Identification of PCPE-2 as the endogenous specific inhibitor of human BMP-1/tolloid-like proteinases.

BMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-ß superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.

Microrheology and structural quantification of hypercoagulable clots.

Hypercoagulability is a pathology that remains difficult to explain today in most cases. It is likely due to a modification of the conditions of polymerization of the fibrin, the main clot component. Using passive microrheology, we measured the mechanical properties of clots and correlated them under the same conditions with structural information obtained with confocal microscopy. We tested our approach with known alterations: an excess of fibrinogen and of coagulation Factor VIII. We observed simultaneously a rigidification and densification of the fibrin network, showing the potential of microrheology for hypercoagulability diagnosis.

Development and qualification of clinical grade decellularized and cryopreserved human esophagi.

Tissue engineering is a promising alternative to current full thickness circumferential esophageal replacement methods. The aim of our study was to develop a clinical grade Decellularized Human Esophagus (DHE) for future clinical applications. After decontamination, human esophagi from deceased donors were placed in a bioreactor and decellularized with sodium dodecyl sulfate (SDS) and ethylendiaminetetraacetic acid (EDTA) for 3 days. The esophagi were then rinsed in sterile water and SDS was eliminated by filtration on an activated charcoal cartridge for 3 days. DNA was removed by a 3-hour incubation with DNase. A cryopreservation protocol was evaluated at the end of the process to create a DHE cryobank. The decellularization was efficient as no cells and nuclei were observed in the DHE. Sterility of the esophagi was obtained at the end of the process. The general structure of the DHE was preserved according to immunohistochemical and scanning electron microscopy images. SDS was efficiently removed, confirmed by a colorimetric dosage, lack of cytotoxicity on Balb/3T3 cells and mesenchymal stromal cell long term culture. Furthermore, DHE did not induce lymphocyte proliferation in-vitro. The cryopreservation protocol was safe and did not affect the tissue, preserving the biomechanical properties of the DHE. Our decellularization protocol allowed to develop the first clinical grade human decellularized and cryopreserved esophagus.

On the structural origin of the anisotropy in the myocardium: Multiscale modeling and analysis.

Due to structural heterogeneities within the tissue, the myocardium displays an orthotropic material behavior. However, the link between the microstructure and the macroscopic mechanical properties is still not fully established. In particular, if it is admitted that the cardiomyocyte organization induces a transversely isotropic symmetry, the relative role in the observed orthotropic symmetry of cardiomyocyte orientation variation and perimysium collagen "sheetlet" structure, two mechanisms occurring at different scales, is still a matter of debate. In order to shed light on this question, we designed a multiscale model of the myocardium, bridging the cell, sheetlet and tissue scales. More precisely, we compared the macroscopic anisotropy obtained by homogenization of different mesostructures consisting in cardiomyocytes and extracellular collageneous layers, also taking into account the variation of cardiomyocyte and sheetlet orientations on the macroscale, to available experimental data. This study confirms the importance of sheetlets layers in assuring the tissue's anisotropic response, as cardiomyocytes-only mesostructures cannot reproduce the observed anisotropy. Moreover, our model shows the existence of a size effect in the myocardial tissue shear properties, which will require further experimental analysis.

Cellular transduction of mechanical oscillations in plants by the plasma-membrane mechanosensitive channel MSL10.

Plants spend most of their life oscillating around 1-3 Hz due to the effect of the wind. Therefore, stems and foliage experience repetitive mechanical stresses through these passive movements. However, the mechanism of the cellular perception and transduction of such recurring mechanical signals remains an open question. Multimeric protein complexes forming mechanosensitive (MS) channels embedded in the membrane provide an efficient system to rapidly convert mechanical tension into an electrical signal. So far, studies have mostly focused on nonoscillatory stretching of these channels. Here, we show that the plasma-membrane MS channel MscS-LIKE 10 (MSL10) from the model plant Arabidopsis thaliana responds to pulsed membrane stretching with rapid activation and relaxation kinetics in the range of 1 s. Under sinusoidal membrane stretching MSL10 presents a greater activity than under static stimulation. We observed this amplification mostly in the range of 0.3-3 Hz. Above these frequencies the channel activity is very close to that under static conditions. With a localization in aerial organs naturally submitted to wind-driven oscillations, our results suggest that the MS channel MSL10, and by extension MS channels sharing similar properties, represents a molecular component allowing the perception of oscillatory mechanical stimulations by plants.

