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Microfluidic-based tissue-on-a-chip devices have generated significant research interest for biomedical applications, such as pharmaceutical development, as they can be used for small volume, high throughput studies on the effects of therapeutics on tissue-mimics. Tissue-on-a-chip devices are evolving from basic 2D cell cultures incorporated into microfluidic devices to complex 3D approaches, with modern designs aimed at recapitulating the dynamic and mechanical environment of the native tissue. Thus far, most tissue-on-a-chip research has concentrated on organs involved with drug uptake, metabolism and removal (e.g., lung, skin, liver, and kidney); however, models of the drug metabolite target organs will be essential to provide information on therapeutic efficacy. Here, we develop an osteogenesis-on-a-chip device that comprises a 3D environment and fluid shear stresses, both important features of bone. This inexpensive, easy-to-fabricate system based on a polymerized High Internal Phase Emulsion (polyHIPE) supports proliferation, differentiation and extracellular matrix production of human embryonic stem cell-derived mesenchymal progenitor cells (hES-MPs) over extended time periods (up to 21 days). Cells respond positively to both chemical and mechanical stimulation of osteogenesis, with an intermittent flow profile containing rest periods strongly promoting differentiation and matrix formation in comparison to static and continuous flow. Flow and shear stresses were modeled using computational fluid dynamics. Primary cilia were detectable on cells within the device channels demonstrating that this mechanosensory organelle is present in the complex 3D culture environment. In summary, this device aids the development of 'next-generation' tools for investigating novel therapeutics for bone in comparison with standard laboratory and animal testing.

International audience; Mechanical forces and 3D topological environment can be used to control differentiation of mesenchymal stem cells (MSCs). However, the effects of physical and mechanical cues of the microenvironment on MSC fate determination have not yet been fully understood. This study investigates and compares the effect of mechanical stimulations on soft cellular microspheres when subjected to dynamic fluid compression. Microspheres were produced by gelation of bovine collagen type I with concentrations of 2 mg/ml and 1000–2000 hES-MP cells per 5 μl droplet. A loading condition of 10% dynamic loading was applied by a BOSE BioDynamic bioreactor for 15 and 40 min/day for 5 and 10 days on the cell-seeded collagen microspheres. Cell viability and proliferation, alkaline phosphatase activity and mineralization were compared with controls. Monitoring alkaline phosphatase level reported a significant increase in the enzyme activity by day 14 in loaded samples of 40 min/day loading protocol compared with other experimental conditions. Mineralization was assessed by measuring calcium, phosphorous concentrations and intensity of H&E and alizarin red S staining and showed the highest mineral accumulation in the loaded samples on day 28 post encapsulation. This study indicated that loading of very low cell number seeded on soft natural scaffold can encourage osteogenesis of cells by enhancing both early stage bone marker and mineralization. Self-assembled cell/collagen microspheres present exceptional cell delivery model in bone healing/repair process and field of regenerative medicine.

Early detection right at epidemic areas can prevent infectious diseases from propagation. Currently, the most common nucleic acid test—polymerase chain reaction (PCR) is time-consuming, complex, expensive and thermocycler required, thus limiting its utility in poor laboratory conditions or even non-laboratory condition of epidemic areas. Loop-mediated isothermal amplification (LAMP) is quick, cheap, sensitive and isothermal assay could be combined with a simple DNA extraction method to integrate into Lab-on-a-chip (LOC) device. Here, we attempted to improve LAMP method for malaria diagnosis on portable microfluidics chip platform by optimizing DNA extraction using boil and spin method and altering Tris-containing amplification buffer for ascertaining changing in pH of reaction solution. Basically, blood sample was mixed with extraction buffer containing Sodium Dedocyl Sulfate (SDS) concentration and treated under high temperature condition. Four concentrations of SDS (0, 0.4, 0.8 and 1%) were tested along with different temperature (65 and 95 °C) to adapt into LOC platform and avoid denaturation of LAMP reagent. All samples treated at 65 °C showed the presence of DNA after extraction. Furthermore, DNA amplification buffer was minimized Tris concentration to facilitate result read-out step. The releasing of hydrogen ion from amplification reaction causes increasing in pH which could be recognized by color of pH indicator paper or dye, for example, phenolphthalein. Throughout a series of experiments, LAMP is demonstrated that it can also occur in low-Tris buffer with pH indicator dye, efficiently. The positive sample will have a change from pink to transparent in solution color, otherwise, the negative sample will maintain pink. These improvements allowed us to adapt LAMP technique into Point-of-care (POC) devices in which the whole process run under isothermal condition (65 °C) and non-instrument required visual detection. The LAMP microfluidics chip will be potential tool for early detection infectious diseases and several other diseases in non-laboratory condition.

