Tatsuya Koike

Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Wnt/ β -catenin signaling stimulates matrix catabolic genes and activity in articular chondrocytes: its possible role in joint degeneration

A fine balance between anabolic and catabolic mechanisms maintains extracellular matrix homeostasis in articular cartilage, and shifts toward degradation are associated with joint conditions such as osteoarthritis. To test the possible involvement, relevance and significance of the Wnt/β-catenin-signaling pathway in those catabolic shifts, rabbit articular chondrocyte cultures were subjected to experimental activation of β-catenin signaling by Wnt3A treatment or forced expression of constitutive-active β-catenin (CA-β-catenin). Both interventions provoked strong gelatinase activity and stimulated gene expression of matrix metalloprotease-3 and -13 and a disintegrin-like and metalloprotease with thrombospondin motif (ADAMTS)-4 and -5 proteases. Furthermore, Wnt3A treatment additively enhanced the effects of intereukin-1β, a well-known catabolic culprit of proteoglycan matrix loss. To determine whether Wnt/β-catenin signaling is associated with age-associated osteoarthritic changes in articular cartilage in vivo, we analyzed the presence and intracellular distribution of β-catenin in a spontaneous guinea pig osteoarthritis model. Healthy articular chondrocytes in young guinea pig knees contained barely detectable levels of β-catenin. In contrast, the protein was highly abundant in osteoarthritic-like chondrocytes present in older guinea pig joints, and was localized not only in the cytoplasm but also the nucleus, a clear reflection of activated Wnt signaling. These and other data suggest that Wnt/β-catenin signaling is a powerful stimulator of chondrocyte matrix catabolic action and may be part of mechanisms leading to excessive remodeling and degradation of cartilage matrix in age-associated joint pathologies.

Potent mitogenic effects of parathyroid hormone (PTH) on embryonic chick and rabbit chondrocytes. Differential effects of age on growth, proteoglycan, and cyclic AMP responses of chondrocytes to PTH.

The effect of PTH on chondrocyte proliferation as a function of cartilage age was examined. PTH[1-34] induced a 12- to 15-fold increase in the efficiency of colony formation in soft agar by chondrocytes from embryonic 13- to 19-d-old chickens and fetal 25-d-old rabbits with a 10-fold increase in their DNA content. It also caused a 2.5-fold increase in [3H]thymidine incorporation into DNA in fetal 25-d-old rabbit chondrocytes. No mitogenic responses to PTH were observed, however, in postnatal 7- to 21-d-old chick chondrocytes or postnatal 21-d-old rabbit chondrocytes. This age dependency was observed only with PTH: fibroblast growth factor, epidermal growth factor, and insulin stimulated chondrocyte proliferation irrespective of cartilage age. The absence of a mitogenic effect in postnatal chondrocytes was not due to a decrease in number or a reduction in affinity of receptors for PTH. PTH also increased [35S]sulfate incorporation into proteoglycans and the cyclic AMP level in fetal and postnatal chondrocytes, but at 100-fold higher concentrations (10(-8)-10(-7) M) than those (10(-10)-10(-9) M) required for the stimulation of cell division. These results suggest that PTH is a potent mitogen for embryonic chondrocytes, and that its mitogenic effect disappears selectively after birth.

Local Application of Basic Fibroblast Growth Factor Minipellet Induces the Healing of Segmental Bony Defects in Rabbits

Abstract. Fibroblast growth factor (FGF) has been reported to increase the volume of callus in a fracture model of rats. There are, however, no reports of successful repair of segmental bony defects by application of an FGF solution. In this study, the effects of basic FGF on the repair of segmental bony defects in the rabbit femur were examined. Minipellet, a new drug delivery system using atelocollagen, was employed to ensure effective delivery of FGF. Segmental bony defects (10 mm in length) were created in the right femurs of 19 rabbits. In pilot studies, no defects of this size healed spontaneously within 6 weeks. Bones were stabilized with miniexternal fixators. Minipellets containing basic FGF were implanted between fragments so as to bridge the two fragments. The healing processes were monitored radiographically and studied histologically. In rabbits in which FGF was added to the defect site at doses of 1.4 μg or higher, approximately 90% of the defects were filled with new bone and cartilage within 6 weeks after minipellet implantation. In rabbits receiving placebo minipellets, however, approximately 15% of the defects were filled by callus within 6 weeks. Furthermore, this callus did not change into mature bone. An injection of 2 μg of FGF solution to bony defects had no effect on the repair of segmental bony defects. These findings suggest that FGF plays a role in the production of adequate volumes of callus particularly in the initial stages of fracture healing and that sustained local release enables FGF to be effective at a low dose. In summary, large segmental bony defects healed after insertion of low-dose FGF minipellets. An adequate dose of FGF and an appropriate delivery system are required for successful healing of large bony defects. These findings imply the potential value of FGF minipellets in clinical practice.

