Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

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.

Sinking while you swim: A dual role for CCR7 in leukocyte migration.

The CCR7 receptor allows dendritic cells to sense the chemokine CCL19. It also depletes CCL19 from the environment by endocytosis, leading to local gradients that can steer cells accurately and robustly through tissues, even over long distances (see related Research Article by Alanko et al.).

Surface-Modified Piezoelectric Copolymer Poly(vinylidene fluoride-trifluoroethylene) Supporting Physiological Extracellular Matrixes to Enhance Mesenchymal Stem Cell Adhesion for Nanoscale Mechanical Stimulation.

There is an unmet clinical need to provide viable bone grafts for clinical use. Autologous bone, one of the most commonly transplanted tissues, is often used but is associated with donor site morbidity. Tissue engineering strategies to differentiate an autologous cell source, such as mesenchymal stromal cells (MSCs), into a potential bone-graft material could help to fulfill clinical demand. However, osteogenesis of MSCs can typically require long culture periods that are impractical in a clinical setting and can lead to significant cost. Investigation into strategies that optimize cell production is essential. Here, we use the piezoelectric copolymer poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE), functionalized with a poly(ethyl acrylate) (PEA) coating that drives fibronectin network formation, to enhance MSC adhesion and to present growth factors in the solid phase. Dynamic electrical cues are then incorporated, via a nanovibrational bioreactor, and the MSC response to electromechanical stimulation is investigated.

Cotransfer of antigen and contextual information harmonizes peripheral and lymph node conventional dendritic cell activation.

T cell responses against infections and cancer are directed by conventional dendritic cells (cDCs) in lymph nodes distant from the site of challenge. Migratory cDCs, which travel from the tissue to the lymph node, not only drive initial T cell activation but also transfer antigen to lymph node-resident cDCs. These resident cells have essential roles defining the character of the resulting T cell response; however, it is unknown how they can appropriately process and present antigens to suitably direct responses given their spatial separation. Here, using a novel strain of influenza A and a modified melanoma model, we show that tissue and lymph node cDC activation is harmonized and that this is driven by cotransfer of contextual cues. In the tumor, incomplete cDC activation in the tumor microenvironment is mirrored by lymph node-resident cDCs, whereas during influenza infection, pathogen-associated molecular patterns cotransferred with antigen drive TLR signaling in resident cDCs and their subsequent robust activation. This cotransfer mechanism explains how individual antigens can be handled distinctly by resident cDCs and how signals driving poor tumoral cDC activation further impact the lymph node. Our findings clarify how tissue context dictates antigenic and, consequently, T cell fate in the lymph node.

Fibronectin matrix assembly and TGFß1 presentation for chondrogenesis of patient derived pericytes for microtia repair.

Tissue engineered cartilage for external ear reconstruction of congenital deformities, such as microtia or resulting from trauma, remains a significant challenge for plastic and reconstructive surgeons. Current strategies involve harvesting autologous costal cartilage or expanding autologous chondrocytes ex vivo. However, these procedures often lead to donor site morbidity and a cell source with limited expansion capacity. Stromal stem cells such as perivascular stem cells (pericytes) offer an attractive alternative cell source, as they can be isolated from many human tissues, readily expanded in vitro and possess chondrogenic differentiation potential. Here, we successfully isolate CD146+ pericytes from the microtia remnant from patients undergoing reconstructive surgery (Microtia pericytes; MPs). Then we investigate their chondrogenic potential using the polymer poly(ethyl acrylate) (PEA) to unfold the extracellular matrix protein fibronectin (FN). FN unfolding exposes key growth factor (GF) and integrin binding sites on the molecule, allowing tethering of the chondrogenic GF transforming growth factor beta 1 (TGFß1). This system leads to solid-phase, matrix-bound, GF presentation in a more physiological-like manner than that of typical chondrogenic induction media (CM) formulations that tend to lead to off-target effects. This simple and controlled material-based approach demonstrates similar chondrogenic potential to CM, while minimising proclivity toward hypertrophy, without the need for complex induction media formulations.

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.

Material-driven fibronectin and vitronectin assembly enhances BMP-2 presentation and osteogenesis.

