Gel Compaction Research Articles

Polymeric reinforcements for cellularized collagen-based vascular wall models: influence of the scaffold architecture on the mechanical and biological properties.

A previously developed cellularized collagen-based vascular wall model showed promising results in mimicking the biological properties of a native vessel but lacked appropriate mechanical properties. In this work, we aim to improve this collagen-based model by reinforcing it using a tubular polymeric (reinforcement) scaffold. The polymeric reinforcements were fabricated exploiting commercial poly (ε-caprolactone) (PCL), a polymer already used to fabricate other FDA-approved and commercially available devices serving medical applications, through 1) solution electrospinning (SES), 2) 3D printing (3DP) and 3) melt electrowriting (MEW). The non-reinforced cellularized collagen-based model was used as a reference (COL). The effect of the scaffold's architecture on the resulting mechanical and biological properties of the reinforced collagen-based model were evaluated. SEM imaging showed the differences in scaffolds' architecture (fiber alignment, fiber diameter and pore size) at both the micro- and the macrolevel. The polymeric scaffold led to significantly improved mechanical properties for the reinforced collagen-based model (initial elastic moduli of 382.05 ± 132.01kPa, 100.59 ± 31.15kPa and 245.78 ± 33.54kPa, respectively for SES, 3DP and MEW at day 7 of maturation) compared to the non-reinforced collagen-based model (16.63 ± 5.69kPa). Moreover, on day 7, the developed collagen gels showed stresses (for strains between 20% and 55%) in the range of [5-15] kPa for COL, [80-350] kPa for SES, [20-70] kPa for 3DP and [100-190] kPa for MEW. In addition to the effect on the resulting mechanical properties, the polymeric tubes' architecture influenced cell behavior, in terms of proliferation and attachment, along with collagen gel compaction and extracellular matrix protein expression. The MEW reinforcement resulted in a collagen gel compaction similar to the COL reference, whereas 3DP and SES led to thinner and longer collagen gels. Overall, it can be concluded that 1) the selected processing technique influences the scaffolds' architecture, which in turn influences the resulting mechanical and biological properties, and 2) the incorporation of a polymeric reinforcement leads to mechanical properties closely matching those of native arteries.

Spectrin-Based Regulation of Cardiac Fibroblast Cell-Cell Communication.

Cardiac fibroblasts (CFs) maintain the fibrous extracellular matrix (ECM) that supports proper cardiac function. Cardiac injury induces a transition in the activity of CFs to promote cardiac fibrosis. CFs play a critical role in sensing local injury signals and coordinating the organ level response through paracrine communication to distal cells. However, the mechanisms by which CFs engage cell-cell communication networks in response to stress remain unknown. We tested a role for the action-associated cytoskeletal protein βIV-spectrin in regulating CF paracrine signaling. Conditioned culture media (CCM) was collected from WT and βIV-spectrin deficient (qv4J) CFs. WT CFs treated with qv4J CCM showed increased proliferation and collagen gel compaction compared to control. Consistent with the functional measurements, qv4J CCM contained higher levels of pro-inflammatory and pro-fibrotic cytokines and increased concentration of small extracellular vesicles (30-150 nm diameter, exosomes). Treatment of WT CFs with exosomes isolated from qv4J CCM induced a similar phenotypic change as that observed with complete CCM. Treatment of qv4J CFs with an inhibitor of the βIV-spectrin-associated transcription factor, STAT3, decreased the levels of both cytokines and exosomes in conditioned media. This study expands the role of the βIV-spectrin/STAT3 complex in stress-induced regulation of CF paracrine signaling.

PH and Thrombin Concentration Are Decisive in Synthesizing Stiff, Stable, and Open-Porous Fibrin-Collagen Hydrogel Blends without Chemical Cross-Linker.

