chondrogenic differentiation of marrow-derived mesenchymal stem cells (MSCs). The purpose of this study was to evaluate the effectiveness of intra-articular administration of BMP-2 into bone marrow-derived MSCs recruited to defects using original collagen hydrogel in rabbits at various time points. Full-thickness defects were created in both knees, then collagen hydrogels were transplanted, and BMP-2 was supplied for 1-week periods, as follows. BMP-2 was administered immediately after the operation for 1 week (BMP0-1 group), and BMP-2 was administered between weeks 1 and 2 after the operation (BMP1-2 group). BMP2 was administered between weeks 2 and 3 (BMP2-3 group). Specimens were then obtained, and bromodeoxyuridine (BrdU)-positive cells were enumerated and histologic grading was also performed. In addition, the gene expression analysis was performed using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Enumeration of BrdU-positive cells showed a significant increase in the BMP0-1 group compared with the other groups. Similarly, histologic scores in the BMP0-1 group were superior for up to 8 weeks. Finally, RT-PCR findings revealed that immediate BMP-2 administration enhanced chondrogenic differentiation.

The COX-2 Selective Blocker Etodolac Inhibits TNFα-Induced Apoptosis in Isolated Rabbit Articular Chondrocytes

Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA). Recently, we reported that activation of volume-sensitive Cl− current (ICl,vol) mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX) blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of ICl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl− conductance. The TNFα-evoked Cl− current exhibited electrophysiological and pharmacological properties similar to those of ICl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD) and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

Activation of a chondrocyte volume-sensitive Cl− conductance prior to macroscopic cartilage lesion formation in the rabbit knee anterior cruciate ligament transection osteoarthritis model

Summary Objective The anterior cruciate ligament transection (ACLT) rabbit osteoarthritis (OA) model confers permanent knee instability and induces joint degeneration. The degeneration process is complex, but includes chondrocyte apoptosis and OA-like loss of cartilage integrity. Previously, we reported that activation of a volume-sensitive Cl− current (ICl,vol) can mediate cell shrinkage and apoptosis in rabbit articular chondrocytes. Our objective was therefore to investigate whether ICl,vol was activated in the early stages of the rabbit ACLT OA model. Design Adult Rabbits underwent unilateral ACLT and contralateral arthrotomy (sham) surgery. Rabbits were euthanized at 2 or 4 weeks. Samples were analyzed histologically and with assays of cell volume, apoptosis and electrophysiological characterization of ICl,vol. Results At 2 and 4 weeks post ACLT cartilage appeared histologically normal, nevertheless cell swelling and caspase 3/7 activity were both significantly increased compared to sham controls. In cell-volume experiments, exposure of chondrocytes to hypotonic solution led to a greater increase in cell size in ACLT compared to controls. Caspase-3/7 activity, an indicator of apoptosis, was elevated in both ACLT 2wk and 4wk. Whole-cell currents were recorded with patch clamp of chondrocytes in iso-osmotic and hypo-osmotic external solutions under conditions where Na+, K+ and Ca2+ currents were minimized. ACLT treatment resulted in a large increase in hypotonic-activated chloride conductance. Conclusion Changes in chondrocyte ion channels take place prior to the onset of apparent cartilage loss in the ACLT rabbit model of OA. Further studies are needed to investigate if pharmacological inhibition of ICl,vol decreases progression of OA in animal models.

P2X7 ionotropic receptor is functionally expressed in rabbit articular chondrocytes and mediates extracellular ATP cytotoxicity

Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E2 (PGE2) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE2 release and was attenuated by inhibition of either phospholipase A2 or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic target in chondrocyte death associated with cartilage injury and disorders including osteoarthritis.

Total knee arthroplasty for treatment of osteoarthritis with prolonged patellar dislocation

Prolonged dislocation of the patella is a rare condition and is often related to severe osteoarthritis (OA) of the femorotibial (FT) joint. For this conditions treatment, numerous surgical techniques using total knee arthroplasty (TKA) have been published. To the best of our knowledge, this case report is the first description of the use of lateral release alone to treat recurrent patellar subluxation with TKA. An interesting point in this case is that the patient had a good recovery after TKA in spite of quite a long-term (a duration of almost 55 years) dislocation of her patella and development of secondary OA. We describe a case that we treated by TKA for FT-OA with a prolonged patellar dislocation. We were able to obtain good patellar reduction without additional surgery by performing adequate lateral release of the patellar retinaculum. This clinical case indicates the usefulness of lateral patellar retinaculum release for obtaining stable patellar tracking in TKA for FT-OA with remaining lateral patellar dislocation. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

AB0509 Hip Joint Protection and Falling Numbers of Total Hip Arthroplasties in Cases of Rheumatoid Arthritis: Does Medication Work?

