Building bones for blood and beyond: the growing field of bone marrow niche model development.

The bone marrow (BM) niche is a complex microenvironment that provides the signals required for regulation of hematopoietic stem cells (HSCs) and the process of hematopoiesis they are responsible for. Bioengineered models of the BM niche incorporate various elements of the in vivo BM microenvironment, including cellular components, soluble factors, a three-dimensional environment, mechanical stimulation of included cells, and perfusion. Recent advances in the bioengineering field have resulted in a spate of new models that shed light on BM function and are approaching precise imitation of the BM niche. These models promise to improve our understanding of the in vivo microenvironment in health and disease. They also aim to serve as platforms for HSC manipulation or as preclinical models for screening novel therapies for BM-associated disorders and diseases.

Nanotopography reveals metabolites that maintain the immunomodulatory phenotype of mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes.

Materials-driven fibronectin assembly on nanoscale topography enhances mesenchymal stem cell adhesion, protecting cells from bacterial virulence factors and preventing biofilm formation.

Post-operative infection is a major complication in patients recovering from orthopaedic surgery. As such, there is a clinical need to develop biomaterials for use in regenerative surgery that can promote mesenchymal stem cell (MSC) osteospecific differentiation and that can prevent infection caused by biofilm-forming pathogens. Nanotopographical approaches to pathogen control are being identified, including in orthopaedic materials such as titanium and its alloys. These topographies use high aspect ratio nanospikes or nanowires to prevent bacterial adhesion but these features also significantly reduce MSC adhesion and activity. Here, we use a poly (ethyl acrylate) (PEA) polymer coating on titanium nanowires to spontaneously organise fibronectin (FN) and to deliver bone morphogenetic protein 2 (BMP2) to enhance MSC adhesion and osteospecific signalling. Using a novel MSC-Pseudomonas aeruginosa co-culture, we show that the coated nanotopographies protect MSCs from cytotoxic quorum sensing and signalling molecules, enhance MSC adhesion and osteoblast differentiation and reduce biofilm formation. We conclude that the PEA polymer-coated nanotopography can both support MSCs and prevent pathogens from adhering to a biomaterial surface, thus protecting from biofilm formation and bacterial infection, and supporting osteogenic repair.

Nanovibrational Stimulation of Mesenchymal Stem Cells Induces Therapeutic Reactive Oxygen Species and Inflammation for Three-Dimensional Bone Tissue Engineering.

There is a pressing clinical need to develop cell-based bone therapies due to a lack of viable, autologous bone grafts and a growing demand for bone grafts in musculoskeletal surgery. Such therapies can be tissue engineered and cellular, such as osteoblasts, combined with a material scaffold. Because mesenchymal stem cells (MSCs) are both available and fast growing compared to mature osteoblasts, therapies that utilize these progenitor cells are particularly promising. We have developed a nanovibrational bioreactor that can convert MSCs into bone-forming osteoblasts in two- and three-dimensional, but the mechanisms involved in this osteoinduction process remain unclear. Here, to elucidate this mechanism, we use increasing vibrational amplitude, from 30 nm (N30) to 90 nm (N90) amplitudes at 1000 Hz and assess MSC metabolite, gene, and protein changes. These approaches reveal that dose-dependent changes occur in MSCs' responses to increased vibrational amplitude, particularly in adhesion and mechanosensitive ion channel expression and that energetic metabolic pathways are activated, leading to low-level reactive oxygen species (ROS) production and to low-level inflammation as well as to ROS- and inflammation-balancing pathways. These events are analogous to those that occur in the natural bone-healing processes. We have also developed a tissue engineered MSC-laden scaffold designed using cells' mechanical memory, driven by the stronger N90 stimulation. These mechanistic insights and cell-scaffold design are underpinned by a process that is free of inductive chemicals.

Control of cell behaviour through nanovibrational stimulation: nanokicking.

Mechanical signals are ubiquitous in our everyday life and the process of converting these mechanical signals into a biological signalling response is known as mechanotransduction. Our understanding of mechanotransduction, and its contribution to vital cellular responses, is a rapidly expanding field of research involving complex processes that are still not clearly understood. The use of mechanical vibration as a stimulus of mechanotransduction, including variation of frequency and amplitude, allows an alternative method to control specific cell behaviour without chemical stimulation (e.g. growth factors). Chemical-independent control of cell behaviour could be highly advantageous for fields including drug discovery and clinical tissue engineering. In this review, a novel technique is described based on nanoscale sinusoidal vibration. Using finite-element analysis in conjunction with laser interferometry, techniques that are used within the field of gravitational wave detection, optimization of apparatus design and calibration of vibration application have been performed. We further discuss the application of nanovibrational stimulation, or 'nanokicking', to eukaryotic and prokaryotic cells including the differentiation of mesenchymal stem cells towards an osteoblast cell lineage. Mechanotransductive mechanisms are discussed including mediation through the Rho-A kinase signalling pathway. Optimization of this technique was first performed in two-dimensional culture using a simple vibration platform with an optimal frequency and amplitude of 1 kHz and 22 nm. A novel bioreactor was developed to scale up cell production, with recent research demonstrating that mesenchymal stem cell differentiation can be efficiently triggered in soft gel constructs. This important step provides first evidence that clinically relevant (three-dimensional) volumes of osteoblasts can be produced for the purpose of bone grafting, without complex scaffolds and/or chemical induction. Initial findings have shown that nanovibrational stimulation can also reduce biofilm formation in a number of clinically relevant bacteria. This demonstrates additional utility of the bioreactor to investigate mechanotransduction in other fields of research.This article is part of a discussion meeting issue 'The promises of gravitational-wave astronomy'.

The use of microarrays and fluorescence in situ hybridization for the study of mechanotransduction from topography.

The combination of transcriptomic analysis and fluorescence in situ hybridization (FISH) provides a robust methodology to study genomic changes in different biological conditions. Microarrays allow a global study of gene expression in response to the conditions of interest, with comparison between control(s) and one or more test condition(s). The messenger RNA amplification step permits detection of even low abundance transcripts, a critical advantage for applications such as biomaterials research, where the starting material may be limited. Different types of microarrays are commercially available that allow the investigation of specific features, such as exon arrays, microRNA arrays, and gene arrays. Microarrays are available for different model organisms, but we use Affymetrix ® HuGene ® ST (Sense Target) arrays, a type of gene array for analysis of human samples. FISH involves fluorescent detection of probe DNA hybridized to an in situ chromosomal target that can be either whole chromosomes or chromosomal segments. The overall hybridization is similar to labeling with radioactive probes but the incorporation of fluorescent detection of the probe sequences allows for high sensitivity in a simple and quick assay. FISH can be applied to a variety of specimen types depending on the study of interest. In this chapter, we describe the methodologies of these two techniques and provide technical tips that should help overcome challenges in carrying them out.