Thomas Kok Hiong Teh

Aligned hybrid silk scaffold for enhanced differentiation of mesenchymal stem cells into ligament fibroblasts.

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.

Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties

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.

In vitro generation of a multilayered osteochondral construct with an osteochondral interface using rabbit bone marrow stromal cells and a silk peptide-based scaffold.

Tissue engineering of a biological osteochondral multilayered construct with a cartilage‐interface subchondral bone layer is a key challenge. This study presented a rabbit bone marrow stromal cell (BMSC)/silk fibroin scaffold‐based co‐culture approach to generate tissue‐engineered osteochondral grafts with an interface. BMSC‐seeded scaffolds were first cultured separately in osteogenic and chondrogenic stimulation media. The two differentiated pieces were then combined using an RADA self‐assembling peptide and subsequently co‐cultured. Gene expression, histological and biochemical analyses were used to evaluate the multilayered structure of the osteochondral graft. A complete osteochondral construct with a cartilage‐subchondral bone interface was regenerated and BMSCs were used as the only cell source for the osteochondral construct and interface regeneration. Furthermore, in the intermediate region of co‐cultured samples, hypertrophic chondrogenic gene markers type X collagen and MMP‐13 were found on both chondrogenic and osteogenic section edges after co‐culture. However, significant differences gene expression profile were found in distinct zones of the construct during co‐culture and the section in the intermediate region had significantly higher hypertrophic chondrocyte gene expression. Biochemical analyses and histology results further supported this observation. This study showed that specific stimulation from osteogenic and chondrogenic BMSCs affected each other in this co‐culture system and induced the formation of an osteochondral interface. Moreover, this system provided a possible approach for generating multilayered osteochondral constructs. Copyright

Novel Silk Scaffolds for Ligament Tissue Engineering Applications

Scaffold technology is integral in advancing tissue engineering and one of the tissues of interest here is the tendon/ligament. Advancement in the tissue engineering of tendon/ligament has become very much a materials engineering problem than ever, with the selection of appropriate biomaterial and scaffold architecture. Such is the key to successful tendon/ligament tissue regeneration construct. Popular materials used in recent years include various poly (l-lactic) biomaterials and collagen. However, shortcomings of these materials, in terms of poor mechanical strength or short degradation period, are yet overcome. Bombyx mori silk, though used in biomedical sutures for decades due to its excellent mechanical properties, has been overlooked for applications in ligament tissue engineering, only until recently. This is largely due to previous misconceptions in its biocompatibility and biodegradability characteristics. This paper describes the use of a silk-based scaffold with knitted architecture and investigates its strengths as compared to previous PLGA-based knitted scaffolds. An electrospun nanofiber surface on knitted microfiber architecture is adopted and it is found to have better composite-material integrity, in vitro degradation resistance, and encourages cell adhesion and proliferation.

Controlled Bioactive Molecules Delivery Strategies for Tendon and Ligament Tissue Engineering using Polymeric Nanofibers

The interest in polymeric nanofibers has escalated over the past decade given its promise as tissue engineering scaffolds that can mimic the nanoscale structure of the native extracellular matrix. With functionalization of the polymeric nanofibers using bioactive molecules, localized signaling moieties can be established for the attached cells, to stimulate desired biological effects and direct cellular or tissue response. The inherently high surface area per unit mass of polymeric nanofibers can enhance cell adhesion, bioactive molecules loading and release efficiencies, and mass transfer properties. In this review article, the application of polymeric nanofibers for controlled bioactive molecules delivery will be discussed, with a focus on tendon and ligament tissue engineering. Various polymeric materials of different mechanical and degradation properties will be presented along with the nanofiber fabrication techniques explored. The bioactive molecules of interest for tendon and ligament tissue engineering, including growth factors and small molecules, will also be reviewed and compared in terms of their nanofiber incorporation strategies and release profiles. This article will also highlight and compare various innovative strategies to control the release of bioactive molecules spatiotemporally and explore an emerging tissue engineering strategy involving controlled multiple bioactive molecules sequential release. Finally, the review article concludes with challenges and future trends in the innovation and development of bioactive molecules delivery using polymeric nanofibers for tendon and ligament tissue engineering.

