Yasuo Yoshihara

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis

OBJECTIVE Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA. METHODS Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [3H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation withp-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated. RESULTS Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF. CONCLUSIONS Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.

Joint fluid carboxy-terminal type II procollagen peptide as a marker of cartilage collagen biosynthesis

Joint fluid levels of carboxy-terminal type II procollagen peptide (pCOL II-C) were measured in osteoarthritis, rheumatoid arthritis and traumatic arthritis by a newly developed one-step enzyme immunoassay (EIA). The detection limit of the new method was as low as 0.2 ng/ml. The levels of pCOL II-C were significantly (P < 0.001) higher in osteoarthritis and traumatic arthritis than in rheumatoid arthritis. In osteoarthritis, pCOL II-C levels were higher in moderately afflicted patients. Since type II collagen is a unique component of cartilage, pCOL II-C levels in joint fluids could reflect the synthetic activity of type II collagen of chondrocytes in the diseased joint and therefore could be utilized as a simple marker of type II collagen synthesis in articular cartilage in joint diseases.

Cementless total hip replacement: past, present, and future

Cementless total hip replacement (THR) is rapidly being accepted as the surgery for arthritic diseases of the hip joint. The bone-ingrowth rate in porous-type cementless implants was about 90% over 10 years after surgery, showing that biological fixation of cementless THR was well maintained on both the stem and cup sides. As for the stress shielding of the femur operated using a distal fixation-type stem, severe bone resorption was observed. The severe bone resorption group showed continuous progression for more than 10 years after surgery. Stem loosening directly caused by stress shielding has been considered less likely; however, close attention should be paid to bone resorption-associated disorders including femoral fracture. Cementless cups have several specific problems. It is difficult to decide whether a cup should be placed in the physiological position for the case of acetabular dysplasia by bone grafting or at a relatively higher position without bone grafting. The bone-ingrowth rate was lower in the group with en bloc bone grafting, and the reactive line was frequently noted in the bone-grafted region. Although no data indicated that en bloc bone grafting directly led to poor outcomes, such as loosening, cup placement at a higher site without bone grafting is now selected by most operators. The polyethylene liner in a cementless cup is thinned due to the metal cup thickness; however, it has been suggested that the apparent relation between the cup size and the wear rate was absent as long as a cementless cup is used. Comparative study indicated cementless THR was inferior with regard to the yearly polyethylene wear rate and incidence of osteolysis on both the stem and cup sides. Meta-analysis study on the survival rate between cement and cementless THR reported that cemented THR was slightly superior. It should be considered that specific problems for cementless THR, especially with regard to polyethylene wear, do occur.

Optimal Photosensitizers for Photodynamic Therapy of Infections Should Kill Bacteria but Spare Neutrophils

Photodynamic therapy (PDT) for localized microbial infections exerts its therapeutic effect both by direct bacterial killing and also by the bactericidal effects of host neutrophils stimulated by PDT. Therefore, PDT‐induced damage to neutrophils must be minimized, while direct photoinactivation of bacteria is maintained to maximize the therapeutic efficacy of antimicrobial PDT in vivo. However, there has been no study in which the cytocidal effect of PDT on neutrophils was investigated. In this study, the cytocidal effects of PDT on neutrophils were evaluated using different antimicrobial photosensitizers to find suitable candidate photosensitizers for antimicrobial PDT. PDT on murine peripheral‐blood neutrophils was performed in vitro using each photosensitizer at a concentration that exerted a maximum bactericidal effect on methicillin‐resistant Staphylococcus aureus, and morphological alteration and viability of neutrophils were studied. Most neutrophils were viable (>80%) after PDT using toluidine blue‐O (TB) or methylene blue (MB), while neutrophils showed morphological change and their viabilities were decreased (<70%) after PDT using other photosensitizers (erythrosine B, rose bengal, crystal violet, Photofrin, new methylene blue and Laserphyrin). These results suggest that PDT using TB or MB can preserve host neutrophils while exerting a significant therapeutic effect on in vivo localized microbial infection.

