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1.  Characterization of Porcine Aortic Valvular Interstitial Cell ‘Calcified’ Nodules 
PLoS ONE  2012;7(10):e48154.
Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-β1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-β1 (OST+TGF-β1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-β1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-β1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that ‘calcified’ nodules formed from PAVICs grown in OST+TGF-β1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.
doi:10.1371/journal.pone.0048154
PMCID: PMC3482191  PMID: 23110195
2.  Anisotropic Fibrous Scaffolds for Articular Cartilage Regeneration 
Tissue Engineering. Part A  2012;18(19-20):2073-2083.
Articular cartilage lesions, which can progress to osteoarthritis, are a particular challenge for regenerative medicine strategies, as cartilage function stems from its complex depth-dependent microstructural organization, mechanical properties, and biochemical composition. Fibrous scaffolds offer a template for cartilage extracellular matrix production; however, the success of homogeneous scaffolds is limited by their inability to mimic the cartilage's zone-specific organization and properties. We fabricated trilaminar scaffolds by sequential electrospinning and varying fiber size and orientation in a continuous construct, to create scaffolds that mimicked the structural organization and mechanical properties of cartilage's collagen fibrillar network. Trilaminar composite scaffolds were then compared to homogeneous aligned or randomly oriented fiber scaffolds to assess in vitro cartilage formation. Bovine chondrocytes proliferated and produced a type II collagen and a sulfated glycosaminoglycan-rich extracellular matrix on all scaffolds. Furthermore, all scaffolds promoted significant upregulation of aggrecan and type II collagen gene expression while downregulating that of type I collagen. Compressive testing at physiological strain levels further demonstrated that the mechanical properties of trilaminar composite scaffolds approached those of native cartilage. Our results demonstrate that trilaminar composite scaffolds mimic key organizational characteristics of native cartilage, support in vitro cartilage formation, and have superior mechanical properties to homogenous scaffolds. We propose that these scaffolds offer promise in regenerative medicine strategies to repair articular cartilage lesions.
doi:10.1089/ten.tea.2011.0606
PMCID: PMC3463280  PMID: 22655795
3.  Effects of Fiber Orientation on the Frictional Properties and Damage of Regenerative Articular Cartilage Surfaces 
Tissue Engineering. Part A  2013;19(19-20):2300-2310.
Articular cartilage provides a low-friction, wear-resistant surface for diarthrodial joints. Due to overloading and overuse, articular cartilage is known to undergo significant wear and degeneration potentially resulting in osteoarthritis (OA). Regenerative medicine strategies offer a promising solution for the treatment of articular cartilage defects and potentially localized early OA. Such strategies rely on the development of materials to restore some aspects of cartilage. In this study, microfibrous poly(ɛ-caprolactone) scaffolds of varying fiber orientations (random and aligned) were cultured with bovine chondrocytes for 4 weeks in vitro, and the mechanical and frictional properties were evaluated. Mechanical properties were quantified using unconfined compression and tensile testing techniques. Frictional properties were investigated at physiological compressive strains occurring in native articular cartilage. Scaffolds were sheared along the fiber direction, perpendicular to the fiber direction and in random orientation. The evolution of damage as a result of shear was evaluated via white light interferometry and scanning electron microscopy. As expected, the fiber orientation strongly affected the tensile properties as well as the compressive modulus of the scaffolds. Fiber orientation did not significantly affect the equilibrium frictional coefficient, but it was, however, a key factor in dictating the evolution of surface damage on the surface. Scaffolds shear tested perpendicular to the fiber orientation displayed the highest surface damage. Our results suggest that the fiber orientation of the scaffold implanted in the joint could strongly affect its resistance to damage due to shear. Scaffold fiber orientation should thus be carefully considered when using microfibrous scaffolds.
doi:10.1089/ten.tea.2012.0580
PMCID: PMC3761558  PMID: 23688110
4.  Stimulation of Chondrogenic Differentiation of Adult Human Bone Marrow-Derived Stromal Cells by a Moderate-Strength Static Magnetic Field 
Tissue Engineering. Part A  2014;20(11-12):1612-1620.