Characterization of Electrospun Substrates for Ligament Regeneration using Bone Marrow Stromal Cells

Challenges persist in the tissue engineering and regeneration of ligaments. Of the various aspects contributing to the success of ligament tissue engineering (such as the scaffold, cell source and stimulatory biochemical and mechanical cues), architecture and material involved in scaffold design remain as a significant area of study. Essentially, the scaffold for ligament regeneration, especially the cell-seed substrate, should be viable for cell attachment, promotes nutrient and waste transfer, be mechanically viable, and stimulates initial cell proliferation and ECM production. In this study, electrospun substrates using the silk fibroin (SF) and PLGA material, with different electrospun fiber arrangements (aligned (AL) and random (RD)) were compared for their ability to promote MSC attachment and subsequent differentiation down the ligament fibroblast cell lineage. The rationale for such characterizations lies in the hypothesis that SF, being a natural protein, provides significantly more favorable surface chemistry for cell attachment and differentiation than synthetic polymers such as PLGA. On the other hand, the aligned electrospun fiber arrangement will mimic the native ligament ECM environment, to guide MSCs down the fibroblast lineage. Electrospun PLGA and SF substrates were fabricated by first dissolving the respective material in HFIP (7–10% w/v). Following which, aligned PLGA and SF substrates were collected from a rotational electrospin setup, while the random types were collected from a grounded plate. These four groups of substrates (SF-AL, SF-RD, PLGA-AL, PLGA-RD) were then characterized in terms of cell adhesion, proliferation, viability and function via post-seed cell counting, AlamarBlueTM assay, Fluorescence Microscopy, Sircol® Collagen Assay and SEM. Results from this characterization show that cells remained viable when cultured on these substrates, with the AL types being mechanically stronger and promoted increased proliferation and collagen production as compared to the RD types. SF substrates promoted cell attachment and differentiation, as inferred from the increased collagen production.

Ligament-to-bone Interface Tissue Regeneration Using a Functionalized Biphasic Silk Fibroin Scaffold

The emergence of the tissue engineering approach has shown to be a game changer in ligament reconstruction, making it possible to create multi-phasic scaffold structures for multi-specific tissue regeneration. To regenerate the bone-ligament-bone tissue, mimicking hard to soft tissue transition, we propose the use of a functionalized biphasic silk scaffold. Hydroxyapatite nanoparticles (nHA) and bone morphogenetic protein 2 (BMP2) were loaded in the ends of Bombyx mori silk fibroin (SF) scaffold system to enhance enthesis regeneration and bone tunnel healing, while the central onethird supported ligament regeneration. Two groups of biphasic scaffolds, distinguished by the different ends’ additive, were fabricated: nHA only (Ctrl) and n-HA/BMP2 (Exp). A series of bench work, small animal study and large animal preclinical trial was performed using MSC-seeded constructs for the reconstruction of excised ACL. The bioactivity of BMP2 was ascertained and shown to be eluting with an initial burst, followed by a lowered sustained release. Osteogenic genes were upregulated in both groups compared to pure SF. By 24 weeks in vivo, the ACL was regenerated and bone tunnel narrowing was observed with histological evidences indicating new bone and enthesis regeneration in Exp. Better graft to bone integration was observed in Exp compared to Ctrl. From this study, it was demonstrated that the BMP2 eluting biphasic silk scaffold is promising as an advanced tissue engineering treatment modality for complete bone-ligament-bone reconstruction.

Stem cell-derived cell-sheets for connective tissue engineering

ABSTRACT Cell-sheet technology involves the recovery of cells with its secreted ECM and cell–cell junctions intact, and thereby harvesting them in a single contiguous layer. Temperature changes coupled with a thermoresponsive polymer grafted culture plate surface are typically used to induce detachment of this cell–matrix layer by controlling the hydrophobicity and hydrophilicity properties of the culture surface. This review article details the genesis and development of this technique as a critical tissue-engineering tool, with a comprehensive discussion on connective tissue applications. This includes applications in the myocardial, vascular, cartilage, bone, tendon/ligament, and periodontal areas among others discussed. In particular, further focus will be given to the use of stem cells-derived cell-sheets, such as those involving bone marrow-derived and adipose tissue-derived mesenchymal stem cells. In addition, some of the associated challenges faced by approaches using stem cells-derived cell-sheets will also be discussed. Finally, recent advances pertaining to technologies forming, detaching, and manipulating cell-sheets will be covered in view of the potential impact they will have on shaping the way cell-sheet technology will be utilized in the future as a tissue-engineering technique.