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

<p>Abstract</p> <p>Background</p> <p>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 (<it>Ovis aries</it>) 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.</p> <p>Results</p> <p>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., <it>n </it>= 48) at 0 weeks to 0.51 g/ml ± 0.10 g/ml (<it>n </it>= 46) at 72 weeks. Maximum collagen density in the deeper cartilage increases from 0.39 g/ml ± 0.08 g/ml (<it>n </it>= 48) at 0 weeks to 0.91 g/ml ± 0.13 g/ml (<it>n </it>= 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.</p> <p>Conclusions</p> <p>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.</p

Risk factors for developing heel ulcers for bedridden patients: A finite element study

Background: The heel is one of the most common sites of pressure ulcers and the anatomical location with the highest prevalence of deep tissue injury. Several finite element modeling studies investigate heel ulcers for bedridden patients. In the current study we have added the implementation of the calf structure to the current heel models. We tested the effect of foot posture, mattress stiffness, and a lateral calcaneus displacement to the contact pressure and internal maximum shear strain occurring at the heel. Methods: A new 3D finite element model is created which includes the heel and calf structure. Sensitivity analyses are performed for the foot orientation relative to the mattress, the Young's modulus of the mattress, and a lateral displacement of the calcaneus relative to the other soft tissues in the heel. Findings: The models predict that a stiffer mattress results in higher contact pressures and internal maximum shear strains at the heel as well as the calf. An abducted foot posture reduces the internal strains in the heel and a lateral calcaneus displacement increases the internal maximum shear strains. A parameter study with different mattress-skin friction coefficients showed that a coefficient below 0.4 decreases the maximum internal shear strains in all of the used loading conditions. Interpretation: In clinical practice, it is advised to avoid internal shearing of the calcaneus of patients, and it could be taken into consideration by medical experts and nurses that a more abducted foot position may reduce the strains in the heel

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

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

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.ISSN:2045-232

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

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

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.ISSN:2701-019