Fibrin-collagen hydrogel blends exhibit high potential for tissue engineering applications. However, it is still unclear whether the underlying cross-linking mechanisms are of chemical or physical nature. It is here hypothesized that chemical cross-linkers play a negligible role and that instead pH and thrombin concentration are decisive for synthetizing blends with high stiffness and hydrolytic stability. Different fibrin-collagen formulations (pure and with additional transglutaminase) are used and the blends' compaction rate, hydrolytic stability, compressive strength, and hydrogel microstructure are investigated. The effect of thrombin concentration on gel compaction is examined and the importance of pH control during synthesis observed. It is revealed that transglutaminase impairs gel stability and it is deduced that fibrin-collagen blends mainly cross-link by mechanical interactions due to physical fibril entanglement as opposed to covalent bonds from chemical cross-linking. High thrombin concentrations and basic pH during synthesis reduce gel compaction and enhance stiffness and long-term stability. Scanning electron microscopy reveals a highly interpenetrating fibrous network with unique, interconnected open-porous microstructures. Endothelial cells proliferate on the blends and form a confluent monolayer. This study reveals the underlying cross-linking mechanisms and presents enhanced fibrin-collagen blends with high stiffness, hydrolytic stability, and large, interconnected pores; findings that offer high potential for advanced tissue engineering applications.

Physically-based structural modeling of a typical regenerative tissue analog bridges material macroscale continuum and cellular microscale discreteness and elucidates the hierarchical characteristics of cell-matrix interaction

This paper presents a comprehensive physically-based structural modelling for the passive and active biomechanical processes in a typical engineered tissue – namely, cell-compacted collagen gel. First, it introduces a sinusoidal curve analog for quantifying the mechanical response of the collagen fibrils and a probability distribution function of the characteristic crimp ratio for taking into account the fibrillar geometric entropic effect. The constitutive framework based on these structural characteristics precisely reproduces the nonlinearity, the viscoelasticity, and fairly captures the Poisson effect exhibiting in the macroscale tensile tests; which, therefore, substantially validates the structural modelling for the analysis of the cell-gel interaction during collagen gel compaction. Second, a deterministic molecular clutch model specific to the interaction between the cell pseudopodium and the collagen network is developed, which emphasizes the dependence of traction force on clutch number altering with the retrograde flow velocity, actin polymeric velocity, and the deformation of the stretched fibril. The modelling reveals the hierarchical features of cellular substrate sensing, i.e. a biphasic traction force response to substrate elasticity begins at the level of individual fibrils and develops into the second biphasic sensing by means of the fibrillar number integration at the whole-cell level. Singular in crossing the realms of continuum and discrete mechanics, the methodologies developed in this study for modelling the filamentous materials and cell-fibril interaction deliver deep insight into the temporospatially dynamic 3D cell-matrix interaction, and are able to bridge the cellular microscale and material macroscale in the exploration of related topics in mechanobiology.

Autological fibrin matrixs: prospect surgery use

The article presents a technique for obtaining autologous carriers of tissue micrografts, or scaffolds, based on the interaction of fibrin and cells of the implanted material, as well as a method for the controlled production of autologous tissue matrices of the required volume, which are easily modeled. The processes of formation of a fibrin clot, its main physical characteristics have been investigated. The process of matrix gel compaction is considered, which significantly stimulates the attachment of tissue micrografts to the scaffold surface, which promotes metabolic processes.

