Tracking single adatoms in liquid in a transmission electron microscope.

Single atoms or ions on surfaces affect processes from nucleation1 to electrochemical reactions2 and heterogeneous catalysis3. Transmission electron microscopy is a leading approach for visualizing single atoms on a variety of substrates4,5. It conventionally requires high vacuum conditions, but has been developed for in situ imaging in liquid and gaseous environments6,7 with a combined spatial and temporal resolution that is unmatched by any other method-notwithstanding concerns about electron-beam effects on samples. When imaging in liquid using commercial technologies, electron scattering in the windows enclosing the sample and in the liquid generally limits the achievable resolution to a few nanometres6,8,9. Graphene liquid cells, on the other hand, have enabled atomic-resolution imaging of metal nanoparticles in liquids10. Here we show that a double graphene liquid cell, consisting of a central molybdenum disulfide monolayer separated by hexagonal boron nitride spacers from the two enclosing graphene windows, makes it possible to monitor, with atomic resolution, the dynamics of platinum adatoms on the monolayer in an aqueous salt solution. By imaging more than 70,000 single adatom adsorption sites, we compare the site preference and dynamic motion of the adatoms in both a fully hydrated and a vacuum state. We find a modified adsorption site distribution and higher diffusivities for the adatoms in the liquid phase compared with those in vacuum. This approach paves the way for in situ liquid-phase imaging of chemical processes with single-atom precision.

Antitumor Immunity Mediated by Photodynamic Therapy Using Injectable Chitosan Hydrogels for Intratumoral and Sustained Drug Delivery.

Photodynamic therapy (PDT) is an anticancer therapy with proven efficacy; however, its application is often limited by prolonged skin photosensitivity and solubility issues associated with the phototherapeutic agents. Injectable hydrogels which can effectively provide intratumoral delivery of photosensitizers with sustained release are attracting increased interest for photodynamic cancer therapies. However, most of the hydrogels for PDT applications are based on systems with high complexity, and often, preclinical validation is not provided. Herein, we provide a simple and reliable pH-sensitive hydrogel formulation that presents appropriate rheological properties for intratumoral injection. For this, Temoporfin (m-THPC), which is one of the most potent clinical photosensitizers, was chemically modified to introduce functional groups that act as cross-linkers in the formation of chitosan-based hydrogels. The introduction of -COOH groups resulted in a water-soluble derivative, named PS2, that was the most promising candidate. Although PS2 was not internalized by the target cells, its extracellular activation caused effective damage to the cancer cells, which was likely mediated by lipid peroxidation. The injection of the hydrogel containing PS2 in the tumors was monitored by high-frequency ultrasounds and in vivo fluorescence imaging which confirmed the sustained release of PS2 for at least 72 h. Following local administration, light exposure was conducted one (single irradiation protocol) or three (multiple irradiation protocols) times. The latter delivered the best therapeutic outcomes, which included complete tumor regression and systemic anticancer immune responses. Immunological memory was induced as ∼75% of the mice cured with our strategy rejected a second rechallenge with live cancer cells. Additionally, the failure of PDT to treat immunocompromised mice bearing tumors reinforces the relevance of the host immune system. Finally, our strategy promotes anticancer immune responses that lead to the abscopal protection against distant metastases.

Highly Enhanced Chloride Adsorption Mediates Efficient Neutral CO2 Electroreduction over a Dual-Phase Copper Catalyst.

Electrocatalytic carbon dioxide reduction (CO2R) in neutral electrolytes can mitigate the energy and carbon losses caused by carbonate formation but often experiences unsatisfied multicarbon selectivity and reaction rates because of the kinetic limitation to the critical carbon monoxide (CO)-CO coupling step. Here, we describe that a dual-phase copper-based catalyst with abundant Cu(I) sites at the amorphous-nanocrystalline interfaces, which is electrochemically robust in reducing environments, can enhance chloride-specific adsorption and consequently mediate local *CO coverage for improved CO-CO coupling kinetics. Using this catalyst design strategy, we demonstrate efficient multicarbon production from CO2R in a neutral potassium chloride electrolyte (pH ∼6.6) with a high Faradaic efficiency of 81% and a partial current density of 322 milliamperes per square centimeter. This catalyst is stable after 45 h of operation at current densities relevant to commercial CO2 electrolysis (300 mA per square centimeter).

Substrate Stiffness Modulates the Crosstalk Between Mesenchymal Stem Cells and Macrophages.

