Nanoscale vibration could promote tenogenic differentiation of umbilical cord mesenchymal stem cells.

Regulation of mesenchymal stem cell (MSC) fate for targeted cell therapy applications has been a subject of interest, particularly for tissues such as tendons that possess a marginal regenerative capacity. Control of MSCs' fate into the tendon-specific lineage has mainly been achieved by implementation of chemical growth factors. Mechanical stimuli or 3-dimensional (D) scaffolds have been used as an additional tool for the differentiation of MSCs into tenocytes, but oftentimes, they require a sophisticated bioreactor or a complex scaffold fabrication technique which reduces the feasibility of the proposed method to be used in practice. Here, we used nanovibration to induce the differentiation of MSCs toward the tenogenic fate solely by the use of nanovibration and without the need for growth factors or complex scaffolds. MSCs were cultured on 2D cell culture dishes that were connected to piezo ceramic arrays to apply nanovibration (30-80 nm and 1 kHz frequency) over 7 and 14 d. We observed that nanovibration resulted in significant overexpression of tendon-related markers in both gene expression and protein expression levels, while there was no significant differentiation into adipose and cartilage lineages. These findings could be of assistance in the mechanoregulation of MSCs for stem cell engineering and regenerative medicine applications.

3D-printed capillaric ELISA-on-a-chip with aliquoting.

Sandwich immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have been miniaturized and performed in a lab-on-a-chip format, but the execution of the multiple assay steps typically requires a computer or complex peripherals. Recently, an ELISA for detecting antibodies was encoded structurally in a chip thanks to the microfluidic chain reaction (Yafia et al. Nature, 2022, 605, 464-469), but the need for precise pipetting and intolerance to commonly used surfactant concentrations limit the potential for broader adoption. Here, we introduce the ELISA-on-a-chip with aliquoting functionality that simplifies chip loading and pipetting, accommodates higher surfactant concentrations, includes barrier channels that delay the contact between solutions and prevent undesired mixing, and that executed a quantitative, high-sensitivity assay for the SARS-CoV-2 nucleocapsid protein in 4×-diluted saliva. Upon loading the chip using disposable pipettes, capillary flow draws each reagent and the sample into a separate volumetric measuring reservoir for detection antibody (70 µL), enzyme conjugate (50 µL), substrate (80 µL), and sample (210 µL), and splits washing buffer into 4 different reservoirs of 40, 40, 60, and 20 µL. The excess volume is autonomously drained via a structurally encoded capillaric aliquoting circuit, creating aliquots with an accuracy of >93%. Next, the user click-connects the assay module, comprising a nitrocellulose membrane with immobilized capture antibodies and a capillary pump, to the chip which triggers the step-by-step, timed flow of all aliquoted solutions to complete the assay in 1.5 h. A colored precipitate forming a line on a nitrocellulose strip serves as an assay readout, and upon digitization, yielded a binding curve with a limit of detection of 54 and 91 pg mL-1 for buffer and diluted saliva respectively, vastly outperforming rapid tests. The ELISA chip is 3D-printed, modular, adaptable to other targets and assays, and could be used to automate ELISA in the lab; or as a diagnostic test at the point of care with the convenience and form factor of rapid tests while preserving the protocol and performance of central laboratory ELISA.

Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

A Bilayered, Electrospun Poly(Glycerol-Sebacate)/Polyurethane-Polyurethane Scaffold for Engineering of Endothelial Basement Membrane.

In the cardiovascular system, heart valves and vessels are subjected to continuous cyclic mechanical loadings due to the pulsatile nature of blood flow. Hence, in leveraging tissue engineering (TE) strategies to regenerate such a system, the candidate scaffold should not only be biocompatible with the desired biodegradation rate, but it should also be mechanically competent to provide a supportive structure for facilitating stem cells retention, growth, and differentiation. To this end, herein, we introduced a novel scaffold composed of poly(glycerol-sebacate) (PGS) and polyurethane (PU), which comprises of two layers: an electrospun pure PU layer beneath another electrospun PGS/PU layer with a different ratio of PGS to PU (3:2, 1:1, 2:3 Wt:Wt). The electrospun PGS/PU-PU scaffold was mechanically competent and showed intended hydrophilicity and a good biodegradation rate. Moreover, the PGS/PU-PU scaffold indicated cell viability and proliferation within ten days of in vitro cell culture and upon 7 day vascular endothelial growth factor (VEGF) stimulation, supported endothelial differentiation of mesenchymal stem cells by significant overexpression of platelet-endothelial cell adhesion molecule-1, von Willebrand factor, and VEGF receptor 2. The results of this study could be implemented in cardiovascular TE strategies when regeneration of blood vessel or heart valve is desired.

