Takahito Yuasa

A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis

The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/beta-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.

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.

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP‐2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein‐2 (Bmp‐2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino‐terminal portion of SHH (rSHH‐N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3‐E1 cells. rSHH‐N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH‐N‐induced ALP activity in C3H10T1/2 and MC3T3‐E1 cells, indicating that BMPs are involved in SHH‐induced osteoblast differentiation. Simultaneous supplement with rSHH‐N and BMP‐2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH‐N for 6 h enhanced the response to BMP‐2 by increasing ALP activity in C3H10T1/2 and MC3T3‐E1 cells. Stimulatory effects of rSHH‐N and additive effects with rSHH‐N and BMP‐2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP‐2 (1 μg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r‐SHH‐N (1–5 μg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.

Roles of β -catenin signaling in phenotypic expression and proliferation of articular cartilage superficial zone cells

The superficial zone (SFZ) of articular cartilage has unique structural and biomechanical features, is thought to promote self-renewal of articular cartilage, and is thus important for joint long-term function, but the mechanisms regulating its properties remain unclear. Previous studies revealed that Wnt/β-catenin signaling is continuously active in SFZ, indicating that it may be essential for SFZ function. Thus, we examined whether Wnt/β-catenin signaling regulates proliferation and phenotypic expression in SFZ cells. Using transgenic mice, we found that acute activation of Wnt/β-catenin signaling increases SFZ thickness, Proteoglycan 4 (Prg4, also called lubricin) expression and the number of slow-cell cycle cells, whereas conditional ablation of β-catenin causes the opposite. We developed a novel method to isolate SFZ cell-rich populations from the epiphyseal articular cartilage of neonatal mice, and found that the SFZ cells in culture exhibit a fibroblastic cytoarchitecture and higher Prg4 and Ets-related gene (Erg) expression and lower aggrecan expression compared with chondrocyte cultures. Gene array analyses indicated that SFZ cells have distinct gene expression profiles compared with underlying articular chondrocytes. Treatment of Wnt3a strongly stimulated SFZ cell proliferation and maintained strong expression of Prg4 and Erg, whereas ablation of β-catenin strongly impaired proliferation and phenotypic expression. When the cells were transplanted into athymic mice, they formed Prg4- and aggrecan-expressing cartilaginous masses attesting to their autonomous phenotypic capacity. Ablation of β-catenin caused a rapid loss of Prg4 gene expression and strong increases in expression of aggrecan and collagen 10, the latter being a trait of hypertrophic chondrocytes. Together, the data reveal that Wnt/β-catenin signaling is a key regulator of SFZ cell phenotype and proliferation, and may be as important for articular cartilage long-term function.

Wnt/β-Catenin and Retinoic Acid Receptor Signaling Pathways Interact to Regulate Chondrocyte Function and Matrix Turnover

Activation of the Wnt/β-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/β-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/β-catenin reporter activity and β-catenin nuclear accumulation. This stimulation was accompanied by increased gene expression of Wnt proteins and receptors and was inhibited by co-treatment with Dickkopf-related protein-1, an extracellular inhibitor of Wnt/β-catenin signaling, suggesting that RA modulates Wnt signaling at Wnt cell surface receptor level. RA also enhanced matrix loss triggered by Wnt/β-catenin signaling, whereas treatment with a retinoid antagonist reduced it. Interestingly, overexpression of retinoic acid receptor γ (RARγ) strongly inhibited Wnt/β-catenin signaling in retinoid-free cultures, whereas small interfering RNA-mediated silencing of endogenous RARγ expression strongly increased it. Small interfering RNA-mediated silencing of RARα or RARβ had minimal effects. Co-immunoprecipitation and two-hybrid assays indicated that RARγ interacts with β-catenin and induces dissociation of β-catenin from lymphoid enhancer factor in retinoid-free cultures. The N-terminal domain (AF-1) of RARγ but not the C-terminal domain (AF-2) was required for association with β-catenin, whereas both AF-1 and AF-2 were necessary for inhibition of β-catenin transcriptional activity. Taken together, our data indicate that the Wnt and retinoid signaling pathways do interact in chondrocytes, and their cross-talks and cross-regulation play important roles in the regulation of cartilage matrix homeostasis.

Transient Activation of Wnt/β-Catenin Signaling Induces Abnormal Growth Plate Closure and Articular Cartilage Thickening in Postnatal Mice

Wnt/beta-catenin signaling is required for skeletal development and organization and for function of the growth plate and articular cartilage. To further clarify these roles and their possible pathophysiological importance, we created a new transgenic mouse model in which Wnt/beta-catenin signaling can be activated in cartilage for specific periods of time. These transgenic mice expressed a constitutive active form of beta-catenin fused to a modified estrogen receptor ligand-binding domain under the control of cartilage-specific collagen 11alpha2 promoter/enhancer. Transient Wnt/beta-catenin signaling activation in young adult mice by tamoxifen injections induced growth retardation and severe deformities in knee joints. Tibial and femoral growth plates displayed an excessive number of apoptotic cells and eventually underwent abnormal regression. Articular cartilage exhibited an initial acute loss of proteoglycan matrix that was followed by increases in thickness, cell density, and cell proliferation. In reciprocal studies, we found that conditional ablation of beta-catenin in postnatal mice using a Col2-CreER strategy led to hypocellularity in articular cartilage, growth plate disorganization, and a severe reduction in bone volume. Together, these data provide evidence that Wnt/beta-catenin signaling has important and distinct roles in growth plate and articular cartilage and that postnatal dysregulation of this signaling pathway causes diverse structural and functional changes in the two cartilaginous structures.