Upon implantation of a biomaterial, mesenchymal stem cells (MSCs) and macrophages contribute to the wound healing response and the regeneration cascade. Although biomaterial properties are known to direct MSC differentiation and macrophage polarization, the role of biomaterial cues, specifically stiffness, in directing the crosstalk between the two cell types is still poorly understood. This study aimed to elucidate the role of substrate stiffness in modulating the immunomodulatory properties of MSCs and to shed light on their complex interactions with macrophages when presented with diverse biomaterial stiffness cues, a situation analogous to the implant environment where multiple cell types interact with an implanted biomaterial to determine regenerative outcomes. We show that MSCs do not play an immunomodulatory role in the absence of an inflammatory stimulus. Using collagen-coated polyacrylamide gels of varying stiffness values, we demonstrate that the immunomodulatory capability of MSCs in the presence of an inflammatory stimulus is not dependent on the stiffness of the underlying substrate. Moreover, using paracrine and direct contact culture models, we show that a bidirectional crosstalk between MSCs and macrophages is necessary for promoting anti-inflammatory responses and positive immunomodulation, which is dependent on the stiffness of the underlying substrate. We finally show that direct cell-cell contact is not essential for this effect, with paracrine interactions promoting immunomodulatory interactions between MSCs and macrophages. Together, these results demonstrate that biophysical cues such as stiffness that are presented by biomaterials can be tuned to promote positive interactions between MSCs and macrophages which can in turn direct the downstream regenerative response.

Atomic Resolution Imaging of CrBr3 Using Adhesion-Enhanced Grids.

Suspended specimens of 2D crystals and their heterostructures are required for a range of studies including transmission electron microscopy (TEM), optical transmission experiments, and nanomechanical testing. However, investigating the properties of laterally small 2D crystal specimens, including twisted bilayers and air-sensitive materials, has been held back by the difficulty of fabricating the necessary clean suspended samples. Here we present a scalable solution that allows clean free-standing specimens to be realized with 100% yield by dry-stamping atomically thin 2D stacks onto a specially developed adhesion-enhanced support grid. Using this new capability, we demonstrate atomic resolution imaging of defect structures in atomically thin CrBr3, a novel magnetic material that degrades in ambient conditions.

Raman Fingerprints of Graphene Produced by Anodic Electrochemical Exfoliation.

Electrochemical exfoliation is one of the most promising methods for scalable production of graphene. However, limited understanding of its Raman spectrum as well as lack of measurement standards for graphene strongly limit its industrial applications. In this work, we show a systematic study of the Raman spectrum of electrochemically exfoliated graphene, produced using different electrolytes and types of solvents in varying amounts. We demonstrate that no information on the thickness can be extracted from the shape of the 2D peak as this type of graphene is defective. Furthermore, the number of defects and the uniformity of the samples strongly depend on the experimental conditions, including postprocessing. Under specific conditions, the formation of short conductive trans-polyacetylene chains has been observed. Our Raman analysis provides guidance for the community on how to get information on defects coming from electrolyte, temperature, and other experimental conditions, by making Raman spectroscopy a powerful metrology tool.

Solid-State Phase Transformation and Self-Assembly of Amorphous Nanoparticles into Higher-Order Mineral Structures.

Materials science has been informed by nonclassical pathways to crystallization, based on biological processes, about the fabrication of damage-tolerant composite materials. Various biomineralizing taxa, such as stony corals, deposit metastable, magnesium-rich, amorphous calcium carbonate nanoparticles that further assemble and transform into higher-order mineral structures. Here, we examine a similar process in abiogenic conditions using synthetic, amorphous calcium magnesium carbonate nanoparticles. Applying a combination of high-resolution imaging and in situ solid-state nuclear magnetic resonance spectroscopy, we reveal the underlying mechanism of the solid-state phase transformation of these amorphous nanoparticles into crystals under aqueous conditions. These amorphous nanoparticles are covered by a hydration shell of bound water molecules. Fast chemical exchanges occur: the hydrogens present within the nanoparticles exchange with the hydrogens from the surface-bound H2O molecules which, in turn, exchange with the hydrogens of the free H2O molecule of the surrounding aqueous medium. This cascade of chemical exchanges is associated with an enhanced mobility of the ions/molecules that compose the nanoparticles which, in turn, allow for their rearrangement into crystalline domains via solid-state transformation. Concurrently, the starting amorphous nanoparticles aggregate and form ordered mineral structures through crystal growth by particle attachment. Sphere-like aggregates and spindle-shaped structures were, respectively, formed from relatively high or low weights per volume of the same starting amorphous nanoparticles. These results offer promising prospects for exerting control over such a nonclassical pathway to crystallization to design mineral structures that could not be achieved through classical ion-by-ion growth.