Mesenchymal stem cells (MSCs) are widely implicated for their potential use as a cell source for tissue engineering of skeletal tissue in regenerative medicine and tissue engineering. Mechanical forces from the microenvironment have a significant influence on differentiation of MSCs, and the resulting mechanotransduction would provide crucial adjuncts to standard biochemical signalling pathways. Combining microfluidic systems with mechanical stimulation for osteogenesis represents both a scientific and technological innovation that would greatly impact the field of regenerative medicine. We demonstrate a microfluidic chamber design for mechanical stimulation of flexible cellular microspheres and possibly a high-throughput microfluidic system for parallel processing of stem cell aggregation. We also showed that collagen microspheres serve an efficient cell delivery device supporting cell viability and migration post encapsulation.

The increased need to accelerate the healing process of critical size defects in the bone led to the study of optimal combination of cells, materials and external stimuli to obtain fully differentiated tissue to the injured site. Bioreactors play a crucial role in the control over the development of functional tissue allowing control over the surrounding chemical and mechanical environment. This chapter aims to review bioreactor systems currently available for monitoring mesenchymal stem cells (MSCs) behaviour under mechanical stimuli and to give an insight of their effect on cellular commitment. Shear stress, mechanical strain and pulsed electromagnetic field bioreactors are presented, and the effect of multiple conditions under varying parameters such as amplitude, frequency or duration of the stimuli on bone progenitor cells differentiation is considered and extensively discussed with particular focus on osteogenic and chondrogenic commitment.

Three-dimensional (3D) scaffolds are increasingly employed as support for studies on cellular activities. They are widely shown to enhance cell survival and are a promising approach to be employed to mimic the in vivo conditions due to their controlled architecture. Moreover, 3D stiff structures fabricated by additive manufacturing are able to bear mechanical stimuli finding a role in the investigation of the effect of mechanical forces on cell proliferation and commitment. With this purpose, we propose a combination of a 3D polycaprolactone (PCL) scaffold and collagen soft gel as support for studying the response of mesenchymal stem cells following mechanical compression. This chapter focuses on the characterization of 3D Insert® PCL scaffolds behaviour under mechanical compression. After defining mechanical properties and variability due to boundary effects, the focus moves on the development of a new composite scaffold made of a stiff PCL structure acting as support for cell activities and able to bear mechanical compression while embedding a soft collagen gel matrix responsible to provide an environment enhancing cellular activities as well as to transmit the stress resulting from the mechanical stimulation from the stiff matrix to the seeded cells. Finally, the last section focuses on the effect of low mechanical strain applied on seeded scaffolds and how the cellular response varies to bursts of compression applied at different time points.

Mesenchymal stem cells are widely implicated as a cell source for tissue engineering of skeletal tissue in cell-based therapy. Physical and mechanical cues are potent controlling factors in cell differentiation and can be implemented as a guide to study cellular response, matrix production and tissue regeneration. Microspheres were produced by gelation of bovine collagen type I with concentration of 2 mg/mL and 1,000-2,000 cells per droplet. Short and long term cell viability of human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) and MG-63 osteoblastic cells as well as collagen microstructure and contraction were monitored during 28 days post encapsulation (pc). Results indicated that collagen concentration, hence mechanical properties of cell’s extracellular micro-environment are important in cell proliferation and differentiation. Contraction of cell-embedded microspheres was found to be vital in cell adaptation and the remodelling of their new environment. It was also found that collagen concentration of 2 mg/mL supports proliferation of hES-MPs while higher collagen concentration promoted the viability of MG-63s. Results of hES-MPs characterization in 3D soft environment and mechanically stimulated hES-MPs collagen microspheres can be used in cells/therapeutic carriers, implants in bone and cartilage healing applications. The microspheres developed in this study can also be used as a tool to build more optimised construct to transfer mechanically stimulated stem cells to the specific area of a defective bone which would add significant benefit to the field of bone regeneration.

3D polymeric scaffolds are increasingly used for in vitro experiments aiming to mimic the environment found in vivo, to support for cellular growth and to induce differentiation through the application of external mechanical cues. In research, experimental results must be shown to be reproducible to be claimed as valid and the first clause to ensure consistency is to provide identical initial experimental conditions between trials. As a matter of fact, 3D structures fabricated in batch are supposed to present a highly reproducible geometry and consequently, to give the same bulk response to mechanical forces. This study aims to measure the overall mechanical response to compression of commercially available 3D Insert PCL scaffolds (3D PCL) fabricated in series by fuse deposition and evaluate how small changes in the architecture of scaffolds affect the mechanical response. The apparent elastic modulus (Ea) was evaluated by performing quasi-static mechanical tests at various temperatures showing a decrease in material stiffness from 5 MPa at 25 °C to 2.2 MPa at 37 °C. Then, a variability analysis revealed variations in Ea related to the repositioning of the sample into the testing machine, but also consistent differences comparing different scaffolds. To clarify the source of the differences measured in the mechanical response, the same scaffolds previously undergoing compression, were scanned by micro computed tomography (μCT) to identify any architectural difference. Eventually, to clarify the contribution given by differences in the architecture to the standard deviation of Ea, their mechanical response was qualitatively compared to a compact reference material such as polydimethylsiloxane (PDMS). This study links the geometry, architecture and mechanical response to compression of 3D PCL scaffolds and shows the importance of controlling such parameters in the manufacturing process to obtain scaffolds that can be used in vitro or in vivo under reproducible conditions.

