Noriyuki Kanzaki

Role of p53 in human chondrocyte apoptosis in response to shear strain.

OBJECTIVE Chondrocyte apoptosis plays an important role in cartilage degeneration in osteoarthritis (OA), and mechanical injury to cartilage induces chondrocyte apoptosis. In response to DNA damage, p53 expression is up-regulated, transcription activity is increased, and apoptosis signals are initiated. The p53-regulated apoptosis-inducing protein 1 (p53AIP-1) is one of the p53-regulated genes, and is activated in response to DNA damage. This study was undertaken to analyze p53 function after induction of apoptosis by shear strain in chondrocytes. METHODS OA cartilage samples were obtained from subjects undergoing total knee replacement surgery, and normal cartilage samples were obtained from subjects undergoing surgery for femoral neck fracture. Chondrocytes were isolated from human cartilage and cultured. Expression of p53 and p53AIP in chondrocytes was detected by reverse transcriptase-polymerase chain reaction and Western blotting. Shear strain was introduced in normal human knee chondrocytes. To explore p53 function, normal human knee chondrocytes were pretreated with pifithrin-alpha or p53 small interfering RNA (siRNA) before induction of shear strain. Chondrocyte apoptosis was detected by expression of cleaved caspase 9 with Western blotting and TUNEL staining. Expression of p53 and p53AIP-1 was analyzed by Western blotting. RESULTS OA and normal chondrocytes expressed p53. OA chondrocytes showed much higher expression of p53 and p53AIP-1 than did normal chondrocytes. TUNEL-positive cells and expression of p53, p53AIP-1, and cleaved caspase 9 were increased by shear strain, but chondrocyte apoptosis was suppressed after pretreatment with pifithrin-alpha or p53 siRNA. CONCLUSION Our findings indicate that p53 and p53AIP-1 play important roles in human chondrocyte apoptosis. Down-regulation of p53 expression prevents cartilage from undergoing apoptosis introduced by shear strain.

Regulation of p38 MAPK phosphorylation inhibits chondrocyte apoptosis in response to heat stress or mechanical stress

Activation of p38 MAPK has been associated with a stress response and with apoptotic processes. However, the function of p38 MAPK in chondrocytes is not clearly understood. In this study, we analyzed the expression of p38 MAPK in chondrocytes and investigated the function of p38 MAPK in response to heat stress and mechanical stress. Chondrocytes were isolated from human cartilage and cultured. Expression of p38 and phosphorylated p38 in cartilage of patients with osteoarthritis (OA) was compared to those in normal cartilage by immunohistochemistry and Western blotting. Human knee chondrocytes were exposed to heat stress or mechanical stress. Normal knee chondrocytes were pre-treated with SB203580 or p38 small interfering RNA (siRNA) before induction of heat stress or mechanical stress. Chondrocyte apoptosis was detected by TUNEL staining and Western blotting of cleaved caspases. OA and normal chondrocytes expressed p38; however, OA chondrocytes showed much higher phosphorylated p38 compared to normal chondrocytes. Heat stress or mechanical stress induced apoptosis and increased phosphorylated p38 in normal chondrocytes. The TUNEL positive cells and expression levels of phosphorylated p38 in response to stress decreased when chondrocytes were incubated with SB203580 or transfected with siRNA against p38. In conclusion, we have demonstrated that heat stress or mechanical stress increased chondrocyte apoptosis via phosphorylation of p38. Stress-induced chondrocyte apoptosis decreased due to inhibition of p38 MAPK activation. In contrast, the phosphorylation of p38 MAPK increased in OA chondrocytes. Our results show that down-regulation of p38 MAPK activation inhibits chondrocyte death induced by heat stress or mechanical stress.

Akt phosphorylation in human chondrocytes is regulated by p53R2 in response to mechanical stress

