Shigeki Asada

Hydrogen peroxide induces apoptosis of chondrocytes; involvement of calcium ion and extracellular signal-regulated protein kinase.

Abstract:Objective: Recent observations demonstrated that reactive oxygen species facilitate cartilage degradation. We demonstrated that hydrogen peroxide (H2O2) caused inhibition of proteoglycan synthesis, induction of apoptosis and stimulation of extracellular signal-regulated protein kinase (ERK) of the chondrocytes (Inflamm Res 48: 399-403, 1999). To determine whether activation of ERK is involved in the induction of chondrocyte apoptosis, we examined the signal transduction pathways in this hydrogen peroxide induced apoptosis.¶Design: Bovine articular chondrocytes were cultured. To determine the induction of apoptosis, Annexin V staining and terminal deoxynucleotidyl transferase were used. The activity of caspase-3 was measured using an apopain assay kit. Intracellular Ca2+ imaging was observed after fura2-AM loading.¶Results: Hydrogen peroxide enhanced annexin V positive apoptotic cells and caspase-3 activity, which is an executor of apoptosis. Hydrogen peroxide also enhanced intracellular Ca2+ and preincubation with the intracellular Ca2+ chelator protected chondrocytes against hydrogen peroxide-induced cell apoptosis, indicating that an increase in the cytosolic Ca2+ plays a decisive role in this action. When ERK activity was blocked with geldanamycin and PD098059, increased apoptosis was evident.¶Conclusion: Hydrogen peroxide induces chondrocyte apoptosis via Ca2+ signaling, and ERK is involved in these signal transduction pathways.¶

Hyaluronic acid inhibits interleukin-1-induced superoxide anion in bovine chondrocytes

Abstract.Objective: To examine the effect of hyaluronic acid (HA) on the induction of superoxide anion by IL-1 in chondrocytes. ¶Materials and Methods: Bovine articular chondrocytes were treated with different concentrations of IL-1. A chemiluminescent probe (L-012) was added to the medium and chemiluminescence detection was used to measure superoxide anion. ¶Results: IL-1 caused induction of superoxide anions in a dose-dependent manner. HA (10–100 g/ml) countered superoxide induction caused by 20 ng/ml of IL-1. ¶Conclusion: HA can afford protection against cartilage degradation, probably acting as a free-radical scavenger.

Concurrent generation of nitric oxide and superoxide inhibits proteoglycan synthesis in bovine articular chondrocytes : involvement of peroxynitrite

OBJECTIVE Nitric oxide (NO), widely assumed to be a mediator of interleukin 1 (IL-1), inhibits proteoglycan synthesis in articular chondrocytes. IL-1 also produces superoxide anion. We hypothesized that the IL-1 inhibited proteoglycan synthesis is the result of peroxynitrite formed by the reaction of NO with superoxide. METHODS Bovine articular chondrocytes were cultured in the presence of SIN-1, which leads to simultaneous generation of both NO and superoxide. Proteoglycan synthesis was measured based on the incorporation of [35S] sulfate, and the presence of peroxynitrite was confirmed using immunohistochemistry. RESULTS SIN-1 inhibited proteoglycan synthesis and superoxide dismutase reversed SIN-1 inhibited proteoglycan synthesis, indicating the simultaneous generation of superoxide is essential to inhibit proteoglycan synthesis. IL-1 induced peroxynitrite in articular chondrocytes and addition of peroxynitrite inhibited proteoglycan synthesis. CONCLUSION The concurrent generation of superoxide anion and NO is required for the action of IL-1 to inhibit proteoglycan synthesis. Peroxynitrite is a candidate for this underlying mechanism.

Cyclic tensile stretch on bovine articular chondrocytes inhibits protein kinase C activity

Osteoarthrosis, a common pathway of joint deterioration, is caused by mechanical stress loaded on articular cartilage. We previously demonstrated the involvement of protein kinase C (PKC) in the development of osteoarthritis in vitro. In this study, we examined the effect of mechanical stress on chondrocyte metabolism and the activity of PKC in vitro. Low frequency and magnitude of cyclic tensile stretch loaded on chondrocytes increased proteoglycan synthesis. However, high frequency and magnitude of stress decreased its synthesis. In this condition, activity of PKC was reduced. These results suggest an involvement of PKC in the stress-mediated inhibition of proteoglycan synthesis.