Effects of Leptin to Cultured Growth Plate Chondrocytes

Objective: This study aimed to evaluate whether leptin has any effect on growth plate chondrocytes. Methods: We studied the effects of exogenous leptin on cultured rabbit growth plate chondrocytes. This involved assessing [3H]thymidine incorporation, alkaline phosphatase (ALP) activity, proteoglycan production, leptin receptor (Ob-R) activity, and detection of Ob-R using Western blot analysis. Results: The existence of Ob-R in growth plate chondrocytes was revealed by Western blot and Ob-R activity. Prior to semiconfluence, leptin increased [3H]thymidine incorporation while at the semiconfluent and early confluent stages, leptin promoted ALP activity and tended to promote proteoglycan production. Conclusion: Growth plate chondrocytes possess Ob-Rs, and leptin enhance chondrocyte proliferation and subsequent cell differentiation.

Risk factors for severe nonsteroidal anti-inflammatory drug-induced small intestinal damage

BACKGROUND Few studies have assessed the risk factors associated with nonsteroidal anti-inflammatory drugs (NSAIDs)-induced small intestinal damage. AIMS To evaluate the risk factors for NSAID-induced enteropathy in patients with rheumatoid arthritis. METHODS A cross-sectional study using capsule endoscopy was conducted. A total of 113 patients who took NSAIDs for over 3 months underwent capsule endoscopies. Endoscopic findings were scored as (0) normal, (1) red spots, (2) 1-4 erosions, (3) >4 erosions, or (4) large erosions/ulcers. Initial scores were grouped into 3 categories: No damage (0-1), mild damage (2), and severe damage (3-4), and the potential risk factors for damage development were assessed. RESULTS Five patients were excluded because of incomplete visualization of the entire small intestine. Fifty-two (47.2%) and 27 (25%) patients had no damage and mild damage, respectively, while the remaining 30 patients (27.8%) had severe damage and significantly decreased hemoglobin levels. In a multivariate logistic regression analysis, ages of 65 years or more (odds ratio [OR], 4.16; 95% confidence interval [CI], 1.51-11.47), proton pump inhibitor usage (OR, 5.22; 95% CI, 1.36-20.11), and histamine H2 receptor antagonist usage (OR, 3.95; 95% CI, 1.28-12.25) were independent risk factors for severe damage. CONCLUSIONS Elderly patients and acid suppressant users are more likely to develop severe NSAID-induced enteropathy.

Evaluation of rotator cuff tears with magnetic resonance arthrography.

The size and morphologic features of rotator cuff tears may influence treatment selection and affect final outcomes. Magnetic resonance arthrography allows observation of these features and other intraarticular structures. To assess the utility of magnetic resonance imaging in assessing size and morphologic features, we retrospectively reviewed observations on 41 shoulders in 37 consecutive surgically treated patients (mean age, 63.2 years) who had magnetic resonance imaging followed by magnetic resonance arthrography. The maximum rotator cuff defect size in the anteroposterior direction defined transverse size, and the maximum rotator cuff defect size in the mediolateral direction defined longitudinal size. Sensitivities for detecting full-thickness rotator cuff tears by magnetic resonance imaging and magnetic resonance arthrography were 90.2% and 100%, respectively. Maximum longitudinal and transverse dimensions of the tear as shown by magnetic resonance arthrography correlated better with intraoperative measurements (r2 = 0.85 transversely, 0.92 longitudinally) than magnetic resonance imaging measurements (r2 = 0.47 transversely, 0.26 longitudinally). The reproducibility of the two methods is similar. Magnetic resonance arthrography also allowed morphologic classification of the torn tendon as blunt end, tapering end, indistinct end, horizontal tear, and global tear. There was good agreement in classifying torn edges; the imaging findings agreed with findings at surgery. Magnetic resonance arthrography was more accurate in evaluating rotator cuff tear size and morphologic features than conventional magnetic resonance imaging. Level of Evidence: Diagnostic study, Level II (development of diagnostic criteria on basis of consecutive patients-with universally applied reference “gold” standard). See the Guidelines for Authors for a complete description of levels of evidence.

