Yasuharu Nakashima

Signaling Pathways for Tumor Necrosis Factor-α and Interleukin-6 Expression in Human Macrophages Exposed to Titanium-Alloy Particulate Debris in Vitro*

BACKGROUND Loosening of the implant after total joint arthroplasty remains a serious problem. The activation of macrophages by wear debris from implants, mediated by the release of cytokines that elicit bone resorption, may lead to loosening. The purpose of the present study was to elucidate the mechanisms of macrophage activation by titanium particles from the components of implants and to identify the signaling pathways involved in particle-mediated release of cytokines. METHODS Macrophages were isolated from mononuclear leukocytes obtained from healthy human donors and were exposed to titanium-alloy particles that had been obtained from periprosthetic membranes collected at revision total joint arthroplasties and then enzymatically prepared. The experimental protocols included examination of the effects of the inhibition of phagocytosis and the binding of antibodies to macrophage complement receptors on particle-induced macrophage activation. The release of the proinflammatory cytokines TNF-alpha (tumor necrosis factor-alpha) and IL-6 (interleukin-6) was used to assess macrophage activation. The signaling pathways involved in the induction of cytokine release were analyzed by identification of phosphorylated proteins with use of the Western blot technique and by translocation of the transcription factors nuclear factor-kappa B (NF-kappaB) and nuclear factor-interleukin-6 (NF-IL-6) into the nuclear protein fraction with use of electrophoretic mobility shift assays. The role of serine/threonine and tyrosine kinase pathways in the activation of nuclear factors and the release of cytokines was examined with use of selective pharmacological agents. RESULTS Exposure of macrophages to titanium-alloy particles in vitro for forty-eight hours resulted in a fortyfold increase in the release of TNF-alpha and a sevenfold increase in the release of IL-6 (p<0.01). Phagocytosis of particles occurred in approximately 73 percent of the macrophages within one hour of exposure. Pretreatment of the macrophages with cytochalasin B reduced phagocytosis by 95 percent but did not reduce the release of TNF-alpha or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF-alpha or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF-alpha and IL-6. Antibodies to CD11b/ CD18 receptors (macrophage Mac-1 receptors) reduced phagocytosis of particles by 50 percent (p<0.05). (The CD11b/CD18 macrophage receptor is the macrophage receptor for the complement component CR3bi. The CD11b/CD18 macrophage receptor can also bind to ICAM-1 and ICAM-2. CD is the abbreviation for cluster of differentiation, and ICAM is the abbreviation for intercellular adhesion molecule.) Inhibition of phagocytosis was not accompanied by a decrease in the release of TNF-alpha and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF-alpha and IL-6 from the macrophages. Macrophage release of TNF-alpha and IL-6 in response to particles coincided with increased tyrosine phosphorylation and mitogen-activated protein kinase activation. Inhibition of tyrosine and serine/threonine kinase activity decreased the particle-induced release of cytokines. Exposure of macrophages to either titanium-alloy particles or to antibodies to the receptor proteins CD11b and CD18 for thirty minutes activated the transcription factors NF-kappaB and NF-IL-6. Inhibition of particle phagocytosis did not block activation of the transcription factors. However, inhibition of tyrosine and serine/threonine kinase activity decreased the activation of NF-kappaB and NF-IL-6. CONCLUSIONS These data suggest that particle induced macrophage release of TNF-alpha and IL-6 does not require phagocytosis but is dependent on tyrosine and serine/threonine kinase activity culminating in activation of

Constitutively Activated ALK2 and Increased SMAD1/5 Cooperatively Induce Bone Morphogenetic Protein Signaling in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.

Effect of surface roughness of hydroxyapatite—coated titanium on the bone-implant interface shear strength

We have investigated the bone-implant interface shear strength of hydroxyapatite (HA)-coated Ti-6Al-4V (HA-coating A) (roughness average, Ra = 3.4 +/- 0.5 microns) and HA-coated Ti-6Al-4V with a rougher surface (HA-coating B) (Ra = 8.4 +/- 1.8 microns). There was no significant difference between HA-coating A and HA-coating B implants with respect to the bone-implant interface shear strength as determined in push-out tests using the transcortical model in adult dogs. The bone-implant interface shear strength of bead-coated porous Ti-6Al-4V was significantly greater than that of both HA-coating A and HA-coating B implants. The failure site, as determined by scanning electron microscopy, was the coating-substrate interface, not the coating-bone interface. This indicates a need to protect the HA coating from the direct shear forces. HA coating enhances early bone growth into the porous surface of the implant. Long-term fixation should depend on bone anchoring to this porous surface. Hydroxyapatite coatings must be developed which do not obstruct the pores of the surface of the implant.

