Eiji Isoya

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.

Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes

Background and purpose Three-dimensionally (3D-) embedded chondrocytes have been suggested to maintain the chondrocytic phenotype. Furthermore, mechanical stress and growth factors have been found to be capable of enhancing cell proliferation and ECM synthesis. We investigated the effect of mechanical loading and growth factors on reactivation of the 3D-embedded chondrocytes. Methods Freshly isolated chondrocytes from rat articular cartilage were grown in monolayer cultures and then in collagen gel. Real-time RT-PCR and histological analysis for aggrecan and type II and type I collagen was performed to evaluate their chondrocytic activity. Then, the 3D-embedded chondrocytes were cultured under either mechanical loading alone or in combination with growth factor. The dynamic compression (5% compression, 0.33 Hz) was loaded for 4 durations: 0, 10, 60, and 120 min/day. The growth factor administered was either basic fibroblast growth factor (bFGF) or bone morphogenetic protein-2 (BMP-2). Results Mechanical loading statistically significantly reactivated the aggrecan and type II collagen expression with loading of 60 min/day as compared to the other durations. The presence of BMP-2 and bFGF clearly enhanced the aggrecan and type II collagen expression of 3D-embedded chondrocytes. Unlike previous reports using monolayer chondrocytes, however, BMP-2 or bFGF did not augment the chondrocytic phenotype when applied together with mechanical loading. Interpretation Dynamic compression effectively reactivated the dedifferentiated chondrocytes in 3D culture. However, the growth factors did not play any synergistic role when applied with dynamic compressive loading, suggesting that growth factors should be administered at different time points during regeneration of the transplantation-ready cartilage.

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.

Exogenous collagen-enhanced recruitment of mesenchymal stem cells during rabbit articular cartilage repair

Background Despite the well-known effect of type-I collagen in promoting cartilage repair, the mechanism still remains unknown. In this study we investigated this mechanism using a rabbit model of cartilage defects. Animals and methods 5-mm-diameter full-thickness defects were created on both patellar grooves of 53 Japanese white rabbits (approximately 13 weeks old). The left defect was filled with collagen gel and the right defect was left empty. The rabbits were killed and examined morphometrically until the twenty-fourth postoperative week, by (1) evaluation of matrix production, (2) enumeration of the total number of cells engaged in cartilage repair, (3) enumeration of the proliferating cells, (4) localization of mesenchymal stem cells, and (v) localization of apoptotic cells. Results We found that type-I collagen enhances cell recruitment, and thereby increases the number of proliferating cells. A considerable proportion of the proliferating cells were identified as bone marrow-derived mesenchymal stem cells. However, type-I collagen does not prevent the chondrocyte precursors from undergoing apoptotic disengagement from the chondrogenic lineage. Interpretation Type-I collagen promotes cartilage repair by enhancing recruitment of bone marrowderived mesenchymal stem cells. Additional use of agent(s) that sustain mesenchymal stem cells along the chondrogenic path of differentiation may constitute an appropriate environment for cartilage repair.

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.

A case of post-streptococcal reactive arthritis and acute nephritis after bacterial endophthalmitis due to Streptococcus pyogenes