Microstructural deformation observed by Mueller polarimetry during traction assay on myocardium samples.

Despite recent advances, the myocardial microstructure remains imperfectly understood. In particular, bundles of cardiomyocytes have been observed but their three-dimensional organisation remains debated and the associated mechanical consequences unknown. One of the major challenges remains to perform multiscale observations of the mechanical response of the heart wall. For this purpose, in this study, a full-field Mueller polarimetric imager (MPI) was combined, for the first time, with an in-situ traction device. The full-field MPI enables to obtain a macroscopic image of the explored tissue, while providing detailed information about its structure on a microscopic scale. Specifically it exploits the polarization of the light to determine various biophysical quantities related to the tissue scattering or anisotropy properties. Combined with a mechanical traction device, the full-field MPI allows to measure the evolution of such biophysical quantities during tissue stretch. We observe separation lines on the tissue, which are associated with a fast variation of the fiber orientation, and have the size of cardiomyocyte bundles. Thus, we hypothesize that these lines are the perimysium, the collagen layer surrounding these bundles. During the mechanical traction, we observe two mechanisms simultaneously. On one hand, the azimuth shows an affine behavior, meaning the orientation changes according to the tissue deformation, and showing coherence in the tissue. On the other hand, the separation lines appear to be resistant in shear and compression but weak against traction, with a forming of gaps in the tissue.

Ultrasounds could be considered as a future tool for probing growing bone properties.

Juvenile bone growth is well described (physiological and anatomical) but there are still lacks of knowledge on intrinsic material properties. Our group has already published, on different samples, several studies on the assessment of intrinsic material properties of juvenile bone compared to material properties of adult bone. The purpose of this study was finally to combine different experimental modalities available (ultrasonic measurement, micro-Computed Tomography analysis, mechanical compression tests and biochemical measurements) applied on small cubic bone samples in order to gain insight into the multiparametric evaluation of bone quality. Differences were found between juvenile and adult groups in term of architectural parameters (Porosity Separation), Tissue Mineral Density (TMD), diagonal stiffness coefficients (C33, C44, C55, C66) and ratio between immature and mature cross-links (CX). Diagonal stiffness coefficients are more representative of the microstructural and biochemical parameters of child bone than of adult bone. We also found that compression modulus E was highly correlated with several microstructure parameters and CX in children group while it was not at all correlated in the adult group. Similar results were found for the CX which was linked to several microstructure parameters (TMD and E) only in the juvenile group. To our knowledge, this is the first time that, on a same sample, ultrasonic measurements have been combined with the assessment of mechanical and biochemical properties. It appears that ultrasonic measurements can provide relevant indicators of child bone quality (microstructural and biochemical parameters) which is promising for clinical application since, B-mode ultrasound is the preferred first-line modality over other more constraining imaging modalities (radiation, parent-child accessibility and access to the patient's bed) for pediatric patients.

Calcium and plasma membrane force-gated ion channels behind development.

During development, tissues are submitted to high variation of compression and tension forces. The roles of the cell wall, the cytoskeleton, the turgor pressure and the cell geometry during this process have received due attention. In contrast, apart from its role in the establishment of turgor pressure, the involvement of the plasma membrane as a transducer of mechanical forces during development has been under studied. Force-gated (FG) or Mechanosensitive (MS) ion channels embedded in the bilayer represent 'per se' archetypal mechanosensor able to directly and instantaneously transduce membrane forces into electrical and calcium signals. We discuss here how their fine-tuning, combined with their ability to detect micro-curvature and local membrane tension, allows FG channels to transduce mechanical cues into developmental signals.

