Y. Muramatsu

Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration

Mechanical stress and aging are major risk factors of cartilage degeneration. Human studies have previously reported that oxidative damage increased, while SOD2 protein was reciprocally downregulated in osteoarthritic degenerated cartilage. However, it remains unclear whether mitochondrial superoxide imbalance in chondrocytes causes cartilage degeneration. We herein demonstrate that mechanical loading promoted mitochondrial superoxide generation and selective Sod2 downregulation in chondrocytes in vivo and that mitochondrial superoxide inducer also downregulated Sod2 expression in chondrocytes in vitro. A genetically manipulated model revealed that Sod2 deficiency in chondrocytes also resulted in mitochondrial superoxide overproduction and dysfunction, thus leading to cartilage degeneration. Intra-articular injection of a permeable antioxidant effectively suppressed the mechanical loading-induced mitochondrial superoxide generation and cartilage degeneration in mice. Our findings demonstrate that mitochondrial superoxide plays a pivotal role in the development and progression of osteoarthritis, and the mitochondrial superoxide balance may therefore be a promising target for the treatment of cartilage degeneration.

Hidden osteophyte formation on plain X-ray is the predictive factor for development of knee osteoarthritis after 48 months – data from the Osteoarthritis Initiative

OBJECTIVE To examine whether the detection of osteophytes anywhere in the knee could serve as a pre-radiographic biomarker for osteoarthritis (OA) development. METHODS Baseline magnetic resonance imaging (MRIs) of 132 participants in the Osteoarthritis Initiative (OAI) were studied. Based on radiographs, 66 knees were assessed as osteoarthritis-free (no-osteoarthritis [NOA], or Kellgren/Lawrence [K/L] severity grade 0/1 both at baseline and 48 months), and another 66 knees were assessed as having radiographic OA changes (pre-radiographic osteoarthritis [PROA], or with K/L grade 0/1 at baseline and grade ≥ 2 at 48 months). Using baseline MRI data, we examined eight sites of osteophyte formation: the medial and lateral femoral condyle (MFC and LFC, respectively); medial and lateral tibial plateau (MTP and LTP, respectively); medial and lateral facets of the patellofemoral joint (PM and PL, respectively); tibial spine (TS); and femoral intercondylar notch (IC). Knee joint osteophyte size was assessed via the 8-point marginal osteophytes item of the whole-organ magnetic resonance imaging score (WORMS). The frequencies and distributions of osteophytes were compared between groups. RESULTS Mild-size osteophytes (defined as score ≥ 2) were observed more frequently at the MFC (P = 0.00278), MTP (P = 0.0046), TS (P = 0.0146), PM (P < 0.0001), PL (P = 0.0012), and IC (P < 0.0001) in PROA knees than in NOA knees. Moderate-size osteophytes (defined as score ≥ 4) were more frequently observed in PROA knees than in NOA knees only at the IC (P < 0.0001). CONCLUSION Knees with osteophyte formation at the IC, even those of K/L severity grade 0/1, are at risk for the development of radiographic OA by 48 months.

Effective knock down of matrix metalloproteinase-13 by an intra-articular injection of small interfering RNA (siRNA) in a murine surgically-induced osteoarthritis model

This study investigated the effect of MMP‐13 gene knock down on cartilage degradation by injecting small interfering RNA (siRNA) into knee joints in a mouse model of osteoarthritis (OA). OA was induced in male C57BL/6 mice by destabilization of medial meniscus (DMM) surgery. Change of Mmp13 expression over time was determined by qPCR analysis from 3 days to 6 weeks after surgery. Mmp13 and control chemically modified siRNA were injected into the knee joint 1 week after surgery and expression levels were assessed in synovium by qPCR 48 h later. Cartilage degradation was histologically assessed 8 weeks after DMM surgery according to OARSI recommendations. Mmp13 expression levels were elevated 1 week after surgery and peaked at 77 fold at 2 weeks compared to expression at 3 days. A 55% decrease of Mmp13 levels in cartilage was observed 48 h after injection of Mmp13 siRNA (p = 0.05). Significant reduction in the histological score at 8 weeks after surgery was observed in the Mmp13 siRNA‐treated group compared to the control siRNA group (p < 0.001). Intra‐articular injection of Mmp13 siRNA at the early phase of OA development resulted in effective knock down of Mmp13 expression and delay in cartilage degradation in vivo.