The identification of articular cartilage and growth plate extracellular matrix-specific proteins supportive of either osteogenesis or stable chondrogenesis of stem cells.

Tissue-specific extracellular matrix (ECM) proteins can play a key role in regulating the fate of stem cells and can potentially be utilized for therapeutic applications. Realising this potential requires further characterization of the diversity of biomolecules present in tissue-specific ECMs and an evaluation of their role as regulatory cues for regenerative medicine applications. The goal of this study was to identify specific soluble factors within the ECM of articular cartilage (AC) and growth plate (GP) that may impart chondro-inductivity or osteo-inductivity respectively. To this end, the significantly different proteins between both matrisomes were searched against the STRING database platform, from which C-type lectin domain family-11 member-A (CLEC11A) and S100 calcium-binding protein-A10 (S100A10) were identified as potential candidates for supporting osteogenesis, and Gremlin-1 (GREM1) and TGF-ß induced gene human clone-3 (ßIGH3) were identified as potential candidates for supporting stable chondrogenesis. Stimulation of chondrogenically-primed bone marrow-derived stem cells (BMSCs) with the AC-specific proteins GREM1 and ßIGH3 had no noticeable effect on the deposition of collagen-II, a marker of chondrogenesis, but appeared to suppress the production of the hypertrophic marker collagen-X, particularly for higher concentrations of GREM1. Stimulation with GREM1 was also found to suppress the direct osteoblastic differentiation of BMSCs. In contrast, stimulation with the GP-specific factors CLEC11A and S100A10 was found to enhance osteogenesis of BMSCs, increasing the levels of mineralization, particularly for higher concentration of CLEC11A. Together these results demonstrate that AC- and GP-specific proteins may play a key role in developing novel strategies for engineering phenotypically stable articular cartilage or enhancing the regeneration of critically-sized bone defects.

The development of natural polymer scaffold-based therapeutics for osteochondral repair.

Due to the limited regenerative capacity of cartilage, untreated joint defects can advance to more extensive degenerative conditions such as osteoarthritis. While some biomaterial-based tissue-engineered scaffolds have shown promise in treating such defects, no scaffold has been widely accepted by clinicians to date. Multi-layered natural polymer scaffolds that mimic native osteochondral tissue and facilitate the regeneration of both articular cartilage (AC) and subchondral bone (SCB) in spatially distinct regions have recently entered clinical use, while the transient localized delivery of growth factors and even therapeutic genes has also been proposed to better regulate and promote new tissue formation. Furthermore, new manufacturing methods such as 3D bioprinting have made it possible to precisely tailor scaffold micro-architectures and/or to control the spatial deposition of cells in requisite layers of an implant. In this way, natural and synthetic polymers can be combined to yield bioactive, yet mechanically robust, cell-laden scaffolds suitable for the osteochondral environment. This mini-review discusses recent advances in scaffolds for osteochondral repair, with particular focus on the role of natural polymers in providing regenerative templates for treatment of both AC and SCB in articular joint defects.

Porous Silica-Pillared MXenes with Controllable Interlayer Distances for Long-Life Na-Ion Batteries.

MXenes are a recently discovered class of two-dimensional materials that have shown great potential as electrodes in electrochemical energy storage devices. Despite their promise in this area, MXenes can still suffer limitations in the form of restricted ion accessibility between the closely spaced multistacked MXene layers causing low capacities and poor cycle life. Pillaring, where a secondary species is inserted between layers, has been used to increase interlayer spacings in clays with great success but has had limited application in MXenes. We report a new amine-assisted pillaring methodology that successfully intercalates silica-based pillars between Ti3C2 layers. Using this technique, the interlayer spacing can be controlled with the choice of amine and calcination temperature, up to a maximum of 3.2 nm, the largest interlayer spacing reported for an MXene. Another effect of the pillaring is a dramatic increase in surface area, achieving BET surface areas of 235 m2 g-1, a sixty-fold increase over the unpillared material and the highest reported for MXenes using an intercalation-based method. The intercalation mechanism was revealed by different characterization techniques, allowing the surface chemistry to be optimized for the pillaring process. The porous MXene was tested for Na-ion battery applications and showed superior capacity, rate capability and remarkable stability compared with those of the nonpillared materials, retaining 98.5% capacity between the 50th and 100th cycles. These results demonstrate the applicability and promise of pillaring techniques applied to MXenes providing a new approach to optimizing their properties for a range of applications, including energy storage, conversion, catalysis, and gas separations.

