Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue

Restricted oxygen diffusion can result in central cell necrosis in engineered tissue, a problem that is exacerbated when engineering large tissue constructs for clinical application. Here we show that pre-treating human mesenchymal stem cells (hMSCs) with synthetic membrane-active myoglobin-polymer–surfactant complexes can provide a reservoir of oxygen capable of alleviating necrosis at the centre of hyaline cartilage. This is achieved through the development of a new cell functionalization methodology based on polymer–surfactant conjugation, which allows the delivery of functional proteins to the hMSC membrane. This new approach circumvents the need for cell surface engineering using protein chimerization or genetic transfection, and we demonstrate that the surface-modified hMSCs retain their ability to proliferate and to undergo multilineage differentiation. The functionalization technology is facile, versatile and non-disruptive, and in addition to tissue oxygenation, it should have far-reaching application in a host of tissue engineering and cell-based therapies

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM

High-Resolution Patterned Cellular Constructs by Droplet-Based 3D Printing.

Bioprinting is an emerging technique for the fabrication of living tissues that allows cells to be arranged in predetermined three-dimensional (3D) architectures. However, to date, there are limited examples of bioprinted constructs containing multiple cell types patterned at high-resolution. Here we present a low-cost process that employs 3D printing of aqueous droplets containing mammalian cells to produce robust, patterned constructs in oil, which were reproducibly transferred to culture medium. Human embryonic kidney (HEK) cells and ovine mesenchymal stem cells (oMSCs) were printed at tissue-relevant densities (10(7) cells mL(-1)) and a high droplet resolution of 1 nL. High-resolution 3D geometries were printed with features of ≤200 μm; these included an arborised cell junction, a diagonal-plane junction and an osteochondral interface. The printed cells showed high viability (90% on average) and HEK cells within the printed structures were shown to proliferate under culture conditions. Significantly, a five-week tissue engineering study demonstrated that printed oMSCs could be differentiated down the chondrogenic lineage to generate cartilage-like structures containing type II collagen

Size-tunable nanoneedle arrays for influencing stem cell morphology, gene expression and nuclear membrane curvature

High-aspect-ratio nanostructures have emerged as versatile platforms for intracellular sensing and biomolecule delivery. Here, we present a microfabrication approach in which a combination of reactive ion etching protocols was used to produce high-aspect-ratio, nondegradable silicon nanoneedle arrays with tip diameters that can be finely tuned between 20 and 700 nm. We used these arrays to guide the long-term culture of human mesenchymal stem cells (hMSCs). Notably, we used the nanoneedle tip diameter to control the morphology, nuclear size and F-actin alignment of interfaced hMSCs, and to regulate the expression of nuclear lamina genes, Yes-associated protein (YAP) target genes and focal adhesion genes. These topography-driven changes were attributed to signaling by Rho-family GTPase pathways, differences in the effective stiffness of the nanoneedle arrays and the degree of nuclear membrane impingement, with the latter clearly visualized using focused-ion beam scanning electron microscopy (FIB-SEM). Our approach to design high-aspect-ratio nanostructures will be broadly applicable to design biomaterials and biomedical devices used for long-term cell stimulation and monitoring

Enhancing Biological and Biomechanical Fixation of Osteochondral Scaffold: A Grand Challenge

Osteoarthritis (OA) is a degenerative joint disease, typified by degradation of cartilage and changes in the subchondral bone, resulting in pain, stiffness and reduced mobility. Current surgical treatments often fail to regenerate hyaline cartilage and result in the formation of fibrocartilage. Tissue engineering approaches have emerged for the repair of cartilage defects and damages to the subchondral bones in the early stage of OA and have shown potential in restoring the joint's function. In this approach, the use of three-dimensional scaffolds (with or without cells) provides support for tissue growth. Commercially available osteochondral (OC) scaffolds have been studied in OA patients for repair and regeneration of OC defects. However, some controversial results are often reported from both clinical trials and animal studies. The objective of this chapter is to report the scaffolds clinical requirements and performance of the currently available OC scaffolds that have been investigated both in animal studies and in clinical trials. The findings have demonstrated the importance of biological and biomechanical fixation of the OC scaffolds in achieving good cartilage fill and improved hyaline cartilage formation. It is concluded that improving cartilage fill, enhancing its integration with host tissues and achieving a strong and stable subchondral bone support for overlying cartilage are still grand challenges for the early treatment of OA

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

Re-Engineering Extracellular Vesicles as Smart Nanoscale Therapeutics

In the past decade, extracellular vesicles (EVs) have emerged as a key cell-free strategy for the treatment of a range of pathologies, including cancer, myocardial infarction, and inflammatory diseases. Indeed, the field is rapidly transitioning from promising in vitro reports toward in vivo animal models and early clinical studies. These investigations exploit the high physicochemical stability and biocompatibility of EVs as well as their innate capacity to communicate with cells via signal transduction and membrane fusion. This review focuses on methods in which EVs can be chemically or biologically modified to broaden, alter, or enhance their therapeutic capability. We examine two broad strategies, which have been used to introduce a wide range of nanoparticles, reporter systems, targeting peptides, pharmaceutics, and functional RNA molecules. First, we explore how EVs can be modified by manipulating their parent cells, either through genetic or metabolic engineering or by introducing exogenous material that is subsequently incorporated into secreted EVs. Second, we consider how EVs can be directly functionalized using strategies such as hydrophobic insertion, covalent surface chemistry, and membrane permeabilization. We discuss the historical context of each specific technology, present prominent examples, and evaluate the complexities, potential pitfalls, and opportunities presented by different reengineering strategies

Three-dimensional representations of powder mixedness using a positron emission tracking technique

The mixing of powders is a long established practice and is undertaken in most industrial applications where powders are handled. The quality of the mixture has a significant impact on the end product; for example the efficacy of Pharmaceuticals or the strength of a sintered metal component. The measurement of the quality of a mixture is not without debate, and many ways of determining 'mixedness' have been proposed. Tomographic techniques have been used extensively to follow process behaviour in dynamic solid/liquid and solid/gas systems, but the evaluation of batch solid/solid blending systems has been under represented. This paper presents some initial tomographic studies to evaluate mixedness in batch blenders with a view to developing comparisons with established techniques for evaluation of blend quality and determination of end point

Three-dimensional representations of powder mixedness using a positron emission tracking technique

The mixing of powders is a long established practice and is undertaken in most industrial applications where powders are handled. The quality of the mixture has a significant impact on the end product; for example the efficacy of Pharmaceuticals or the strength of a sintered metal component. The measurement of the quality of a mixture is not without debate, and many ways of determining 'mixedness' have been proposed. Tomographic techniques have been used extensively to follow process behaviour in dynamic solid/liquid and solid/gas systems, but the evaluation of batch solid/solid blending systems has been under represented. This paper presents some initial tomographic studies to evaluate mixedness in batch blenders with a view to developing comparisons with established techniques for evaluation of blend quality and determination of end point