SFAlab: image-based quantification of mechano-active ventral actin stress fibers in adherent cells.

Ventral actin stress fibers (SFs) are a subset of actin SFs that begin and terminate at focal adhesion (FA) complexes. Ventral SFs can transmit forces from and to the extracellular matrix and serve as a prominent mechanosensing and mechanotransduction machinery for cells. Therefore, quantitative analysis of ventral SFs can lead to deeper understanding of the dynamic mechanical interplay between cells and their extracellular matrix (mechanoreciprocity). However, the dynamic nature and organization of ventral SFs challenge their quantification, and current quantification tools mainly focus on all SFs present in cells and cannot discriminate between subsets. Here we present an image analysis-based computational toolbox, called SFAlab, to quantify the number of ventral SFs and the number of ventral SFs per FA, and provide spatial information about the locations of the identified ventral SFs. SFAlab is built as an all-in-one toolbox that besides analyzing ventral SFs also enables the identification and quantification of (the shape descriptors of) nuclei, cells, and FAs. We validated SFAlab for the quantification of ventral SFs in human fetal cardiac fibroblasts and demonstrated that SFAlab analysis i) yields accurate ventral SF detection in the presence of image imperfections often found in typical fluorescence microscopy images, and ii) is robust against user subjectivity and potential experimental artifacts. To demonstrate the usefulness of SFAlab in mechanobiology research, we modulated actin polymerization and showed that inhibition of Rho kinase led to a significant decrease in ventral SF formation and the number of ventral SFs per FA, shedding light on the importance of the RhoA pathway specifically in ventral SF formation. We present SFAlab as a powerful open source, easy to use image-based analytical tool to increase our understanding of mechanoreciprocity in adherent cells.

Shape-Morphing Photoresponsive Hydrogels Reveal Dynamic Topographical Conditioning of Fibroblasts.

The extracellular environment defines a physical boundary condition with which cells interact. However, to date, cell response to geometrical environmental cues is largely studied in static settings, which fails to capture the spatiotemporally varying cues cells receive in native tissues. Here, a photoresponsive spiropyran-based hydrogel is presented as a dynamic, cell-compatible, and reconfigurable substrate. Local stimulation with blue light (455 nm) alters hydrogel swelling, resulting in on-demand reversible micrometer-scale changes in surface topography within 15 min, allowing investigation into cell response to controlled geometry actuations. At short term (1 h after actuation), fibroblasts respond to multiple rounds of recurring topographical changes by reorganizing their nucleus and focal adhesions (FA). FAs form primarily at the dynamic regions of the hydrogel; however, this propensity is abolished when the topography is reconfigured from grooves to pits, demonstrating that topographical changes dynamically condition fibroblasts. Further, this dynamic conditioning is found to be associated with long-term (72 h) maintenance of focal adhesions and epigenetic modifications. Overall, this study offers a new approach to dissect the dynamic interplay between cells and their microenvironment and shines a new light on the cell's ability to adapt to topographical changes through FA-based mechanotransduction.

Single-Cell Profiling Reveals Functional Heterogeneity and Serial Killing in Human Peripheral and Ex Vivo-Generated CD34+ Progenitor-Derived Natural Killer Cells.

Increasing evidence suggests that natural killer (NK) cells are composed of distinct functional subsets. This multifunctional role has made them an attractive choice for anticancer immunotherapy. A functional NK cell repertoire is generated through cellular education, resulting in a heterogeneous NK cell population with distinct capabilities responding to different stimuli. The application of a high-throughput droplet-based microfluidic platform allows monitoring of NK cell-target cell interactions at the single-cell level and in real-time. A variable response of single NK cells toward different target cells is observed, and a distinct population of NK cells (serial killers) capable of inducing multiple target lysis is identified. By assessing the cytotoxic dynamics, it is shown that single umbilical cord blood-derived CD34+ hematopoietic progenitor (HPC)-NK cells display superior antitumor cytotoxicity. With an integrated analysis of cytotoxicity and cytokine secretion, it is shown that target cell interactions augment cytotoxic as well as secretory behavior of NK cells. By providing an integrated assessment of NK cell functions by microfluidics, this study paves the way to further functionally characterize NK cells ultimately aimed to improve cancer immunotherapy.