Synovial fluid concentrations of the C-propeptide of type II collagen correlate with body mass index in primary knee osteoarthritis

OBJECTIVE To explore in a cross sectional study in patients with primary knee osteoarthritis (OA) the relations between body mass index (BMI), disease stage, and the concentrations of a putative joint fluid marker of type II collagen synthesis, procollagen II C-propeptide. PATIENTS AND METHODS The study included 142 patients with knee OA (median age 68, median BMI 24.1). OA was staged radiologically. The concentrations in synovial fluid of procollagen II C-propeptide were measured by a sandwich enzyme immunoassay. RESULTS Joint fluid concentrations of procollagen II C-propeptide were increased in knees with OA (median 3.7 ng/ml), compared with published reference values for knees in healthy adult volunteers (median 1.3 ng/ml). The concentrations of procollagen II C-propeptide were independently related to both OA stage and BMI (r s = 0.343, p < 0.0001 andr s = 0.253, p = 0.002, respectively). CONCLUSIONS Joint fluid concentrations of this putative marker of collagen II synthesis are high in early and mid-stage OA, but decrease in end stage disease. In addition and for the first time it was shown that the concentrations in synovial fluid of procollagen II C-propeptide increase with increasing BMI in primary knee OA. The increased joint fluid values of this marker in patients with primary knee OA and a high BMI, may reflect increased rates of collagen synthesis in their joint cartilage and could relate to the previously shown increased risk for disease progression in such patients.

Photodynamic Therapy Can Induce a Protective Innate Immune Response against Murine Bacterial Arthritis via Neutrophil Accumulation

Background Local microbial infections induced by multiple-drug-resistant bacteria in the orthopedic field can be intractable, therefore development of new therapeutic modalities is needed. Photodynamic therapy (PDT) is a promising alternative modality to antibiotics for intractable microbial infections, and we recently reported that PDT has the potential to accumulate neutrophils into the infected site which leads to resolution of the infection. PDT for cancer has long been known to be able to stimulate the innate and adaptive arms of the immune system. Methodology/Principal Findings In the present study, a murine methicillin-resistant Staphylococcus aureus (MRSA) arthritis model using bioluminescent MRSA and polystyrene microparticles was established, and both the therapeutic (Th-PDT) and preventive (Pre-PDT) effects of PDT using methylene blue as photosensitizer were examined. Although Th-PDT could not demonstrate direct bacterial killing, neutrophils were accumulated into the infectious joint space after PDT and MRSA arthritis was reduced. With the preconditioning Pre-PDT regimen, neutrophils were quickly accumulated into the joint immediately after bacterial inoculation and bacterial growth was suppressed and the establishment of infection was inhibited. Conclusions/Significance This is the first demonstration of a protective innate immune response against a bacterial pathogen produced by PDT.

Photodynamic therapy using intra-articular photofrin for murine MRSA arthritis: Biphasic light dose response for neutrophil-mediated antibacterial effect

Bacterial arthritis does not respond well to antibiotics and moreover multidrug resistance is spreading. We previously tested photodynamic therapy (PDT) mediated by systemic Photofrin® in a mouse model of methicillin‐resistant Staphylococcus aureus (MRSA) arthritis, but found that neutrophils were killed by PDT and therefore the infection was potentiated.

Skeletal unloading induces a full-thickness patellar cartilage defect with increase of urinary collagen II CTx degradation marker in growing rats

