Study on the Expansion Dynamics of MDCK Epithelium by Interstitial Flow Using a Traction Force-Measurable Microfluidic Chip

The movement of collective cells is affected through changes in physical interactions of cells in response to external mechanical stimuli, including fluid flow. Most tissues are affected by fluid flow at the interstitial level, but few studies have investigated the physical effects in collective cells affected by a low flow rate. In this study, collective cell migration of Madin–Darby canine kidney (MDCK) epithelial cells was investigated under static or interstitial flow (0, 0.1, and 1 μL/min) using a traction microfluidic device. The optimization of calculation of cellular traction forces was first achieved by changing interrogation window size from the fluorescent bead images. Migration analysis of cell collectives patterned with a 700 μm circular shape reveals that cells under the slow flow (0.1 and 1 μL/min) showed the inhibitory migration by decreasing cell island size and cellular speed compared to that of static condition. Analysis of cellular forces shows that level of traction forces was lower in the slow flow condition (~20 Pa) compared to that of static condition (~50 Pa). Interestingly, the standard deviation of traction force of cells was dramatically decreased as the flow rate increased from 0 to 1 μL/min, which indicates that flow affects the distribution of cellular traction forces among cell collectives. Cellular tension was increased by 50% in the cells under the fluid flow rate of 1 μL/min. Treatment of calcium blocker increased the migratory speed of cells under the flow condition, whereas there is little change of cellular forces. In conclusion, it has been shown that the interstitial flow inhibited the collective movement of epithelial cells by decreasing and re-distributing cellular forces. These findings provide insights into the study of the effect of interstitial flow on cellular behavior, such as development, regeneration, and morphogenesis

Thermoresponsive Behavior of Magnetic Nanoparticle Complexed pNIPAm-co-AAc Microgels

Characterization of responsive hydrogels and their enhancement with novel moieties have improved our understanding of functional materials. Hydrogels coupled with inorganic nanoparticles have been sought for novel types of responsive materials, but the efficient routes for the formation and the responsivity of complexed materials remain for further investigation. Here, we report that responsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) hydrogel microparticles (microgels) are tunable by varying composition of co-monomer and crosslinker as well as by their complexation with magnetic nanoparticles in aqueous dispersions. Our results show that the hydrodynamic diameter and thermoresponsivity of microgels are closely related with the composition of anionic co-monomer, AAc and crosslinker, N,N′-Methylenebisacrylamide (BIS). As a composition of hydrogels, the higher AAc increases the swelling size of the microgels and the volume phase transition temperature (VPTT), but the higher BIS decreases the size with no apparent effect on the VPTT. When the anionic microgels are complexed with amine-modified magnetic nanoparticles (aMNP) via electrostatic interaction, the microgels decrease in diameter at 25 °C and shift the volume phase transition temperature (VPTT) to a higher temperature. Hysteresis on the thermoresponsive behavior of microgels is also measured to validate the utility of aMNP-microgel complexation. These results suggest a simple, yet valuable route for development of advanced responsive microgels, which hints at the formation of soft nanomaterials enhanced by inorganic nanoparticles

Energetic Contributions Including Gender Differences and Metabolic Flexibility in the General Population and Athletes

Metabolic flexibility includes the ability to perform fat and carbohydrate oxidation, as well as oxidative capacity, which is associated with mitochondrial function, energetic contributions, and physical health and performance. During a session of graded incremental exercise testing (GIET), we investigated metabolic flexibility, the contributions of three energy systems, and performances of individuals with different metabolic characteristics. Fifteen general population (GP; n = 15, male n = 7, female n = 8) and 15 national-level half-marathon and triathlon athletes (A; n = 15, male n = 7, female n = 8) participated in this study. During GIET, heart rate (HR), oxygen uptake (V˙O2mean and V˙CO2mean), metabolic equivalents (METs) in V˙O2mean, and blood glucose and lactate concentrations (La−) were measured. Furthermore, jogging/running speeds (S) at specific La−, fat and carbohydrate oxidations (FATox and CHOox), and energetic contributions (oxidative; WOxi, glycolytic; WGly, and phosphagen; WPCr) were calculated. The percentages of HRmax, relative V˙O2mean, V˙CO2mean, and METs in V˙O2mean were all lower in A than they were in GP. FATox values were lower in GP than in A, while CHOox and La− were higher in GP than in A. Negative correlations between La− and FATox were also observed in both groups. Contributions of WOxi, WGly, and WPCr were higher in GP than in A during GIET. Moreover, values of WGly, and WPCr were significantly lower and higher, respectively, in male GP than in female GP. Furthermore, S at specific La− were higher in A than in GP. It is suggested that an individualized low-intensity recovery exercise program be established, to achieve increased metabolic flexibility and oxidative capacity (aerobic base), such as public health improvements and a greater volume of higher exercise intensities; this is the type of exercise that elite athletes worldwide mostly perform during their training period and progression. This may prevent cardiac/metabolic diseases in GP