Effect of hydrogen peroxide on the metabolism of articular chondrocytes

Abstract.Objective: To examine the effect of hydrogen peroxide on chondrocyte metabolism.¶Materials and Methods: Bovine articular chondrocytes were used. Proteoglycan (PG) synthesis was measured with [35S] sulfate incorporation. For detection of apoptosis, the TdT-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V assay were used. Extracellular-regulated protein kinase (ERK) activity was measured using a mitogen-activated protein kinase assay system.¶Results: Addition of hydrogen peroxide resulted in the inhibition of PG synthesis, apoptosis, and enhanced ERK activity.¶Conclusion: Hydrogen peroxide plays an important role in regulating the metabolism of chondrocytes.

Isolation and characterization of side population stem cells in articular synovial tissue

BackgroundAutologous chondrocyte implantation is an established technique for the repair of degenerated articular cartilage. Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 (Ho) fluorescence dye, has attracted attention as a method of stem cell isolation. Although SP cells from synovial tissue were expected to be an excellent source for this tissue engineering, their precise character in the synovial tissue has not been determined.MethodsSynovial tissues from bovine metacarpophalangeal joints were used as a stem cell source. For efficient collection of stem cells, we first prepared a preculture before sorting in medium containing FBS at variable concentrations for 4 days. Using a cell sorter and the Ho-dye, a poorly stained population enriched with stem cells was then isolated. To determine the characteristics of the stem cells, specific marker genes such as CD34, Flk-1, c-Kit, Abcg-2 were identified by real-time PCR. Sorted SP cells were cultured in a stem cell medium supplemented with bFGF, SCF and fibronectin, and evaluated for their differentiation potentials into chondrocytes, osteocytes and myocytes.ResultsSP cells of synovium tissue were increased from 2% of the total cell population to approximately 10% of the total cells by preculture in the 1%FBS contained medium. Sorted SP cells expressed CD34, Flk-1, c-Kit, Abcg-2 and Mdr-1 -all are important marker genes for stem cell characteristics. The SP cells could be further expanded ex vivo while maintaining stem cell potentials such as marker gene expression, Ho-dye efflux potential and multiple differentiation potentials into chondrocyte, osteocyte and myocyte.ConclusionIn the present study, we demonstrated that the cells with outstanding stem cell properties were efficiently collected as a SP fraction from bovine synovial membrane. Furthermore, we have described an efficient isolation method and the culture conditions for ex vivo expansion that maintains their important characteristics. Our results suggest that the SP cells of synovium tissue might be important candidates as sources for cell transplantation.

Hypoxia-induced nitric oxide protects chondrocytes from damage by hydrogen peroxide

AbstractObjective:Because articular cartilage has no vascular supply, chondrocytes are hypoxic under normal physiological conditions. Nitric oxide (NO) plays an important role in chondrocyte damage, such as apoptosis. Although oxygen stress with hydrogen peroxide was found to cause chondrocyte damage, these data were obtained under normoxic (21% O2) conditions. We investigated the effects of hypoxia on hydrogen peroxide-induced chondrocyte damage Methods:Bovine articular chondrocytes were used in this study. Proteoglycan (PG) synthesis and the induction of apoptosis were analyzed with [35S]-sulfate incorporation and annexin V staining, respectively. The induction of NO was examined using a fluorescent probe and RT-PCR. Results:Cells maintained at 5% O2 had the maximum PG synthesis. Under normoxic conditions, hydrogen peroxide inhibited PG synthesis and induced annexin V positive cells in a dose-dependent fashion. However, in those cells cultured under hypoxic (5%) conditions, the hydrogen peroxide-induced annexin V expression was attenuated. Chondrocytes exposed to hypoxia showed induction of NO. When the hypoxia-induced NO was inhibited, the hypoxia-enhanced PG synthesis was abolished and hydrogen peroxide clearly induced cell damage. Conclusions:Endogenous NO induced by hypoxia protects chondrocytes from apoptosis induced by an oxidative stress.