Root Hair Sizer: an algorithm for high throughput recovery of different root hair and root developmental parameters.

BACKGROUND: The root is an important organ for water and nutrient uptake, and soil anchorage. It is equipped with root hairs (RHs) which are elongated structures increasing the exchange surface with the soil. RHs are also studied as a model for plant cellular development, as they represent a single cell with specific and highly regulated polarized elongation. For these reasons, it is useful to be able to accurately quantify RH length employing standardized procedures. Methods commonly employed rely on manual steps and are therefore time consuming and prone to errors, restricting analysis to a short segment of the root tip. Few partially automated methods have been reported to increase measurement efficiency. However, none of the reported methods allow an accurate and standardized definition of the position along the root for RH length measurement, making data comparison difficult. RESULTS: We developed an image analysis algorithm that semi-automatically detects RHs and measures their length along the whole differentiation zone of roots. This method, implemented as a simple automated script in ImageJ/Fiji software that we termed Root Hair Sizer, slides a rectangular window along a binarized and straightened image of root tips to estimate the maximal RH length in a given measuring interval. This measure is not affected by heavily bent RHs and any bald spots. RH length data along the root are then modelled with a sigmoidal curve, generating several biologically significant parameters such as RH length, positioning of the root differentiation zone and, under certain conditions, RH growth rate. CONCLUSIONS: Image analysis with Root Hair Sizer and subsequent sigmoidal modelling of RH length data provide a simple and efficient way to characterize RH growth in different conditions, equally suitable to small and large scale phenotyping experiments.

Combination of Traction Assays and Multiphoton Imaging to Quantify Skin Biomechanics.

An important issue in tissue biomechanics is to decipher the relationship between the mechanical behavior at macroscopic scale and the organization of the collagen fiber network at microscopic scale. Here, we present a protocol to combine traction assays with multiphoton microscopy in ex vivo murine skin. This multiscale approach provides simultaneously the stress/stretch response of a skin biopsy and the collagen reorganization in the dermis by use of second harmonic generation (SHG) signals and appropriate image processing.

Monitoring dynamic collagen reorganization during skin stretching with fast polarization-resolved second harmonic generation imaging.

The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization-resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line-to-line switching of polarization using an electro-optical modulator and works in epi-detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.

How aging impacts skin biomechanics: a multiscale study in mice.

Skin aging is a complex process that strongly affects the mechanical behavior of skin. This study aims at deciphering the relationship between age-related changes in dermis mechanical behavior and the underlying changes in dermis microstructure. To that end, we use multiphoton microscopy to monitor the reorganization of dermal collagen during mechanical traction assays in ex vivo skin from young and old mice. The simultaneous variations of a full set of mechanical and microstructural parameters are analyzed in the framework of a multiscale mechanical interpretation. They show consistent results for wild-type mice as well as for genetically-modified mice with modified collagen V synthesis. We mainly observe an increase of the tangent modulus and a lengthening of the heel region in old murine skin from all strains, which is attributed to two different origins that may act together: (i) increased cross-linking of collagen fibers and (ii) loss of water due to proteoglycans deterioration, which impedes inner sliding within these fibers. In contrast, the microstructure reorganization upon stretching shows no age-related difference, which can be attributed to opposite effects of the decrease of collagen content and of the increase of collagen cross-linking in old mice.

Recent advances in studying single bacteria and biofilm mechanics.

Bacterial biofilms correspond to surface-associated bacterial communities embedded in hydrogel-like matrix, in which high cell density, reduced diffusion and physico-chemical heterogeneity play a protective role and induce novel behaviors. In this review, we present recent advances on the understanding of how bacterial mechanical properties, from single cell to high-cell density community, determine biofilm tri-dimensional growth and eventual dispersion and we attempt to draw a parallel between these properties and the mechanical properties of other well-studied hydrogels and living systems.

A novel microstructural interpretation for the biomechanics of mouse skin derived from multiscale characterization.