Intervertebral disc development is regulated by Wnt/β-catenin signaling.

Study Design. Histologic analysis of intervertebral disc (IVD) in three types of transgenic mice. Objective. To investigate the role of Wnt/&bgr;-catenin signaling in regulation of IVD development and organization. Summary of Background Data. -catenin dependent Wnt signaling is one of the central regulators in cartilage development during limb skeletal formation. Little is known, however, about the physiologic relevance of this signaling pathway to IVD development and organization. Methods. Temporal-spatial distribution of Wnt/&bgr;-catenin signaling activity was examined in IVD using Wnt/&bgr;-catenin reporter (TOPGAL) mice. The structural changes in the mouse IVD components such as the nucleus pulposus (NP), endplate (EP), annulus fibrosus (AF), and the growth plate (GP) of the vertebral body were analyzed after transient activation of Wnt/&bgr;-catenin signaling or deletion of &bgr;-catenin in the mice. Results. Activity of Wnt/&bgr;-catenin signaling was high in EP, AF, and GP in the embryonic stages and decreased at the postnatal stage; it was undetectable in the embryonic NP but upregulated after birth. The transient activation of Wnt/&bgr;-catenin signaling caused severe deterioration of the GP and the AF, whereas deficiency of &bgr;-catenin accelerated bone formation in between EP and GP. Conclusion. The findings in this study suggest that proper regulation of Wnt/&bgr;-catenin signaling is required for development and organization of IVD.

Seven Years of Chronological Changes of Serum Chromium Levels After Metasul Metal-On-Metal Total Hip Arthroplasty

Although many authors have reported the serum concentrations of metal ions in patients who had metal-on-metal coupling prostheses, most of the studies were not longitudinal, and the follow-up periods were short. We evaluated the longitudinal changes of serum chromium levels in 44 patients who had undergone unilateral metal-on-metal total hip arthroplasty for a minimum of 7 years postoperatively. Although there was a consistent increase in the mean serum chromium level until 3 years after implantation, there was little difference in the levels from years 3 to 7 postoperatively. Although the serum chromium concentration was low throughout postoperative follow-up for 7 years in about 25% of patients, the serum chromium level stayed high or showed gradual elevation in 16.3% of our patients.

Surgical Outcome for Hip Fractures in Patients with and without Parkinson's Disease:

Purpose. To compare surgical outcome of hip fractures (in terms of the ability to walk and complications) in patients with and without Parkinsons disease (PD). Methods. Records of 207 patients who underwent surgery for femoral neck fractures were reviewed. Of whom, 9 men and 26 women with PD aged 58 to 89 (mean, 76.8) years underwent hemiarthroplasty for subcapital fractures (n=22) or internal fixation for intertrochanteric fractures (n=13). The remaining 36 men and 136 women with no PD aged 61 to 96 (mean, 81.8) years underwent hemiarthroplasty (n=108) or internal fixation (n=64) for subcapital (n=120) or intertrochanteric (n=52) fractures. None of the patients had pathological fractures, and all had been ambulatory prior to the fracture. According to the Columbia classification system, severity of PD was grade II in 11, grade III in 11, and grade IV in 13 patients. Results. Respectively for the patients with and without PD at the one-year follow-up, 68.2% and 79.6% of those treated with hemiarthroplasty and 46.2% and 62.5% of those treated with internal fixation were able to walk with or without assistive devices. 81.8%, 63.6%, and 38.5% of the patients with grade-II, -III, and -IV PD, respectively, were able to walk at the one-year follow-up, compared to 73.3% of the patients without PD. Conclusion. Surgical treatment for hip fractures was appropriate for patients with grade-II or -III PD.

Original methods to move femoral head medially and caudally after rotational acetabular osteotomy: especially to use ceramic spacer

The operative procedures chosen to treat arthrosis in patients with severe acetabular dysplasia vary among orthopaedic surgeons, particularly in younger patients. We operated on 450 hips with acetabular dysplasia by using the rotational acetabular osteotomy (RAO) method of Ninomiya and Tagawa by Ninomiya (Clin Orthop 247:127–137, 1989). In this report, we describe our technique of performing RAO with hydroxyapatite tricalcium phosphate-composite (HAP-TCP) blocks to achieve good acetabular coverage and the results of this procedure in 16 hips with severe dysplasia, including nine with advanced arthrosis. Adequate coverage of the osteotomized acetabulum was maintained and symptoms, especially pain, showed marked improvement at the latest follow up.