Stem cell-derived cell-sheets for connective tissue engineering.

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.

Anisotropic silk fibroin/gelatin scaffolds from unidirectional freezing.

Recent studies have underlined the importance of matching scaffold properties to the biological milieu. Tissue, and thus scaffold, anisotropy is one such property that is important yet sometimes overlooked. Methods that have been used to achieve anisotropic scaffolds present challenges such as complicated fabrication steps, harsh processing conditions and toxic chemicals involved.

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.

Characterization and mechanical performance study of silk/PVA cryogels: towards nucleus pulposus tissue engineering.

Poly (vinyl) alcohol (PVA) cryogels are reported in the literature for application in nucleus pulposus (NP) replacement strategies. However, these studies are mainly limited to acellular approaches-in part due to the high hydrophilicity of PVA gels that renders cellular adhesion difficult. Silk is a versatile biomaterial with excellent biocompatibility.

Enhancing analysis of cells and proteins by fluorescence imaging on silk-based biomaterials: modulating the autofluorescence of silk.

Silk is a versatile and established biomaterial for various tissue engineering purposes. However, it also exhibits strong autofluorescence signals-thereby hindering fluorescence imaging analysis of cells and proteins on silk-derived biomaterials. Sudan Black B (SB) is a lysochrome dye commonly used to stain lipids in histology.

Three-dimensional spatial configuration of tumour cells confers resistance to chemotherapy independent of drug delivery.

Anticancer drug discovery has been hampered by the lack of reliable preclinical models, which routinely use cells grown in two-dimensional (2D) culture systems. However, many of the characteristics of cells in 2D culture do not translate into the findings in animal xenografts. Three-dimensional (3D) growth may be responsible for some of these changes, and models using cells grown in 3D may form a more representative step in tumouricidal validation prior to animal implantation and human testing.

The dominant role of IL-8 as an angiogenic driver in a three-dimensional physiological tumor construct for drug testing.

The induction of angiogenesis and the promotion of tumor growth and invasiveness are processes critical to metastasis, and are dependent on reciprocal interactions between tumor cells and their microenvironment. The formation of a clinically relevant tumor requires support from the surrounding stroma, and it is hypothesized that three-dimensional (3D) tumor coculture models offer a microenvironment that more closely resembles the physiological tumor microenvironment. In this study, we investigated the effects of tissue-engineered 3D architecture and tumor-stroma interaction on the angiogenic factor secretion profiles of U2OS osteosarcoma cells by coculturing the tumor cells with immortalized fibroblasts or human umbilical vein endothelial cells (HUVECs).

A novel compact compliant actuator design for rehabilitation robots.

Rehabilitation robots have direct physical interaction with human body. Ideally, actuators for rehabilitation robots should be compliant, force controllable, and back drivable due to safety and control considerations. Various designs of Series Elastic Actuators (SEA) have been developed for these applications.

Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets.

Cell-sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold-free constructs. Adipose tissue-derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow-derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC-CSs) is non-existent in the literature.

In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells, employing a two-chambered co-culture well design.

The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk-RADA peptide scaffold in a specially designed two-chambered co-culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two-chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold.

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.

Aligned fibrous scaffolds for enhanced mechanoresponse and tenogenesis of mesenchymal stem cells.

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.

Enhanced osteoinductivity and osteoconductivity through hydroxyapatite coating of silk-based tissue-engineered ligament scaffold.

Hybrid silk scaffolds combining knitted silk fibers and silk sponge have been recently developed for use as ligament-alone grafts. Incorporating an osteoinductive phase into the ends of a ligament scaffold may potentially generate an integrated "bone-ligament-bone" graft and improve graft osteointegration with host bone. To explore the possible application of hydroxyapatite (HA) coating in the fabrication of osteoinductive ends of silk-based scaffold, HA was coated on the hybrid silk scaffold and the effects to the bone-related cells were evaluated.

In vitro ligament-bone interface regeneration using a trilineage coculture system on a hybrid silk scaffold.

The ligament-bone interface is a complex structure that comprises ligament, fibrocartilage, and bone. We hypothesize that mesenchymal stem cells cocultured in between ligament and bone cells, on a hybrid silk scaffold with sections suitable for each cell type, would differentiate into fibrocartilage. The section of scaffold for osteoblast seeding was coated with hydroxyapatite.

Osteochondral interface generation by rabbit bone marrow stromal cells and osteoblasts coculture.