Acetabular retroversion in developmental dysplasia of the hip

BACKGROUND Patients with developmental dysplasia of the hip are prone to the development of degenerative changes in the affected hip. The aim of this study was to evaluate the prevalence, morphological features, and clinical relevance of acetabular retroversion in these patients. METHODS We investigated the version and morphological features of the acetabulum using pelvic radiographs and computed tomography images of ninety-six hips in fifty-nine patients with developmental dysplasia of the hip. A diagnosis of acetabular retroversion was based on the presence of a positive cross-over sign on the pelvic radiograph. Using computed tomography images, we determined the acetabular anteversion angle at various levels in the axial plane. The acetabular sector angle served as an indicator of acetabular coverage of the femoral head. We evaluated the association between acetabular version and the patients age at the onset of pain. Fifty normal hips were examined as controls. RESULTS We observed acetabular retroversion in 18% (seventeen) of the ninety-six hips in the patients with developmental dysplasia of the hip. The mean acetabular anteversion angle in the hips with acetabular retroversion was significantly smaller, at all levels, than that in the hips with acetabular anteversion; this tendency was more evident at proximal levels. There was significantly less posterior and posterosuperior coverage in the hips with acetabular retroversion than in those with acetabular anteversion, but superior acetabular coverage did not differ between the groups. Multivariate analysis showed that the onset of pain occurred at a significantly earlier age in patients with acetabular retroversion (27.9 years) than in those with acetabular anteversion (40.5 years), regardless of the severity of the dysplasia (p = 0.003). CONCLUSIONS In patients with developmental dysplasia of the hip, acetabular retroversion results from relatively deficient coverage by the posterior portion of the acetabulum. Developmental dysplasia with acetabular retroversion is associated with an earlier onset of pain than is developmental dysplasia with anteversion, suggesting a correlation between deficiency of the posterior acetabular wall and the earlier onset of pain.

Effects of the Femoral Offset and the Head Size on the Safe Range of Motion in Total Hip Arthroplasty

The purpose of this study was to quantify the effects of femoral offset and head size on range of motion (ROM) after total hip arthroplasty. Modular prostheses were implanted into 11 cadaveric hips using a posterolateral approach and tested for ROM with 3 different offsets and 5 different femoral head sizes. Increasing the femoral offset to 4 and 8 mm resulted in 21.1 degrees and 26.7 degrees of improved flexion, and 13.7 degrees and 21.2 degrees of improved internal rotation, respectively. The ROM improved in a head size-dependent manner primarily because of increasing the jumping distance of the femoral head rather than delaying any impingement. In contrast, the effectiveness of femoral offset was driven by delayed osseous impingement.

Hydroxyapatite‐coating on titanium arc sprayed titanium implants

We developed a new titanium spray technique using an inert gas shielded arc spray (titanium arc spray). Hydroxyapatite (HA)-coating can be applied to the implant without any surface pore obstruction after the rough surface is made by this technique. Scanning electron microscopy (SEM) of various porous implant surfaces after HA-coating revealed that the bead and fiber metal-coated implants had either a pore obstruction or an uneven HA-coating. On the other hand, the titanium arc sprayed implant demonstrated an even HA-coating all the way to the bottom of the surface pore. In the first set of animal experiments (Exp. 1), the interfacial shear strength to bone of four kinds of cylindrical Ti-6A1-4V (Ti) implants were compared using a canine transcortical push-out model 4 and 12 weeks after implantation. The implant surfaces were roughened by titanium arc spray (group A-C) and sand blasting (group D) to four different degrees (roughness average, Ra = group A: 56.1, B: 44.9, C: 28.3, D: 3.7 microns). The interfacial shear strength increased in a surface roughness-dependent manner at both time periods. However, the roughest implants (group A) showed some failed regions in the sprayed layers after pushout test. In the second set of animal experiments (Exp. 2), four kinds of Ti implants; HA-coated smooth Ti (sHA) with Ra of 3.4 microns, bead-coated Ti (Beads), titanium arc sprayed Ti (Ti-spray) with Ra of 38.1 microns and HA-coated Ti-spray (HA + Ti-spray) with Ra of 28.3 microns were compared using the same model as that in Exp. 1. The interfacial shear strength of HA + Ti-spray was significantly greater than that of sHA and Beads at both time periods, and that of Ti-spray at 4 weeks. Although a histological examination revealed that HA-coating enhanced bone ingrowth, sHA showed the lowest shear strength at both time periods. SEM after pushout test showed that sHA consistently demonstrated some regional failure at the HA-implant substrate interface. HA + Ti-spray had many failed regions either at the HA-bone interface or within the bone tissue rather than at the HA-implant substrate interface. These results suggested that the HA-coated smooth surfaced implants had a mechanical weakness at the HA-substrate interface. Therefore, HA should be coated on the rough surfaced implants to avoid a detachment of the HA-coating layer from the substrate and thus obtain a mechanical anchoring strength to bone. HA-coating on this new type of surface morphology may thus lead to a solution to the problems of conventional HA-coated and porous-coated implants.

