Akihiko Yonekura

Primary human bone marrow adipocytes support TNF-α-induced osteoclast differentiation and function through RANKL expression

PURPOSE In previous reports, it was demonstrated that bone marrow adipocytes were related to steroid osteoporosis through osteoclastogenesis induced by Receptor Activator of Nuclear factor κ-B Ligand (RANKL) expression. The purpose of this study was to evaluate the effect of Tumor necrosis factor-alpha (TNF-α) on RANKL expression in bone marrow adipocytes, and osteoclast differentiation supported by human bone marrow adipocytes. METHODS RANKL, osteoprotegerin (OPG), and macrophage-colony stimulating factor (M-CSF) mRNA expression in bone marrow adipocytes and their regulation by TNF-α treatment were measured by real-time RT-PCR. Co-cultures of bone marrow adipocytes and osteoclast precursors were performed with or without TNF-α, and osteoclast differentiation was evaluated morphologically and functionally. RESULTS RANKL expression and an increase in the RANKL/OPG ratio in bone marrow adipocytes were stimulated by TNF-α treatment. In co-culture of bone marrow adipocytes and osteoclast precursors with TNF-α, the number of TRAP-positive multinuclear cells and resorption cavity formations of calcium phosphate film were increased. Osteoclast differentiation was suppressed by anti-RANKL antibody treatment. In co-culture with non-cell-contact conditions, no TRAP-positive cells or resorption cavity formations were observed. CONCLUSIONS TNF-α increased RANKL expression in primary human bone marrow adipocytes. TNF-α induced the ability of bone marrow adipocytes to promote osteoclast differentiation and activity in a manner directly related to RANKL expression.

Measurement of advanced glycation endproducts in skin of patients with rheumatoid arthritis, osteoarthritis, and dialysis-related spondyloarthropathy using non-invasive methods.

Advanced glycation endproducts (AGEs) are the products of non-enzymatic glycation and oxidation of proteins and lipids. Low-turnover tissues such as articular cartilage seem to be susceptible to the accumulation of AGEs, which might lead to cartilage degradation. Recently, a non-invasive method for measuring skin AGE accumulation was developed by using the Autofluorescence Reader (AFR). To examine the usefulness of measuring skin AGE in patients with bone and joint diseases, we examined autofluorescence (AF) levels in skin of patients with osteoarthritis (OA), rheumatoid arthritis (RA), and dialysis-related spondyloarthropathy (DRSA). Ninety-three patients with RA, 24 patients with OA, and 29 patients with DRSA were examined, and 43 healthy volunteers were used as controls. Skin AF was assessed on the lower arm with the AGE-Reader. Mean AF was significantly higher in the patients with RA (median 2.13 and range 1.25-2.94) or with DRSA (median 2.21 and range 1.29–3.88) than in the patients with OA (median 1.63 and range 1.07–2.31) or in the controls (median 1.74 and range 1.10–2.46). There was no significant difference between OA and the controls, or between RA and DRSA. These findings suggest that differences of AGE accumulation in the skin might reflect the different pathologies of these diseases.

Early Staphylococcal Biofilm Formation on Solid Orthopaedic Implant Materials: In Vitro Study

Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4V), commercially pure titanium (cp-Ti) and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984) was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR) was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2–4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P<0.05). The absorbance value determined in the crystal violet assay and the number of viable cells on Co-Cr-Mo were not significantly lower than for the other materials (P>0.05). These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness.

Biofilm-Forming Staphylococcus epidermidis Expressing Vancomycin Resistance Early after Adhesion to a Metal Surface

