Kyosuke Fujikawa

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.

Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source.

Adult bone marrow contains mesenchymal stem cells (bone marrow-derived mesenchymal stem cells; BMSCs) which contribute to the generation of mesenchymal tissue such as bone, cartilage, muscle and adipose. However, using bone marrow as a source of stem cells has the limitation of a low cell number. An alternate source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Human adipose tissue obtained by liposuction was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). In this study, we compared the osteogenic differentiation of ATSCs with that of BMSCs. Both cell types were cultured in atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS scaffold) for three-dimensional culturing in a specific osteogenic induction medium. Optimal osteogenic differentiation in both cell types, as determined by alkaline phosphatase cytochemistry, secretion of osteocalcin, mineral (calcium phosphate) deposition and scanning electron microscopy, was obtained with the same three-dimensional culture. Furthermore, osteoblastic lining in vivowas examined using ATSC-seeded or BMSC-seeded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts to that of BMSCs.

An Experimental Study of the Regeneration of the Intervertebral Disc With an Allograft of Cultured Annulus Fibrosus Cells Using a Tissue-Engineering Method

Study Design. Cultured annulus fibrosus cells within an atelocollagen honeycomb-shaped scaffold with a membrane seal were allografted into the lacunas of intervertebral discs of which the nucleus pulposus had been vaporized using an indocyanine green dye-enhanced laser. Regeneration of the intervertebral disc was assessed based on the viability and histologic status of the allografted annulus fibrosus cells, as well as the prevention of narrowing disc space. Objectives. To study the regeneration of intervertebral disc after laser discectomy using tissue-engineering methods. Summary of Background Data. Intervertebral disc is the most avascular tissue in the human body, and its ability to regenerate is as low as that of articular cartilage. When nucleotomy is carried out, little regeneration of the annulus fibrosus is observed; consequently, intervertebral disc degeneration is inevitable. Methods. Annulus fibrosus cells isolated from 20 Japanese white rabbits were labeled with a PKH-26 fluorescent dye and seeded within an atelocollagen honeycomb-shaped scaffold with a membrane seal. Annulus fibrosus cells cultured in atelocollagen honeycomb-shaped scaffold with a membrane seal for 1 week were allografted into the lacunas of intervertebral discs of recipient rabbit of which the nucleus pulposus had been vaporized using an ICG dye-enhanced laser. Soft radiograph photographs of the lumbar spine of these anesthetized rabbits were taken, the disc space measured, and the lumbar spine extracted 2, 4, 8, and 12 weeks after the operation. The proliferation of allografted annulus fibrosus cells with 5-bromo-2′-deoxyuridine/PKH-26 fluorescent labels was assessed using consecutive frozen sections, and safranin-O staining carried out for histologic evaluation. Results. The allografted annulus fibrosus cells were viable and showed proliferation activity with a hyaline-like cartilage being produced. The narrowing of the intervertebral disc space of the cell translation group was significantly prevented, as shown, up to 12 postoperative weeks. Conclusion. The annulus fibrosus cells cultured in an ACHMS-scaffold were allografted into the lacunae of nucleus pulposus (obtained using laser vaporization), as well as the hole of annulus fibrosus (obtained by laser fiber insertion) of rabbit intervertebral discs. These cells were viable and showed cell proliferation in the disc tissues of recipients.