Notch signaling regulates strain-mediated phenotypic switching of vascular smooth muscle cells.

Mechanical stimuli experienced by vascular smooth muscle cells (VSMCs) and mechanosensitive Notch signaling are important regulators of vascular growth and remodeling. However, the interplay between mechanical cues and Notch signaling, and its contribution to regulate the VSMC phenotype are still unclear. Here, we investigated the role of Notch signaling in regulating strain-mediated changes in VSMC phenotype. Synthetic and contractile VSMCs were cyclically stretched for 48 h to determine the temporal changes in phenotypic features. Different magnitudes of strain were applied to investigate its effect on Notch mechanosensitivity and the phenotypic regulation of VSMCs. In addition, Notch signaling was inhibited via DAPT treatment and activated with immobilized Jagged1 ligands to understand the role of Notch on strain-mediated phenotypic changes of VSMCs. Our data demonstrate that cyclic strain induces a decrease in Notch signaling along with a loss of VSMC contractile features. Accordingly, the activation of Notch signaling during cyclic stretching partially rescued the contractile features of VSMCs. These findings demonstrate that Notch signaling has an important role in regulating strain-mediated phenotypic switching of VSMCs.

An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip.

Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Here, we present a fully automated droplet-based microfluidic platform that allowed the real-time monitoring of effector-target cell interactions and killing, allowing the screening of over 60,000 droplets identifying 2000 individual cellular interactions monitored over 10 h. During the course of incubation, we observed that the dynamics of cytotoxicity within the Natural Killer (NK) cell population varies significantly over the time. Around 20% of the total NK cells in droplets showed positive cytotoxicity against paired K562 cells, most of which was exhibited within first 4 h of cellular interaction. Using our single cell analysis platform, we demonstrated that the population of NK cells is composed of individual cells with different strength in their effector functions, a behavior masked in conventional studies. Moreover, the versatility of our platform will allow the dynamic and resolved study of interactions between immune cell types and the finding and characterization of functional sub-populations, opening novel ways towards both fundamental and translational research.

Biomaterial screening of protein coatings and peptide additives: towards a simple synthetic mimic of a complex natural coating for a bio-artificial kidney.

Bio-artificial kidneys require conveniently synthesized membranes providing signals that regulate renal epithelial cell function. Therefore, we aimed to find synthetic analogues for natural extracellular matrix (ECM) protein coatings traditionally used for epithelial cell culturing. Two biomaterial libraries, based on natural ECM-coatings and on synthetic supramolecular small molecule additives, were developed. The base material consisted of a bisurea (BU) containing polymer, providing supramolecular BU-additives to be incorporated via specific hydrogen bonding interactions. This system allows for a modular approach and therefore easy fractional factorial based screening. A natural coating on the BU-polymer material with basement membrane proteins, laminin and collagen IV, combined with catechols was shown to induce renal epithelial monolayer formation. Modification of the BU-polymer material with synthetic BU-modified ECM peptide additives did not result in monolayer formation. Unexpectedly, simple BU-catechol additives induced monolayer formation and presented similar levels of epithelial markers and apical transporter function as on the laminin, collagen IV and catechol natural coating. Importantly, when this BU-polymer material was processed into fibrous e-spun membranes the natural coating and the BU-catechol additive were shown to perfectly function. This study clearly indicates that complex natural ECM-coatings can be replaced by simple synthetic additives, and displays the potency of material libraries based on design of experiments in combination with modular, supramolecular chemistry.

Cellular Contact Guidance Emerges from Gap Avoidance.

In the presence of anisotropic biochemical or topographical patterns, cells tend to align in the direction of these cues-a widely reported phenomenon known as "contact guidance." To investigate the origins of contact guidance, here, we created substrates micropatterned with parallel lines of fibronectin with dimensions spanning multiple orders of magnitude. Quantitative morphometric analysis of our experimental data reveals two regimes of contact guidance governed by the length scale of the cues that cannot be explained by enforced alignment of focal adhesions. Adopting computational simulations of cell remodeling on inhomogeneous substrates based on a statistical mechanics framework for living cells, we show that contact guidance emerges from anisotropic cell shape fluctuation and "gap avoidance," i.e., the energetic penalty of cell adhesions on non-adhesive gaps. Our findings therefore point to general biophysical mechanisms underlying cellular contact guidance, without the necessity of invoking specific molecular pathways.

