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Diamond-like carbon (DLC) is a biocompatible material that has many potential biomedical applications, including in orthopaedics. DLC layers doped with Cr at atomic percent (at.%) of 0, 0.9, 1.8, 7.3, and 7.7 at.% were evaluated with reference to their osteoinductivity with human bone marrow mesenchymal stromal cells (hMSCs), immune activation potential with RAW 264.7 macrophage-like cells, and their effect on apoptosis in Saos-2 human osteoblast-like cells and neonatal human dermal fibroblasts (NHDFs). At mRNA level, hMSCs on DLC doped with 0.9 and 7.7 at.% of Cr reached higher maximum values of both RUNX2 and alkaline phosphatase. An earlier onset of mRNA production of type I collagen and osteocalcin was also observed on these samples; they also supported the production of both type I collagen and osteocalcin. RAW 264.7 macrophages were screened using a RayBio™ Human Cytokine Array for cytokine production. 10 cytokines were at a concentration more than 2 × as high as the concentration of a positive control, but the values for the DLC samples were only moderately higher than the values on glass. NHDF cells, but not Saos-2 cells, had a higher expression of pro-apoptotic markers Bax and Bim and a lower expression of anti-apoptotic factor BCL-XL in proportion to the Cr content. Increased apoptosis was also proven by annexin V staining. These results show that a Cr-doped DLC layer with a lower Cr content can act as an osteoinductive material with relatively low immunogenicity, but that a higher Cr content can induce cell apoptosis.

Marketa Bacakova,1,2 Julia Pajorova,1,2 Denisa Stranska,3 Daniel Hadraba,1,4 Frantisek Lopot,4 Tomas Riedel,5 Eduard Brynda,5 Margit Zaloudkova,6 Lucie Bacakova1 1Department of Biomaterials and Tissue Engineering, Institute of Physiology, Czech Academy of Sciences, 2Second Faculty of Medicine,Charles University, Prague, 3InStar Technologies, Liberec, 4Department of Anatomy and Biomechanics, Faculty of Physical Education and Sport, Charles University, 5Department of Chemistry and Physics of Surfaces and Biointerfaces, Institute of Macromolecular Chemistry, 6Department of Composites and Carbon Materials, Institute of Rock Structure and Mechanics, Czech Academy of Sciences, Prague, Czech Republic Abstract: 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. Keywords: skin-tissue engineering, nanocoating, nanofibers, skin cells, fibrin, collagen

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.

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

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.

This report is about composites used for bone grafting. Targets the composites compact of organic chemical compounds (biopolymers) and anorganic chemical compounds (nanoparticles). These composites must be, among others, biocompatible, nontoxic for organism and their mechanical properties must be near to mechanical properties of bone. This report focused on biodegradable composites based on gelatine and nanopowder of hydroxyapatite in nanofiber and particle form.

Marketa Bacakova,1,2 Jana Musilkova,1 Tomas Riedel,3 Denisa Stranska,4 Eduard Brynda,3 Margit Zaloudkova,5 Lucie Bacakova1 1Department of Biomaterials and Tissue Engineering, Institute of Physiology, Czech Academy of Sciences, 2Second Faculty of Medicine, Charles University in Prague, 3Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, 4InStar Technologies, Liberec, 5Institute of Rock Structure and Mechanics, Czech Academy of Sciences, Prague, Czech Republic Abstract: Fibrin plays an important role during wound healing and skin regeneration. It is often applied in clinical practice for treatment of skin injuries or as a component of skin substitutes. We prepared electrospun nanofibrous membranes made from poly(L-lactide) modified with a thin fibrin nanocoating. Fibrin surrounded the individual fibers in the membrane and also formed a thin fibrous mesh on several places on the membrane surface. The cell-free fibrin nanocoating remained stable in the cell culture medium for 14days and did not change its morphology. Onmembranes populated with human dermal fibroblasts, the rate of fibrin degradation correlated with the degree of cell proliferation. The cell spreading, mitochondrial activity, and cell population density were significantly higher on membranes coated with fibrin than on nonmodified membranes, and this cell performance was further improved by the addition of ascorbic acid in the cell culture medium. Similarly, fibrin stimulated the expression and synthesis of collagen I in human dermal fibroblasts, and this effect was further enhanced by ascorbic acid. The expression of beta1-integrins was also improved by fibrin, and on pure polylactide membranes, it was slightly enhanced by ascorbic acid. In addition, ascorbic acid promoted deposition of collagen I in the form of a fibrous extracellular matrix. Thus, the combination of nanofibrous membranes with a fibrin nanocoating and ascorbic acid seems to be particularly advantageous for skin tissue engineering. Keywords: electrospun nanofibers, nanocoating, skin tissue engineering, fibroblasts, fibrin, ascorbic acid, nanotechnology, nanomedicine, collagen I synthesis, beta1-integrins