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Biological soft tissue is a non-linear and viscoelastic material and its mechanical properties can greatly affect quality of life. Many external mechanical factors can alter the tissue, for example the tissue of talipes equinovarus congenitus, also known as clubfoot, which is the most frequent congenital deformity affecting lower extremities with pathological changes of connective tissue. In clubfoot, the presence of disc-like mass of fibrous tissue, resembling intervertebral disc tissue, is described to be between the medial malleolus and the medial side of the navicular bone. The clubfoot tissue is often referred to be stiffer or rigid by clinicians, or it is referred to as contracted and less contracted tissue, however relevant evidence about mechanical properties is missing. Therefore, the description "disc-like" is informing only about relative mechanical properties of clubfoot tissue. We aim to prepare methodical approach to quantify mechanical properties of biological tissue with uniaxial tensile stress-relaxation test, in order to help clinicians and scientist to identify precisely the mechanical properties of normal and pathological tissue and their structural behaviour during mechanical testing. In this study, we test and tune the uniaxial tensile stress-relaxation test on biological tissue with high content of connective tissue such as collagen. The model tissue is porcine pericardium. The tissue has clear collagen fibres aligning parallel to the force applied. Modulus of elasticity measured here is comparable to other studies.

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.

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (similar to 7 days) to elderly (similar to 38 months) rabbits were monitored in intact (n(tendons) = 24) and microtome sectioned (ntendons = 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 (Lambda) started at very low values (A = 2.0 +/- 0.6 mu m, Lambda = 19 +/- 4 mu m) 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. The project was mainly supported by the Grant Agency of Charles University (grant no. GAUK 956213), Czech Science Foundation, grant no. 16-14758S, by the Ministry of Health of the Czech Republic, grant no. 16-28637A, by the Ministry of Education, Youth and Sports within National Sustainability Programme I, project no. LO1309, by MEYS (LM2015062 Czech-BioImaging), by ERDF OPPK Brain-View CZ.2.16/3.1.00/21544, and D.H. acknowledges the support from the Flemish Government and Hasselt University for a research stay at Hasselt University.

Autologous and allogenic human pericardia used as biomaterials for cardiovascular surgery are traditionally crosslinked with glutaraldehyde. In this work, we have evaluated the resistivity to collagenase digestion and the cytotoxicity of human pericardium crosslinked with various concentrations of glutaraldehyde in comparison with pericardium crosslinked by genipin, nordihydroguaiaretic acid, tannic acid, and in comparison with unmodified pericardium. Crosslinking retained the wavy-like morphology of native pericardium visualized by second harmonic generation microscopy. The collagenase digestion products were analyzed using SDS-PAGE, capillary electrophoresis, and a hydroxyproline assay. Glutaraldehyde and genipin crosslinking protected the native pericardium efficiently against digestion with collagenase III. Only low protection was provided by the other crosslinking agents. The cytotoxicity of crosslinked pericardium was evaluated using xCELLigence by monitoring the viability of porcine valve interstitial cells cultured in eluates from crosslinked pericardium. The highest cell index, reflecting both the number and the shape of the monitored cells was observed in eluates from genipin. Crosslinking pericardium grafts with genipin therefore seems to be a promising alternative procedure to the traditional crosslinking with glutaraldehyde, because it provides similarly high protection against degradation with collagenase, without cytotoxic effects.

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.

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.

Various types of nanofibers are increasingly used in tissue engineering, mainly for their ability to mimic the archi- tecture 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 poly- styrene. Staining with a live/dead kit showed that the viability of cells on all nanofibrous scaffolds was very high and compa- rable 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 behav- ior of the scaffolds in their dry state. Thus, nanofibrous PLA scaffolds have potential for bone tissue engineering, particu- larly those with 15 wt % of HA. V

The inflation-extension test was performed in order to obtain the mechanical response (stress-strain curves) of the human vein - vena saphena magna (usually used for coronary artery bypass graft surgery). Tubular samples of the vein were inflated four times up to the pressure approx. 4 kPa (vein pressure) and then four times up to approx. 16 kPa (systolic pressure). The experiments were recorded by the CCD camera. The longitudinal and circumferential deformations of the tube were evaluated using the edge detection method. The experimental data were fitted by anisotropic, nonlinear, constitutive model in order to obtain model parameters, especially the parameter which can be explained as collagen fibres orientation approximation. This parameter was then compared with the findings from histology. The histology analyses based on label-free imaging were performed additionally to the mechanical testing. Collagen (most important load-bearing component of the vein wall) was visualized using second harmonic generation imaging (SHG, excitation at 860 nm by a tunable IR pulse laser, detection at 430±10 nm). This method enabled us to observe collagen through the vein wall. It was found that the collagen fibres are helically aligned within the vein at an angle 37±6° measured from circumferential axis. The results of collagen orientation angle show a good agreement of findings obtained from histology and from constitutive model.

Jana Liskova,1 Oleg Babchenko,2 Marian Varga,2 Alexander Kromka,2 Daniel Hadraba,1 Zdenek Svindrych,1 Zuzana Burdikova,1 Lucie Bacakova1 1Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 2Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Abstract: 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. Keywords: nanocrystalline diamond film, osteoblast, Saos-2, collagen, SHG

Biological prostheses of human heart valves are prepared from autologous heart valves and from xenogeneic heart valves or pericardium. Xenogenous and allogenous biological prostheses are associated with adverse immune reactions, thrombosis and degeneration, and thus they have a high rate of reoperation. An optimum autologous tissue for heart valve grafts therefore needs to be found. Human pericardium was assessed as a potential source, and was compared with native human heart valves and porcine and bovine pericardium and heart valves. The tissues were evaluated for their mechanical properties, their collagen content and structure, and their histological structure. We observed differences in mechanical properties, collagen fibre orientation, the shape of the collagen bundles, and the collagen content among the pericardium samples and heart valves of different origin.

The function of the pelvic floor is fundamentally influenced by the behaviour of several organs and the organ-linked processes. The aim of this work is to study the properties and changes of the women’s pelvic floor. The motive arises from the fact that pelvic floor dysfunctions badly influence the quality of life. The loss of the proper function in the pelvic floor results in a wide range of problems from asymptomatic and anatomic defects to vaginal eversion. All the aforementioned problems are frequently followed by urinating and defecating difficulties together with sexual dysfunctions.