Tissue-engineering strategies for the treatment of osteoarthritis would benefit from the ability to induce chondrogenesis in precursor cells. One such cell source is bone marrow-derived stromal cells (BMSCs). Here, we examined the effects of moderate-strength static magnetic fields (SMFs) on chondrogenic differentiation in human BMSCs in vitro. Cells were cultured in pellet form and exposed to several strengths of SMFs for various durations. mRNA transcript levels of the early chondrogenic transcription factor SOX9 and the late marker genes ACAN and COL2A1 were determined by reverse transcription–polymerase chain reaction, and production of the cartilage-specific macromolecules sGAG, collage type 2 (Col2), and proteoglycans was determined both biochemically and histologically. The role of the transforming growth factor (TGF)-β signaling pathway was also examined. Results showed that a 0.4 T magnetic field applied for 14 days elicited a strong chondrogenic differentiation response in cultured BMSCs, so long as TGF-β3 was also present, that is, a synergistic response of a SMF and TGF-β3 on BMSC chondrogenic differentiation was observed. Further, SMF alone caused TGF-β secretion in culture, and the effects of SMF could be abrogated by the TGF-β receptor blocker SB-431542. These data show that moderate-strength magnetic fields can induce chondrogenesis in BMSCs through a TGF-β-dependent pathway. This finding has potentially important applications in cartilage tissue-engineering strategies.
doi:10.1089/ten.tea.2013.0307
PMCID: PMC4029136  PMID: 24506272
5.  The Changing Integrin Expression and a Role for Integrin β8 in the Chondrogenic Differentiation of Mesenchymal Stem Cells 
PLoS ONE  2013;8(11):e82035.
Many cartilage tissue engineering approaches aim to differentiate human mesenchymal stem cells (hMSCs) into chondrocytes and develop cartilage in vitro by targeting cell-matrix interactions. We sought to better inform the design of cartilage tissue engineering scaffolds by understanding how integrin expression changes during chondrogenic differentiation. In three models of in vitro chondrogenesis, we studied the temporal change of cartilage phenotype markers and integrin subunits during the differentiation of hMSCs. We found that transcript expression of most subunits was conserved across the chondrogenesis models, but was significantly affected by the time-course of differentiation. In particular, ITGB8 was up-regulated and its importance in chondrogenesis was further established by a knockdown of integrin β8, which resulted in a non-hyaline cartilage phenotype, with no COL2A1 expression detected. In conclusion, we performed a systematic study of the temporal changes of integrin expression during chondrogenic differentiation in multiple chondrogenesis models, and revealed a role for integrin β8 in chondrogenesis. This work enhances our understanding of the changing adhesion requirements of hMSCs during chondrogenic differentiation and underlines the importance of integrins in establishing a cartilage phenotype.
doi:10.1371/journal.pone.0082035
PMCID: PMC3842320  PMID: 24312400
6.  Human Periosteum Is a Source of Cells for Orthopaedic Tissue Engineering: A Pilot Study 
Background
Periosteal cells are important in embryogenesis, fracture healing, and cartilage repair and could provide cells for osteochondral tissue engineering.
Questions/purpose
We determined whether a population of cells isolated from human periosteal tissue contains cells with a mesenchymal stem cell (MSC) phenotype and whether these cells can be expanded in culture and used to form tissue in vitro.
Methods
We obtained periosteal tissue from six patients. Initial expression of cell surface markers was assessed using flow cytometry. Cells were cultured over 10 generations and changes in gene expression evaluated to assess phenotypic stability. Phenotype was confirmed using flow cytometry and colony-forming ability assays. Mineral formation was assessed by culturing Stro-1− and unsorted cells with osteogenic supplements. Three cell culture samples were used for a reverse transcription–polymerase chain reaction, four for flow cytometry, three for colony-forming assay, and three for mineralization.
Results
Primary cultures, containing large numbers of hematopoietic cells were replaced initially by Stro-1 and ALP-expressing immature osteoblastic cell types and later by ALP-expressing cells, which lacked Stro-1 and which became the predominant cell population during subculture. Approximately 10% of the total cell population continued to express markers for Stro1+/ALP− cells throughout.
Conclusions
These data suggest periosteum contains a large number of undifferentiated cells that can differentiate into neotissue and persist despite culture in noncell-specific media for over 10 passages.
Clinical Relevance
Cultured periosteal cells may contribute to tissue formation and may be applicable for tissue engineering applications.
doi:10.1007/s11999-011-1895-x
PMCID: PMC3183217  PMID: 21547415

Results 1-6 (6)