Mesenchymal stem cell (MSC)-based tissue engineering strategies are of interest in the field of bone tissue regenerative medicine. MSCs are commonly investigated in combination with growth factors (GFs) and biomaterials to provide a regenerative environment for the cells. However, optimizing how biomaterials interact with MSCs and efficiently deliver GFs, remains a challenge. Here, via plasma polymerization, tissue culture plates are coated with a layer of poly (ethyl acrylate) (PEA), which is able to spontaneously permit fibronectin (FN) to form fibrillar nanonetworks. However, vitronectin (VN), another important extracellular matrix (ECM) protein forms multimeric globules on the polymer, thus not displaying functional groups to cells. Interestingly, when FN and VN are co-absorbed onto PEA surfaces, VN can be entrapped within the FN fibrillar nanonetwork in the monomeric form providing a heterogeneous, open ECM network. The combination of FN and VN promote MSC adhesion and leads to enhanced GF binding; here we demonstrate this with bone morphogenetic protein-2 (BMP2). Moreover, MSC differentiation into osteoblasts is enhanced, with elevated expression of osteopontin (OPN) and osteocalcin (OCN) quantified by immunostaining, and increased mineralization observed by von Kossa staining. Osteogenic intracellular signalling is also induced, with increased activity in the SMAD pathway. The study emphasizes the need of recapitulating the complexity of native ECM to achieve optimal cell-material interactions.

Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids.

Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance, a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precise editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12, L482F) or cleavage and polyadenylation specificity factor 3 (N232H), in the Trypanosoma cruzi proteasome ß5 subunit (G208S), or in Leishmania donovani CRK12 (G572D). We further implemented oligo targeting for site saturation mutagenesis, targeting codon G492 in T. brucei CRK12. This approach, combined with amplicon sequencing for codon variant scoring, revealed fourteen resistance conferring G492 edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy.

Current insights into the bone marrow niche: From biology in vivo to bioengineering ex vivo.

Hematopoietic stem cells (HSCs) are fundamental to the generation of the body's blood and immune cells. They reside primarily within the bone marrow (BM) niche microenvironment, which provides signals responsible for the regulation of HSC activities. While our understanding of these signalling mechanisms continues to improve, our ability to recapitulate them in vitro to harness the clinical potential of the HSC populations is still lacking. Recent studies have applied novel engineering techniques combined with traditional in vitro work to establish ex vivo BM niche models. These models exhibit promising potential for research and clinical applications. In this review, BM niche factors that regulate the HSCs in vivo are discussed and their applications in the engineering of BM biomaterial-based platforms are considered. Many questions remain regarding the heterogeneity of niche components and the interactions of HSCs with their microenvironment. A greater understanding of the niche would help to elucidate these remaining questions, leading to the development of novel therapeutic tools.

The influence of nanotopography on cell behaviour through interactions with the extracellular matrix - A review.

Nanotopography presents an effective physical approach for biomaterial cell manipulation mediated through material-extracellular matrix interactions. The extracellular matrix that exists in the cellular microenvironment is crucial for guiding cell behaviours, such as determination of integrin ligation and interaction with growth factors. These interactions with the extracellular matrix regulate downstream mechanotransductive pathways, such as rearrangements in the cytoskeleton and activation of signal cascades. Protein adsorption onto nanotopography strongly influences the conformation and distribution density of extracellular matrix and, therefore, subsequent cell responses. In this review, we first discuss the interactive mechanisms of protein physical adsorption on nanotopography. Secondly, we summarise advances in creating nanotopographical features to instruct desired cell behaviours. Lastly, we focus on the cellular mechanotransductive pathways initiated by nanotopography. This review provides an overview of the current state-of-the-art designs of nanotopography aiming to provide better biomedical materials for the future.

Biophysical phenotyping of mesenchymal stem cells along the osteogenic differentiation pathway.

Mesenchymal stem cells represent an important resource, for bone regenerative medicine and therapeutic applications. This review focuses on new advancements and biophysical tools which exploit different physical and chemical markers of mesenchymal stem cell populations, to finely characterize phenotype changes along their osteogenic differentiation process. Special attention is paid to recently developed label-free methods, which allow monitoring cell populations with minimal invasiveness. Among them, quantitative phase imaging, suitable for single-cell morphometric analysis, and nanoindentation, functional to cellular biomechanics investigation. Moreover, the pool of ion channels expressed in cells during differentiation is discussed, with particular interest for calcium homoeostasis.Altogether, a biophysical perspective of osteogenesis is proposed, offering a valuable tool for the assessment of the cell stage, but also suggesting potential physiological links between apparently independent phenomena.