Effect of bFGF and fibroblasts combined with hyaluronic acid-based hydrogels on soft tissue augmentation: an experimental study in rats

Background Hyaluronic acid (HA) has been applied as a primary biomaterial for temporary soft tissue augmentation and as a carrier for cells and the delivery of growth factors to promote tissue regeneration. Although HA derivatives are the most versatile soft tissue fillers on the market, they are resorbed early, within 3 to 12 months. To overcome their short duration, they can be combined with cells or growth factors. The purpose of this study was to investigate the stimulating effects of human fibroblasts and basic fibroblast growth factors (bFGF) on collagen synthesis during soft tissue augmentation by HA hydrogels and to compare these with the effects of a commercial HA derivative (Restylane®). Methods The hydrogel group included four conditions. The first condition consisted of hydrogel (H) alone as a negative control, and the other three conditions were bFGF-containing hydrogel (HB), human fibroblast-containing hydrogel (HF), and human fibroblast/bFGF-containing hydrogel (HBF). In the Restylane® group (HGF), the hydrogel was replaced with Restylane® (R, RB, RF, RBF). The gels were implanted subdermally into the back of each nude mouse at four separate sites. Twelve nude mice were used for the hydrogel (n = 6) and Restylane® groups (n = 6). The specimens were harvested 8 weeks after implantation and assessed histomorphometrically, and collagen synthesis was evaluated by RT-PCR. Results The hydrogel group showed good biocompatibility with the surrounding tissues and stimulated the formation of a fibrous matrix. HBF and HF showed significantly higher soft tissue synthesis compared to H (p < 0.05), and human collagen type I was well expressed in HB, HF, and HBF; HBF showed the strongest expression. The Restylane® filler was surrounded by a fibrous capsule without any soft tissue infiltration from the neighboring tissue, and collagen synthesis within the Restylane® filler could not be observed, even though no inflammatory reactions were observed. Conclusion This study revealed that HA-based hydrogel alone or hydrogel combined with fibroblasts and/or bFGF can be effectively used for soft tissue augmentation

Activation of Calf Intestinal Alkaline Phosphatase by Trifluoroethanol

Alkaline phosphatase is a stable enzyme which is strongly resistant to urea, guanidine hydrochloride, acid pH, and heat. But there have been few studies on the effect of organic cosolvents on the activity and structure of alkaline phosphatase. The activity of calf intestinal alkaline phosphatase (CIAP) is markedly increased when incubated in solutions with elevated trifluoroethanol (TFE) concentrations. The activation is a time dependent course. There is a very fast phase in the activation kinetics in the mixing dead time (30 s) using convential methods. Further activation after the very fast phase follows biphasic kinetics. The structural basis of the activation has been monitored by intrinsic fluorescence and far ultraviolet circular dichroism. TFE (0-60%) did not lead to any significant change in the intrinsic fluorescence emission maximum, indicating no significant change in the tertiary structure of CIAP. But TFE did significantly change the secondary structure of CIAP, especially increasing α content. We conclude that the activation of CIAP is due to its secondary structural change. The time for the secondary structure change induced by TFE preceds that of the activity increase. These results suggest that a rapid conformational change of CIAP induced by TFE results in the enhancement of CIAP activity, followed by further increase of this activity because of the further slightly slower rearrangements of the activated conformation. It is concluded that the higher catalytic activity of CIAP can be attained with various secondary structures

