Faculty Bibliography

OBJECTIVE: To determine the role of ANK/Myb-binding protein 1a (MYBBP1a) and sphingosine kinase 1 (SPHK1) interactions in catabolic events of articular chondrocytes. METHOD: ANK/MYBBP1a and SPHK1 interactions were identified using yeast two-hybrid screening and co-immunoprecipitation. To determine the role of these interactions in catabolic events of articular chondrocytes, ank/ank and wild type mouse chondrocytes transfected with full-length or mutant ank expression vectors or femoral heads were treated with interleukin-1beta (IL-1beta) in the absence or presence of SPHK inhibitor. Catabolic marker mRNA levels were analyzed by real time PCR; proteoglycan loss using safranin O staining and MMP-13 immunostaining were determined in femoral head explants; NF-kappaB activity was determined by transfecting chondrocytes with a NF-kappaB-specific luciferase reporter and analyzing nuclear translocation of p65 by immunoblotting; MYBBP1a nuclear or cytoplasmic amounts were determined by immunohistochemistry and immunoblotting. RESULTS: The ANK N-terminal region interacted with SPHK1, whereas a cytoplasmic C-terminal loop interacted with MYBBP1a. Lack of ANK/MYBBP1a and SPHK1 interactions in ank/ank chondrocytes resulted in increased MYBBP1a nuclear amounts and decreased SPHK1 activity, and consequently decreased NF-kappaB activity, catabolic marker mRNA levels, proteoglycan loss, and MMP-13 immunostaining in IL-1beta-treated articular chondrocytes or femoral heads. Transfection with full-length ank expression vector reduced nuclear MYBBP1a amounts and fully restored SPHK and NF-kappaB activities in IL-1beta-treated ank/ank chondrocytes, whereas transfection with P5L or F376del mutant ank reduced nuclear MYBBP1a or increased SPHK activity, respectively, and consequently either transfection only partially restored NF-kappaB activity. CONCLUSION: ANK/MYBBP1a and SPHK1 interactions stimulate catabolic events in IL-1beta-mediated cartilage degradation.

Objective. To determine the actions of lithium chloride (LiCl) on catabolic events in articular chondrocytes and cartilage degradation after interleukin (IL)-1beta(beta) treatment and after surgically induced osteoarthritis (OA) in mice. Methods. The expression levels of catabolic genes in human articular chondrocytes treated with LiCl followed by IL-1beta were determined by real time PCR. To understand the mechanism of how LiCl affects catabolic events in articular chondrocytes after IL-1beta treatment, the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) was determined using luciferase reporter assays, and the activities of mitogen-activated protein kinases (MAPK) and signal transducer and activator of transcription 3 (Stat3) signaling pathway were determined by immunoblot analysis of total cell lysates. Mouse femoral head explant cultures treated with IL-1beta and a surgically induced OA mouse model were used to determine the effect of LiCl on proteoglycan loss and cartilage degradation. Results. LiCl treatment resulted in decreased catabolic marker mRNA levels and activation of NF-kappaB, p38 MAPK, and Stat3 signaling in IL-1beta -treated chondrocytes. Furthermore, LiCl directly inhibited IL-6-stimulated activation of Stat3 signaling. Consequently, loss of proteoglycan and cartilage destruction in LiCl-treated knee joints 8 weeks after OA-induced surgery or in LiCl-treated femoral head explants after IL-1beta treatment were markedly reduced compared to vehicle-treated joints or explants. Conclusion. LiCl reduced catabolic events in IL-1beta -treated human articular chondrocytes and cartilage destruction in IL-1beta -treated mouse femoral head explants and in surgically induced OA mouse models via the inhibition of NF-kappaB, p38 and Stat3 signaling pathway activities. (c) 2014 American College of Rheumatology.

