Daniel Hadraba

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.

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.

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.

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.

Constitutive Modelling and Histology of Vena saphena

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.

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.

Automated System for Remote Defect Inspection

The paper deals with development and application of a method which allows to perform automatic check the surface integrity of the monitored component. In our case, it is the bearing race of the rotating cylindrical container of the lime regeneration line in the paper mill factory. By visual checks, some cracks have been discovered on the circular surface of the race. These cracks have needed to be monitored to prevent fatal failure. To measure the cracks manually, it was always necessary to stop the cylinder. However, these breaks have caused momentous problems in the operation of the line, which is conceived for continuous work due to the nature of the production process. This was the main reason for the development and deployment of the automated system. Because the problem takes of low-frequency bearing run (less than 1 Hz) moreover used in outdoor conditions an image processing technology was chosen to create a tracking system to determine the immediate crack length at specified times.

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.

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.

Collagen structures in pericardium and aortic heart valves and their significance for tissue engineering

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.