Integrating finite element modelling and 3D printing to engineer biomimetic polymeric scaffolds for tissue engineering.

The suitability of a scaffold for tissue engineering is determined by a number of interrelated factors. The biomaterial should be biocompatible and cell instructive, with a porosity and pore interconnectivity that facilitates cellular migration and the transport of nutrients and waste products into and out of the scaffolds. For the engineering of load bearing tissues, the scaffold may also be required to possess specific mechanical properties and/or ensure the transfer of mechanical stimuli to cells to direct their differentiation. Achieving these design goals is challenging, but could potentially be realised by integrating computational tools such as finite element (FE) modelling with three-dimensional (3D) printing techniques to assess how scaffold architecture and material properties influence the performance of the implant. In this study we first use Fused Deposition Modelling (FDM) to modulate the architecture of polycaprolactone (PCL) scaffolds, exploring the influence of varying fibre diameter, spacing and laydown pattern on the structural and mechanical properties of such scaffolds. We next demonstrate that a simple FE modelling strategy, which captures key aspects of the printed scaffold's actual geometry and material behaviour, can be used to accurately model the mechanical characteristics of such scaffolds. We then show the utility of this strategy by using FE modelling to help design 3D printed scaffolds with mechanical properties mimicking that of articular cartilage. In conclusion, this study demonstrates that a relatively simple FE modelling approach can be used to inform the design of 3D printed scaffolds to ensure their bulk mechanical properties mimic specific target tissues.

Two-Dimensional Covalent Crystals by Chemical Conversion of Thin van der Waals Materials.

Most of the studied two-dimensional (2D) materials have been obtained by exfoliation of van der Waals crystals. Recently, there has been growing interest in fabricating synthetic 2D crystals which have no layered bulk analogues. These efforts have been focused mainly on the surface growth of molecules in high vacuum. Here, we report an approach to making 2D crystals of covalent solids by chemical conversion of van der Waals layers. As an example, we used 2D indium selenide (InSe) obtained by exfoliation and converted it by direct fluorination into indium fluoride (InF3), which has a nonlayered, rhombohedral structure and therefore cannot  possibly be obtained by exfoliation. The conversion of InSe into InF3 is found to be feasible for thicknesses down to three layers of InSe, and the obtained stable InF3 layers are doped with selenium. We study this new 2D material by optical, electron transport, and Raman measurements and show that it is a semiconductor with a direct bandgap of 2.2 eV, exhibiting high optical transparency across the visible and infrared spectral ranges. We also demonstrate the scalability of our approach by chemical conversion of large-area, thin InSe laminates obtained by liquid exfoliation, into InF3 films. The concept of chemical conversion of cleavable thin van der Waals crystals into covalently bonded noncleavable ones opens exciting prospects for synthesizing a wide variety of novel atomically thin covalent crystals.

Nanometer Resolution Elemental Mapping in Graphene-Based TEM Liquid Cells.

We demonstrate a new design of graphene liquid cell consisting of a thin lithographically patterned hexagonal boron nitride crystal encapsulated on both sides with graphene windows. The ultrathin window liquid cells produced have precisely controlled volumes and thicknesses and are robust to repeated vacuum cycling. This technology enables exciting new opportunities for liquid cell studies, providing a reliable platform for high resolution transmission electron microscope imaging and spectral mapping. The presence of water was confirmed using electron energy loss spectroscopy (EELS) via the detection of the oxygen K-edge and measuring the thickness of full and empty cells. We demonstrate the imaging capabilities of these liquid cells by tracking the dynamic motion and interactions of small metal nanoparticles with diameters of 0.5-5 nm. We further present an order of magnitude improvement in the analytical capabilities compared to previous liquid cell data with 1 nm spatial resolution elemental mapping achievable for liquid encapsulated bimetallic nanoparticles using energy dispersive X-ray spectroscopy (EDXS).

Chondrocyte-based intraoperative processing strategies for the biological augmentation of a polyurethane meniscus replacement.