Cancer cell migration in collagen-hyaluronan composite extracellular matrices

Hyaluronan (HA) is a key component in the tumor microenvironment (TME) that participates in cancer growth and invasiveness. While the molecular weight (MW) dependent properties of HA can cause tumor-promoting and -repressing effects, the elevated levels of HA in the TME impedes drug delivery. The degradation of HA using hyaluronidases (HYALs), resulting in fragments of HA, is a way to overcome this, but the consequences of changes in HA molecular weight and concentration is currently unknown. Therefore, it is critical to understand the MW-dependent biological effects of HA. Here we examine the influence of HA molecular weight on biophysical properties that regulate cell migration and extracellular matrix (ECM) remodeling. In our study, we used vLMW, LMW and HMW HA at different physiologically relevant concentrations, with a particular interest in correlating the mechanical and structural properties to different cell functions. The elastic modulus, collagen network pore size and collagen fiber diameter increased with increasing HA concentration. Although the collagen network pore size increased, these pores were filled with the bulky HA molecules. Consequently, cell migration decreased with increase in HA concentration due to multiple, long-lived and unproductive protrusions, suggesting the influence of steric factors. Surprisingly, even though elastic modulus increased with HA molecular weight and concentration, gel compaction assays showed an increased degree of ECM compaction among HMW HA gels at high concentrations (2 and 4 mg mL−1 [0.2 and 0.4%]). These results were not seen in collagen gels that lacked HA, but had similar stiffness. HA appears to have the effect of decreasing migration and increasing collagen network contraction, but only at high HA molecular weight. Consequently, changes in HA molecular weight can have relatively large effects on cancer cell behavior. Statement of significanceHyaluronan (HA) is a critical component of the tumor microenvironment (TME). Overproduction of HA in the TME results in poor prognosis and collapse of blood vessels, inhibiting drug delivery. Hyaluronidases have been used to enhance drug delivery. However, they lead to low molecular weight (MW) HA, altering the mechanical and structural properties of the TME and cancer cell behavior. Understanding how HA degradation affects cancer cell behavior is critical for uncovering detrimental effects of this therapy. Very little is known about how HA MW affects cancer cell behavior in tumor-mimicking collagen-HA composite networks. Here we examine how MW and HA content in collagen-HA networks alter structural and mechanical properties to regulate cell migration and matrix remodeling in 3D TME-mimicking environments.

Protein gel shrinkage during solvent exchange: Quantification of gel compaction, mass transfer and compressive strength

In this work, we experimentally investigate into one step solvent exchange of cylindrical shaped whey protein isolate (WPI) gels, where the pore fluid water inside the pores is replaced by ethanol. Solvent exchange has three major impacts on macroscopic gel properties, leading to an increase in compressive strength, shrinkage of the gel cylinders diameter and consequently a change in the spatial protein distribution. 1D-Raman spectroscopy was employed for in-situ investigation of pore fluid composition and protein density distribution, together with a conventional camera to track the diameter change of the gel during solvent exchange. Compressive strength of the gel cylinders is measured ex-situ using a universal testing machine. For gels with three different protein concentrations, we report comparable shrinkage of the diameter to about 85% of the initial size, with most shrinkage taking place during the first stages of solvent exchange, when ethanol is detected in the outermost layers only. Further shrinkage is prevented by a sharp increase in mechanical stiffness of the gels. Solvent exchange leads to an inhomogeneous protein density distribution for most gels, with a compaction towards the radial center. We showed a complex interaction of solvent exchange kinetics and pore fluid dependent stiffness of the gel matrix controls the final size and density distribution of the gel.

Spontaneous Lung Fibrosis Resolution Reveals Novel Antifibrotic Regulators

Fibroblast activation is transient in successful wound repair but persistent in fibrotic pathologies. Understanding fibroblast deactivation during successful wound healing may provide new approaches to therapeutically reverse fibroblast activation. To characterize the gene programs that accompany fibroblast activation and reversal during lung fibrosis resolution, we used RNA sequencing analysis of flow sorted Col1α1-GFP-positive and CD45-, CD31-, and CD326-negative cells isolated from the lungs of young mice exposed to bleomycin. We compared fibroblasts isolated from control mice with those isolated at Days 14 and 30 after bleomycin exposure, representing the peak of extracellular matrix deposition and an early stage of fibrosis resolution, respectively. Bleomycin exposure dramatically altered fibroblast gene programs at Day 14. Principal component and differential gene expression analyses demonstrated the predominant reversal of these trends at Day 30. Upstream regulator and pathway analyses of reversing "resolution" genes identified novel candidate antifibrotic genes and pathways. Two genes from these analyses that were decreased in expression at Day 14 and reversed at Day 30, Aldh2 and Nr3c1, were selected for further analysis. Enhancement of endogenous expression of either gene by CRISPR activation in cultured human idiopathic pulmonary fibrosis fibroblasts was sufficient to reduce profibrotic gene expression, fibronectin deposition, and collagen gel compaction, consistent with roles for these genes in fibroblast deactivation. This combination of RNA sequencing analysis of freshly sorted fibroblasts and hypothesis testing in cultured idiopathic pulmonary fibrosis fibroblasts offers a path toward identification of novel regulators of lung fibroblast deactivation, with potential relevance to understanding fibrosis resolution and its failure in human disease.

