Measurement of Liver Stiffness using Atomic Force Microscopy Coupled with Polarization Microscopy.

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level. To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young's (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice. A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

Influence of Biomimetically Mineralized Collagen Scaffolds on Bone Cell Proliferation and Immune Activation.

Collagen, as the main component of connective tissue, is frequently used in various tissue engineering applications. In this study, porous sponge-like collagen scaffolds were prepared by freeze-drying and were then mineralized in a simulated body fluid. The mechanical stability was similar in both types of scaffolds, but the mineralized scaffolds (MCS) contained significantly more calcium, magnesium and phosphorus than the unmineralized scaffolds (UCS). Although the MCS contained a lower percentage (~32.5%) of pores suitable for cell ingrowth (113-357 µm in diameter) than the UCS (~70%), the number of human-osteoblast-like MG-63 cells on days 1, 3 and 7 after seeding was higher on MCS than on UCS, and the cells penetrated deeper into the MCS. The cell growth in extracts prepared by eluting the scaffolds for 7 days in a cell culture medium was also markedly higher in the MCS extracts, as indicated by real-time monitoring in the sensory xCELLigence system for 7 days. From this point of view, MCS are more promising for bone tissue engineering than UCS. However, MCS evoked a more pronounced inflammatory response than UCS, as indicated by the production of tumor necrosis factor-alpha (TNF-α) in macrophage-like RAW 264.7 cells in cultures on these scaffolds.

Accelerated in vitro recellularization of decellularized porcine pericardium for cardiovascular grafts.

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient's cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

Cellulose Mesh with Charged Nanocellulose Coatings as a Promising Carrier of Skin and Stem Cells for Regenerative Applications.

Engineering artificial skin constructs is an ongoing challenge. An ideal material for hosting skin cells is still to be discovered. A promising candidate is low-cost cellulose, which is commonly fabricated in the form of a mesh and is applied as a wound dressing. Unfortunately, the structure and the topography of current cellulose meshes are not optimal for cell growth. To enhance the surface structure and the physicochemical properties of a commercially available mesh, we coated the mesh with wood-derived cellulose nanofibrils (CNFs). Three different types of mesh coatings are proposed in this study as a skin cell carrier: positively charged cationic cellulose nanofibrils (cCNFs), negatively charged anionic cellulose nanofibrils (aCNFs), and a combination of these two materials (c+aCNFs). These cell carriers were seeded with normal human dermal fibroblasts (NHDFs) or with human adipose-derived stem cells (ADSCs) to investigate cell adhesion, spreading, morphology, and proliferation. The negatively charged aCNF coating significantly improved the proliferation of both cell types. The positively charged cCNF coating significantly enhanced the adhesion of ADSCs only. The number of NHDFs was similar on the cCNF coatings and on the noncoated pristine cellulose mesh. However, the three-dimensional (3D) structure of the cCNF coating promoted cell survival. The c+aCNF construct proved to combine benefits from both types of CNFs, which means that the c+aCNF cell carrier is a promising candidate for further application in skin tissue engineering.

Human decellularized and crosslinked pericardium coated with bioactive molecular assemblies.

Decellularized human pericardium is under study as an allogenic material for cardiovascular applications. The effects of crosslinking on the mechanical properties of decellularized pericardium were determined with a uniaxial tensile test, and the effects of crosslinking on the collagen structure of decellularized pericardium were determined by multiphoton microscopy. The viability of human umbilical vein endothelial cells seeded on decellularized human pericardium and on pericardium strongly and weakly crosslinked with glutaraldehyde and with genipin was evaluated by means of an MTS assay. The viability of the cells, measured by their metabolic activity, decreased considerably when the pericardium was crosslinked with glutaraldehyde. Conversely, the cell viability increased when the pericardium was crosslinked with genipin. Coating both non-modified pericardium and crosslinked pericardium with a fibrin mesh or with a mesh containing attached heparin and/or fibronectin led to a significant increase in cell viability. The highest degree of viability was attained for samples that were weakly crosslinked with genipin and modified by means of a fibrin and fibronectin coating. The results indicate a method by which in vivo endothelialization of human cardiac allografts or xenografts could potentially be encouraged.