Evaluation of Small Bowel Injury in Patients with Rheumatoid Arthritis by Capsule Endoscopy: Effects of Anti-Rheumatoid Arthritis Drugs

Background and Aim: The medical treatment of rheumatoid arthritis (RA) includes nonsteroidal anti-inflammatory drugs (NSAIDs), low-dose corticosteroids, and disease-modifying antirheumatic drugs (DMARDs). We evaluated the incidence of small bowel injury in RA patients who were taking anti-RA drugs with or without concomitant NSAIDs by capsule endoscopy. Methods: A total of 28 RA patients who took low-dose corticosteroids and/or DMARDs for more than 1 year were enrolled. Results: The incidence of red spots did not differ between the 2 groups: 14 of 16 patients (87.5%) in the NSAID group and 11 of 12 patients (91.7%) in the non-NSAID group. In contrast, the incidence of mucosal breaks was significantly higher in the NSAID group than in the non-NSAID group: mucosal breaks were detected in 13 of 16 patients (81.3%) and 4 of 12 patients (33.3%) in the NSAID and non-NSAID groups, respectively. In the NSAID group, mucosal breaks developed in users of preferential cyclooxygenase-2 inhibitors at a frequency similar to that in users of traditional NSAIDs. Conclusion: Patients taking anti-RA drugs may have an increased frequency of small bowel injury regardless of NSAID use, and NSAID use may be associated with an increased incidence of severe small bowel injury.

External hip protectors are effective for the elderly with higher-than-average risk factors for hip fractures

SummaryIn our cluster randomised controlled trial for efficacy of hip protector with 672 ambulatory elderly women, a hip protector was more effective for prevention of hip fractures in residents with fall history (n = 202; hazard ratio (HR), 0.375; 95%CI, 0.14–0.98; p = 0.05) and body-mass index (BMI) ≤ 19.0 (n = 206; HR, 0.37; 95%CI, 0.14–0.95; p = 0.04) by a Cox proportional hazards regression model.IntroductionHip fractures result from both osteoporosis and falling. A potentially cost-effective method of preventing hip fractures involves the use of hip protectors but recent studies have revealed the uncertain effectiveness of hip protectors even in institutional settings.MethodsThis study was a cluster randomised controlled trial with nursing homes. We randomly assigned 76 homes with 672 ambulatory but frail elderly women. Several risk factors were assessed at baseline and incorporated into a Cox proportional hazards regression model. UMIN Clinical Trials Registry number is UMIN000000467. Research period was between January 2004 and March 2006.ResultsIn the intervention group, 19 hip fractures occurred (54.0/1,000 person-years), whereas 39 hip fractures occurred in the control group (78.8/1,000 person-years). Hazard ratio of hip fracture in the intervention group was 0.56 (95%CI, 0.31–1.03; p = 0.06) after adjusting for risk factors. In subgroup analysis, hip protectors were more effective for prevention of hip fractures in residents with fall history (n = 202; HR, 0.375; 95%CI, 0.14–0.98; p = 0.05) and BMI ≤ 19.0 (n = 206; HR, 0.37; 95%CI, 0.14–0.95; p = 0.04). Overall compliance with use of hip protectors was 79.7%.ConclusionRisk of hip fracture can be reduced by hip protectors among elderly women with fall history and low BMI.

Cyclic AMP enhances Smad-mediated BMP signaling through PKA-CREB pathway.

We present experimental results indicating involvement of cyclic AMP (cAMP)-mediated signaling in bone morphogenetic protein (BMP)-induced osteoblastic gene expression at the transcriptional level by luciferase activity assay in C2C12 cells using the promoter sequence of the Id1 gene, an early-response gene to BMPs, which contains both a BMP-responsive element (BRE) and a cAMP-response element (CRE). In cells transfected with luciferase gene driven by wild-type Id1 promoter, treatment with BMP-4 increased luciferase expression, which was further enhanced by the addition of dibutyryl cAMP (dbcAMP). This dbcAMP-enhanced luciferase expression was significantly suppressed when the CRE site in the Id1 promoter was replaced by mutated CRE or endogenous CRE-binding protein (CREB) was knocked down by transfection of CREB RNAi. Pretreatment of cells with protein kinase A (PKA) inhibitor, H89, also dramatically reduced dbcAMP-enhanced luciferase expression. Immunoprecipitation assay showed phosphorylated-Smad1/5/8, phosphorylated-CREB, and CREB-binding protein (CBP) formed the transcriptional complex. These data indicate that cAMP-PKA/CREB/CRE signaling potentially enhances BMP-induced transcription through the BRE in the promoter of the BMP-responsive gene through a PKA-mediated pathway.