Intraarticular findings in symptomatic developmental dysplasia of the hip

Background: The purpose of this study was to examine intraarticular pathology in patients younger than 20 years with symptomatic developmental dysplasia of the hip. Methods: We performed hip arthroscopy during corrective osteotomy in 23 hips in 22 patients. All patients were female, and the average age at operation was 16.4 years. Eighteen hips were in a prearthritic stage, and 5 hips were in an early stage. The presence and location of cartilage degeneration and labral tears were evaluated. Second-look arthroscopy was performed in 13 hips in 12 patients. Results: Fourteen hips (77.8%) in the prearthritic stage had cartilage degeneration. Cartilage lesions were more frequent in the acetabulum than in the femoral head (72.2% vs 16.7%). Sixty-one percent of acetabular lesions were located at the anterosuperior area. Labral tears were observed in 77.8% of hips in prearthritic stages located at the anterosuperior (72.2%) and superior (44.4%) areas. The degree of cartilage and labral lesions in the early stage was more severe than in the prearthritic stage. On second-look arthroscopy, there were no changes in the state of the cartilage and labrum in the majority (84.6%) of hips. Conclusions: The incidence of intraarticular lesions in developmental dysplasia of the hip was high, even in the prearthritic stage. These lesions tended to originate in the anterosuperior area of the acetabulum and were generally progressive. Level of evidence: Therapeutic study, level IV.

Subchondral changes in transient osteoporosis of the hip.

Abstract Objective. To review the subchondral changes on MR imaging in transient osteoporosis of the hip (TOH) and to consider the pathophysiology. Design and patients. MR images of 12 hips of 11 consecutive patients with TOH were retrospectively studied. The diagnoses of TOH were confirmed on the basis of previously published criteria, including decreased bone density of the femoral head and/or neck on radiographs, bone marrow edema (BME) pattern on MR images, spontaneous resolution of the symptoms and a return to normal radiodensity. Results. All 12 hips showed a BME pattern in the femoral head and/or neck. Linear patterns of very low signal intensity were identified on T1-weighted images in the subchondral area within the diffuse low signal intensity area in all 12 hips. On T2-weighted images, a low signal intensity line was observed in the corresponding area in eight hips only. These linear patterns were thought to represent subchondral fracture lines. Conclusions. The presence of a subchondral fracture may be important when considering the pathophysiology of TOH.

MRI Evaluation of Collapsed Femoral Heads in Patients 60 Years Old or Older: Differentiation of Subchondral Insufficiency Fracture From Osteonecrosis of the Femoral Head

OBJECTIVE The purpose of this article is to verify the hypothesis that osteonecrosis and subchondral insufficiency fracture of the femoral head can be differentiated on the basis of their appearance on MRI. SUBJECTS AND METHODS Between May 1998 and February 2009, we reviewed 30 consecutive hips in 30 patients, 60 years old or older at the time of onset of hip pain, with radiologic evidence of subchondral collapse of the femoral head and with both MR images and histologic results available. Both clinical and radiologic appearances were investigated. The patients were divided into two groups according to the shape of low-intensity bands on T1-weighted images. The first group showed concavity of the articular surface, which is characteristic of osteonecrosis, and the second group showed an irregular convexity of the articular surface, which is characteristic of subchondral insufficiency fracture. RESULTS Sixteen hips (53.3%) showed evidence of osteonecrosis, and 14 (46.7%) showed evidence of subchondral insufficiency fracture, which was consistent with the corresponding histopathologic diagnoses. In all cases of osteonecrosis, the patient had a history of either corticosteroid intake or alcohol abuse. Among patients with subchondral insufficiency fracture, the proportion of women was significantly higher than that among patients with osteonecrosis. A crescent sign (subchondral fracture) was present radiographically in about half of all cases in both groups. CONCLUSION The results of the present study suggest that the shape of the low-intensity band on MRI is useful for the differentiating subchondral insufficiency fracture from osteonecrosis. In addition, among osteoporotic elderly women without any history of corticosteroid intake or alcohol abuse, a diagnosis of subchondral insufficiency fracture should be considered.

Effect of intra-articular lesions on the outcome of periacetabular osteotomy in patients with symptomatic hip dysplasia

In order to clarify how intra-articular lesions influence the survival of a periacetabular osteotomy in patients with dysplasia of the hip, we performed an observational study of 121 patients (121 hips) who underwent a transposition osteotomy of the acetabulum combined with an arthroscopy. Their mean age was 40.2 years (13 to 64) and the mean follow-up was 9.9 years (2 to 18). Labral and cartilage degeneration tended to originate from the anterosuperior part of the acetabulum, followed by the femoral side. In all, eight hips (6.6%) had post-operative progression to Kellgren-Lawrence grade 4 changes, and these hips were associated with the following factors: moderate osteoarthritis, decreased width of the joint space, joint incongruity, and advanced intra-articular lesions (subchondral bone exposure in the cartilage and a full-thickness labral tear). Multivariate analysis indicated subchondral bone exposure on the femoral head as an independent risk factor for progression of osteoarthritis (p = 0.003). In hips with early stage osteoarthritis, femoral subchondral bone exposure was a risk factor for progression of the grade of osteoarthritis. Although the outcome of transposition osteotomy of the acetabulum was satisfactory, post-operative progression of osteoarthritis occurred at a high rate in hips with advanced intra-articular lesions, particularly in those where the degenerative process had reached the point of femoral subchondral bone exposure.