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. In this study, unidirectional freezing was employed to fabricate anisotropic silk fibroin/gelatin scaffolds in a simple and mild manner. Morphological, mechanical, chemical and cellular compatibility properties were investigated, as well as the effect of the addition of gelatin to certain properties of the scaffold. It was shown that scaffold properties were suitable for cell proliferation and that mesenchymal stem cells were able to align themselves along the directed fibers. The fabricated scaffolds present a platform that can be used for anisotropic tissue engineering applications such as cardiac patches.

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. 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.

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. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC-CSs development; and (b) the multipotentiality of ASC-CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid-supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC-CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT-PCR and histology at multiple time points. ASC-CSs remained healthy up to 3 weeks of hyperconfluent culture. One week-old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non-hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC-CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.

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. For the 3D model, we cultured 143.98.2, SaOS2 or U2OS osteosarcoma cells seeded in porous Bombyx mori silk sponges. We conducted real-time PCR on cells grown in 2D culture and 3D scaffolds for the proliferation markers cyclin B1 and E2F1 and the actin regulator RhoA, and found a significant decrease in expression levels for the 3D tumour models (p = 0.02, < 0.001 and 0.008 for cyclin B1, E2F1 and RhoA for 143.98.2; p = 0.02, 0.002 and 0.02 for cyclin B1, E2F1 and RhoA for U2OS, respectively). In contrast, p21 was upregulated when SaOS2 and U2OS were cultured in the 3D scaffolds (p < 0.001) and there was no increase in DNA quantity during the culture period. We correspondingly observed G1 arrest when cell cycle analysis was conducted. Cytotoxicity results for cells treated with serial dilutions of doxorubicin and cisplatin showed that cells in 3D scaffolds were less sensitive to drug treatment than in 2D culture, and the difference was more pronounced for cell cycle specific agents. Copyright © 2013 John Wiley & Sons, Ltd.

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.

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. We demonstrated that this co-culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage-like and subchondral bone-like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single-step approach that highlights the feasibility of rabbit BMSCs as a single-cell source for multilayered osteochondral construct generation in vitro.

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.

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. It has also been reported to be able to quench autofluorescence of tissues in histology and has been tested on artificial biomedical polymers in recent years. It was hypothesized that SB would exert similar quenching effects on silk, modulating the autofluorescence signals, and thereby enabling improved imaging analysis of cells and molecules of interests. The quenching effect of SB on the intrinsic fluorescence properties of silk and on commercial fluorescent dyes were first investigated in this study. SB was then incorporated into typical fluorescence-based staining protocols to study its effectiveness in improving fluorescence-based imaging of the cells and proteins residing with the silk-based biomaterials. Silk processed into various forms of biomaterials (e.g., films, sponges, fibers, and electrospun mats) was seeded with cells and cultured in vitro. At sacrificial time points, specimens were harvested, fixed, and prepared for fluorescence staining. SB, available commercially as a powder, was dissolved in 70% ethanol (0.3% [w/v]) to form staining solutions. SB treatment was introduced at the last step of typical immunofluorescence staining protocols for 15-120 min. For actin staining protocols by phalloidin toxin, SB staining solutions were added before and after permeabilization with Triton-X for 15-30 min. Results showed that ideal SB treatment duration is about 15 min. Apart from being able to suppress the autofluorescence of silk, this treatment duration was also not too long to adversely affect the fluorescent labeling probes used. The relative improvement brought about by SB treatment was most evident in the blue and green emission wavelengths compared with the red emission wavelength. This study has showed that the use of SB is a cost and time effective approach to enhance fluorescence-based imaging analyses of cell-seeded silk biomaterials, which otherwise would have been hindered by the unmodulated autofluorescence signals.

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. We hypothesize that the incorporation of silk with PVA will (i) improve the cell-hosting abilities of PVA cryogels and (ii) allow better tailoring of physical properties of the composite cryogels for an NP tissue engineering purpose. 5% (wt/vol) PVA is blended with 5% silk fibroin (wt/vol) to investigate the effect of silk : PVA ratios on the cryogels' physical properties. Results show that the addition of silk results in composite cryogels that are able to swell to more than 10 times its original dry weight and rehydrate to at least 70% of its original wet weight. Adding at least 20% silk significantly improves surface hydrophobicity and is correlated with an improvement in cell-hosting abilities. Cell-seeded cryogels also display an increment in compressive modulus and hoop stress values. In all, adding silk to PVA creates cryogels that can be potentially used as NP replacements.