OBJECTIVE The p53 tumor-suppressor protein p53R2 is activated in response to various stressors that act on cell signaling. When DNA is damaged, phosphorylation of p53 at its Ser 15 residue induces p53R2 production. The role of p53R2 in chondrocytes remains poorly understood. In this study, we evaluated in chondrocytes, p53R2 expression and its regulation in response to mechanical stress. Furthermore, we investigated the function of p53R2 in relation to mechanotransduction. METHODS Osteoarthritis (OA) cartilage obtained from total knee replacements and normal cartilage obtained from femoral neck fractures was used to measure p53R2 expression by using immunohistochemistry, western blotting, and real-time polymerase chain reaction (PCR). The OA chondrocytes were subjected to a high magnitude of cyclical tensile strain by using an FX-2000 Flexercell system. Next, sulfated glycosaminoglycan (sGAG) production was quantified in these cells. Protein expression of p53R2, and phosphorylation of Akt, p38MAPK, ERK1/2, and JNK was also detected using western blotting. Moreover, Akt phosphorylation was detected after transfecting the cells with p53R2-specific small interfering RNA (siRNA). RESULTS Expression of p53R2 was significantly increased in OA chondrocytes and in chondrocytes after applying 5% tensile strain to the cells. However, Akt phosphorylation was down-regulated in OA chondrocytes after the strain, and was up-regulated after transfection of p53R2. sGAG protein as well as collagen type II and aggrecan mRNA was increased following transfection of p53R2-specific siRNA after 5% tensile strain. CONCLUSIONS p53R2 could regulate matrix synthesis via Akt phosphorylation during chondrocyte mechanotransduction. Down-regulation of p53R2 may be a new therapeutic approach in OA therapy.

Oxidative stress-induced apoptosis and matrix loss of chondrocytes is inhibited by eicosapentaenoic acid

Eicosapentaenoic acid (EPA) is an antioxidant and n‐3 polyunsaturated fatty acid that reduces the production of inflammatory cytokines. We evaluated the role of EPA in chondrocyte apoptosis and degeneration. Normal human chondrocytes were treated with EPA and sodium nitroprusside (SNP). Expression of metalloproteinases (MMPs) was detected by real‐time polymerase chain reaction (PCR) and that of apoptosis‐related proteins was detected by western blotting. Chondrocyte apoptosis was detected by flow cytometry. C57BL/6J mice were used for the detection of MMP expression by immunohistochemistry and for investigation of chondrocyte apoptosis. EPA inhibited SNP‐induced chondrocyte apoptosis, caspase 3 and poly(ADP‐ribose) polymerase cleavage, phosphorylation of p38 MAPK and p53, and expression of MMP3 and MMP13. Intra‐articular injection of EPA prevented the progression of osteoarthritis (OA) by inhibiting MMP13 expression and chondrocyte apoptosis. EPA treatment can control oxidative stress‐induced OA progression, and thus may be a new approach for OA therapy.

Obese patients may have more soft tissue impingement following primary total hip arthroplasty

PurposeSeveral studies have reported a risk of dislocation in obese patients after total hip arthroplasty. In this study, we evaluated the interaction between obesity and dislocation by kinematic analysis using a navigation system.MethodsThe intraoperative range of motion (ROM) and postoperative impingement-free ROM were measured in 38 patients, and we compared the impingement-free ROM in obese and non obese patients.ResultsThe postoperatively simulated ROM was similar in the obese and non obese groups. The intraoperative ROM was smaller in the obese group. The difference values between the intraoperative ROM and postoperatively simulated ROM were larger in the obese group. These results indicate that obese patients have less ROM following primary total hip arthroplasty even when the implant positioning is performed correctly.ConclusionsDislocations are multifactorial problems including soft tissue impingement. Therefore, the risk of dislocation caused by soft tissue impingement in obese patients may be increased.

DcR3 induces cell proliferation through MAPK signaling in chondrocytes of osteoarthritis

