A proteomic analysis of engineered tendon formation under dynamic mechanical loading in vitro

Department of Plastic and Reconstructive Surgery, Shanghai 9th People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Tissue Engineering Research, 639 Zhi Zao Ju Road, Shanghai 200011, PR China.
Biomaterials (Impact Factor: 8.56). 03/2011; 32(17):4085-95. DOI: 10.1016/j.biomaterials.2011.02.033
Source: PubMed


Previous studies have demonstrated the beneficial effect of mechanical loading on in vitro tendon engineering. To understand the mechanism, human tenocytes and polyglycolic acid long fibers were used for in vitro tendon engineering in a bioreactor system for 12 weeks with and without dynamic loading. The engineered neo-tendons were subjected to proteomic analysis using mass spectrometry along with shotgun strategy. As expected, mechanical loading resulted in a more mature tendon tissue characterized by a firmer tissue texture and densely deposited matrices which formed longitudinally aligned collagen fibers in a highly compact fashion. In contrast, non-loaded neo-tendon revealed loosely and less deposited matrices in a relatively less organized pattern. Proteins isolated from two groups of tissues exhibited similar distribution of isoeletric point and molecular weight indicating the similarity and comparability of the tissue specimens. Further, proteomic analysis showed that total 758 proteins were identified from both groups with 194 and 177 proteins uniquely presented in loaded and non-loaded tendons, respectively. Comparison of loaded and non-loaded tendons revealed 195 significantly up-regulated proteins and 189 significantly down-regulated proteins. The differentially expressed proteins could generally be classified into the categories of extracellular matrix, intra-cellular signaling, cytoskeleton and inflammatory response. Among them, significantly up-regulated collagens I and VI, MMP-14, WNT5A, microfilament molecules and some inflammatory factors suggest that the possible mechanism for this particular biological phenomenon may involve increased production of tendon specific matrices, enhanced cross-link of collagens and other matrix molecules, proper matrix remodeling for tissue maturation and mechanotransduction (including non-canonical Wnt signal pathway) mediated other biological processes.

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    • "Differential proteins under mechanical stretch are also profiled, especially for those events undetectable in transcriptomic analysis. Cyclic biaxial stretch initiates multiple phosphorylations associated with the newly-identified mechanosensitive proteins in chondrosarcoma cells (Piltti et al., 2008) and promotes 194 and 177 differential proteins related to ECM production, intracellular signaling, cytoskeletal remodeling, and inflammatory response from tenocytes cultured on polyglycolic acid long fibers, respectively (Jiang et al., 2011). Cyclic equiaxial stretch uncovers a few consistent major proteins (TGF-β1, TNF, CASP3, and p53) to hub at the center of the interacting network with the newlyprofiled proteins of interest (BAG5, NO66, and eIF-5A) in activated lamina cribrosa cells (Rogers et al., 2012), recapitulating the importance of MAPK and TGF-β signaling pathways in mechanotransduction. "
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    ABSTRACT: Cells sense various in vivo mechanical stimuli, which initiate downstream signaling to mechanical forces. While a body of evidences is presented on the impact of limited mechanical regulators in past decades, the mechanisms how biomechanical responses globally affect cell function need to be addressed. Complexity and diversity of in vivo mechanical clues present distinct patterns of shear flow, tensile stretch, or mechanical compression with various parametric combination of its magnitude, duration, or frequency. Thus, it is required to understand, from the viewpoint of mechanobiology, what mechanical features of cells are, why mechanical properties are different among distinct cell types, and how forces are transduced to downstream biochemical signals. Meanwhile, those in vitro isolated mechanical stimuli are usually coupled together in vivo, suggesting that the different factors that are in effect individually could be canceled out or orchestrated with each other. Evidently, omics analysis, a powerful tool in the field of system biology, is advantageous to combine with mechanobiology and then to map the full-set of mechanically sensitive proteins and transcripts encoded by its genome. This new emerging field, namely mechanomics, makes it possible to elucidate the global responses under systematically-varied mechanical stimuli. This review discusses the current advances in the related fields of mechanomics and elaborates how cells sense external forces and activate the biological responses.
    Full-text · Article · Apr 2014 · Protein & Cell
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    • "Static mechanical stress has been shown to improve the fiber arrangement of engineered tendon constructs, but did not produce a compacted tendon-like collagen structure in vitro (Chen et al., 2007). More promisingly, engineered tendon under cyclic tensile strain exhibited a well-organized collagen fiber arrangement and structure and enhanced tensile strength, suggesting that PMS is preferable for engineered tendon formation (Jiang et al., 2011). Even though the importance of providing PMS in tendon bioreactor has been recognized previously (Brown and Carson, 1999; Wang et al., 2012), the optimal cyclic tensile stimulation parameters have not been defined. "
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    ABSTRACT: Identification of functional programmable mechanical stimulation (PMS) on tendon not only provides the insight of the tendon homeostasis under physical/pathological condition, but also guides a better engineering strategy for tendon regeneration. The aims of the study are to design a bioreactor system with PMS to mimic the in vivo loading conditions, and to define the impact of different cyclic tensile strain on tendon. Rabbit Achilles tendons were loaded in the bioreactor with/without cyclic tensile loading (0.25Hz for 8 hours/day, 0∼9% for 6 days). Tendons without loading lost its structure integrity as evidenced by disorientated collagen fiber, increased type III collagen expression, and increased cell apoptosis. Tendons with 3% of cyclic tensile loading had moderate matrix deterioration and elevated expression levels of MMP-1, 3 and 12, whilst exceeded loading regime of 9% caused massive rupture of collagen bundle. However, 6% of cyclic tensile strain was able to maintain the structural integrity and cellular function. Our data indicated that an optimal PMS is required to maintain the tendon homeostasis and there is only a narrow range of tensile strain that can induce the anabolic action. The clinical impact of this study is that optimized eccentric training program is needed to achieve maximum beneficial effects on chronic tendinopathy management. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
    Full-text · Article · May 2013 · Biotechnology and Bioengineering
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    • "Three dimensional environments for cells have proved to be necessary to achieve in vitro significant tendon-like matrix production (Garvin et al., 2003), and such 3D scaffolds are also likely to make part of any successful in vivo strategy. Synthetic materials available for scaffold fabrication belong to the standard families of polyesthers (polylactic and polyglycolic acid and their copolymers, polycaprolactone), and they have been assayed with different morphologies, including knitted and electrospun versions (Ouyang et al., 2003; Guo & Spector, 2006; Thorfinn et al., 2010; Ladd et al., 2011; Jiang et al., 2011). None of these synthetic materials, however, possesses mechanical properties approaching those of natural tendon; the less so when they have a porous or microfibrilar structure. "

    Full-text · Chapter · Aug 2011
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