We investigated biofilm formation and time of vancomycin (VCM) resistance expression after adhesion to a metal surface in Staphylococcus epidermidis. Biofilm-forming Staphylococcus epidermidis with a VCM MIC of 1 μg/mL was used. The bacteria were made to adhere to a stainless steel washer and treated with VCM at different times and concentrations. VCM was administered 0, 2, 4, and 8 hours after adhesion. The amount of biofilm formed was evaluated based on the biofilm coverage rates (BCRs) before and after VCM administration, bacterial viability in biofilm was visually observed using the fluorescence staining method, and the viable bacterial count in biofilm was measured. The VCM concentration required to decrease BCR significantly compared with that of VCM-untreated bacteria was 4 μg/mL, even in the 0 hr group. In the 4 and 8 hr groups, VCM could not inhibit biofilm growth even at 1,024 μg/mL. In the 8 hr group, viable bacteria remained in biofilm at a count of 104 CFU even at a high VCM concentration (1,024 μg/mL). It was suggested that biofilm-forming Staphylococcus epidermidis expresses resistance to VCM early after adhesion to a metal surface. Resistance increased over time after adhesion as the biofilm formed, and strong resistance was expressed 4–8 hours after adhesion.

Steroid changes adipokine concentration in the blood and bone marrow fluid

Our previous study has shown that plasminogen activator inhibitor 1 (PAI-1) gene expression and secretion from bone marrow adipocytes increased markedly with dexamethasone administration. The purpose of the present study was to measure the secretion of various adipokines from human bone marrow and blood, and investigate how adipokine secretion changes in a steroid environment. Human blood and bone marrow fluid were collected from a steroid treatment group and a control group during hip replacement surgery, and an enzyme-linked immunosorbent assay (ELISA) was used to measure the adiponectin, leptin, and PAI-1 levels. Adiponectin and leptin showed no significant differences between bone marrow and blood levels, but PAI-1 was significantly higher in bone marrow. The steroid treatment group had higher levels of leptin and PAI-1 in both the blood and bone marrow than the control group. PAI-1 was present at high concentrations in the bone marrow and increased by steroid treatment. High levels of PAI-1 in bone marrow may influence intraosseous hemodynamics and may induce necrotic bone disorders.

Double-bundle anterior cruciate ligament reconstruction improves tibial rotational instability: analysis of squatting motion using a 2D/3D registration technique

BackgroundThe anterior cruciate ligament-deficient (ACLD) knee requires appropriate treatment for the patient to return to sports. The purpose of this study was to clarify the kinematics of the anterior cruciate ligament-deficient knee in squatting motion before and after double-bundle anterior cruciate ligament reconstruction (DB-ACLR) using a 2D/3D registration technique.MethodsThe subjects of this study were 10 men with confirmed unilateral ACL rupture who underwent DB-ACLR. Computed tomography (CT) of the knee joints was performed before DB-ACLR. Fluoroscopic imaging of the knee motion in squatting before and after DB-ACLR was also performed. The 2D/3D registration technique is a method of calculating positional relationships by projecting the 3D bone model created from the CT data onto the image extracted from the fluoroscopic images. The tibial anteroposterior (AP) and rotational positions were analyzed with reference to the femur.ResultsThe tibial AP position of the ACLD knees was significantly anterior to the contralateral knees (p = 0.015). The tibial rotational position of the ACLD knees was significantly internally rotated compared to the contralateral knees (p < 0.001). Both tibial AP and rotational positions improved after DB-ACLR (p < 0.001), with no significant differences compared to the contralateral knees.ConclusionDB-ACLR improved not only tibial AP instability but also tibial rotational instability at knee flexion with weight-bearing. DB-ACLR appears to be a useful technique for normalizing the knee joint kinematics of ACLD knees.

Investigating interindividual variations in cortical bone quality: analysis of the morphotypes of secondary osteons and their population densities in the human femoral diaphysis

Osteons are the primary sites of cortical bone lesions. However, many aspects of osteon microstructure remain poorly understood. This study aimed to explores interindividual differences in the osteon morphotype distributions in the human femoral diaphysis by evaluating the secondary osteon distributions in samples from human femurs. Two anonymized bone fragments from two modern Japanese femurs were examined. Twelve continuous transverse femoral diaphysis specimens were prepared from each fragment. Imaging examinations were conducted using a circularly polarized light microscope, and cross-sectional images were rendered using graphical synthesis software. Osteons in the images were identified as either bright-type osteons, dark-type osteons, or an others type. The two femurs were compared, and the secondary osteon morphotype distributions in different regions of their cross-sections were analyzed. When the two femurs were compared, significant differences in osteon density were observed in some regions and cross-sections. The dark-type osteon presence was strongest in the anterior and posterior regions of the femurs. The analytical method used in this study was found to be able to evaluate osteon microstructure. The results suggest that examining additional specimens and analyzing the biomechanical underpinnings of interindividual differences in osteon distribution patterns may help to improve our understanding of osteon microstructure.