Purpose/aim of study: Menisectomies account for over 1.5 million surgical interventions in Europe annually, and there is a growing interest in regenerative strategies to improve outcomes in meniscal replacement. The overall objective of this study was to evaluate the role of intraoperatively applied fresh chondrocyte (FC) isolates compared to minced cartilage (MC) fragments, used without cell isolation, to improve bioactivity and tissue integration when combined with a polyurethane replacement. MATERIALS AND METHODS: First, to optimize the intraoperative cell isolation protocol, caprine articular cartilage biopsies were digested with 750 U/ml or 3000 U/ml collagenase type II (ratio of 10 ml per g of tissue) for 30 min, 1 h or 12 h with constant agitation and compared to culture-expanded chondrocytes in terms of matrix deposition when cultured on polyurethane scaffolds. Finally, FCs and MC-augmented polyurethane scaffolds were evaluated in a caprine meniscal explant model to assess the potential enhancements on tissue integration strength. RESULTS: Adequate numbers of FCs were harvested using a 30 min chondrocyte isolation protocol and were found to demonstrate improved matrix deposition compared to standard culture-expanded cells in vitro. Upon evaluation in a meniscus explant defect model, both FCs and MC showed improved matrix deposition at the tissue-scaffold interface and enhanced push-out strength, fourfold and 2.5-fold, respectively, compared with the acellular implant. CONCLUSIONS: Herein, we have demonstrated a novel approach that could be applied intraoperatively, using FCs or MC for improved tissue integration with a polyurethane meniscal replacement.

A Short History of Bioengineering Research in Ireland.

In July 2018, Ireland will host the World Congress of Biomechanics in Dublin. This Congress is held once every 4 yr and is the premier meeting worldwide in its field, with over 3000 people expected to visit Dublin in July. The awarding of the 2018 Congress to Ireland is a reflection of the strength of biomechanics and bioengineering research in this country. To mark this event, herein we describe the development of biomechanics and bioengineering research in Ireland over the past 40 yr, which has grown in parallel with the medical device industry as well as the expansion of Government investment in science, innovation, and a knowledge-based economy. The growth of this activity has resulted in Ireland becoming established as a global hub in the field.

Chondrogenesis of embryonic limb bud cells in micromass culture progresses rapidly to hypertrophy and is modulated by hydrostatic pressure.

Chondrogenesis in vivo is precisely controlled in time and space. The entire limb skeleton forms from cells at the core of the early limb bud that condense and undergo chondrogenic differentiation. Whether they form stable cartilage at the articular surface of the joint or transient cartilage that progresses to hypertrophy as endochondral bone, replacing the cartilage template of the skeletal rudiment, is spatially controlled over several days in the embryo. Here, we follow the differentiation of cells taken from the early limb bud (embryonic day 11.5), grown in high-density micromass culture and show that a self-organising pattern of evenly spaced cartilage nodules occurs spontaneously in growth medium. Although chondrogenesis is enhanced by addition of BMP6 to the medium, the spatial pattern of nodule formation is disrupted. We show rapid progression of the entire nodule to hypertrophy in culture and therefore loss of the local signals required to direct formation of stable cartilage. Dynamic hydrostatic pressure, which we have previously predicted to be a feature of the forming embryonic joint region, had a stabilising effect on chondrogenesis, reducing expression of hypertrophic marker genes. This demonstrates the use of micromass culture as a relatively simple assay to compare the effect of both biophysical and molecular signals on spatial and temporal control of chondrogenesis that could be used to examine the response of different types of progenitor cell, both adult- and embryo-derived.

Biofabrication of soft tissue templates for engineering the bone-ligament interface.

Regenerating damaged tissue interfaces remains a significant clinical challenge, requiring recapitulation of the structure, composition, and function of the native enthesis. In the ligament-to-bone interface, this region transitions from ligament to fibrocartilage, to calcified cartilage and then to bone. This gradation in tissue types facilitates the transfer of load between soft and hard structures while minimizing stress concentrations at the interface. Previous attempts to engineer the ligament-bone interface have utilized various scaffold materials with an array of various cell types and/or biological cues. The primary goal of this study was to engineer a multiphased construct mimicking the ligament-bone interface by driving differentiation of a single population of mesenchymal stem cells (MSCs), seeded within blended fibrin-alginate hydrogels, down an endochondral, fibrocartilaginous, or ligamentous pathway through spatial presentation of growth factors along the length of the construct within a custom-developed, dual-chamber culture system. MSCs within these engineered constructs demonstrated spatially distinct regions of differentiation, adopting either a cartilaginous or ligamentous phenotype depending on their local environment. Furthermore, there was also evidence of spatially defined progression toward an endochondral phenotype when chondrogenically primed MSCs within this construct were additionally exposed to hypertrophic cues. The study demonstrates the feasibility of engineering spatially complex soft tissues within a single MSC laden hydrogel through the defined presentation of biochemical cues. This novel approach represents a new strategy for engineering the ligament-bone interface. Biotechnol. Bioeng. 2017;114: 2400-2411. © 2017 Wiley Periodicals, Inc.