Dear Editor Post-streptococcal reactive arthritis (PSRA) has been deWned as a reactive arthritis following streptococcal infection in the absence of suYcient Jones criteria for acute rheumatic fever [1, 2]. We describe a patient with PSRA, who presented with acute nephritis. A 61-year-old Japanese man was transferred to our department because of severe bilateral knee pain, marked peripheral edema and renal dysfunction. He had a 20-year history of well-controlled diabetes mellitus, but he had never been noted to have arthritis, urinary abnormalities or renal dysfunction. One week previous to the transfer, he had noticed acute pain in his left eye and visual disturbance, and he was admitted to the Department of Ophthalmology in our hospital. He showed fever (38.0°C), marked leukocytosis (3.27 £ 10/L), inXammatory signs [C-reactive protein (CRP), 0.436 g/L), normocomplementemia (C3, 0.116 g/L; C4, 0.03 g/L), and renal impairment [creatinine, 238.68 mol/L; blood urea nitrogen (BUN), 20.35 mmol/L]. The diagnosis of bacterial endophthalmitis was made, and emergent left ophthalmectomy was performed. Blood and urine cultures were sterile, but vitreous samples from his left eye were positive for Streptococcus pyogenes. During hospitalization, he was treated with antibiotics; however, his knee pain and renal function worsened; thus, he was transferred to our department. On transfer, he complained of marked bilateral knee pain. Physical examination revealed bilateral knee arthritis and leg edema. His heart sounds were regular without murmur and no skin lesions were observed. Urinalysis revealed proteinuria (0.64 g/day), hematuria (>100 red blood cells/ high-power Weld) and pyuria (20–30 white blood cells/ high-power Weld) with red blood cell/white cell casts. Full blood count revealed mild anemia and leukocytosis with normal peripheral blood smear and coagulation screen. He showed mild hypoproteinemia, normal liver biochemistry including creatine kinase, and progressive renal dysfunction (creatinine, 270.3 mol/L; blood urea nitrogen, 33.56 mmol/L), marked inXammatory signs (CRP, 0.174 g/L) and development of hypocomplementemia (C3, 0.20 g/L and C4, 0.19 g/L). Further laboratory tests (i.e., blood/urine cultures, hepatitis B/C antigen/antibodies, rheumatoid factor, anti-nuclear antibodies, anti-double-stranded-DNA antibodies, anti-neutrophil cytoplasmic antibodies and antibasement membrane antibodies) showed negative/normal results, but anti-streptolysin O antibodies were elevated to a titer of 283 IU/ml (normal range, <70). Electrocardiograms and echocardiograms were normal. His bilateral knee eVusions had enlarged and were aspirated, showing inXammatory Xuid without any crystals, and synovial Xuid cultures were sterile. Thus, oral prednisolone at 15 mg/day was initiated on day 4 and his arthritis rapidly improved with corticosteroid therapy. On day 12, improvement of leg edema, urinary abnormalities (no hematuria without any remarkable casts), renal function (creatinine, 182.2 mol/L; BUN, 23.2 mmol/L) and inXammatory signs (CRP, 0.04 g/L) were observed, and percutaneous renal biopsy was performed on day 14. On light microscopic examination, all glomeruli showed mild mesangial proliferation T. Sugimoto (&) · N. Takeda · M. Sakaguchi · T. Koyama · Y. Yagi · T. Uzu · A. Kashiwagi Department of Internal Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan e-mail: toshiro@belle.shiga-med.ac.jp

CONGENITAL UNILATERAL UPPER LIMB MUSCULAR HYPERTROPHY ASSOCIATED WITH CONTRACTURE OF AN EXTRINSIC EXTENSOR TENDON

We report a case of congenital unilateral upper limb muscular hypertrophy which presented with loss of extensor tendon excursion of a single digit in late adolescence. The reason for the problem arising is not clear and is not clearly explained by the underlying congenital pathology.

156 THE ROLE OF THE VOLUME-SENSITIVE CL- CURRENT IN THE PROCESS OF REGULATORY VOLUME DECREASE (RVD) IN FRESHLY ISOLATED RABBIT ARTICULAR CHONDROCYTES

differences were also apparent. In mature articular cartilage, the geneexpression level of Sox 9 was lower in the metacarpal than in either the shoulder or the knee joint; that of type X collagen was lower in the metacarpal than in the shoulder joint, but higher in the metacarpal than in the knee joint; that of type IX collagen was lower in the metacarpal and the knee joints than in that of the shoulder. Conclusions: In the immature articular cartilage of the three synovial joint types, the activity profiles of the key genes were almost identical, with but one exception (type I collagen). Hence, during the early phase of growth, a common type of cartilage is formed in the different joints. During its maturation, articular cartilage undergoes a process of complete structural reorganization (Hunziker et al., Osteoarthritis Cartilage 15:403, 2007). The gene-activity profiles also change, and in a jointspecific manner. This finger-printing may reflect the differing mechanical needs of the three joint types. The terminal structure of mature articular cartilage is achieved via the activity of committed chondrocytes within the superficial zone, whereas immature articular cartilage is formed from a “less” committed pool of chondroprogenitor cells.

176 EFFECTS OF CYCLIC COMPRESSIVE LOADING OR IN COMBINATION WITH GROWTH FACTOR ON PHENOTYPIC CHANGES IN CHONDROCYTES IN THREE DIMENSIONAL SCAFFOLD

dose dependent suppressive effect on chondrocyte proliferation that was greatest at their highest concentrations and was significant at all time points (see figures 2 and 3). CGRP showed no significant effect on proliferation or proteoglycan production. Conclusions: Substance P showed a reliable stimulation of chondrocyte proliferation while NPY and VIP showed dosedependent depressive effects. These findings support the idea that the peripheral nervous system, through neuropeptides, exerts direct influence on articular chondrocytes. This may provide some insight into the pathophysiology of inflammatory and degenerative arthritis and provide targets for modifying the repair response of articular cartilage.