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Title: Tissue Engineering of Ligament through Rehabilitative Mechanical Conditioning of Mechano-active Hybrid Silk Scaffolds
Keywords: Ligament, Tissue Engineering, Silk, Scaffold, Mechano-active, Mechanical Conditioning
Issue Date: 5-Jan-2011
Citation: TEH KOK HIONG THOMAS (2011-01-05). Tissue Engineering of Ligament through Rehabilitative Mechanical Conditioning of Mechano-active Hybrid Silk Scaffolds. ScholarBank@NUS Repository.
Abstract: The use of appropriate mechanically viable scaffold and the provision of appropriate biophysical environment has always been one of the keys to successful regeneration of ligament tissues. With the aim to biomimic the native environment, an aligned hybrid silk fibroin (SF) scaffold and a rehabilitative mechanical conditioning regime were studied. It was hypothesized that the mechano-active hybrid SF scaffold (AL) consisting of knitted SF integrated with aligned SF electrospun fibers (AL-SFEF) could enhance tissue regeneration by first promoting cellular alignment, which in turn facilitated effective mechano-transduction when the cell-seeded AL scaffolds were mechanically conditioned rehabilitatively. The study was grouped into four stages: (i) design and development of the SF knit, (ii) development of the AL scaffold, (iii) in vitro characterization of the AL scaffold, and (iv) rehabilitative mechanical conditioning of the AL scaffolds. The first stage involved evaluation of the SF mechanical properties as an initial step to the design of the SF knit. Upon selecting the mechanical properties of the optimally degummed SF fibers, design of the SF knit revealed that 240 SF count was necessary. The designed silk knits were subsequently optimally degummed for overall structural/mechanical properties retention and effective sericin removal. The second stage then involved electrospinning SFEF meshes and physically incorporating them to the knitted SF. Highly aligned SFEF meshes were obtained by using a customized electrospinning setup. The meshes were subsequently integrated physically with the SF knit via sequential and localized application of methanol to produce inherent contractile forces of the SFEF meshes. Characterization of the completed hybrid SF scaffolds revealed that the AL scaffolds had SFEF meshes well-integrated with the knitted structure and were mechanically superior. The third stage involved in vitro characterization of the AL scaffolds using rabbit mesenchymal stem cells (MSCs). It was shown that the AL scaffolds stimulated increased proliferation and collagen synthesis via providing favorable topographical conditions for cell and ECM alignment. Consequently, cells expressed up-regulation of ligament-related genes and deposition of the related ECM components, which were indicative of a differentiative phase. Mechanically superior AL constructs were obtained after 14 days of culture. These effects were intensified synergistically when the mechano-active AL scaffolds were dynamically cultured. The fourth stage involved the optimization of the mechanical stimulation approach to further enhance tenogenic differentiation. Dynamic conditioning was also performed over a longer duration to examine its prolonged effect on MSC differentiation and development in the AL hybrid SF scaffold. Leveled mechanical stimulation regimes were used to compare with the rehabilitative approach, which in contrast with level state stimulations, involved gradual application of dynamic cues with increasing intensities in terms of cyclic frequency. Through the up-regulation and deposition of ligament-related genes and ECM components, it was shown that the rehabilitative approach to dynamic conditioning AL scaffolds allowed timely introduction of appropriate stimulation intensities, which allowed early introduction of the synergistic mechanical cues to the MSC-seeded mechano-active AL scaffold to effect an accelerated tenogenic profile.
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