Abstract-We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of ␤-catenin.In this study we demonstrate directly that growth factors stimulate ␤-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether ␤-catenin/TCF signaling regulates VSMC proliferation via modulation of the ␤-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited ␤-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of ␤-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by ␤-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting ␤-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and ␤-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 ␤-catenin/TCF responsive genes.

The extracellular matrix (ECM) of articular cartilage is comprised of complex networks of proteins and glycoproteins, all of which are expressed by its resident cell, the chondrocyte. Cartilage is a unique tissue given its complexity and ability to resist repeated load and deformation. The mechanisms by which articular cartilage maintains its integrity throughout our lifetime is not fully understood, however there are numerous regulatory pathways known to govern ECM turnover in response to mechanical stimuli. To further our understanding of this field, we envision that proteomic analysis of the secretome will provide information on how the chondrocyte remodels the surrounding ECM in response to load, in addition to providing information on the metabolic state of the cell. In this review, we attempt to summarize the recent mass spectrometry-based proteomic discoveries in healthy and diseased cartilage and chondrocytes, to facilitate the discovery of novel biomarkers linked to degenerative pathologies, such as osteoarthritis (OA).

Objective. The influence of the cruciate ligaments in spontaneous osteoarthritis (OA) is not understood, although ligament rupture is known to cause secondary OA. Additionally, femoral notch narrowing at the anterior cruciate ligament (ACL) insertion site is associated with disease severity, but it is unknown whether ligament deterioration precedes or follows osteophyte formation. We examined cruciate ligament mechanics and metabolism and the intercondylar notch width in OAprone Dunkin-Hartley (DH) guinea pigs at ages up to and including the age at OA onset (24 weeks), and compared the data with those in age-matched controls (Bristol strain 2 [BS2] guinea pigs).Methods. Guinea pigs were assessed at 3, 6, 9, 12, 16, 20, 24, and 36 weeks of age. ACLs were mechanically tested, and the intercondylar notch width index (NWI) was determined. Cruciate ligament metabolism was determined by measuring the following markers of collagen turnover: matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinases 2, C-terminal type I procollagen propeptide (PICP), and the immature collagen-derived crosslink dihydroxylysinonorleucine (DHLNL).Results. DH guinea pigs had significantly laxer ACLs than did BS2 guinea pigs, at 12, 16, and 24 weeks. We observed elevated levels of pro and active MMP-2, PICP, and DHLNL in the cruciate ligaments of DH animals at most ages, compared with BS2 guinea pigs. The NWI in DH animals was significantly lower than that in BS2 guinea pigs at 24 and 36 weeks.Conclusion. In DH guinea pigs, laxer ACLs, which are associated with increased collagen turnover, may cause joint instability and predispose these animals to the early onset of OA. Decreased intercondylar notch width in the DH animals indicates that bone remodeling at the ACL insertion site is a response to elevated ACL laxity.

Objectives Bioreactor‐based production systems have the potential to overcome limitations associated with conventional tissue engineering manufacturing methods, facilitating regulatory compliant and cost‐effective production of engineered grafts for widespread clinical use. In this work, we established a bioreactor‐based manufacturing system for the production of cartilage grafts. Materials & Methods All bioprocesses, from cartilage biopsy digestion through the generation of engineered grafts, were performed in our bioreactor‐based manufacturing system. All bioreactor technologies and cartilage tissue engineering bioprocesses were transferred to an independent GMP facility, where engineered grafts were manufactured for two large animal studies. Results The results of these studies demonstrate the safety and feasibility of the bioreactor‐based manufacturing approach. Moreover, grafts produced in the manufacturing system were first shown to accelerate the repair of acute osteochondral defects, compared to cell‐free scaffold implants. We then demonstrated that grafts produced in the system also facilitated faster repair in a more clinically relevant chronic defect model. Our data also suggested that bioreactor‐manufactured grafts may result in a more robust repair in the longer term. Conclusion By demonstrating the safety and efficacy of bioreactor‐generated grafts in two large animal models, this work represents a pivotal step towards implementing the bioreactor‐based manufacturing system for the production of human cartilage grafts for clinical applications. Read the Editorial for this article on doi:10.1111/cpr.12625