Physiological osteochondral interface regeneration is a significant challenge. This study aims to investigate the effect of the coculture of chondrogenic rabbit bone marrow stromal cells (rBMSCs) with rabbit osteoblasts in a specially designed two-dimensional (2D)-three-dimensional (3D) co-interface culture to develop the intermediate osteochondral region in vitro. The 2D-3D coculture system was set up by first independently culturing chondrogenic rBMSCs on a scaffold and osteoblasts in cell culture plates, and subsequently placed in contact and cocultured.

A hybrid silk/RADA-based fibrous scaffold with triple hierarchy for ligament regeneration.

While silk-based microfibrous scaffolds possess excellent mechanical properties and have been used for ligament tissue-engineering applications, the microenvironment in these scaffolds is not biomimetic. We hypothesized that coating a hybrid silk scaffold with an extracellular matrix (ECM)-like network of self-assembling peptide nanofibers would provide a biomimetic three-dimensional nanofibrous microenvironment and enhance ligament tissue regeneration after bone marrow-derived mesenchymal stem cell (BMSC)-seeding. A novel scaffold possessing a triple structural hierarchy comprising macrofibrous knitted silk fibers, a silk microsponge, and a peptide nanofiber mesh was developed by coating self-assembled RADA16 peptide nanofibers on a silk microfiber-reinforced-sponge scaffold.

Bioengineering education @ NUS: a design-centered curriculum.

In resonance with the NUS Mission, the aim of the Bioengineering undergraduate degree program is to produce engineers with a strong foundation in the relevant engineering, sciences and technology, who are able to contribute to the biomedical sciences through innovation, enterprise and leadership. Our educational program in Bioengineering is characterised by a strong emphasis on scientific and engineering fundamentals and a high degree of flexibility which can provide a wide diversity of educational experiences. By providing graduates with a combination of broad-based fundamentals and specialized knowledge, the Bioengineering program strives to graduate versatile engineers who would be best positioned to lead and be an integral part of the Bioengineering industries in the future.

Effects of radial compression on a novel simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell cell-sheets for annulus fibrosus regeneration.

Study Design: The aim of this study was to develop a tissue engineering approach in regenerating the annulus fibrosus (AF) as part of an overall strategy to produce a tissue-engineered intervertebral disc (IVD) replacement.

Objective: To determine whether a rehabilitative simulation regime on bone marrow–derived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF.

Summary Of Background Data: No previous study has used bone marrow–derived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF.

Simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell sheets and silk scaffolds for annulus fibrosus regeneration.

Most studies on the intervertebral disc (IVD) focus on the regeneration of the nucleus pulposus (NP). However, without a proper strategy to regenerate the damaged annulus fibrosus (AF), the NP replacements are bound to fail. Therefore the objective of this study was to investigate whether the use of bone marrow-derived mesenchymal stem cells (BMSCs) to form cell sheets, and incorporating them onto silk scaffolds, has the potential to regenerate the annulus fibrosus.

Interface tissue engineering: next phase in musculoskeletal tissue repair.

Increasing incidence of musculoskeletal injuries coupled with limitations in the current treatment options have necessitated tissue engineering and regenerative medicine- based approaches. Moving forward from engineering isolated musculoskeletal tissues, research strategies are now being increasingly focused on repairing and regenerating the interfaces between dissimilar musculoskeletal tissues with the aim to achieve seamless integration of engineered musculoskeletal tissues. This article reviews the state-of-the-art in the tissue engineering of musculoskeletal tissue interfaces with a focus on Singapore's contribution in this emerging field.

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.

PLGA nanofiber-coated silk microfibrous scaffold for connective tissue engineering.

A modified degumming technique, involving boiling in 0.25% Na2CO3 with addition of 1% sodium dodecyl sulphate and intermittent ultrasonic agitation, was developed for knitted silk scaffolds. Sericin was efficiently removed, while mechanical and structural properties of native silk fibroin were preserved.

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).

Bio-electrospraying: A potentially safe technique for delivering progenitor cells.

Bio-electrospraying is fast becoming an attractive tool for in situ cell delivery into scaffolds for tissue engineering applications, with several cell types been successfully electrosprayed. Bone marrow derived mesenchymal progenitor/stem cells (BMSC), which are an important cell source for tissue engineering, have not been explored in detail and the effect of electrospraying on their "stemness" is not known. This study therefore investigates the effects of electrospraying on BMSC viability, proliferation, and multilineage differentiation potential.

In vitro study of stem cell communication via gap junctions for fibrocartilage regeneration at entheses.

Background: Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. To replace a severely damaged ligament, a tissue-engineered graft preinstalled with interfacial fibrocartilage, which is being regenerated from stem cells, appears to be more promising than ligament-alone graft. Such a concept can be realized by a biomimetic approach of establishing a dynamic communication of stem cells with bone cells and/or ligament fibroblasts in vitro.