Direct UV-Triggered Thiol-ene Cross-Linking of Electrospun Polyester Fibers from Unsaturated Poly(macrolactone)s and Their Drug Loading by Solvent Swelling.

Electrospinning is considered a relatively simple and versatile technique to form high porosity porous scaffolds with micron to nanoscale fibers for biomedical applications. Here, electrospinning of unsaturated aliphatic polyglobalide (PGl) into well-defined fibers with an average diameter of 9 µm is demonstrated. Addition of a dithiol cross-linker and a photoinitiator to the polymer solution enabled the UV-triggered intracross-linking of the fibers during the spinning process. The in situ cross-linking of the fibers resulted in amorphous material able to swell up to 14% in tetrahydrofurane (THF) without losing the fiber morphology. Seeding mesenchymal stem cells (MSCs) onto both cross-linked and non-cross-linked PGl fibers proved their compatibility with MSCs and suitability as scaffolds for cell growth and proliferation of MSCs. Moreover, the ability to directly load cross-linked PGl with hydrophobic molecules by soaking the fiber mesh in solution is shown with Rhodamine B and Indomethacin, a hydrophobic anti-inflammatory drug. This marks an advantage over conventional aliphatic polyesters and opens opportunities for the design of drug loaded polyester scaffolds for biomedical applications or tissue engineering.

Mechanical regulation of mesenchymal stem cell differentiation.

Biophysical cues play a key role in directing the lineage commitment of mesenchymal stem cells or multipotent stromal cells (MSCs), but the mechanotransductive mechanisms at play are still not fully understood. This review article first describes the roles of both substrate mechanics (e.g. stiffness and topography) and extrinsic mechanical cues (e.g. fluid flow, compression, hydrostatic pressure, tension) on the differentiation of MSCs. A specific focus is placed on the role of such factors in regulating the osteogenic, chondrogenic, myogenic and adipogenic differentiation of MSCs. Next, the article focuses on the cellular components, specifically integrins, ion channels, focal adhesions and the cytoskeleton, hypothesized to be involved in MSC mechanotransduction. This review aims to illustrate the strides that have been made in elucidating how MSCs sense and respond to their mechanical environment, and also to identify areas where further research is needed.

Engineering articular cartilage-like grafts by self-assembly of infrapatellar fat pad-derived stem cells.

Well documented limitations associated with primary chondrocytes for cartilage tissue engineering applications have led to increased interest in the use of multi-potent stem/progenitor cells. The objective of this study was to firstly investigate if infrapatellar fat pad-derived stem cells (FPSCs) could be used to engineer cartilage-like tissues through a self-assembly (SA) process, and secondly to compare the properties of such grafts to those engineered by agarose hydrogel encapsulation (AE). Self-assembled cartilaginous tissues were first engineered by geometrically confining FPSCs on tissue culture plastic, and then either continuously or transiently supplementing these constructs with transforming growth factor-b3 (TGF-b3). Transient supplementation with TGF-b3 (for the first 21 days of culture) enhanced the development of self-assembled grafts, with sGAG accumulation reaching levels of 8.4 ± 1.5% w/w after 6 weeks of culture. While overall levels of matrix synthesis were higher with AE compared to SA, when normalized to tissue wet weight, ECM accumulation was significantly greater in the lighter SA constructs. A potential drawback with the SA approach on tissue culture plastic was that it often led to the development of contracted,geometrically inconsistent tissues.We therefore next explored if SA on polyethylene terephthalate (PET) transwell membranes would lead to the development of more morphologically stable and homogenous tissues. At high seeding densities, SA on such transwell membranes led to the formation of geometrically uniform constructs that underwent minimal contraction during culture. In conclusion, the results of this study demonstrate the potential of SA using FPSCs for cartilage tissue engineering, with grafts attaining relatively high levels of sGAG content within clinically relevant timeframes. Such an approach is easily scalable and may lend itself to treating large, full thickness cartilage defects.