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). We also carried out Transwell migration assays for U2OS cells grown in monoculture or fibroblast coculture systems to study the physiological effect of upregulated angiogenic factors on endothelial cell migration. Anti-IL-8 and anti-vascular endothelial growth factor (VEGF)-A therapies were tested out on these models to investigate the role of 3D culture and the coculture of tumor cells with immortalized fibroblasts on the efficacy of antiangiogenic treatments. The coculture of U2OS cells with immortalized fibroblasts led to the upregulation of IL-8 and VEGF-A, especially in 3D culture. Conversely, coculture with endothelial cells resulted in the downregulation of VEGF-A for cells seeded in 3D scaffolds. The migration of HUVECs through the Transwell polycarbonate inserts increased for the 3D and immortalized fibroblast coculture models, and the targeted inhibition of IL-8 greatly reduced HUVEC migration despite the presence of VEGF-A. A similar effect was not observed when anti-VEGF-A neutralizing antibody was used instead, suggesting that IL-8 plays a more critical role in endothelial cell migration than VEGF-A, with significant implications on the clinical utility of antiangiogenic therapy targeting VEGF-A.

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. However, current SEA designs face a common performance limitation due to the compromise on the spring stiffness selection. This paper presents a novel compact compliant force control actuator design for portable rehabilitation robots to overcome the performance limitations in current SEAs. Our design consists of a servomotor, a ball screw, a torsional spring between the motor and the ball screw, and a set of translational springs between the ball screw nut and the external load. The soft translational springs are used to handle the low force operation and reduce output impedance, stiction, and external shock load. The torsional spring, being in the high speed range, has high effective stiffness and improves the system bandwidth in large force operation when the translational springs are fully compressed. This design is also more compact due to the smaller size of the springs. We explain the construction and the working principle of our new design, followed by the dynamic modeling and analysis of the actuator. We also show the preliminary testing results of a prototype actuator designed for a lower limb exoskeleton for gait rehabilitation.

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. 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.

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. HA could be coated in a uniform and controlled manner on the silk sponge, using an alternate soaking technology, with the amount deposited being dependent on the number of soaking cycles. HA coating also progressively reduced the hydrophobicity of silk surface (decreasing water contact angle from 87° to 42-76°, after 1-3 soaking cycles), making the HA-coated silk scaffold less favorable for initial cell attachments; but the attached cells showed viability and sustained proliferation on the HA-coated scaffold. As demonstrated by real-time polymerase chain reaction and alkaline phosphatase assay, the osteoinductivity of HA-coated silk scaffolds resulted in the osteogenic differentiation of bone marrow mesenchymal stem cells, and the osteoconductivity of HA-coated silk scaffolds supported osteoblasts growth and maintained the properties of mature osteoblasts. These properties of HA-coating demonstrated its possible application in fabricating osteoinductive ends of the silk-based ligament graft to potentially enhance graft-to-host bone integration.

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. A trilineage coculture system (osteoblasts-BMSCs-fibroblasts) on a hybrid silk scaffold was established. RT-PCR results and immunohistochemistry results demonstrated that BMSCs cocultured between fibroblasts and osteoblasts had differentiated into the fibrocartilaginous lineage. The morphological change was also observed by SEM observation. A gradual transition from the uncalcified to the calcified region was formed in the cocultured BMSCs from the region that directly interacted with fibroblasts to the region that directly interacted with osteoblasts. The role of transforming growth factor ß3 (TGF-ß3) in this trilineage coculture model was also investigated by supplementing the coculture system with 10 ng/mL TGF-ß3. The TGF-treated group showed similar results of fibrocartilaginous differentiation of BMSCs with coculture group without TGF-ß3 supplement. However, no calcium deposition was found in the cocultured BMSCs in the TGF-treated group. This may indicate TGF-ß3 delayed the mineralization process of chondrocytes.