There is an individual tolerance to mechanical loading in compression induced deep tissue injury.

BACKGROUND: Deep tissue injury is a type of pressure ulcer which originates subcutaneously due to sustained mechanical loading. The relationship between mechanical compression and damage development has been extensively studied in 2D. However, recent studies have suggested that damage develops beyond the site of indentation. The objective of this study was to compare mechanical loading conditions to the associated damage in 3D. METHODS: An indentation test was performed on the tibialis anterior muscle of rats (n = 39). Changes in the form of oedema and structural damage were monitored with MRI in an extensive region. The internal deformations were evaluated using MRI based 3D finite element models. FINDINGS: Damage propagates away from the loaded region. The 3D analysis indicates that there is a subject specific tolerance to compression induced deep tissue injury. INTERPRETATION: Individual tolerance is an important factor when considering the mechanical loading conditions which induce damage.

Image-based analysis of uniaxial ring test for mechanical characterization of soft materials and biological tissues.

Uniaxial ring test is a widely used mechanical characterization method for a variety of materials, from industrial elastomers to biological materials. Here we show that the combination of local material compression, bending, and stretching during uniaxial ring test results in a geometry-dependent deformation profile that can introduce systematic errors in the extraction of mechanical parameters. We identify the stress and strain regimes under which stretching dominates and develop a simple image-based analysis approach that eliminates these systematic errors. We rigorously test this approach computationally and experimentally, and demonstrate that we can accurately estimate the sample mechanical properties for a wide range of ring geometries. As a proof of concept for its application, we use the approach to analyze explanted rat vascular tissues and find a clear temporal change in the mechanical properties of these explants after graft implantation. The image-based approach can therefore offer a straightforward, versatile, and accurate method for mechanically characterizing new classes of soft and biological materials.

MRI based 3D finite element modelling to investigate deep tissue injury.

Pressure ulcers occur due to sustained mechanical loading. Deep tissue injury is a severe type of pressure ulcer, which is believed to originate in subcutaneous tissues adjacent to bony prominences. In previous experimental-numerical studies the relationship between internal tissue state and damage development was investigated using a 2D analysis. However, recent studies suggest that a local analysis is not sufficient. In the present study we developed a method to create animal-specific 3D finite element models of an indentation test on the tibialis anterior muscle of rats based on MRI data. A detailed description on how the animal specific models are created is given. Furthermore, two indenter geometries are compared and the influence of errors in determining the indenter orientation on the resulting internal strain distribution in a defined volume of tissue was investigated. We conclude that with a spherically-shaped indenter errors in estimating the indenter orientation do not unduly influence the results of the simulation.

Behavior of CMPCs in unidirectional constrained and stress-free 3D hydrogels.

Cardiomyocyte progenitor cells (CMPCs) are a candidate cell source for cardiac regenerative therapy. However, like other stem cells, after transplantation in the heart, cell retention and differentiation capacity of the CMPCs are low. Combining cells with biomaterials might overcome this problem. By serving as a (temporal) environment, the biomaterial can retain the cells and provide signals that enhance survival, proliferation and differentiation of the cells. To gain more insight into the effect that the encapsulation of CMPCs in a biomaterial has on their behavior, we cultured CMPCs in unidirectional constrained and stress-free collagen/Matrigel hydrogels. CMPCs cultured in 3D hydrogels stay viable and keep their cardiomyogenic profile independent of the application of strain. Moreover, the increased expression of Nkx2.5, myocardin and cTnT in 3D hydrogels compared to 2D cultures, suggests enhanced cardiomyogenic differentiation capacity of cells in 3D. Furthermore, increased expression of collagen I, collagen III, elastin and fibronectin and of the matrix remodeling enzymes MMP-1, MMP-2, MMP-9, and TIMP-1 and TIMP-2 in the 3D hydrogels is indicative of an enhanced matrix remodeling capacity of CMPCs in a 3D environment, independent of the application of strain. Interestingly, the additional application of static strain to the 3D hydrogels, as imposed by hydrogel constrainment, stabilized CMPC viability and proliferation, resulted in enhanced cardiac marker protein expression and appeared crucial for cellular organization and morphology. More specifically, CMPCs cultured in 3D collagen/Matrigel constrained hydrogels became readily mechanosensitive, had a rod-shaped morphology, and responded to the applied strain by orienting in the direction of the constraint. Overall, our data demonstrate the applicability of CMPCs in a 3D environment since encapsulation of CMPCs may stabilize survival and proliferation, can enhance the differentiation and remodeling capacity of the cells, and could induce cellular re-organization, which all may contribute to an improved efficiency of cardiac stem cell therapy.

