Tomoatsu Kimura

Maturational disturbance of chondrocytes in Cbfa1-deficient mice.

Cbfa1, a transcription factor that belongs to the runt‐domain gene family, plays an essential role in osteogenesis. Cbfa1‐deficient mice completely lacked both intramembranous and endochondral ossification, owing to the maturational arrest of osteoblasts, indicating that Cbfa1 has a fundamental role in osteoblast differentiation. However, Cbfa1 was also expressed in chondrocytes, and its expression was increased according to the maturation of chondrocytes. Terminal hypertrophic chondrocytes expressed Cbfa1 extensively. The significant expression of Cbfa1 in hypertrophic chondrocytes was first detected at embryonic day 13.5 (E13.5), and its expression in hypertrophic chondrocytes was most prominent at E14.5–16.5. In Cbfa1‐deficient mice, whose entire skeleton was composed of cartilage, the chondrocyte differentiation was disturbed. Calcification of cartilage occurred in the restricted parts of skeletons, including tibia, fibula, radius, and ulna. Type X collagen, BMP6, and Indian hedgehog were expressed in their hypertrophic chondrocytes. However, osteopontin, bone sialoprotein, and collagenase 3 were not expressed at all, indicating that they are directly regulated by Cbfa1 in the terminal hypertrophic chondrocytes. Chondrocyte differentiation was severely disturbed in the rest of the skeleton. The expression of PTH/PTHrP receptor, Indian hedgehog, type X collagen, and BMP6 was not detected in humerus and femur, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes. These findings demonstrate that Cbfa1 is an important factor for chondrocyte differentiation. Dev Dyn 1999;214:279–290.

Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel

In an attempt to repair articular cartilage, allograft articular chondrocytes embedded in collagen gel, were transplanted into full-thickness defects in rabbit articular cartilage. Twenty-four weeks after the transplantation, the defects were filled with hyaline cartilage, specifically synthesising Type II collagen. These chondrocytes were autoradiographically proven to have originated from the transplanted grafts. Assessed histologically the success rate was about 80%, a marked improvement over the results reported in previous studies on chondrocyte transplantation without collagen gel. By contrast, the defects without chondrocyte transplantation healed with fibrocartilage. Immunological enhancement induced by transplanted allogenic chondrocytes or collagen was not significant at eight weeks after treatment, so far as shown by both direct and indirect blastformation reactions. Thus, allogenic transplantation of isolated chondrocytes embedded in collagen gel appears to be one of the most promising methods for the restoration of articular cartilage.

The Association of Lumbar Disc Disease with Vitamin-D Receptor Gene Polymorphism

Background: Although the etiology of lumbar disc disease is unknown, it has been suggested that a genetic factor contributes to its development. Recently, some genetic polymorphisms have been found to be related to clinical disorders. We investigated the association between vitamin-D receptor gene and estrogen receptor gene polymorphisms and lumbar disc disease in young adults.Methods: The participants included 205 young adults (166 women and thirty-nine men) with or without low-back problems. A magnetic resonance imaging scan of the lumbar spine was performed for all subjects, and the grade of disc degeneration was determined, according to the four-grade classification system of Schneiderman et al. The presence or absence of disc herniation was also evaluated. Genomic DNA was extracted from peripheral blood samples. The polymorphisms of the vitamin-D receptor and estrogen receptor genes were detected with use of a polymerase-chain-reaction assay. The restriction fragment length polymorphisms (RFLPs) for the vitamin-D receptor gene were analyzed by TaqI and ApaI restriction enzymes. XbaI and PvuII restriction enzymes were used for the estrogen receptor gene analysis. The distribution of polymorphism in subjects with disc degeneration and/or disc herniation was compared with that in the normal subjects.Results: The allelic frequencies of both vitamin-D receptor gene and estrogen receptor gene polymorphisms were similar to those in previous analyses of Japanese subjects. The allelic variation in the vitamin-D receptor gene was associated with multilevel and severe disc degeneration and disc herniation. The Tt allele was found to be more frequently associated with multilevel disc disease, severe disc degeneration, and disc herniation than was the TT allele. No additional associations were found.Conclusions: This study revealed that the Tt allele of the vitamin-D receptor gene was more frequently associated with multilevel and severe disc degeneration and disc herniation than was the TT allele, pointing to an increased risk of disc disease at an early age in subjects with the Tt allele in the vitamin-D receptor gene.

Regulation of proliferation and osteochondrogenic differentiation of periosteum-derived cells by transforming growth factor-beta and basic fibroblast growth factor.