Leveraging the thermoresponsiveness of fluorinated poly(N-isopropylacrylamide) copolymers as a sensing tool for perfluorooctane sulfonate.

Due to mounting evidence of the negative health effects of persistent perfluoroalkyl acids (PFAAs) with long (i.e., >C7) tails, there is a need for convenient systems capable of sensing these contaminants at dilute aqueous concentrations. To address this concern, a thermoresponsive polymeric network composed of poly(N-isopropylacrylamide) copolymerized with fluorinated comonomers was studied to characterize the gel's physical response to fluorosurfactants in solution. Incorporating fluorinated comonomers into the polymer backbone raised their swelling in fluorocontaminant solutions relative to water - gels synthesized with 10.0 mol% 2,2,2-trifluoroethyl acrylate (TFEA) displayed a heightened maximum water-analyte swelling difference of 3761 ± 147% compared to 3201 ± 466% for non-fluorinated gels in the presence of 1 mM tetraethylammonium perfluorooctane sulfonate (TPFOS). The normalized area under the curve for gels with 12.5 mol% TFEA was further raised to 1.77 ± 0.09, indicating a broadened response window for the contaminant, but at the cost of reducing the overall swelling ratio to 3227 ± 166% and elongating the time required to reach swelling equilibrium. Overall, a copolymer fed with 10.7 mol% TFEA was predicted to maximize both the swelling and response window of the polymer toward TPFOS. Equilibration times followed a logarithmic increase as the percentage of comonomer was raised, noting gradual fluorosurfactant penetration into the gels impeded by initial gel compaction caused by the addition of fluorinated comonomers. Comparative study of gels containing 1H,1H,7H-dodecafluoroheptyl acrylate, TFEA, or 1,1,1,3,3,3-hexafluoroisopropyl acrylate identified careful selection of fluorinated comonomers and their feed ratios as useful tools for tailoring the network's swelling response to TPFOS.

A human hair-based platform for long-term maintenance of 3D engineered cardiac tissues in vitro

In vitro cardiac models constructed with human cardiomyocytes and appropriate biomaterial scaffolds are still in pressing need for drug testing and disease modeling. Here, we describe a platform based on human hair to generate 3D cardiac tissues, termed cardiowire, from human induced pluripotent stem cell-derived cardiomyocytes. The cardiowire was cultivated for an extended period of time (up to 118 days). To monitor the contractile motion of the cardiac tissue, video-based analysis was performed throughout the time course of cultivation. After an appreciably long time (52 days) of culture, the cardiowire remained its strong contractility. Although the gel compaction went on with time, the width of the cardiowire was as large as 1 mm after 100 days. Moreover, it was demonstrated that human hair not only could support cell attachment, but also had long-term mechanical strength to sustain the highly repetitive mechanical stress of the cardiac tissue contraction. This platform could be easily combined with electrical and mechanical stimulation and serve as an in vitro cardiac model for drug screening and other therapeutic applications.

Matrix stiffness regulates microvesicle-induced fibroblast activation.