Colorful protein-based fluorescent probes for collagen imaging.

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Deformation thresholds for chondrocyte death and the protective effect of the pericellular matrix.

In cartilage tissue engineering studies, the stimulatory effect of mechanical perturbation declines after the first 2 weeks of culture. Similarly, it is known that chondrocyte-agarose constructs should not be loaded within the first days after seeding, to prevent considerable cell death, suggesting a mechanical threshold. This study aims to establish a relationship between chondrocyte deformation and death, and to evaluate the protective effect of the pericellular matrix (PCM) that is formed in 3D cultures. Chondrocyte viability was monitored every hour for 24 h after applying a strain range of 0% to 25% to agarose constructs containing chondrocytes, cultured for 1, 3, 5, 7, or 10 days. At these culture time points, the PCM thickness and chondrocyte deformation were assessed by means of histology and assayed for biochemical contents. Inverse finite element (FE) simulations were used to evaluate the change of mechanical properties of the chondrocyte and PCM over the 10-day culture duration. Chondrocyte death was demonstrated to be dependent on both the magnitude and duration of straining. The highest cell death was observed at day 1 (43%), reducing over culture duration (15% at day 3 and 2.5% at day 10). Cell deformation at 25% compression decreased significantly over culture duration (aspect ratio of 2.24±0.67 at day 1 and 1.45±0.24 at day 3) and with increased matrix production. Inverse FE simulations showed an increasing PCM Young's modulus of 45 kPa at day 3 to 162 kPa at day 10. The current results provide evidence for a mechanical threshold for chondrocyte death and for the protective effect of the PCM. As such, these insights may help in establishing mechanical loading protocols for cartilage tissue engineering studies.

Contribution of postnatal collagen reorientation to depth-dependent mechanical properties of articular cartilage.

The collagen fibril network is an important factor for the depth-dependent mechanical behaviour of adult articular cartilage (AC). Recent studies show that collagen orientation is parallel to the articular surface throughout the tissue depth in perinatal animals, and that the collagen orientations transform to a depth-dependent arcade-like structure in adult animals. Current understanding on the mechanobiology of postnatal AC development is incomplete. In the current paper, we investigate the contribution of collagen fibril orientation changes to the depth-dependent mechanical properties of AC. We use a composition-based finite element model to simulate in a 1-D confined compression geometry the effects of ten different collagen orientation patterns that were measured in developing sheep. In initial postnatal life, AC is mostly subject to growth and we observe only small changes in depth-dependent mechanical behaviour. Functional adaptation of depth-dependent mechanical behaviour of AC takes place in the second half of life before puberty. Changes in fibril orientation alone increase cartilage stiffness during development through the modulation of swelling strains and osmotic pressures. Changes in stiffness are most pronounced for small stresses and for cartilage adjacent to the bone. We hypothesize that postnatal changes in collagen fibril orientation induce mechanical effects that in turn promote these changes. We further hypothesize that a part of the depth-dependent postnatal increase in collagen content in literature is initiated by the depth-dependent postnatal increase in fibril strain due to collagen fibril reorientation.

Postnatal development of depth-dependent collagen density in ovine articular cartilage.