Quantitative Assessment of Tendon Healing by Using MR T2 Mapping in a Rabbit Achilles Tendon Transection Model Treated with Platelet-rich Plasma.

PURPOSE To determine if magnetic resonance (MR) imaging T2 mapping can be used to quantify histologic tendon healing by using a rabbit Achilles tendon transection model treated with platelet-rich plasma (PRP). MATERIALS AND METHODS Experiments were approved by the Institutional Animal Care and Use Committee. The Achilles tendons of 24 New Zealand white rabbits (48 limbs) were surgically transected, and PRP (in the test group) or saline (in the control group) was injected into the transection site. The rabbits were sacrificed 2, 4, 8, and 12 weeks after surgery. Thereafter, T2 mapping and histologic evaluations were performed by using the Bonar scale. A mixed-model multivariate analysis of variance was used to test the effects of time and PRP treatment on the T2 value and Bonar grade, respectively. The correlation between the T2 value and Bonar grade was also assessed by using the Spearman correlation coefficient. RESULTS The Bonar scale values decreased in both groups during tendon healing. The T2 value also shortened over time (P < .001 for both groups). The T2 values were positively correlated with the Bonar grade (ρ = 0.78, P < .001). Both the T2 value and Bonar scale value were lower in the PRP group than in the control group at 4, 8, and 12 weeks; however, there was no significant effect of PRP treatment on the T2 value or Bonar grade. CONCLUSION The T2 value changes reflected histologic tendon healing. While T2 and Bonar grade were lower at all time points in tendons treated with PRP, there was no significant difference between the treatment and control tendons.

Accuracy of high-resolution ultrasound in the detection of meniscal tears and determination of the visible area of menisci.

BACKGROUND Imaging is of great importance in diagnosing meniscal tears. The aim of this study was to assess the accuracy of high-resolution ultrasound in the diagnosis of meniscal tears, with arthroscopic examination as the standard reference, after resolution was confirmed with a target-mounted reference phantom. An additional goal was to elucidate the area of the meniscus that could be visualized with the same ultrasound machine after placement of markers into the menisci of cadaveric knees. METHODS Seventy patients were included for the assessment of the accuracy of a high-resolution ultrasound machine with a 14.0 to 6.0-MHz linear transducer. The preoperative ultrasound diagnosis, in terms of the presence and type of tear, was compared with that in the surgical reports. In the cadaveric studies, nine needles were placed in the peripheral zone of the menisci at regular intervals and the number of needles that could be observed with the system was recorded. RESULTS The overall sensitivity, specificity, positive predictive value, and negative predictive value of ultrasound examination for meniscal tears were 88%, 85%, 85%, and 88%, respectively. These statistical parameters did not differ significantly between the medial and lateral menisci. The sensitivity for diagnosing horizontal, vertical, radial, flap, bucket-handle, and complex tears and for detecting discoid lateral menisci was 83%, 64%, 0%, 64%, 54%, 90%, and 80%, respectively. Ten percent of the lateral menisci could not be evaluated because of poor images. The cadaveric studies revealed that the ultrasound visualized the entire meniscus except for the anterior horn. CONCLUSIONS The findings of this study suggest that ultrasound examination may be suitable for screening for meniscal tears. The fact that almost 10% of the lateral menisci could not be evaluated because of poor images appears to be a weakness of ultrasound.