The cooperative effects of micro-grooved topography and TGF-ß1 on the vascular smooth muscle cell contractile protein expression of the mesenchymal stem cells.

Cell morphological changes induced by micro-grooved topography have been shown to be an important regulator of smooth muscle (SM) differentiation of mesenchymal stem cells (MSCs). In addition to the micro-grooved topography, transforming growth factor-ß1 (TGF-ß1) can also modulate MSCs differentiation towards smooth muscle cells (SMCs) through alterations in cell morphological characteristics. Thus, it can be hypothesized that substrate topography and TGF-ß1 may interact to facilitate differentiation of MSCs into SMCs. In this study, we investigated the time-course cooperative effects of substrate topography and TGF-ß1 in the regulation of SM differentiation of human MSCs. Western blotting, followed by image analysis, was performed to assess the protein expression of α-actin, h1-calponin and gelsolin. Three-way analysis of variance was employed to investigate the main effect of each independent variable, i.e. TGF-ß1 conditioning, substrate topography and culture time, along with the interactions of these variables. Each of TGF-ß1, substrate topography and culture time significantly affected the protein expression of α-actin, h1-calponin and gelsolin. Overall, TGF-ß1 conditioning of the cells and culturing the cells on the micro-grooved substrate resulted in greater protein expression of α-actin and h1-calponin, and lesser protein expression of gelsolin. In addition to the isolated effects of the variables, treatment type interacted with substrate topography and culture time to regulate the expression of the above-mentioned proteins. This study indicated the feasibility of promoting SM differentiation of human MSCs by simultaneous recruitment of micro-grooved topography and TGF-ß1. The findings could be of assistance when effective utilization of chemo-physical cues is needed to achieve functional SMC-like MSCs in vitro.

The Effect of Mandibular Flexure on Stress Distribution in the All-on-4 Treated Edentulous Mandible: A Comparative Finite-Element Study Based on Mechanostat Theory.

The All-on-4 treatment concept is a felicitous approach for treatment of edentulous mandible. Mandibular flexure plays a decisive role in several restorative failures-for instance, screw loosening, particularly in widely separated implant supports such as those utilized in All-on-4 treatment methods. We investigated the effect of mandibular flexure on stress distribution and likelihood of bone loss or growth in the implanted mandible using two frequently used All-on-4 methods of implantation: parallel and tilted. Three-dimensional finite-element models of mandible and dental implants together with their compartments were developed. Assuming sagittal symmetry for the mandible, only half of the full geometry was considered. In the parallel model, two dental implants were inserted into the mandible perpendicular to the occlusal plane. In the tilted model, the posterior implant was rotated 30° around the buccal-lingual axis. In both models, maximum stress was detected at the neck region of the posterior implant. This maximum stress was greater in the tilted model than in the parallel model. However, since the corresponding strain was considerably lower in the parallel model, according to mechanostat theory several elements in the parallel model were at risk of bone loss. In contrast, the greater strain in the tilted model decreased the likelihood of bone loss. These findings suggest that use of tilted implants in the treatment of edentulous mandible would reduce the probability of bone loss in vulnerable parts of the osseous tissue surrounding dental implants.

Substrate topography interacts with substrate stiffness and culture time to regulate mechanical properties and smooth muscle differentiation of mesenchymal stem cells.

Substrate stiffness and topography are two powerful means by which mesenchymal stem cells (MSCs) activities can be modulated. The effects of substrate stiffness on the MSCs mechanical properties were investigated previously, however, the role of substrate topography in this regard is not yet well understood. Moreover, in vessel wall, these two physical cues act simultaneously to regulate cellular function, hence it is important to investigate their cooperative effects on cellular activity. Herein, we investigated the combined effects of substrate stiffness, substrate topography and culture time on the mechanical behavior of MSCs. The MSCs were cultured on the stiff and soft substrates with or without micro-grooved topography for 10 days and their viscoelastic properties and smooth muscle (SM) gene expression were investigated on days 2, 6 and 10. In general, substrate topography significantly interacted with substrate stiffness as well as culture time in the modulation of cell viscoelastic behavior and SM gene expression. The micro-grooved, stiff substrates resulted in the maximum cell stiffness and gene expression of α-actin and h1-calponin, and these values were detected to be minimum in the smooth, soft substrates. The findings can be helpful in the mechano-regulation of MSCs for vascular tissue engineering applications.

The effects of short-term uniaxial strain on the mechanical properties of mesenchymal stem cells upon TGF-ß1 stimulation.