A two-layer skin construct consisting of a collagen hydrogel reinforced by a fibrin-coated polylactide nanofibrous membrane.

Background: Repairs to deep skin wounds continue to be a difficult issue in clinical practice. A promising approach is to fabricate full-thickness skin substitutes with functions closely similar to those of the natural tissue. For many years, a three-dimensional (3D) collagen hydrogel has been considered to provide a physiological 3D environment for co-cultivation of skin fibroblasts and keratinocytes. This collagen hydrogel is frequently used for fabricating tissue-engineered skin analogues with fibroblasts embedded inside the hydrogel and keratinocytes cultivated on its surface. Despite its unique biological properties, the collagen hydrogel has insufficient stiffness, with a tendency to collapse under the traction forces generated by the embedded cells. Methods: The aim of our study was to develop a two-layer skin construct consisting of a collagen hydrogel reinforced by a nanofibrous poly-L-lactide (PLLA) membrane pre-seeded with fibroblasts. The attractiveness of the membrane for dermal fibroblasts was enhanced by coating it with a thin nanofibrous fibrin mesh. Results: The fibrin mesh promoted the adhesion, proliferation and migration of the fibroblasts upwards into the collagen hydrogel. Moreover, the fibroblasts spontaneously migrating into the collagen hydrogel showed a lower tendency to contract and shrink the hydrogel by their traction forces. The surface of the collagen was seeded with human dermal keratinocytes. The keratinocytes were able to form a basal layer of highly mitotically-active cells, and a suprabasal layer. Conclusion: The two-layer skin construct based on collagen hydrogel with spontaneously immigrated fibroblasts and reinforced by a fibrin-coated nanofibrous membrane seems to be promising for the construction of full-thickness skin substitute.

Novel contribution to clubfoot pathogenesis: The possible role of extracellular matrix proteins.

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

In vivo assessment of time dependent changes of T2* in medial meniscus under loading at 3T: A preliminary study.

Due to the internal structure of the knee joint, the ability to characterize and quantify the dynamic response of the meniscal tissue directly in vivo is highly problematic. The main purpose of this study was to investigate the behaviour of the meniscus under loading conditions. Four healthy young females were included. To obtain T2* values in the meniscus, the vTE sequence was used with 10 echoes ranging from 0.8 to 10.1 ms. Submilisecond first echo time is a great advantage of vTE sequence allowing for precise mapping of relatively short T2*. The two-parametric least squares fitting procedure was used to calculate T2* pixel-wise. A custom-made diamagnetic apparatus was developed to simulate stress conditions on the lower limb in a conventional MR scanner. vTE T2* was performed in five consecutive scans, 6:10 min apart. Three different compartments of the medial and lateral meniscus were segmented. The differences at the different time-points were calculated. A constant increase of T2* times after compression was statistically significant in the anterior horn of the medial meniscus. T2* mapping with variable echo time sequence might be a satisfactorily sensitive technique to detect the changes of meniscus physiology under loading conditions in vivo.

Morphology of a fibrin nanocoating influences dermal fibroblast behavior.

BACKGROUND: Our study focuses on the fabrication of appropriate scaffolds for skin wound healing. This research brings valuable insights into the molecular mechanisms of adhesion, proliferation, and control of cell behavior through the extracellular matrix represented by synthetic biodegradable nanofibrous membranes coated by biomolecules. METHODS: Nanofibrous polylactic acid (PLA) membranes were prepared by a needle-less electrospinning technology. These membranes were coated with fibrin according to two preparation protocols, and additionally they were coated with fibronectin in order to increase the cell affinity for colonizing the PLA membranes. The adhesion, growth, and extracellular matrix protein production of neonatal human dermal fibroblasts were evaluated on the nanofibrous membranes. RESULTS: Our results showed that fibrin-coated membranes improved the adhesion and proliferation of human dermal fibroblasts. The morphology of the fibrin nanocoating seems to be crucial for the adhesion of fibroblasts, and consequently for their phenotypic maturation. Fibrin either covered the individual fibers in the membrane (F1 nanocoating), or covered the individual fibers and also formed a fine homogeneous nanofibrous mesh on the surface of the membrane (F2 nanocoating), depending on the mode of fibrin preparation. The fibroblasts on the membranes with the F1 nanocoating remained in their typical spindle-like shape. However, the cells on the F2 nanocoating were spread mostly in a polygon-like shape, and their proliferation was significantly higher. Fibronectin formed an additional mesh attached to the surface of the fibrin mesh, and further enhanced the cell adhesion and growth. The relative gene expression and protein production of collagen I and fibronectin were higher on the F2 nanocoating than on the F1 nanocoating. CONCLUSION: A PLA membrane coated with a homogeneous fibrin mesh seems to be promising for the construction of temporary full-thickness skin tissue substitutes.