Preventive effects of hyaluronan from deterioration of gait parameters in surgically induced mice osteoarthritic knee model

OBJECTIVE Osteoarthritis (OA) leads to pain and loss of function in affected joints. Gait disturbance results from these symptoms of OA, and gait analysis can be important to evaluate the progression of OA. The purpose of this study was to analyze gait pattern in a rodent model of OA and to assess the effects of intra-articular injection of hyaluronan (IAI-HA) by gait analysis, along with histological evaluation. DESIGN OA was induced by destabilization of the medial meniscus (DMM) of C57BL/6 mice. IAI-HA started 3 weeks after DMM surgery. Mice were allocated to three groups and were given either 800-kDa HA (800-HA), 6000-kDa HA (6000-HA) or saline. We compared these three groups with a sham group by gait analysis using CatWalk. Histological evaluation was performed to assess articular cartilage changes in the knee joints. RESULTS Mice injected with 800-HA or 6000-HA showed gait patterns similar to that of the sham mice, while the saline-injected group showed gait disturbances 12 and 16 weeks after DMM surgery. Histological changes in articular cartilage were similar among the 800-HA, 6000-HA and saline-treated groups, demonstrating OA progression throughout the experimental time points. Positive gait-related effects of IAI-HA might occur by its pain relieving effect and/or by preventing contracture. CONCLUSION IAI-HA prevented gait disturbances in the DMM model, but did not prevent histological changes associated with OA progression.

Utility of T2 mapping and dGEMRIC for evaluation of cartilage repair after allograft chondrocyte implantation in a rabbit model.

OBJECTIVE To investigate the effectiveness of quantitative Magnetic resonance imaging (MRI) for evaluating the quality of cartilage repair over time following allograft chondrocyte implantation using a three-dimensional scaffold for osteochondral lesions. DESIGN Thirty knees from 15 rabbits were analyzed. An osteochondral defect (diameter, 4 mm; depth, 1 mm) was created on the patellar groove of the femur in both legs. The defects were filled with a chondrocyte-seeded scaffold in the right knee and an empty scaffold in the left knee. Five rabbits each were euthanized at 4, 8, and 12 weeks and their knees were examined via macroscopic inspection, histological and biochemical analysis, and quantitative MRI (T2 mapping and dGEMRIC) to assess the state of tissue repair following allograft chondrocyte implantation with a three-dimensional scaffold for osteochondral lesions. RESULTS Comparatively good regenerative cartilage was observed both macroscopically and histologically. In both chondrocyte-seeded and control knees, the T2 values of repair tissues were highest at 4 weeks and showed a tendency to decrease with time. ΔR1 values of dGEMRIC also tended to decrease with time in both groups, and the mean ΔR1 was significantly lower in the CS-scaffold group than in the control group at all time points. ΔR1 = 1/r (R1post - R1pre), where r is the relaxivity of Gd-DTPA(2-), R1 = 1/T1 (longitudinal relaxation time). CONCLUSION T2 mapping and dGEMRIC were both effective for evaluating tissue repair after allograft chondrocyte implantation. ΔR1 values of dGEMRIC represented good correlation with histologically and biochemically even at early stages after the implantation.

Identification of genes required for the spontaneous repair of partial-thickness cartilage defects in immature rats

ABSTRACT Purpose: Our previous study showed that partial-thickness articular cartilage defects (PTCDs) created in immature rats spontaneously healed to resemble normal hyaline cartilage, but that of mature rats did not. To identify molecules involved in the spontaneous cartilage repair observed in this model, gene expression was compared between PTCD and sham-operated cartilage of immature and mature rats. Materials and Methods: Six sets of gene comparisons were made at 12, 24, and 48 hours after the creation of PTCDs in immature and mature rats using microarrays. All the genes upregulated in immature cartilage at 12 hours were selected for further analysis if their expression pattern was not irregular such that diminished at 24 hours and re-upregulated at 48 hours. Relationships among genes selected through the above steps were analyzed using Ingenuity Pathway Analysis (IPA) software. After deriving networks, important molecules were further narrowed down by location within a network. Genes were regarded as central if they had relationships with more than 10 molecules in a network. Protein localization in tissues was confirmed by immunohistochemistry. Results: Five networks were identified. Their functional annotations were gene expression, cell cycle, growth and proliferation, and cell signaling. Transforming growth factor-beta (TGF-β) was centrally located in the network with the highest IPA score and mothers against decapentaplegic homolog-3 (Smad3) were centrally located in the second highest ranking network. Phosphorylated Smad3 was detected in the nuclei of chondrocytes in immature cartilage. Conclusions: Our data suggest the possible importance of Smad3 in the TGF-β signaling in the spontaneous healing of PTCDs in immature rats.