INTRODUCTION Decoy receptor 3 (DcR3), a soluble receptor belonging to the tumor necrosis factor (TNF) receptor superfamily, competitively binds and inhibits the TNF family including Fas-ligand (Fas-L), lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T-cells (LIGHT) and TNF-like ligand 1A (TL1A). In this study, we investigated the functions of DcR3 on osteoarthritis (OA) chondrocytes. METHODS Expressions of DcR3 in chondrocytes were measured by realtime Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Expression of DcR3 in sera and joint fluids was measured by enzyme-linked immunosorbent assay (ELISA). Chondrocytes were incubated with DcR3-Fc chimera protein (DcR3-Fc) before induction of apoptosis by Fas-L and apoptosis was detected with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling labeling (TUNEL) staining and Western blotting of caspase 8 and poly (ADP-ribose) polymerase (PARP). Chondrocytes were incubated with DcR3-Fc and the proliferation was analyzed by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST) assay. Phosphorylation of Extracellular Signal-Regulated Kinase (ERK), P38 mitogen-activated protein kinase (MAPK) and Jun N-terminal Kinase (JNK) in chondrocytes was measured by Western blotting after incubation with DcR3-Fc, Mitogen-activated protein kinase kinase (MEK1/2) inhibitor, or P38 MAPK inhibitor. Chondrocytes were treated with DcR3-Fc after pre-incubation with blocking antibody of Fas-L, LIGHT and TL1A, and proliferation or phosphorylation of ERK was analyzed. RESULTS DcR3 was expressed in OA and normal chondrocytes. DcR3-Fc protects chondrocytes from Fas-induced apoptosis. DcR3-Fc increased chondrocytes proliferation and induced the phosphorylation of ERK specifically. DcR3-induced chondrocytes proliferation was inhibited by pre-incubation of PD098059 or blocking Fas-L antibody. DcR3 increased chondrocytes proliferation in OA chondrocytes, but did not in normal. CONCLUSION DcR3 regulates the proliferation of OA chondrocytes via ERK signaling and Fas-induced apoptosis.

P21 Deficiency Delays Regeneration of Skeletal Muscular Tissue

The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.

Evaluation of the accuracy of femoral component orientation by the CT-based fluoro-matched navigation system

PurposeAccurate orientation of acetabular and femoral components are important during THA. However, no study has assessed the use of the CT-based fluoro-matched navigation system during THA. Therefore, we have evaluated the accuracy of stem orientation by CT-based fluoro-matched navigation.MethodsThe accuracy of stem orientation by CT-based fluoro-matched navigation was assessed by postoperative CT data. Furthermore, we compared the postoperative stem orientation with the intraoperative registration errors.ResultsThe average antetorsion error of the stem (navigation records − postoperative CT) was −0.5° ± 5.2°. The stem valgus error was 0.4° ± 2.7°. The accuracy of the navigation record for the orientation of the stem valgus was dependent on the intraoperative registration errors.ConclusionsThe clinical accuracy of CT-based fluoro-matched navigation is adequate for stem alignment orientation, and the intraoperative verification of registration errors is valuable for checking the accuracy of stem orientation by navigation.

PTEN regulates matrix synthesis in adult human chondrocytes under oxidative stress.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was identified as an important tumor suppressor gene. PTEN functions as a negative regulator of phosphoinositol‐3‐kinase (PI3K)‐Akt and MEK/ERK signaling. The PI3K‐Akt pathway is critical for cell survival, differentiation, and matrix synthesis. Oxidative stress is considered a critical factor in the onset and progression of osteoarthritis (OA). Therefore, we investigated the function of PTEN in OA chondrocytes under oxidative stress. Chondrocytes were treated with insulin‐like growth factor‐1 (IGF‐1) and/or tert‐butyl hydroperoxide (tBHP), which causes oxidative stress. The expression levels of type2 collagen (Col2a1) and aggrecan were analyzed by real‐time PCR, and phosphorylation of Akt and ERK1/2 was analyzed by Western blotting. Chondrocytes were treated with PTEN‐specific small interfering RNA (siRNA), as well as IGF‐1 and/or tBHP. PTEN and IGF‐1 expressions in OA chondrocytes were increased. The downregulation of PTEN expression increased the expression levels of Col2a1 and aggrecan, and increased proteoglycan synthesis under oxidative stress. Oxidative stress decreased the phosphorylation of Akt and increased that of ERK1/2. The downregulation of PTEN expression increased Akt phosphorylation, but did not increase that of ERK 1/2. Our results suggest that PTEN regulates matrix synthesis via the PI3K‐Akt pathway under oxidative stress.

Computed tomographic measurement of acetabular and femoral component version in total hip arthroplasty

PurposeThe purposes of the present study were (1) to investigate the variation and accuracy of both acetabular and femoral component version on the axial computed tomographic (CT) images, and (2) to better define the associations between the components version and clinical factors.MethodsWe investigated acetabular and femoral component orientation in 1,411 primary total hip arthroplasties that had been performed without computer-assisted navigation. Version of the acetabular and femoral components was measured on the axial CT images.ResultsThe component version was significantly greater than the native version in both acetabular and femoral version. There was a significant correlation between the stem and native femoral versions, but not between the acetabular component and native acetabular versions.ConclusionThis study identifies several features that might help analyse the effect of pre-operative native acetabular and femoral version on the variation of component alignment.