Cyclic tensile stretch inhibition of nitric oxide release from osteoblast-like cells is both G protein and actin-dependent

Recent reports indicate the alteration of nitric oxide (NO) synthesis with mechanical stress loaded on the osteoblast and NO is considered to have a significant role in mechanotransduction. We found the involvement of guanine‐nucleotide‐binding regulatory proteins (G proteins), especially Gi, in stress‐inhibited NO release of osteoblast‐like cells (JOR:17;593–597, 1999). To determine further the mechanism involved in this process, we measured c‐Jun N‐terminal kinase/stress‐activated protein kinase (JNK/SAPK) activity under cyclic tensile stretch loaded on osteoblast‐like cells. Cyclic stretch significantly enhanced JNK/SAPK activity and pertussis toxin clearly reversed stress‐enhanced JNK/SAPK activity. Cytochalasin D, actin microfilament disrupting reagent, also abolished the stress activation of JNK/SAPK. We propose a model for signaling events induced by cyclic tensile stretch, namely a transmembrane mechanosensor which couples Gi‐protein, actin cytoskeleton and finally activates JNK/SAPK activity of osteoblasts.

The Valgus Inclination of the Tibial Component Increases the Risk of Medial Tibial Condylar Fractures in Unicompartmental Knee Arthroplasty

BACKGROUND Medial tibial condylar fractures (MTCFs) are a rare but serious complication after unicompartmental knee arthroplasty. Although some surgical pitfalls have been reported for MTCFs, it is not clear whether the varus/valgus tibial inclination contributes to the risk of MTCFs. METHODS We constructed a 3-dimensional finite elemental method model of the tibia with a medial component and assessed stress concentrations by changing the inclination from 6° varus to 6° valgus. Subsequently, we repeated the same procedure adding extended sagittal bone cuts of 2° and 10° in the posterior tibial cortex. Furthermore, we calculated the bone volume that supported the tibial component, which is considered to affect stress distribution in the medial tibial condyle. RESULTS Stress concentrations were observed on the medial tibial metaphyseal cortices and on the anterior and posterior tibial cortices in the corner of cut surfaces in all models; moreover, the maximum principal stresses on the posterior cortex were larger than those on the anterior cortex. The extended sagittal bone cuts in the posterior tibial cortex increased the stresses further at these 3 sites. In the models with a 10° extended sagittal bone cut, the maximum principal stress on the posterior cortex increased as the tibial inclination changed from 6° varus to 6° valgus. The bone volume decreased as the inclination changed from varus to valgus. CONCLUSION In this finite element method, the risk of MTCFs increases with increasing valgus inclination of the tibial component and with increased extension of the sagittal cut in the posterior tibial cortex.

Comparison of MRI- and CT-based patient-specific guides for total knee arthroplasty

BACKGROUND The patient-specific guide for total knee arthroplasty (TKA) is created from the data provided by magnetic resonance imaging (MRI) or computed tomography (CT) scans. It remains unknown which imaging technology is suitable for the patient-specific guide. The purpose of this study was to compare the accuracy of implant positioning and operative times between the two types of patient-specific guides for TKA. METHODS Forty arthritic knees were divided into two treatment groups using MRI-based (PS-MRI group) or CT-based (PS-CT group) patient-specific guides in this prospective, comparative study. The guide in the PS-MRI group had a cutting slot, whereas that in the PS-CT group only had a pin locator. The operative times were compared between the two groups. The angular error and number of outliers (deviations >3°) of the implant position using pre- and postoperative CT were investigated in both groups. RESULTS The mean operative time was significantly shorter in the PS-MRI group (109.2 ± 16.5 min) than in the PS-CT group (129.5 ± 19.4 min) (p<0.001). There were no significant differences in the accuracy of the implant position regarding the coronal, sagittal, and axial planes between the groups (p>0.05). CONCLUSIONS To reduce the operative time, guides with additional functions, such as cutting and positioning, should be used. Both CT- and MRI-based-guides would result in the same accuracy in three planes but high inaccuracy in the sagittal plane. The use of patient-specific guide based on MRI might not be cost-effective. LEVEL OF EVIDENCE level 2.