Extracellular vesicles released by cancer cells have recently been implicated in the differentiation of stromal cells to their activated, cancer-supporting states. Microvesicles, a subset of extracellular vesicles released from the plasma membrane of cancer cells, contain biologically active cargo, including DNA, mRNA, and miRNA, which are transferred to recipient cells and induce a phenotypic change in behavior. While it is known that microvesicles can alter recipient cell phenotype, little is known about how the physical properties of the tumor microenvironment affect fibroblast response to microvesicles. Here, we utilized cancer cell-derived microvesicles and synthetic substrates designed to mimic the stiffness of the tumor and tumor stroma to investigate the effects of microvesicles on fibroblast phenotype as a function of the mechanical properties of the microenvironment. We show that microvesicles released by highly malignant breast cancer cells cause an increase in fibroblast spreading, α-smooth muscle actin expression, proliferation, cell-generated traction force, and collagen gel compaction. Notably, our data indicate that these phenotypic changes occur only on stiff matrices mimicking the stiffness of the tumor periphery and are dependent on the cell type from which the microvesicles are shed. Overall, these results show that the effects of cancer cell-derived microvesicles on fibroblast activation are regulated by the physical properties of the microenvironment, and these data suggest that microvesicles may have a more robust effect on fibroblasts located at the tumor periphery to influence cancer progression.

Increasing Cell Seeding Density Improves Elastin Expression and Mechanical Properties in Collagen Gel-Based Scaffolds Cellularized with Smooth Muscle Cells.

Vascular tissue engineering combines cells with scaffold materials in vitro aiming the development of physiologically relevant vascular models. For natural scaffolds such as collagen gels, where cells can be mixed with the material solution before gelation, cell seeding density is a key parameter that can affect extracellular matrix deposition and remodeling. Nonetheless, this parameter is often overlooked and densities sensitively lower than those of native tissues, are usually employed. Herein, the effect of seeding density on the maturation of tubular collagen gel-based scaffolds cellularized with smooth muscle cells is investigated. The compaction, the expression, and deposition of key vascular proteins and the resulting mechanical properties of the constructs are evaluated up to 1 week of maturation. Results show that increasing cell seeding density accelerates cell-mediated gel compaction, enhances elastin expression (more than sevenfold increase at the highest density, Day 7) and finally improves the overall mechanical properties of constructs. Of note, the tensile equilibrium elastic modulus, evaluated by stress-relaxation tests, reach values comparable to native arteries for the highest cell density, after a 7-day maturation. Altogether, these results show that higher cell seeding densities promote the rapid maturation of collagen gel-based vascular constructs toward structural and mechanical properties better mimicking native arteries.

Composite Polymer-Clay Hydrogels Based on Bentonite Clay and Acrylates: Synthesis, Characterization and Swelling Capacity

Clay minerals, especially montmorillonite, as well as bentonite minerals with a high content of montmorillonite, have the highest ability to adsorb various organic compounds. In the East Kazakhstan region, the Manyrak deposit has huge deposits of “pink” bentonite clay (BC) with 70% of montmorillonite, extensively studied back in the 70-80-s. Now it is successfully useful in the organo-polymer composition as a mineral filler of polymer composite materials (PCM) and finds the application as domestic sorbents for the group extraction of non-ferrous ions in the purification of industrial sewage and drinking water. This article presents the results of research of creating polymer-clay composite materials based on domestic bentonite clay with improved sorption characteristics. The polymeric matrix of the composites consists of acrylates ‒ polyacrylic and polymethacrylic acids (PAA and PMAA). BC-PAA and BC-PMAA gels were obtained by radical polymerization “in situ” using the intercalation method (and wi thout it). Preliminary intercalation forms more homogeneous and interconnected composite gels. An increase in the content of the clay component and the cross-linking agent in the starting mixture results in a higher cross-linking rate and compaction of the composite gels. The behavior of the swelling of gels under the influence of temperature, pH, ionic strength indicates their polyelectrolyte character with dominant hydrogen bonds and partial hydrophobic interactions (the latter is improved in the case of BC-PMAA). The preparation of composite gels based on local BC and PAA and PMAA expands the range of composite materials and can be used as sorbents for wastewater treatment.