We studied the effects of transforming growth factor-beta and basic fibroblast growth factor on the regulation of proliferation and osteochondrogenic differentiation of periosteum-derived cells, which have the potential to differentiate into bone and hypertrophic cartilage in vitro. Histological observation revealed that transforming growth factor-beta stimulated chondrogenesis of periosteum-derived cells while basic fibroblast growth factor stimulated proliferation of fibroblast-like cells and inhibited osteochondrogenic differentiation. Immunohistochemical studies revealed that basic fibroblast growth factor inhibited the expression of osteocalcin. Transforming growth factor-beta enhanced uronic acid content but decreased DNA content, alkaline phosphatase activity, and calcium content. In contrast, basic fibroblast growth factor enhanced DNA content but decreased alkaline phosphatase activity, calcium content, and uronic acid content. In addition, transforming growth factor-beta shortened the time-course of gene expression of type-X collagen whereas basic fibroblast growth factor inhibited the gene expression. These results indicate that transforming growth factor-beta stimulates osteochondrogenic differentiation of periosteum-derived cells but inhibits proliferation. They also indicate that basic fibroblast growth factor stimulates proliferation of periosteum-derived cells but inhibits osteochondrogenic differentiation.

Reduced expression of the regulatory CD4+ T cell subset is related to Th1/Th2 balance and disease severity in rheumatoid arthritis

OBJECTIVE To elucidate the involvement of the regulatory CD4+ T cells that produce high levels of interleukin-10 (IL-10) and low levels of IL-4 and IL-2 in the pathogenesis of rheumatoid arthritis (RA), we investigated whether the frequency of this type of CD4+ T cell subset in peripheral blood lymphocytes (PBL) or synovial lymphocyte infiltrates of patients with RA correlated with disease severity and histologic features in rheumatoid synovium. METHODS PBL and synovial lymphocyte infiltrates were isolated from peripheral blood samples and synovial tissues obtained from 25 patients with RA. Control specimens were obtained from 18 patients with osteoarthritis (OA) and 10 patients with traumatic injuries of the knee joint. CD4+ T cell subsets were categorized as Th1 (production of interferon-gamma [IFNgamma], but not IL-4), Th2 (production of IL-4, but not IFNgamma), or CD4+ T cell subsets producing IL-10, IL-2, or IL-4. The percentages of these T helper subsets among PBL and among synovial infiltrating lymphocytes were determined by an intracellular staining assay with flow cytometric analysis. RESULTS The level of expression of CD4+ T cells producing IL-10 but not IL-2 and IL-4 in the peripheral blood and synovial tissue was significantly lower in RA patients than in OA patients and trauma patients. In RA patients, the frequency of this type of CD4+ T cell subset among synovial infiltrating CD4+ T cells was inversely correlated with the frequency of Th1 cells and the Th1/Th2 balance in synovial lymphocytes, serum C-reactive protein value, disease activity score, and the degree of synovial lining hyperplasia and lymphocyte infiltration in rheumatoid synovium. There was a reciprocal relationship between the frequency of Thl cells and CD4+ T cells producing IL-10 but not IL-2 and IL-4 in the peripheral blood of RA patients. CONCLUSION In RA, reduced expression of the CD4+ T cell subset producing IL-10 but not IL-2 and IL-4 may be responsible for the dominance of Th1 over Th2 cells at sites of inflamed synovium and in the peripheral blood. Decreases in this type of CD4+ T cell subset may induce the down-regulation of T cell tolerance and exacerbate the inflammatory process in RA.

Progressive degeneration of articular cartilage and intervertebral discs : an experimental study in transgenic mice bearing a type IX collagen mutation

Summary. Transgenic mice expressing mutant α1(IX) collagen were produced and found to develop progressive joint degeneration with age, as well as accelerated intervertebral disc degeneration. Radiological and histological studies showed that cervical and lumbar disc degeneration was more advanced in the transgenic mice than in control litter-mates. The changes included shrinkage or disappearance of the nucleus pulposus, and fissures in the annulus fibrosus which sometimes lead to herniation of disc material and slight osteophyte formation. These findings suggest that mutations of the type IX collagen may cause certain forms of degenerative disease in the spine as well as in joints.Résumé. Nous avons produit des souris transgéniques experimant un collagène mutant α1(IX) et constaté que ces sujets développaient avec l’âge une dégénérescence progressive des articulations alliée à une dégénérescence accélérée des disques intervertébraux. Les examens radiologiques et histologiques ont montré que ce processus e’tait plus avancé sur les souris transgéniques que sur les sujets de référence. La dégénérescence prend la forme du rétrécissement jusqu’à disparition du tissu pulpeux du noyau cellulaire et d’une fissuration du tissue fibreux annulaire avec, dans certains cas, herniation de la matière discale et légère formation d’ostéophytes. Ces constatations nous suggèrent que les mutations de type collagène IX pourraient entraîner certaines affections dégénératives du rachis et des articulations.