Macrophage- and neutrophil-dominant arthritis in human IL-1α transgenic mice

To study the effects of IL-1α in arthritis, we generated human IL-1α (hIL-1α). Transgenic mice expressed hIL-1α mRNA in various organs, had high serum levels of hIL-1α, and developed a severe polyarthritic phenotype at 4 weeks of age. Not only bone marrow cells but also synoviocytes from knee joints produced biologically active hIL-1α. Synovitis started 2 weeks after birth, and 8-week-old mice showed hyperplasia of the synovial lining layer, the formation of hyperplastic synovium (pannus) and, ultimately, destruction of cartilage. Hyperplasia of the synovial lining was due to the accumulation of macrophage-like cells expressing F4/80 molecules. hIL-1α was widely distributed in macrophage- and fibroblast-like cells of the synovial lining cells, as well as synovial fluid monocytes. T and B cells were rare in the synovial fluid, and analysis of marker expression suggests that synoviocytes were directly histolytic and did not act as antigen-presenting cells. In the joints of these mice, we found elevated levels of cells of the monocyte/macrophage and granulocyte lineages and of polymorphonuclear neutrophils (PMNs), most of which expressed Gr-1, indicating that they were mature, tissue-degrading PMNs. Cultured synoviocytes and PMNs from these animals overexpress GM-CSF, suggesting that the hematopoietic changes induced by IL-1 and the consequent PMN activation and joint destruction are mediated by this cytokine.

Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilage.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, 4, 5, 8, 9 and 15, members of the ADAMTS gene family, have the ability to degrade a major cartilage proteoglycan, aggrecan, at the specific sites, and thus are called ‘aggrecanases’. The expression of these ADAMTS species was examined in human osteoarthritic articular cartilage on reverse transcription–polymerase chain reaction. The results demonstrated the predominant expression of ADAMTS4 in osteoarthritic cartilage, while ADAMTS5 was constitutively expressed in osteoarthritic and normal cartilage. ADAMTS9 was expressed mainly in normal cartilage, whereas no or negligible expression of ADAMTS1, 8 and 15 was observed in either osteoarthritic or normal cartilage. In situ hybridization for ADAMTS4 indicated that chondrocytes in osteoarthritic cartilage expressed the mRNA. Two monoclonal antibodies to ADAMTS4 were developed, and immunolocalized ADAMTS4 to chondrocytes in the proteoglycan‐depleted zones of osteoarthritic cartilage, showing a direct correlation with the Mankin scores. Immunoblotting indicated a major protein band of 58 kDa in the chondrocyte culture media and osteoarthritic cartilage tissue homogenates. These data demonstrate that among the six ADAMTS species, ADAMTS4 is mainly expressed in an active form in osteoarthritic cartilage, and suggest that ADAMTS4 may play an important role in the degradation of aggrecan in human osteoarthritic cartilage.

Tissue engineering of the intervertebral disc with cultured annulus fibrosus cells using atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold).

The objective of the study was to investigate the regeneration of intervertebral discs after laser discectomy using tissue engineering procedures. Annulus fibrosus (AF) cells from the intervertebral discs of Japanese white rabbits were cultured in an atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold), to produce a high-density, three-dimensional culture for up to 3 weeks. Although the DNA content in the scaffold increased at a lower rate than that in the monolayer culture, expression of type ll collagen and glycosaminoglycan accumulation in the scaffold were at higher levels than in the monolayer. The AF cells that had been cultured in the scaffold for 7 days were allografted into the lacunae of intervertebral discs of recipients (40 rabbits, 14–16 weeks old; average weight, 3.2kg), whose nucleus pulposus (NP) had been vaporised with an ICG dye-enhanced laser. The allografted cultured AF cells survived and produced hyaline-like cartilage. Furthermore, the narrowing of the intervertebral disc space of the cell-containing scaffold insertion groups was significantly inhibited after 12 post-operative weeks.

Membrane-Associated IL-1 Contributes to Chronic Synovitis and Cartilage Destruction in Human IL-1α Transgenic Mice

IL-1 molecules are encoded by two distinct genes, IL-1α and IL-1β. Both isoforms possess essentially identical activities and potencies, whereas IL-1α, in contrast to IL-1β, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1α (hIL-1α) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys64 to Ala271 of the hIL-1α amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1α. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1α activity or concentration of serum hIL-1α. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1α, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.

Metal ions induce bone-resorbing cytokine production through the redox pathway in synoviocytes and bone marrow macrophages.