Cellular mechanical characteristics represent cell ability to produce tissue-specific metabolites. Therefore, to achieve effective cell therapy, a better understanding of the effects of chemo-mechanical stimuli on the mechanical properties of in vitro-treated cells is essential. Herein, we investigated the effects of uniaxial strain on the mechanical properties of mesenchymal stem cells (MSCs) upon transforming growth factor beta 1 (TGF-ß1) stimulation. The MSCs were categorized into control and test groups. In one test group, the MSCs were treated by TGF-ß1 for 6 d, and in the other, they were additionally subjected to 1-d uniaxial strain on day 2. The cell mechanical properties and smooth muscle (SM) gene expression were assessed on days 2, 4, and 6. During the entire experiment, the MSCs treated by TGF-ß1 ± uniaxial strain were induced to differentiate into SM-like cells by significantly upregulation of α-actin, SM22α, and h1-calponin in respect to the control samples. When the MSCs were treated with TGF-ß1 alone, their stiffness and viscosity decreased significantly on day 2 and then increased by increase in culture time. When the cells were subjected to 1-d uniaxial strain upon TGF-ß1 stimulation, their stiffness and viscosity significantly increased on days 2 and 4 and then decreased on day 6 to a level comparable to that of TGF-ß1 group. Different paths were noticeable among the treated samples to reach nearly similar states on day 6. It seems that uniaxial strain activates mechanobiological cascades by which cellular mechanical behavior can be regulated after its removal. However, these effects are transient and would diminish over time. The findings may be helpful in the chemo-mechanical regulation of MSCs.

Radiation therapy affects the mechanical behavior of human umbilical vein endothelial cells.

Radiation therapy has been widely utilized as an effective method to eliminate malignant tumors and cancerous cells. However, subjection of healthy tissues and the related networks of blood vessels adjacent to the tumor area to irradiation is inevitable. The aim of this study was to investigate the consequent effects of fractionation radiotherapy on the mechanical characteristics of human umbilical vein endothelial cells (HUVECs) through alterations in cytoskeleton organization and cell and nucleus morphology. In order to simulate the clinical condition of radiotherapy, the HUVECs were exposed to the specific dose of 2 Gy for 1-4 times among four groups with incremental total dose from 2 Gy up to 8 Gy. Fluorescence staining was performed to label F-actin filaments and nuclei. Micropipette aspiration and standard linear solid model were employed to evaluate the elastic and viscoelastic characteristics of the HUVECs. Radiotherapy significantly increased cell elastic moduli. Due to irradiation, instantaneous and equilibrium Young's modulus were also increased. Radiotherapy diminished HUVECs viscoelastic behavior and shifted their creep compliance curves downward. Furthermore, gamma irradiation elevated the nuclei sizes and to a lesser extent the cells sizes resulting in the accumulation of F-actin filaments within the rest of cell body. Endothelial stiffening correlates with endothelial dysfunction, hence the results may be helpful when the consequent effects of radiotherapy are the focus of concern.

Stepwise morphological changes and cytoskeletal reorganization of human mesenchymal stem cells treated by short-time cyclic uniaxial stretch.

This study aimed to investigate stepwise remodeling of human mesenchymal stem cells (hMSCs) in response to cyclic stretch through rearrangement and alignment of cells and cytoskeleton regulation toward smooth muscle cell (SMC) fate in different time spans. Image analysis techniques were utilized to calculate morphological parameters. Cytoskeletal reorganization was observed by investigating F-actin filaments using immunofluorescence staining, and expression level of contractile SMC markers was followed by a quantitative polymerase chain reaction method. Applying cyclic uniaxial stretch on cultured hMSCs, utilizing a costume-made device, led to alteration in fractal dimension (FD) and cytoskeleton structure toward continuous alignment and elongation of cells by elevation of strain duration. Actin filaments became more aligned perpendicular to the axis of mechanical stretch by increasing uniaxial loading duration. At first, FD met a significant decrease in 4 h loading duration then increased significantly by further loading up to 16 h, followed by another decrease up to 1 d of uniaxial stretching. HMSCs subjected to 24 h cyclic uniaxial stretching significantly expressed early and intermediate contractile SM markers. It was hypothesized that the increase in FD after 4 h while cells continuously became more aligned and elongated was due to initiation of change in phenotype that influenced arrangement of cells. At this point, change in cell phenotype started leading to change in morphology while mechanical loading still caused cell alignment and rearrangement. Results can be helpful when optimized engineered cells are needed based on mechanical condition for functional engineered tissue and cell therapy.