Topographical cell guidance has been utilized as a tissue-engineering technique to produce aligned cellular orientation in the regeneration of tendon- and ligament-like tissues. Other studies have investigated the effects of dynamic culture to achieve the same end. These works have, however, been limited to two-dimensional cultures, with focus given to the effects from the stimuli independently. The understanding of their combined effects in the tenogenic differentiation of mesenchymal stem cells (MSCs) has also been lacking. This study investigated the synergistic effects of mechanical stimulation on aligned MSCs in a three-dimensional (3D) aligned silk fibroin (SF) hybrid scaffold. Enhanced tenogenesis of seeded MSCs was observed in the scaffold group with aligned SF electrospun fibers (AL) under static culture conditions, as evidenced by the upregulation in expression and production of tendon/ligament-related proteins. The intensity and onset of these differentiative markers were increased and advanced, respectively, under dynamic culture conditions, indicative of an accelerated matrix deposition and remodeling process. Consequently, the tensile properties of dynamically cultured AL were significantly improved. We thus propose that the aligned hybrid SF scaffold facilitates mechanoactivity and tenogenic differentiation of MSCs by intensifying the positive effects of mechanical stimulation in a 3D environment.

The concept of contact guidance utilizes the phenomenon of anchorage dependence of cells on the topography of seeded surfaces. It has been shown in previous studies that cells were guided to align along the topographical alignment of the seeding substrate and produced enhanced amounts of oriented extracellular matrix (ECM). In this study, we aimed to apply this concept to a three-dimensional full silk fibroin (SF) hybrid scaffold system, which comprised of knitted SF and aligned SF electrospun fibers (SFEFs), for ligament tissue engineering applications. Specifically, knitted SF, which contributed to the mechanical robustness of the system, was integrated with highly aligned SFEF mesh, which acted as the initial ECM to provide environmental cues for positive cellular response. Mesenchymal stem cells seeded on the aligned hybrid scaffolds were shown to be proliferative and aligned along the integrated aligned SFEF, forming oriented spindle-shaped morphology and produced an aligned ECM network. Expression and production of ligament-related proteins were also increased as compared to hybrid SF scaffolds with randomly arranged SFEFs, indicating differentiative cues for ligament fibroblasts present in the aligned hybrid SF scaffolds. Consequently, the tensile properties of cultured aligned constructs were significantly improved and superior to the counterpart with randomly arranged SFEF. These results thus show that the aligned hybrid scaffold system is promising for enhancing cell proliferation, differentiation, and function for ligament tissue engineering applications.

In the process of removing sericin (degumming) from a raw silk scaffold, the fibroin structural integrity is often challenged, leading to mechanical depreciation. This study aims to identify the factors and conditions contributing to fibroin degradation during alkaline degumming and to perform an optimization study of the parameters involved to achieve preservation of fibro in structure and properties. The methodology involves degumming knitted silk scaffolds for various durations (5-90 min) and temperatures (60-100 ◦C). Mechanical agitation and use of the refreshed solution during degumming are included to investigate how these factors contribute to degumming efficiency and fibroin preservation. Characterizations of silk fibroin morphology, mechanical properties and protein components are determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS–PAGE),respectively. Sericin removal is ascertained via SEM imaging and a protein fractionation method involving SDS–PAGE. The results show that fibroin fibrillation, leading to reduced mechanical integrity, is mainly caused by prolonged degumming duration. Through a series of optimization, knitted scaffolds are observed to be optimally degummed and experience negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 ◦C.

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