Reconstruction of the anterior cruciate ligament by allogeneic tendon graft. An operation for chronic ligamentous insufficiency

We describe the use of allogeneic human tendon as an intra-articular replacement for the anterior cruciate ligament. Depending on the type and degree of functional instability we recommend the addition, in some cases, of supplementary extra-articular procedures. We have reviewed 31 patients at least two years after operation and have found that 30 of them had been able to return to full sporting activities. The indications for operation and the techniques are discussed and the use of allogeneic tendon is recommended.

Magnetic resonance imaging and histologic evidence of postoperative back muscle injury in rats.

Study Design. Postoperative back muscle injury was evaluated in rats by magnetic resonance imaging and histologic analyses. Objective. To compare the magnetic resonance imaging manifestation of back muscle injury with the histologic findings in rats and to subsequently clarify the histopathologic appearance of the high intensity regions on T2-weighted images in human postoperative back muscles. Summary of Background Data. In a previous study, it was found that the signal intensity on T2-weighted images of the postoperative back muscles was increased in patients who had postsurgical lumbar muscle impairment, especially in those with a prolonged surgery duration. However, the specific histopathologic changes that cause the high signal intensity on T2-weighted images remain unclear. Methods. Rats were divided into three groups: sham operation group, 1-hour retraction group, and 2-hour retraction group. Magnetic resonance imaging and histology of the multifidus muscles were examined before surgery and at 2, 7, and 21 days after surgery. Results. T2-weighted imaging was more useful than T1-weighted imaging to estimate back muscle injury. The high signal intensity of the multifidus muscles on T2-weighted images remained 21 days after surgery only in the 2-hour retraction group. Histologically, the regeneration of the multifidus muscles was complete at 21 days after surgery in the 1-hour retraction group, but the regenerated muscle fibers in the 2-hour retraction group had a small diameter, and the extracellular fluid space remained large. Conclusion. The high signal intensity on T2-weighted images of the postoperative multifidus muscles in the regenerative phase may be due to an increased extracellular space and incomplete muscle fiber regeneration.

Genotype phenotype correlation in achondroplasia and hypochondroplasia

Recent studies of the fibroblast growth factor receptor 3 (FGFR3) gene have established that achondroplasia and hypochondroplasia are allelic disorders of different mutations. To determine whether the genotype could be distinguished on the basis of the phenotype, we analysed height, arm span, and skeletal radiographs from 23 patients with achondroplasia and the G380R mutation of FGFR3 and eight with hypochondroplasia and the N540K mutation. Both conditions share the classical pathological features of micromelic short stature, reduced or unchanged interpedicular distances in the lumbar spine, disproportionately long fibulae, and squared and shortened pelvic ilia. These were significantly more severe in the G380R patients than in the N540K patients. Our findings have shown a firm statistical correlation between the genotype and the phenotype, although there were a few exceptional cases in which there was phenotypic overlap between the two conditions.

Biochemical effect of intra-articular injections of high molecular weight hyaluronate in rheumatoid arthritis patients.

Abstract.Objective: The purpose of this study was to analyze the biochemical characteristics of synovial fluids after treatment of patients with rheumatoid arthritis (RA) with intra-articularly injected hyaluronate (HA).¶Treatment: In a double-blind study, 13 patients received intra-articular injections of 1% HA in phosphate buffered saline (PBS) while 13 other patients were injected 0.01% HA in PBS, as the control group. Injections were administered once a week for five weeks.¶Methods: Clinical efficacy and characteristics of synovial fluid were compared between groups.¶Results: Significant clinical improvement was observed in the HA-treated group compared to the control group. The viscosity of retained synovial fluid increased. Stringing and HA concentration increased significantly whereas the concentrations of protein and chondroitin sulfate 4 and 6 decreased significantly. The HA molecular weight remained the same in both groups.¶Conclusion: Intra-articular HA injection altered the properties of synovial fluid and proved efficacious for patients with RA.