To evaluate the biological reactions to metal ions potentially released from prosthetic implants, we examined the ability of metal ions to produce bone-resorbing cytokines and the underlying mechanism using synoviocytes and bone marrow (BM) macrophages. The cells were incubated with NiCl(2), CoCl(2), CrCl(3) or Fe(2)(SO(4))(3) at optimal concentrations, which are detectable in joint fluid following total joint arthroplasty. The production of interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha were enhanced by all metal ions tested as determined by enzyme-linked immunosorbent assay. From the results of electrophoresis mobility shift assay, all metal ions enhanced the DNA-binding activity of nuclear factor kappaB (NF-kappaB), and p50-p65 heterodimers and p50 homodimers were the major subunits. These effects of the metal ions were considerably blocked by pyrrolidine dithiocarbamate (PDTC) known as a radical scavenger. An electron spin resonance study clearly demonstrated the ability of metal ions to generate activated oxygen species (AOS), especially hydroxyl radicals (*OH), which accounts for PDTC-blockade of metal ion-induced NF-kappaB activation and subsequent cytokine production. Taken together, our data raised the possibility that small amounts of metal ions released from prosthetic implants activate synoviocytes and BM macrophages through the AOS-mediated process (i.e. the redox pathway), and contribute to the initiation of osteolysis at the bone-implant interface.

Tensile load and the metabolism of anterior cruciate ligament cells.

It generally is recognized that tensile load plays a major role in maintaining the homeostasis of the anterior cruciate ligament fibers, but its detailed mechanism remains a matter of controversy. The effects of cyclic tensile load on the metabolism of the anterior cruciate ligament were investigated experimentally using cultured cells from the anterior cruciate ligament of rabbits. Using culture plates with flexible rubber bases, a cyclic tensile load was applied to the cultured cells for 24 hours, and the changes in shape, alignment, and metabolism of the cells were analyzed. Under the cyclic tensile load, the shape of the cells from the anterior cruciate ligament changed to spindle and aligned perpendicularly to the direction of the tensile load. The cyclic tensile load also caused an increase in collagen synthesis by the cells from the anterior cruciate ligament, which was predominant in Type I. The cells from the synovium showed similar changes in shape and alignment under the cyclic tensile load, but no significant change was observed in cell metabolism. These observations suggest that the application of cyclic tensile load on the anterior cruciate ligament cells is an important factor in the regulation of collagen synthesis in the anterior cruciate ligament.

Biochemical markers in the synovial fluid of glenohumeral joints from patients with rotator cuff tear

It is known that rotator cuff tears are sometimes accompanied by joint destruction. Our purpose was to elucidate the pathology with this condition. Thirty‐two synovial fluid (SF) samples aspirated from the glenohumeral joints of patients with rotator cuff tears, including 7 with partial‐thickness and 25 with full‐thickness tears of the rotator cuff (10 massive and 15 isolated supraspinatus tendon (SSp) tears), were examined. Collagenase (MMP‐1), stromelysin 1 (MMP‐3), tissue inhibitor of metalloproteinases‐1 (TIMP‐1) and carboxy‐terminal type II procollagen peptide (pCOL II‐C) were measured in the SF using the respective sandwich enzyme immunoassays. Glycosaminoglycan (GAG) was also quantified with a cationic dye binding method using 1,9‐dimethylmethylene blue. Levels of any molecules except pCOL II‐C in the SF appeared to be higher in full‐thickness tears than those in partial‐thickness tears. Moreover, levels of MMP‐1, MMP‐3 and GAG in the SF were significantly higher in massive tears of the rotator cuff in comparison with those in isolated SSp tears. Such significance was not observed in the levels of TIMP‐1 or pCOL II C in the SF. We examined the relation of those levels with operative findings or clinical parameters from full‐thickness tears, and observed significant correlations of the tear size with the levels of MMP‐1, MMP‐3 and GAG in the SF. Although these marker molecules in SF do not always originate from cartilage, our results may indicate the potential for accelerated cartilage‐degrading activity in the glenohumeral joint in massive tears of the rotator cuff.