Activation of Calf Intestinal Alkaline Phosphatase by Trifluoroethanol

Alkaline phosphatase is a stable enzyme which is strongly resistant to urea, guanidine hydrochloride, acid pH, and heat. But there have been few studies on the effect of organic cosolvents on the activity and structure of alkaline phosphatase. The activity of calf intestinal alkaline phosphatase (CIAP) is markedly increased when incubated in solutions with elevated trifluoroethanol (TFE) concentrations. The activation is a time dependent course. There is a very fast phase in the activation kinetics in the mixing dead time (30 s) using convential methods. Further activation after the very fast phase follows biphasic kinetics. The structural basis of the activation has been monitored by intrinsic fluorescence and far ultraviolet circular dichroism. TFE (0-60%) did not lead to any significant change in the intrinsic fluorescence emission maximum, indicating no significant change in the tertiary structure of CIAP. But TFE did significantly change the secondary structure of CIAP, especially increasing α content. We conclude that the activation of CIAP is due to its secondary structural change. The time for the secondary structure change induced by TFE preceds that of the activity increase. These results suggest that a rapid conformational change of CIAP induced by TFE results in the enhancement of CIAP activity, followed by further increase of this activity because of the further slightly slower rearrangements of the activated conformation. It is concluded that the higher catalytic activity of CIAP can be attained with various secondary structures

Collagen Type I Containing Hybrid Hydrogel Enhances Cardiomyocyte Maturation in a 3D Cardiac Model

In vitro maturation of cardiomyocytes in 3D is essential for the development of viable cardiac models for therapeutic and developmental studies. The method by which cardiomyocytes undergoes maturation has significant implications for understanding cardiomyocytes biology. The regulation of the extracellular matrix (ECM) by changing the composition and stiffness is quintessential for engineering a suitable environment for cardiomyocytes maturation. In this paper, we demonstrate that collagen type I, a component of the ECM, plays a crucial role in the maturation of cardiomyocytes. To this end, embryonic stem-cell derived cardiomyocytes were incorporated into Matrigel-based hydrogels with varying collagen type I concentrations of 0 mg, 3 mg, and 6 mg. Each hydrogel was analyzed by measuring the degree of stiffness, the expression levels of MLC2v, TBX18, and pre-miR-21, and the size of the hydrogels. It was shown that among the hydrogel variants, the Matrigel-based hydrogel with 3 mg of collagen type I facilitates cardiomyocyte maturation by increasing MLC2v expression. The treatment of transforming growth factor &#946;1 (TGF-&#946;1) or fibroblast growth factor 4 (FGF-4) on the hydrogels further enhanced the MLC2v expression and thereby cardiomyocyte maturation

Development of a 3-D Physical Dynamics Monitoring System Using OCM with DVC for Quantification of Sprouting Endothelial Cells Interacting with a Collagen Matrix

The extracellular matrix (ECM) plays a key role during cell migration, proliferation, and differentiation by providing adhesion sites and serving as a physical scaffold. Elucidating the interaction between the cell and ECM can reveal the underlying mechanisms of cellular behavior that are currently unclear. Analysis of the deformation of the ECM due to cell&ndash;matrix interactions requires microscopic, three-dimensional (3-D) imaging methods, such as confocal microscopy and second-harmonic generation microscopy, which are currently limited by phototoxicity and bleaching as a result of the point-scanning approach. In this study, we suggest the use of optical coherence microscopy (OCM) as a live-cell, volumetric, fast imaging tool for analyzing the deformation of fibrous ECM. We optimized such OCM parameters as the sampling rate to obtain images of the best quality that meet the requirements for robust digital volume correlation (DVC) analysis. Visualization and analysis of the mechanical interaction between collagen ECM and human umbilical vein endothelial cells (HUVECs) show that cellular adhesion during protrusion can be analyzed and quantified. The advantages of OCM, such as fine isotropic spatial resolution, fast time resolution, and low phototoxicity, make it the ideal optic tool for 3-D traction force microscopy

Directional migration of mesenchymal stem cells under an SDF-1α gradient on a microfluidic device.

Homing of peripheral stem cells is regulated by one of the most representative homing factors, stromal cell-derived factor 1 alpha (SDF-1α), which specifically binds to the plasma membrane receptor CXCR4 of mesenchymal stem cells (MSCs) in order to initiate the signaling pathways that lead to directional migration and homing of stem cells. This complex homing process and directional migration of stem cells have been mimicked on a microfluidic device that is capable of generating a chemokine gradient within the collagen matrix and embedding endothelial cell (EC) monolayers to mimic blood vessels. On the microfluidic device, stem cells showed directional migration toward the higher concentration of SDF-1α, whereas treatment with the CXCR4 antagonist AMD3100 caused loss of directionality of stem cells. Furthermore, inhibition of stem cell's main migratory signaling pathways, Rho-ROCK and Rac pathways, caused blockage of actomyosin and lamellipodia formation, decreasing the migration distance but maintaining directionality. Stem cell homing regulated by SDF-1α caused directional migration of stem cells, while the migratory ability was affected by the activation of migration-related signaling pathways