Mechanical stress plays an important role in tissue morphogenesis and extracellular matrix metabolism. However, little is known about the effects of reduced loading without restriction of joint motion on the patella. We investigated the effects of long-term skeletal unloading on patellar cartilage and subchondral bone and systemic collagen II metabolism. Nine-week-old male F344/N rats (n=128) were randomly divided into two groups: caged control (C) and tail suspended (TS). Hindlimbs of the TS rats were subjected to unloading for up to 12 weeks. Sequential changes in the patellar cartilage and subchondral bone were analyzed macroscopically, by pathological findings and histomorphologically. All animals received double tidemark fluorochrome labeling prior to sacrifice. Glycosaminoglycan (GAG) content in patellar cartilage, cross-linked C-telopeptide of type II collagen (CTx-II) in 24-h urine and type II procollagen-C-peptide (pCol-II-C) in sera were also measured by DMB assay, ELISA and EIA, respectively. In the TS group, GAG content was significantly reduced only during the first 3 weeks. No further significant decrease was found. Alkaline phosphatase (ALP) activity was increased, especially at the deep zone. Tidemark mineral apposition rate (MAR) was temporally increased, which resulted in an increase in the ratio of calcified cartilage to the entire cartilage. In the medial part, in addition, thickness of the entire cartilage was decreased by temporal acceleration of subchondral ossification advancement and, in the medial margin, a full-thickness cartilage defect was revealed in 88.6% of TS rats. However, the remaining articular surface was free from fibrillation. While urinary CTx-II was significantly increased during the experimental periods, serum pCol-II-C was significantly decreased at the early phase. There were significant correlations between the urinary CTx-II levels and tidemark MAR. Our results provided evidence that skeletal unloading increased ALP activity at the deep zone and temporally accelerated tidemark advancement associated with a decrease in proteoglycan content. In addition, skeletal unloading temporally accelerated subchondral ossification advancement in the medial part of the patella and finally induced a full-thickness patellar cartilage defect without any fibrillation at the remaining articular surface by additional subchondral bone modeling and possible retarded cartilage growth, which was through a different mechanism than overloading.

Influence of intra-articular neutrophils on the effects of photodynamic therapy for murine MRSA arthritis.

Although there have been some reports about the cytotoxic effects of photodynamic therapy (PDT) on multidrug‐resistant bacteria, there have been few reports in which favorable results of PDT on a local infection site are described. This study aimed to verify the hypothesis that the low efficacy of PDT on a local infection site is due to the cytotoxic effect of PDT on leukocytes. PDT using Photofrin® exerted significant cytotoxicity for cultured methicillin‐resistant Staphylococcus aureus (MRSA). Nevertheless, this therapeutic modality was not effective for a murine MRSA arthritis model. Approximately 30% of intra‐articular leukocytes, mainly neutrophils, died immediately after PDT, and a further decrease in the number of intra‐articular leukocytes and atrophy of the synovial tissue were seen 24 h after PDT. Isolated peripheral neutrophils showed significant affinity for Photofrin® and showed significant morphological damage, resulting in cell death, when they were subject to PDT using Photofrin®. These results indicate that intra‐articular neutrophils have an influence on the effects of PDT for MRSA arthritis.

Effects of reloading after simulated microgravity on proteoglycan metabolism in the nucleus pulposus and anulus fibrosus of the lumbar intervertebral disc: an experimental study using a rat tail suspension model.

Study Design. An experiment to measure proteoglycan (PG) content and PG-related gene mRNA expressions in the lumbar intervertebral disc (IVD) of rats tail-suspended (TS) for up to 6 weeks with subsequent reloading. Objective. To assess the effects of reloading after simulated microgravity on PG metabolism in nucleus pulposus (NP) and anulus fibrosus (AF). Summary of Background Data. Although the PG content of rat lumbar IVD is reportedly decreased by low compressive force (due to so-called microgravity) during spaceflight, it is unknown whether it recovers completely on reloading and whether these effects differ between NP and AF. Methods. Eighty-five F344/N rats were divided as follows: caged control (C) or TS for either 3 or 6 weeks, with some TS rats reloaded for 1 or 2 days or 3 weeks after 3 weeks’ suspension (TS+RL-1d, -2d, or -3w). The glycosaminoglycan content and mRNA levels for aggrecan, TIMP1, MMP3, and ADAMTS4 were measured in NP and AF. Results. The glycosaminoglycan contents of NP and AF were significantly decreased (by 27%–42%) in the TS groups, whereas in the TS+RL-3w group recovery was complete in NP, but incomplete in AF, without histologic degenerative changes at any time point. In NP, the aggrecan mRNA level was significantly downregulated in TS-3w, but recovered to control level on reloading (TS+RL-3w). In AF, the MMP3 mRNA level was significantly elevated in TS-6w. In the early (1–2 days) response of PG-related gene expressions to reloading, mRNA levels were significantly increased for aggrecan, TIMP1, and ADAMTS4 in NP and for MMP3 in AF, but significantly decreased for ADAMTS4 in AF (vs. the TS-3w group). Conclusion. Our results suggest that in IVD maintenance against the present type of mechanical stress, modulation of PG plays an important role and may be associated with molecular changes in PG-related genes.