Spontaneous repair of partial thickness linear cartilage injuries in immature rats

Partial thickness articular cartilage injuries (PTCIs) were not previously thought to heal spontaneously. Immature rats have the capacity for spontaneous repair of PTCIs, although it is a long-term process. Our aim has been to examine the spontaneous repair response mechanism in immature rats. Single linear PTCIs were created in 3-week-old and 12-week-old rats in the direction of joint motion. On day 1 and at 1, 2, and 4 weeks after PTCI, evaluations of histological changes and immunohistology at the injury site and in the surrounding cartilage were performed. Anti-CD105 and anti-CD166 antibodies (as stem cell markers to identify mesenchymal stem cells in reparative cartilage tissue) were used for immunohistological evaluations. To determine whether endogenous repair ability existed in articular cartilage, an ex vivo experiment was also carried out. Femoral condyles with PTCIs were incubated in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum for 1 day and for 1 and 2 weeks. Histological changes were subsequently examined. Immature cartilage showed a higher repair response than did mature cartilage, and the response occurred immediately after PTCI. In immature rats, CD105- and CD166-positive cells were found in the superficial and transitional zones of the articular cartilage. Few CD166-positive cells were identified in mature articular cartilage. No significant in vivo differences in the spontaneous repair responses to PTCIs were observed between mature and immature groups. Thus, the repair response to PTCIs seems to be associated not only with CD105- and CD166-positive cells, but also with other perichondral factors.

Quality of Life in Patients With Untreated and Symptomatic Hallux Valgus.

Background: The purposes of this study were to compare the quality of life (QOL) of subjects who had untreated symptomatic hallux valgus with the QOL of the general population and to investigate factors associated with the QOL of the subjects. Methods: One hundred sixteen subjects with previously untreated and symptomatic hallux valgus were surveyed. QOL was assessed using the 36-item Short Form Health Survey (SF-36). Additionally, clinical evaluations (the visual analog scale for pain, Japanese Society for Surgery of the Foot Scale, lesser toe pain, and pain in other parts of the body) and radiographic evaluations (hallux valgus angle, intermetatarsal angle between the first and second metatarsals, and dislocation of the second metatarsophalangeal joint) were performed. Differences in the SF-36 between the subjects and the general population were tested using independent t tests. Correlations between the QOL measurements, clinical evaluations, and radiographic evaluations were assessed using Spearman rank correlation coefficient. Results: All SF-36 subscales and physical component summary scores for the subjects were significantly lower than those of the general population. Notably, the standardized physical function subscale (38.2 ± 15.8, P < .001) and physical component summary scores (38.9 ± 14.5, P < .001) were more than 10 points lower than those of the general population. Most QOL and clinical evaluation parameters were not correlated or were negligibly correlated with radiographic evaluations. Similarly, lesser toe pain or pain in other parts of the body was not correlated with QOL or clinical evaluations. Conclusion: The QOL of untreated and symptomatic hallux valgus subjects was lower than that of the general population. All QOL and clinical evaluation parameters were not significantly or negligibly correlated with the severity of toe deformities. Surgical decision making should not be based on the severity of the deformity alone, but rather patient QOL should also be carefully assessed. Level of Evidence: Level III, comparative series.