BACKGROUND: Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Adult AC is characterised by a depth-dependent composition and structure of the extracellular matrix that results in depth-dependent mechanical properties, important for the functions of adult AC. Collagen is the most abundant solid component and it affects the mechanical behaviour of AC. The current objective is to quantify the postnatal development of depth-dependent collagen density in sheep (Ovis aries) AC between birth and maturity. We use Fourier transform infra-red micro-spectroscopy to investigate collagen density in 48 sheep divided over ten sample points between birth (stillborn) and maturity (72 weeks). In each animal, we investigate six anatomical sites (caudal, distal and rostral locations at the medial and lateral side of the joint) in the distal metacarpus of a fore leg and a hind leg. RESULTS: Collagen density increases from birth to maturity up to our last sample point (72 weeks). Collagen density increases at the articular surface from 0.23 g/ml ± 0.06 g/ml (mean ± s.d., n = 48) at 0 weeks to 0.51 g/ml ± 0.10 g/ml (n = 46) at 72 weeks. Maximum collagen density in the deeper cartilage increases from 0.39 g/ml ± 0.08 g/ml (n = 48) at 0 weeks to 0.91 g/ml ± 0.13 g/ml (n = 46) at 72 weeks. Most collagen density profiles at 0 weeks (85%) show a valley, indicating a minimum, in collagen density near the articular surface. At 72 weeks, only 17% of the collagen density profiles show a valley in collagen density near the articular surface. The fraction of profiles with this valley stabilises at 36 weeks. CONCLUSIONS: Collagen density in articular cartilage increases in postnatal life with depth-dependent variation, and does not stabilize up to 72 weeks, the last sample point in our study. We find strong evidence for a valley in collagen densities near the articular surface that is present in the youngest animals, but that has disappeared in the oldest animals. We discuss that the retardance valley (as seen with polarised light microscopy) in perinatal animals reflects a decrease in collagen density, as well as a decrease in collagen fibril anisotropy.

Postnatal development of collagen structure in ovine articular cartilage.

BACKGROUND: Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries) as our model animal. RESULTS: Predominant collagen orientation is parallel to the articular surface throughout the tissue depth for perinatal cartilage. This remodels to the Benninghoff structure before the sheep reach sexual maturity. Remodelling of predominant collagen orientation starts at a depth just below the future transitional zone. Tissue retardance shows a minimum near the articular surface at all ages, which indicates the presence of zonal differentiation at all ages. The absolute position of this minimum does change between birth and maturity. Between different anatomical sites, we find differences in the dynamics of collagen remodelling, but no differences in adult collagen structure. CONCLUSIONS: The collagen network in articular cartilage remodels between birth and sexual maturity from a network with predominant orientation parallel to the articular surface to a Benninghoff network. The retardance minimum near, but not at, the articular surface at all ages shows that a zonal differentiation is already present in the perinatal animals. In these animals, the zonal differentiation can not be correlated to the collagen network orientation. We find no difference in adult collagen structure in the nearly congruent metacarpophalangeal joint, but we do find differences in the dynamics of collagen network remodelling.

Modeling optical behavior of birefringent biological tissues for evaluation of quantitative polarized light microscopy.

Quantitative polarized light microscopy (qPLM) is a popular tool for the investigation of birefringent architectures in biological tissues. Collagen, the most abundant protein in mammals, is such a birefringent material. Interpretation of results of qPLM in terms of collagen network architecture and anisotropy is challenging, because different collagen networks may yield equal qPLM results. We created a model and used the linear optical behavior of collagen to construct a Jones or Mueller matrix for a histological cartilage section in an optical qPLM train. Histological sections of tendon were used to validate the basic assumption of the model. Results show that information on collagen densities is needed for the interpretation of qPLM results in terms of collagen anisotropy. A parameter that is independent of the optical system and that measures collagen fiber anisotropy is introduced, and its physical interpretation is discussed. With our results, we can quantify which part of different qPLM results is due to differences in collagen densities and which part is due to changes in the collagen network. Because collagen fiber orientation and anisotropy are important for tissue function, these results can improve the biological and medical relevance of qPLM results.