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. As control, samples not cocultured with osteoblasts were used. The regulatory effects exerted by osteoblasts on chondrogenic rBMSCs were quantified by real-time polymerase chain reaction. To study the effect of coculture on cells located in different parts of the scaffold, samples were separated into two parts and significantly different gene expression patterns were found between them. In comparison with the control group, a significant moderate downregulation of chondrogenic marker genes, such as Collagen II and Aggrecan was observed. However, the Sox-9 and Collagen I expression increased. More importantly, chondrogenic rBMSCs in the coculture system were shown to form the osteochondral interface layer by expressing calcified cartilage zone specific extracellular matrix marker Collagen X and the hypertrophic chondrocyte marker MMP-13, which were not observed in the control group. Specifically, only the chondrogenic rBMSC layer in contact with the osteoblasts expressed Collagen X and MMP-13, indicating the positive influence of the coculture upon interface formation. Biochemical analyses, histology results, and immunohistochemical staining further supported this observation. In conclusion, this study revealed that specific regulatory stimulations from osteoblasts in the 2D-3D interface coculture system could induce the formation of ostochondral interface for the purpose of osteochondral tissue engineering.

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. Compared with the uncoated control, RADA-coated scaffolds showed enhanced BMSC proliferation, metabolism, and fibroblastic differentiation during the 3 weeks of culture. BMSC-seeded RADA-coated scaffolds showed an increasing temporal expression of key fibroblastic ECM proteins (collagen type I and III, tenascin-C), with a significantly higher tenascin-C expression compared with the controls. BMSC-seeded RADA-coated scaffolds also showed a temporal increase in total collagen and glycosaminoglycan production (the amount produced being higher than in control scaffolds) during 3 weeks of culture, and possessed 7% higher maximum tensile load compared with the BMSC-seeded control scaffolds. The results indicate that the BMSC-seeded RADA-coated hybrid silk scaffold system has the potential for use in ligament tissue-engineering applications.

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. The BMSC cell sheets and silk scaffolds were wrapped around a silicone NP substitute to form a simulated IVD-like assembly. The simulated IVD-like assembly was cultured for 4 weeks in static conditions and it was shown that the BMSC cell sheets remained viable, with no significant change in cell numbers. Histological analysis showed that the BMSC cell sheets adhered well onto the silk scaffolds and glycosaminoglycans (GAGs) were detected within the extracellular matrix (ECM). The ratio of collagen type I to collagen type II within the ECM of the BMSC cell sheets also decreased significantly over the period of culture. The results suggested that extensive remodelling of the ECM occurred within the simulated IVD-like assembly, and it is suitable for the regeneration of the inner AF.

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 marrow­derived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF. SUMMARY OF BACKGROUND DATA: No previous study has used bone marrow­derived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF. METHODS: The approach was to use bone marrow­derived stem cells to form cell-sheets and incorporating them onto silk scaffolds to simulate the native lamellae of the AF. The in vitro experimental model used to study the efficacy of such a system was made up of the tissue engineering AF construct wrapped around a silicone disc to form a simulated IVD-like assembly. The assembly was cultured within a custom-designed bioreactor that provided a compressive mechanical stimulation onto the silicone disc. The silicone nucleus pulposus would bulge radially and compress the simulated AF to mimic the physiological conditions. The simulated IVD-like assembly was compressed using a rehabilitative regime that lasted for 4 weeks at 0.25 Hz, for 15 minutes each day. RESULTS: With the rehabilitative regime, the cell-sheets remained viable but showed a decrease in cell numbers and viability. Gene expression analysis showed significant upregulation of IVD-related genes and there was an increased ratio of collagen type II to collagen type I found within the extracellular matrix. CONCLUSION: The results suggested that a rehabilitative regime caused extensive remodeling to take place within the simulated IVD-like assembly, producing extracellular matrix similar to that found in the inner AF.

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. Various biomimetic scaffold and cellbased strategies, the use of growth factors, gene therapy and mechanical loading, as well as animal models for functional validation of the tissue engineering strategies are discussed.

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.