Effect of carbon ion implantation on the tribology of metal-on-metal bearings for artificial joints

Metal-on-metal (MoM) bearings have become popular due to a major advantage over metal-on-polymer bearings for total hip arthroplasty in that the larger femoral head and hydrodynamic lubrication of the former reduce the rate of wear. However, concerns remain regarding adverse reactions to metal debris including metallosis caused by metal wear generated at the taper-head interface and another modular junction. Our group has hypothesized that carbon ion implantation (CII) may improve metal wear properties. The purpose of this study was to investigate the wear properties and friction coefficients of CII surfaces with an aim to ultimately apply these surfaces to MoM bearings in artificial joints. CII was applied to cobalt-chromium-molybdenum (Co-Cr-Mo) alloy substrates by plasma source ion implantation. The substrates were characterized using scanning electron microscopy and a 3D measuring laser microscope. Sliding contact tests were performed with a simple geometry pin-on-plate wear tester at a load of 2.5 N, a calculated contact pressure of 38.5 MPa (max: 57.8 MPa), a reciprocating velocity of 30 mm/s, a stroke length of 60 mm, and a reciprocating cycle count of 172,800 cycles. The surfaces of the CII substrates were generally featureless with a smooth surface topography at the same level as untreated Co-Cr-Mo alloy. Compared to the untreated Co-Cr-Mo alloy, the CII-treated bearings had lower friction coefficients, higher resistance to catastrophic damage, and prevented the adhesion of wear debris. The results of this study suggest that the CII surface stabilizes the wear status due to the low friction coefficient and low infiltration of partner materials, and these properties also prevent the adhesion of wear debris and inhibit excessive wear. Carbon is considered to be biologically inert; therefore, CII is anticipated to be applicable to the bearing surfaces of MoM prostheses.

069 BACKGROUND OF GENERALIZED OSTEOARTHRITIS; EPIDEMIOLOGICAL ANALYSIS OF THE DISTRIBUTION OF PERI-ARTICULAR OSTEOPHYTES IN HUMAN SKELETONS

factors. The heritable modes concern the more overall variations in shape, like slenderness of the hips, suggesting that subtle shape variations are due to individual environmental factors. Two of the tested OA susceptibility genes associated with variations in hip shape. FRZB, part of the ubiquitous and for cartilage and bone highly relevant Wnt-signaling pathway, associated with an overall change in shape, ranging from broad to slender hips. The influence of the DIO2 variant on mode 5 appears to link OA susceptibility to shallow placement of the head inside the acetabulum, which resembles dysplasia and has previously been recognized as a predisposing factor of OA. Although independent of OA status, this data shows that SNPs of the tested genes are indeed associated with variations in shape of the hip.

Treatment of Bilateral Fractures of Lower Extremities

We reviewed the treatment of ten patients with bilateral long bone fractures of lower extremities. Six patients had bilateral leg fractures. 2 patients had a leg fracture and a contralateral femoral fracture and 2 patients had bilateral femoral fractures. Seventeen fractures were treated operatively and three conservatively. The average period from surgery to P. W. B. (Partial Weight Bearing) was 85.6 days in comminuted fractures and 38.6 days in non-comminuted fractures. Intra-articular fractures of the ankle which included three comminuted fractures needed 84.0 days before P. W. B and bilateral femoral fractures needed 78.0 days on average. Fractures of the upper extremity and foot affected the starting time of P. W. B.. The average period from P. W. B. to Full Weight Bearing was 45.6 days in comminuted fractures and 17.6 days in non-comminuted fractures. From this study we think that other associated fractures, comminuted fractures and bilateral femoral fractures cause delay in post-operative treatment. For these fractures, nonweight-bearing braces are effective and it is better to use intra-medullary nailing in order to allow P. W. B. earlier.