High numerical aperture Hartmann wave front sensor for extreme ultraviolet spectral range.

We present a novel, to the best of our knowledge, Hartmann wave front sensor for extreme ultraviolet (EUV) spectral range with a numerical aperture (NA) of 0.15. The sensor has been calibrated using an EUV radiation source based on gas high harmonic generation. The calibration, together with simulation results, shows an accuracy beyond λ/39 root mean square (rms) at λ=32nm. The sensor is suitable for wave front measurement in the 10 nm to 45 nm spectral regime. This compact wave front sensor is high-vacuum compatible and designed for in situ operations, allowing wide applications for up-to-date EUV sources or high-NA EUV optics.

The Plot Thickens: The Emerging Role of Matrix Viscosity in Cell Mechanotransduction.

Cell mechanotransduction is an area of intense research focus. Until now, very limited tools have existed to study how cells respond to changes in the extracellular matrix beyond, for example, mechanical deformation studies and twisting cytometry. However, emerging are a range of elastic, viscoelastic and even purely viscous materials that deform and dissipate on cellular length and timescales. This article reviews developments in these materials, typically translating from 2D model surfaces to 3D microenvironments and explores how cells interact with them. Specifically, it focuses on emerging concepts such as the molecular clutch model, how different extracellular matrix proteins engage the clutch under viscoelastic-stress relaxation conditions, and how mechanotransduction can drive transcriptional control through regulators such as YAP/TAZ.

Nanoscale Coatings for Ultralow Dose BMP-2-Driven Regeneration of Critical-Sized Bone Defects.

While new biomaterials for regenerative therapies are being reported in the literature, clinical translation is slow. Some existing regenerative approaches rely on high doses of growth factors, such as bone morphogenetic protein-2 (BMP-2) in bone regeneration, which can cause serious side effects. An ultralow-dose growth factor technology is described yielding high bioactivity based on a simple polymer, poly(ethyl acrylate) (PEA), and mechanisms to drive stem cell differentiation and bone regeneration in a critical-sized murine defect model with translation to a clinical veterinary setting are reported. This material-based technology triggers spontaneous fibronectin organization and stimulates growth factor signalling, enabling synergistic integrin and BMP-2 receptor activation in mesenchymal stem cells. To translate this technology, plasma-polymerized PEA is used on 2D and 3D substrates to enhance cell signalling in vitro, showing the complete healing of a critical-sized bone injury in mice in vivo. Efficacy is demonstrated in a Münsterländer dog with a nonhealing humerus fracture, establishing the clinical translation of advanced ultralow-dose growth factor treatment.

Designing stem cell niches for differentiation and self-renewal.

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries.

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'.

Current approaches for modulation of the nanoscale interface in the regulation of cell behavior.

Regulation of cell behavior in response to nanoscale features has been the focus of much research in recent years and the successful generation of nanoscale features capable of mimicking the natural nanoscale interface has been of great interest in the field of biomaterials research. In this review, we discuss relevant nanofabrication techniques and how they are combined with bioengineering applications to mimic the natural extracellular matrix (ECM) and create valuable nanoscale interfaces.

Bone and cartilage differentiation of a single stem cell population driven by material interface.

Adult stem cells, such as mesenchymal stem cells, are a multipotent cell source able to differentiate towards multiple cell types. While used widely in tissue engineering and biomaterials research, they present inherent donor variability and functionalities. In addition, their potential to form multiple tissues is rarely exploited. Here, we combine an osteogenic nanotopography and a chondrogenic hyaluronan hydrogel with the hypothesis that we can make a complex tissue from a single multipotent cell source with the exemplar of creating a three-dimensional bone-cartilage boundary environment. Marrow stromal cells were seeded onto the topographical surface and the temperature gelling hydrogel laid on top. Cells that remained on the nanotopography spread and formed osteoblast-like cells, while those that were seeded into or migrated into the gel remained rounded and expressed chondrogenic markers. This novel, simple interfacial environment provides a platform for anisotropic differentiation of cells from a single source, which could ultimately be exploited to sort osteogenic and chondrogenic progenitor cells from a marrow stromal cell population and to develop a tissue engineered interface.