Skin is a complex, multi-layered organ, with important functions in the protection of the body. The dermis provides structural support to the epidermal barrier, and thus has attracted a large number of mechanical studies. As the dermis is made of a mixture of stiff fibres embedded in a soft non-fibrillar matrix, it is classically considered that its mechanical response is based on an initial alignment of the fibres, followed by the stretching of the aligned fibres. Using a recently developed set-up combining multiphoton microscopy with mechanical assay, we imaged the fibres network evolution during dermis stretching. These observations, combined with a wide set of mechanical tests, allowed us to challenge the classical microstructural interpretation of the mechanical properties of the dermis: we observed a continuous alignment of the collagen fibres along the stretching. All our results can be explained if each fibre contributes by a given stress to the global response. This plastic response is likely due to inner sliding inside each fibre. The non-linear mechanical response is due to structural effects of the fibres network in interaction with the surrounding non-linear matrix. This multiscale interpretation explains our results on genetically-modified mice with a simple alteration of the dermis microstructure. STATEMENT OF SIGNIFICANCE: Soft tissues, as skin, tendon or aorta, are made of extra-cellular matrix, with very few cells embedded inside. The matrix is a mixture of water and biomolecules, which include the collagen fibre network. The role of the collagen is fundamental since the network is supposed to control the tissue mechanical properties and remodeling: the cells attach to the collagen fibres and feel the network deformations. This paper challenges the classical link between fibres organization and mechanical properties. To do so, it uses multiscale observations combined to a large set of mechanical loading. It thus appears that the behaviour at low stretches is mostly controlled by the network structural response, while, at large stretches, the fibre inner-sliding dominate.

Experimental multiscale measurements for the mechanical identification of a cortical bone by digital image correlation.

The implementation of the experimental methodology by optical measurements of mechanical fields, the development of a test bench, the specimen preparation, the experimental measurements, and the digital image correlation (DIC) method, have already been the object of research in the context of biological materials. Nevertheless, in the framework of the experimental identification of a mesoscopic stochastic model of the random apparent elasticity field, measurements of one specimen is required at both the macroscopic scale and the mesoscopic scale under one single loading. The nature of the cortical bone induces some difficulties, as no single speckled pattern technique is available for simultaneously obtaining the displacement at the macroscopic scale and at the mesoscopic scale. In this paper, we present a multiscale experimental methodology based on (i) an experimental protocol for one specimen of a cortical bone, (ii) its measuring bench, (iii) optical field measurements by DIC method, (iv) the experimental results, and (v) the multiscale experimental identification by solving a statistical inverse problem.

Mechanical Behavior of a Bacillus subtilis Pellicle.

Bacterial biofilms consist of a complex network of biopolymers embedded with microorganisms, and together these components form a physically robust structure that enables bacteria to grow in a protected environment. This structure can help unwanted biofilms persist in situations ranging from chronic infection to the biofouling of industrial equipment, but under certain circumstances it can allow the biofilm to disperse and colonize new niches. Mechanical properties are therefore a key aspect of biofilm life. In light of the recently discovered growth-induced compressive stress present within a biofilm, we studied the mechanical behavior of Bacillus subtilis pellicles, or biofilms at the air-liquid interface, and tracked simultaneously the force response and macroscopic structural changes during elongational deformations. We observed that pellicles behaved viscoelastically in response to small deformations, such that the growth-induced compressive stress was still present, and viscoplastically at large deformations, when the pellicles were under tension. In addition, by using particle imaging velocimetry we found that the pellicle deformations were nonaffine, indicating heterogeneous mechanical properties with the pellicle being more pliable near attachment surfaces. Overall, our results indicate that we must consider not only the viscoelastic but also the viscoplastic and mechanically heterogeneous nature of these structures to understand biofilm dispersal and removal.

Simultaneous microstructural and mechanical characterization of human corneas at increasing pressure.