Porous Heat-Treated Polyacrylonitrile Scaffolds for Bone Tissue Engineering.

Heat-treated polyacrylonitrile (HT-PAN), also referred to as black orlon (BO), is a promising carbon-based material used for applications in tissue engineering and regenerative medicine. To the best of our knowledge, no such complex bone morphology-mimicking three-dimensional (3D) BO structure has been reported to date. We report that BO can be easily made into 3D cryogel scaffolds with porous structures, using succinonitrile as a porogen. The cryogels possess a porous morphology, similar to bone tissue. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation, and mitochondrial activity of human bone-derived cells. This effect was more apparent in scaffolds prepared from a matrix with a higher content of PAN (i.e., 10% rather than 5%). The scaffolds with 10% of PAN also showed enhanced mechanical properties, as revealed by higher compressive modulus and higher compressive strength. Therefore, these scaffolds have a robust potential for use in bone tissue engineering.

Calcaneal Tendon Collagen Fiber Morphometry and Aging.

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (n tendons=24) and microtome sectioned (n tendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.

Valve interstitial cell culture: Production of mature type I collagen and precise detection.

Collagen often acts as an extracellular and intracellular marker for in vitro experiments, and its quality defines tissue constructs. To validate collagen detection techniques, cardiac valve interstitial cells were isolated from pigs and cultured under two different conditions; with and without ascorbic acid. The culture with ascorbic acid reached higher cell growth and collagen deposition, although the expression levels of collagen gene stayed similar to the culture without ascorbic acid. The fluorescent microscopy was positive for collagen fibers in both the cultures. Visualization of only extracellular collagen returned a higher correlation coefficient when comparing the immunolabeling and second harmonic generation microscopy images in the culture with ascorbic acid. Lastly, it was proved that the hydroxyproline strongly contributes to the second-order susceptibility tensor of collagen molecules, and therefore the second harmonic generation signal is impaired in the culture without ascorbic acid.

Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells.

Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide-co-glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films.

Nanocrystalline diamond (NCD) films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination) or oxygen atoms (O-termination). Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers - type I collagen, alkaline phosphatase, and osteocalcin - was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix mineralization, and this is promising for better osteoconductivity of potential bone implant coatings.

Effects of fiber density and plasma modification of nanofibrous membranes on the adhesion and growth of HaCaT keratinocytes.

It may be possible to regulate the cell colonization of biodegradable polymer nanofibrous membranes by plasma treatment and by the density of the fibers. To test this hypothesis, nanofibrous membranes of different fiber densities were treated by oxygen plasma with a range of plasma power and exposure times. Scanning electron microscopy and mechanical tests showed significant modification of nanofibers after plasma treatment. The intensity of the fiber modification increased with plasma power and exposure time. The exposure time seemed to have a stronger effect on modifying the fiber. The mechanical behavior of the membranes was influenced by the plasma treatment, the fiber density, and their dry or wet state. Plasma treatment increased the membrane stiffness; however, the membranes became more brittle. Wet membranes displayed significantly lower stiffness than dry membranes. X-ray photoelectron spectroscopy (XPS) analysis showed a slight increase in oxygen-containing groups on the membrane surface after plasma treatment. Plasma treatment enhanced the adhesion and growth of HaCaT keratinocytes on nanofibrous membranes. The cells adhered and grew preferentially on membranes of lower fiber densities, probably due to the larger area of void spaces between the fibers.