Background Some recent reports indicate that the number of total hip arthroplasty (THA) procedures for RA patients worldwide has been decreasing in recent years. Bone destruction inhibition by use of methotrexate (MTX) and biological drugs has been suggested as a possible reason. However, few studies have investigated whether hip joint destruction in RA patients was altered by treatment with such new drugs. Objectives The purpose of this study was to examine whether the number of RA cases requiring THA has shown a decreasing trend, and to identify any reason for a change. Methods We investigated the records of all patients with RA who underwent a THA procedure at our hospital from January 2000 through December 2014. All cases were sorted by date into 3 groups: the first 5 years (from 2000 to 2004), the middle 5 years (from 2005 to 2009), and the final 5 years (from 2010 to 2015). We noted the number of THA cases each year, age at surgery, duration of RA, and medications administered. In addition, 2 investigators independently evaluated pre-operative X-ray findings using the Larsen classification1 to determine whether the number of severe cases (Larsen grade 5) of hip joint destruction changed during the study period. Results In the 15 year period there were 786 primary THA cases in our hospital, of which 67 (8.5%) were RA cases. The number of THA showed a decreasing tendency, as 29 cases occurred from 2000 to 2005 and 19 from 2009 to 2014, a decrease of approximately 30%. There was little change in patient background in regard to age at surgery and duration of RA. The rate of steroid use decreased from 82.8% to 57.9%, whereas the rate of MTX use increased from 31.0% in the first 5 years to 73.7% in the final 5 years. Furthermore, the rate of biological drug use increased from 0% in the first 5 years to 31.6% in the final 5 years. Cases classified as Larsen grade 5 (Figure 1a) by X-ray evaluation decreased to only two cases in the middle 5 years and a single case in the final 5 years from 8 cases in the first 5 years (Figure 1b). Conclusions The number of patients who underwent THA for RA was decreased and the patient medications administered were also changed. Preoperative X-ray evaluations showed that cases of severe joint destruction (Larsen grade 5) were remarkably decreased. We consider that bone destruction of hip joint can be inhibited by use of MTX and biological drugs. References Larsen A, et al. Radiographic evaluation of rheumatoid arthritis and related conditions by standard reference films. Acta Radiol Diagn 1977;18:481–91. Disclosure of Interest None declared

FRI0179 Reducion in Complement C3 and C4 Levels Greater with Tocilizumab as Compared to Anti-TNF in Patients with Rheumatoid Arthritis

Background A reduction in complement levels during anti-TNF treatment in patients with rheumatoid arthritis (RA) has been described. However, there are no reports regarding changes in complement levels with the use of Tocilizumab (TCZ). Objectives In this study, we investigated changes in complement levels during TCZ treatment and compared them to those with anti-TNF treatment in patients with RA. Methods We measured C3 and C4 levels in 25 patients treated with TCZ and 44 treated with anti-TNF. In addition, major disease activity indicators were monitored before and 1 year after the initiation of treatment. Results Disease activities (DAS28-ESR) were significantly improved in both groups after 1 year. Furthermore, C3 and C4 concentrations were significantly reduced in both after 6 months and 1 year, though those effects were significantly more pronounced in the TCZ group than the anti-TNF group (graphs A and B, p<0.001). Notably, patients in the TCZ group who showed a reduction in C4 to under the lower limit had significantly lower disease activity than others (graph C). Conclusions TCZ more effectively reduces complement C3 and C4 in patients with RA as compared with anti-TNF. Our results also indicate that C4 may be usable as a biomarker in RA patients undergoing TCZ treatment. References Di Mucio G, Perricone C, Ballanti E, et al. 2011. Complement system and rheumatoid arthritis: relationships with autoantibodies, serological, clinical features, and anti-TNF treatment. Int J Immunopathol Pharmacol; 24(2): 357-66. Disclosure of Interest None declared