The cornea, through its shape, is the main contributor to the eye׳s focusing power. Pathological alterations of the cornea strongly affect the eye power. To improve treatments, complex biomechanical models have been developed based on the architecture and mechanical properties of the collagen network in the stroma, the main layer of the cornea. However, direct investigations of the structure of the stroma, as well as its link to the mechanical response, remained limited. We propose here an original set up, associating nonlinear optical imaging and mechanical testing. By using polarization resolved Second Harmonic signals, we simultaneously quantified micrometer (orientation of the collagen lamellae) and nanometer (local disorder within lamellae) scale corneal organization. We showed that the organization of the lamellae changes along the stroma thickness. Then, we measured simultaneously the deformation on the epithelial side of the cornea and the reorientation of the collagen lamellae for increasing intraocular pressure levels, from physiological ones to pathological ones. We showed that the observed deformation is not correlated to initial orientation, but to the reorganization of the lamellae in the stroma. Our results, by providing a direct multi-scale observation, will be useful for the development of more accurate biomechanical models.

Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy.

Soft connective tissues such as skin, tendon or cornea are made of about 90% of extracellular matrix proteins, fibrillar collagens being the major components. Decreased or aberrant collagen synthesis generally results in defective tissue mechanical properties as the classic form of Elhers-Danlos syndrome (cEDS). This connective tissue disorder is caused by mutations in collagen V genes and is mainly characterized by skin hyperextensibility. To investigate the relationship between the microstructure of normal and diseased skins and their macroscopic mechanical properties, we imaged and quantified the microstructure of dermis of ex vivo murine skin biopsies during uniaxial mechanical assay using multiphoton microscopy. We used two genetically-modified mouse lines for collagen V: a mouse model for cEDS harboring a Col5a2 deletion (a.k.a. pN allele) and the transgenic K14-COL5A1 mice which overexpress the human COL5A1 gene in skin. We showed that in normal skin, the collagen fibers continuously align with stretch, generating the observed increase in mechanical stress. Moreover, dermis from both transgenic lines exhibited altered collagen reorganization upon traction, which could be linked to microstructural modifications. These findings show that our multiscale approach provides new crucial information on the biomechanics of dermis that can be extended to all collagen-rich soft tissues.

Bacillus subtilis Bacteria Generate an Internal Mechanical Force within a Biofilm.

A key issue in understanding why biofilms are the most prevalent mode of bacterial life is the origin of the degree of resistance and protection that bacteria gain from self-organizing into biofilm communities. Our experiments suggest that their mechanical properties are a key factor. Experiments on pellicles, or floating biofilms, of Bacillus subtilis showed that while they are multiplying and secreting extracellular substances, bacteria create an internal force (associated with a -80±25 Pa stress) within the biofilms, similar to the forces that self-equilibrate and strengthen plants, organs, and some engineered buildings. Here, we found that this force, or stress, is associated with growth-induced pressure. Our observations indicate that due to such forces, biofilms spread after any cut or ablation by up to 15-20% of their initial size. The force relaxes over very short timescales (tens of milliseconds). We conclude that this force helps bacteria to shape the biofilm, improve its mechanical resistance, and facilitate its invasion and self-repair.

Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration.

Several diseases can lead to opacification of cornea requiring transplantation of donor tissue to restore vision. In this context, transparent collagen I fibrillated matrices have been synthesized at 15, 30, 60 and 90 mg/mL. The matrices were evaluated for fibril organizations, transparency, mechanical properties and ability to support corneal epithelial cell culture. The best results were obtained with 90 mg/mL scaffolds. At this concentration, the fibril organization presented some similarities to that found in corneal stroma. Matrices had a mean Young's modulus of 570 kPa and acellular scaffolds had a transparency of 87% in the 380-780 nm wavelength range. Human corneal epithelial cells successfully colonized the surface of the scaffolds and generated an epithelium with characteristics of corneal epithelial cells (i.e. expression of cytokeratin 3 and presence of desmosomes) and maintenance of stemness during culture (i.e. expression of ΔNp63α and formation of holoclones in colony formation assay). Presence of cultured epithelium on the matrices was associated with increased transparency (89%).