Unpacking compaction: Effect of hydraulic pressure on alginate fouling

High pressure is often considered to be the cause of the high fouling propensity of reverse osmosis (RO) relative to forward osmosis (FO). Several experimental studies have shown that alginate fouling is more susceptible to cleaning in FO than in RO, but the theory that foulant compaction causes this disparity seems to be contradicted by the incompressibility of alginate hydrogels. In addition, the effect of hydraulic pressure on fouling in osmotic membrane desalination has never been experimentally isolated, because fixed-flux comparisons at different hydraulic pressures require different draw solution osmotic pressures. In this study, a new approach to isolating the effect of hydraulic pressure on alginate fouling and cleaning is introduced: operating FO with elevated but equal feed and draw hydraulic pressures. The same concentration of sodium chloride is used as the draw solution in all trials to eliminate possible effects of draw solution osmotic pressure on membrane fouling or cleaning. Theoretical modeling of the effect of alginate foulant compaction on flux reveals that foulant compaction should accelerate flux decline with low salinity feeds but retard flux decline at high salinity. However, in low-salinity alginate fouling trials, for which foulant compaction should accelerate flux decline, the measured flux decline rate was not affected by hydraulic pressure. Furthermore, when fouled membranes were cleaned by increasing the feed velocity and reducing the draw osmotic pressure, there was no apparent relationship between hydraulic pressure and cleaning effectiveness. Finally, in situ visualization of foulant removal during the cleaning process revealed no difference in foulant removal mechanisms between different hydraulic pressures. These findings demonstrate that alginate gel compaction by high feed hydraulic pressure does not occur and suggest that other explanations should be sought for FO's high fouling resistance relative to RO.

Plugging Behavior of Polymer Gel in Fractures by Multi-Slug Injection

In order to understand the plugging mechanism, experiments of multi-slug gel plugging in single fracture and multiple fractures system were simulated by indoor visual fracture models. The experiments showed that there were two stages in the process of multi-slug gel plugging in single fracture: gel filling stage and gel compaction stage. When the total injection of gel is consistent, the brine flooding pressure gradient after multi-slug gel injection increased more than 2 times than the pressure gradient after single slug gel continuous injection. While there were three stages in the process of multi-slug gel plugging in multiple fractures system: Gel selectively entrance stage, gel filling stage and gel compaction stage. However, there would be no gel compaction stage, even no gel filling stage in the process of single cycle gel plugging in multiple fractures system. Key words: Fractured reservoir; Multi-slug; Polymer gel; Plugging process

α-Actinin-4 Is Required for Amoeboid-type Invasiveness of Melanoma Cells

α-Actinin-4 (ACTN4), a key regulator of the actin cytoskeleton, is up-regulated in melanoma, though its role in melanoma remains speculative. We have discovered that in WM1158, a highly aggressive melanoma cell line, down-regulation of ACTN4 by shRNA induces a collagen I-dependent amoeboidal-to-mesenchymal transition. Re-expression of low levels of WT ACTN4 but not similar expression levels of ACTN1 successfully restores the amoeboidal morphology and limits collagen I gel compaction. A truncated ACTN4 mutant 1-890, which lacks the C-terminal tail, fails to rescue the amoeboidal morphology and to compact collagen I gel. Interestingly, in three-dimensional collagen I gels, ACTN4 KD cells are more polarized compared with cells in which scrambled shRNA is expressed. Surprisingly, ACTN4 KD cells migrate faster than the ones expressing the scrambled shRNA on a collagen I gel (two-dimensional) although these two cell lines migrate similarly on tissue culture. Most importantly, down-regulation of ACTN4 significantly reduced invasion of WM1158 cells into the three-dimensional collagen I gel, a representative of the dermis. Taken together, these findings suggest that ACTN4 plays an important role in maintaining the amoeboidal morphology of invasive melanoma and thus promoting dissemination through collagen-rich matrices.