The formation of new blood vessels from existing vasculature, angiogenesis, is driven by coordinated endothelial cell migration and matrix remodelling in response to local signals. Recently, a growing body of evidence has shown that mechanotransduction, along with chemotransduction, is a major regulator of angiogenesis. Mechanical signals, such as fluid shear stress and substrate mechanics, influence sprouting and network formation, but the mechanisms behind this relationship are still unclear. Here, we present cellular traction forces as possible effectors activated by mechanosensing to mediate matrix remodelling, and encourage the use of traction force microscopy to study mechanotransduction in angiogenesis. We also suggest that deciphering the response of endothelial cells to mechanical signals could reveal an optimal angiogenic mechanical environment, and provide insight into development, wound healing, the initiation and growth of tumours, and new strategies for tissue engineering. This article is protected by copyright. All rights reserved.

Stromal-derived factor-1 (SDF-1) is a critical chemokine for endothelial progenitor cell (EPC) recruitment to areas of ischemia, allowing these cells to participate in compensatory angiogenesis. The SDF-1 receptor, CXCR4, is expressed in developing blood vessels as well as on CD34+ EPCs. We describe that picomolar and nanomolar concentrations of SDF-1 differentially influence neovascularization, inducing CD34+ cell migration and EPC tube formation. CD34+ cells isolated from diabetic patients demonstrate a marked defect in migration to SDF-1. This defect is associated, in some but not all patients, with a cell surface activity of CD26/dipeptidyl peptidase IV, an enzyme that inactivates SDF-1. Diabetic CD34+ cells also do not migrate in response to vascular endothelial growth factor and are structurally rigid. However, incubating CD34+ cells with a nitric oxide (NO) donor corrects this migration defect and corrects the cell deformability. In addition, exogenous NO alters vasodilator-stimulated phosphoprotein and mammalian-enabled distribution in EPCs. These data support a common downstream cytoskeletal alteration in diabetic CD34+ cells that is independent of growth factor receptor activation and is correctable with exogenous NO. This inability of diabetic EPCs to respond to SDF-1 may contribute to aberrant tissue vascularization and endothelial repair in diabetic patients.

Purpose: Stent deployment to treat coronary artery disease causes damage and loss of endothelial cells (EC). Repair of injured arteries by EC migration is co-ordinated by Rho family GTPases. It is also regulated by wall shear stress (WSS), a mechanical force exerted by flowing blood on the vessel wall, via mechanisms that are poorly understood. It was hypothesised that stent struts may impede repair of injured endothelium by inducing localised disturbances in WSS. Methods: To simulate a stented artery in vitro, chamber slides were fabricated with ridges (100 μm high) positioned perpendicularly to the flow direction. Confluent EC monolayers seeded on one side of either ridged or non-ridged (control) chambers were exposed to flowing culture medium (Ibidi® system). Flow patterns were determined by computational fluid dynamic (CFD) modelling and particle velocimetry. Live cell imaging and analysis using ImageJ® software enabled quantitation of EC migration velocity and directional persistence (DP). Results: CFD modelling and live cell imaging indicated that EC on the non-ridged chamber slide were exposed to a uniform WSS of 13 dyn/cm2 and migrated relatively uniformly in parallel with the flow direction (average velocity 1.13±0.20 μm/min; DP 0.59±0.14). By contrast, significant spatial differences in WSS were observed over the ridged slide in CFD, with significant spikes above physiological levels (>70 dyn/cm2) at the corners of the ridges and distinctive flow recirculation zone immediately upstream/ downstream from the ridge (-4 dyn/cm2). These features were verified by particle velocimetry using fluorescently labelled polystyrene beads (2 μm diameter). Time-lapse imaging further revealed interrupted EC migration at the ridges. Specifically, although EC could migrate over the ridges, those that reached the recirculation zone downstream from the ridge migrated with non-uniform directionality (DP 0.25±0.06) and displayed a reduction in velocity (0.78±0.18 μm/min). Inhibition of the RhoA/ROCK signalling pathway with ROCK inhibitors (Y27632 or HA1077) promoted EC forward migration within the recirculation zone by significantly elevating DP (0.41±0.04, p=0.01) and velocity (1.16±0.09 μm/min, p=0.01). Conclusions: Disturbed WSS downstream from stent strut-like ridges prevented the forward migration of EC. Inhibition of the RhoA/ROCK signaling pathway promoted EC migration and re-population of these sites. Our data suggest that treatment using a ROCK inhibitor may promote re-endothelialisation of stented arteries; a concept that is currently been tested using a porcine model.