OBJECTIVE: ANXA6, the gene for annexin A6, is highly expressed in osteoarthritic (OA) articular chondrocytes but not in healthy articular chondrocytes. This study was undertaken to determine whether annexin A6 affects catabolic events in these cells. METHODS: Articular chondrocytes were isolated from Anxa6-knockout mice, wild-type (WT) mice, and human articular cartilage in which ANXA6 was overexpressed. Cells were treated with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha), and expression of catabolic genes and activation of NF-kappaB were determined by real-time polymerase chain reaction and luciferase reporter assay. Anxa6(-/-) and WT mouse knee joints were injected with IL-1beta or the medial collateral ligament was transected and partial resection of the medial meniscus was performed to determine the role of Anxa6 in IL-1beta-mediated cartilage destruction and OA progression. The mechanism by which Anxa6 stimulates NF-kappaB activity was determined by coimmunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of IL-1beta-treated Anxa6(-/-) and WT mouse chondrocytes for p65 and Anxa6. RESULTS: Loss of Anxa6 resulted in decreased NF-kappaB activation and catabolic marker messenger RNA (mRNA) levels in IL-1beta- or TNFalpha-treated articular chondrocytes, whereas overexpression of ANXA6 resulted in increased NF-kappaB activity and catabolic marker mRNA levels. Annexin A6 interacted with p65, and loss of Anxa6 caused decreased nuclear translocation and retention of the active p50/p65 NF-kappaB complex. Cartilage destruction in Anxa6(-/-) mouse knee joints after IL-1beta injection or partial medial meniscectomy was reduced as compared to that in WT mouse joints. CONCLUSION: Our data define a role of annexin A6 in the modulation of NF-kappaB activity and in the stimulation of catabolic events in articular chondrocytes.

Purpose: To assess the use of diffusion-tensor (DT) imaging of articular cartilage to detect and grade early cartilage damage in human specimens with early signs of cartilage damage. Materials and Methods: This study was approved by the institutional review board. Forty-three cartilage-on-bone samples drilled from 21 human patellae were examined with 17.6-T magnetic resonance (MR) imaging and a diffusion-weighted spin-echo sequence (spatial resolution, 50 x 100 x 800 mum). Subsequently, samples underwent histologic analysis with safranin O staining. Cartilage damage on safranin O histologic slides was quantified with Osteoarthritis Research Society International (OARSI) grades; grades ranged from 0 (healthy) to 6 (bone remodeling). Maps of longitudinal diffusivity (lambda(l)), transverse diffusivity (lambda(t)), mean diffusivity (MD), and fractional anisotropy (FA) were calculated. Cartilage was segmented, and region of interest (ROI) analysis was performed and compared with histologic findings. Significant differences in MR parameters between the OARSI groups were assessed with the Tukey test. The value of DT imaging in the diagnosis and grading of cartilage damage was assessed with logistic regression analysis. Results: Samples had OARSI grades of 0 (n = 14), 1 (n = 11), 2 (n = 12), 3 (n = 4), and 4 (n = 2). Samples with an OARSI grade greater than 0 had significantly increased lambda(l), lambda(t), and MD (7%-25% increase) in the superficial cartilage growing deeper into cartilage with increasing OARSI grade. Samples with an OARSI grade greater than 0 showed significantly decreased FA in the deep cartilage (-25% to -35% decrease), suggesting that changes in the collagen architecture may occur early in cartilage degradation. DTI showed excellent performance in the detection of cartilage damage (accuracy, 0.95; 41 of 43 samples) and good performance in the grading of cartilage damage (accuracy, 0.74; 32 of 43 samples). Conclusion: DT imaging of articular cartilage can enable physicians to detect and grade early cartilage damage. (c) RSNA, 2012.

Biomineralization is a multifactorial and complex process, which results in the deposition of mineral crystals in the extracellular matrix of various tissues. Physiological mineralization is restricted to tissues, such as bones, teeth, and certain areas of cartilage. Pathological or ectopic mineralization can occur in many soft tissues, including articular cartilage, cardiovascular tissues, kidney, ligaments, and tendons, and can lead to serious problems. Therefore, the understanding of factors and mechanisms that regulate the mineralization process is essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization. This review will discuss some of the mechanisms and factors that regulate physiological mineralization and their potential roles in ectopic mineralization. Finally, potential therapeutic approaches for the treatment of ectopic mineralization are being discussed.

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C alpha (PKCalpha) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCalpha. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCalpha and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.

Stem cell application is a burgeoning field of medicine that is likely to influence the future of orthopaedic surgery. Stem cells are associated with great promise and great controversy. For the orthopaedic surgeon, stem cells may change the way that orthopaedic surgery is practiced and the overall approach of the treatment of musculoskeletal disease. Stem cells may change the field of orthopaedics from a field dominated by surgical replacements and reconstructions to a field of regeneration and prevention. This review will introduce the basic concepts of stem cells pertinent to the orthopaedic surgeon and proceed with a more in depth discussion of current developments in the study of stem cells in fracture healing