The mechanisms of fibroblast-mediated compaction of collagen gels and the mechanical niche around individual fibroblasts

Fibroblast-mediated compaction of collagen gels attracts extensive attention in studies of wound healing, cellular fate processes, and regenerative medicine. However, the underlying mechanism and the cellular mechanical niche still remain obscure. This study examines the mechanical behaviour of collagen fibrils during the process of compaction from an alternative perspective on the primary mechanical interaction, providing a new viewpoint on the behaviour of populated fibroblasts. We classify the collagen fibrils into three types – bent, stretched, and adherent – and deduce the respective equations governing the mechanical behaviour of each type; in particular, from a putative principle based on the stationary state of the instantaneous Hamiltonian of the mechanotransduction system, we originally quantify the stretching force exerted on each stretched fibrils. Via careful verification of a structural elementary model based on this classification, we demonstrate a clear physical picture of the compaction process, quantitatively elucidate the panorama of the micro mechanical niche and reveal an intrinsic biphasic relationship between cellular traction force and matrix elasticity. Our results also infer the underlying mechanism of tensional homoeostasis and stress shielding of fibroblasts. With this study, and sequel investigations on the putative principle proposed herein, we anticipate a refocus of the research on cellular mechanobiology, in vitro and in vivo.

Bioreactor for modulation of cardiac microtissue phenotype by combined static stretch and electrical stimulation

We describe here a bioreactor capable of applying electrical field stimulation in conjunction with static strain and on-line force of contraction measurements. It consisted of a polydimethylsiloxane (PDMS) tissue chamber and a pneumatically driven stretch platform. The chamber contained eight tissue microwells (8.05 mm in length and 2.5 mm in width) with a pair of posts (2.78 mm in height and 0.8 mm in diameter) in each well to serve as fixation points and for measurements of contraction force. Carbon rods, stimulating electrodes, were placed into the PDMS chamber such that one pair stimulated four microwells. For feasibility studies, neonatal rat cardiomyocytes were seeded in collagen gels into the microwells. Following 3 days of gel compaction, electrical field stimulation at 3–4 V cm−1 and 1 Hz, mechanical stimulation of 5% static strain or electromechanical stimulation (field stimulation at 3–4 V cm−1, 1 Hz and 5% static strain) were applied for 3 days. Cardiac microtissues subjected to electromechanical stimulation exhibited elevated amplitude of contraction and improved sarcomere structure as evidenced by sarcomeric α-actinin, actin and troponin T staining compared to microtissues subjected to electrical or mechanical stimulation alone or non-stimulated controls. The expression of atrial natriuretic factor and brain natriuretic peptide was also elevated in the electromechanically stimulated group.

Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo

Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0–20mgml−1), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20mgml−1 HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5mgml−1 HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 20mgml−1 HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.

Agent-Based Modeling Traction Force Mediated Compaction of Cell-Populated Collagen Gels Using Physically Realistic Fibril Mechanics

Agent-based modeling was used to model collagen fibrils, composed of a string of nodes serially connected by links that act as Hookean springs. Bending mechanics are implemented as torsional springs that act upon each set of three serially connected nodes as a linear function of angular deflection about the central node. These fibrils were evaluated under conditions that simulated axial extension, simple three-point bending and an end-loaded cantilever. The deformation of fibrils under axial loading varied <0.001% from the analytical solution for linearly elastic fibrils. For fibrils between 100 μm and 200 μm in length experiencing small deflections, differences between simulated deflections and their analytical solutions were <1% for fibrils experiencing three-point bending and <7% for fibrils experiencing cantilever bending. When these new rules for fibril mechanics were introduced into a model that allowed for cross-linking of fibrils to form a network and the application of cell traction force, the fibrous network underwent macroscopic compaction and aligned between cells. Further, fibril density increased between cells to a greater extent than that observed macroscopically and appeared similar to matrical tracks that have been observed experimentally in cell-populated collagen gels. This behavior is consistent with observations in previous versions of the model that did not allow for the physically realistic simulation of fibril mechanics. The significance of the torsional spring constant value was then explored to determine its impact on remodeling of the simulated fibrous network. Although a stronger torsional spring constant reduced the degree of quantitative remodeling that occurred, the inclusion of torsional springs in the model was not necessary for the model to reproduce key qualitative aspects of remodeling, indicating that the presence of Hookean springs is essential for this behavior. These results suggest that traction force mediated matrix remodeling may be a robust phenomenon not limited to fibrils with a precise set of material properties.