Kinematic and dynamic biomechanical values in relation to muscle activity during contact head impact.

OBJECTIVE: For the evaluation of neck injury the relative distance was observed between a marker placed on the forehead and a marker placed on the shoulder and also by change of the angle. To compare the severity of head injury a value of maximum head acceleration was used, HIC and a 3 ms criterion. All criteria were related to the activity of musculus sternocleidomastoideus and musculus trapezius in a situation of expected or unexpected contact impact. MATERIALS AND METHODS: The situation was recorded using a Qualisys system, head acceleration of probands in three axes was recorded using the accelerometer, activity of neck muscles was monitored by a mobile EMG. RESULTS: Maximum head acceleration was 5.61 g for non-visual and 5.03 g for visual. HIC36 was 6.65 non visual and 5.97 for visual. 3-ms criterion was 5.37 g for non-visual and 4.89 g for visual and max. force was 291 N for non-visual and 314 N for visual. The average time of muscle activation of the observed group without visual perception is 0.355 s after hitting an obstacle, with visual perception 0.085 s before the crash. CONCLUSIONS: Kinematic values indicate more favourable parameters for neck injuries for visual. Head injury criteria show an average decrease of about 10% for visual. We can conclude that the visual perception means a significant increase in pre-activation of the observed muscle group of almost 745% and lower activation in following phase of approximately 90%.

Polylactide nanofibers with hydroxyapatite as growth substrates for osteoblast-like cells.

Various types of nanofibers are increasingly used in tissue engineering, mainly for their ability to mimic the architecture of tissue at the nanoscale. We evaluated the adhesion, growth, viability, and differentiation of human osteoblast-like MG 63 cells on polylactide (PLA) nanofibers prepared by needle-less electrospinning and loaded with 5 or 15 wt % of hydroxyapatite (HA) nanoparticles. On day 7 after seeding, the cell number was the highest on samples with 15 wt % of HA. This result was confirmed by the XTT test, especially after dynamic cultivation, when the number of metabolically active cells on these samples was even higher than on control polystyrene. Staining with a live/dead kit showed that the viability of cells on all nanofibrous scaffolds was very high and comparable to that on control polystyrene dishes. An enzyme-linked immunosorbent assay revealed that the concentration of osteocalcin was also higher in cells on samples with 15 wt % of HA. There was no immune activation of cells (measured by production of TNF-alpha), associated with the incorporation of HA. Moreover, the addition of HA suppressed the creep behavior of the scaffolds in their dry state. Thus, nanofibrous PLA scaffolds have potential for bone tissue engineering, particularly those with 15 wt % of HA.

Pre-activation and muscle activity during frontal impact in relation to whiplash associated disorders.

OBJECTIVE: For the evaluation of neck injury the relative distance was observed between a marker placed on the forehead and a marker placed on the shoulder and also by change of the angle. To compare the severity of head injury a value of maximum head acceleration was used, HIC and a 3 ms criterion. All criteria were related to the activity of musculus sternocleidomastoideus and musculus trapezius in a situation of expected or unexpected impact. MATERIALS AND METHODS: The situation was recorded using a Qualisys system, head acceleration of probands in three axes was recorded using the accelerometer, activity of neck muscles was monitored by a mobile EMG. RESULTS: Maximum head acceleration was 12.1 g for non-visual and 8.2 g for visual. HIC36 was 5.7 non visual and 4.0 for visual. 3-ms criterion was 11.5 g for non-visual and 7.8 g for visual. The average time of muscle activation of the observed group without visual perception is 0.027 s after hitting an obstacle, with visual perception 0.127 s before the crash. CONCLUSIONS: Kinematic values indicate more favourable parameters for neck injuries for visual. Head injury criteria show an average decrease of about 30% for visual. We can conclude that the visual perception means a significant increase in pre-activation of the observed muscle group of almost 400% and lower activation in both following phases of approximately 40%.