Virginija Bukelskienė

The effect of laser‐treated titanium surface on human gingival fibroblast behavior

Surface modification, as a means of enhancing soft tissue integration in titanium would have significant advantages including less marginal bone resorption, predictable esthetic outcome, improved soft tissue stability, and seal against bacterial leakage. The aim of this study was to evaluate the effects of laser-roughened titanium surfaces on human gingival fibroblast (HGF) viability, proliferation, and adhesion. Titanium discs were ablated with impulse laser in four different patterns. Polished and sand-blasted titanium discs were used as control groups. Specimen surface properties were determined using optical profilometry and scanning electron microscopy. HGF behavior on modified surfaces was analyzed using cell adhesion, viability, proliferation, and ELISA assays. Results suggested that modified Ti surfaces did not affect the viability of HGFs and improved adhesion was measured in laser treatment groups after 24 h. However, proliferation study showed that the adsorbance of fibroblast cells after 72 h cultured on polished titanium was higher and comparable with that of control cells. As for focal adhesion kinase (FAK), cells grown on laser modified surfaces had higher expression of FAK as compared with polished titanium. In conclusion, tested laser-treated surfaces seem to favor HGF adhesion. There were no significant differences between different laser treatment groups.

Applications of nonlinear laser nano/microlithography: fabrication from nanophotonic to biomedical components

In this work we present the latest results in the application of multi-photon polymerization for tissue engineering, by fabricating microstructured artificial 3D scaffolds for stem cell growth. Microstructuring of large scale 3D scaffolds is investigated and the direct laser writing technique is supplemented by fabrication by multi-beam interference and micromolding of large scale structures. Within the limitation of our study, we conclude that the proposed nonlinear direct laser writing technique offers rapid and flexible fabrication of biomedical components with required shape, pore size and general porosity. The applications could target biostable and biodegradable implants applied for bone or tissue replacement as well as drug delivery or release agents.

Large Scale Laser Two-Photon Polymerization Structuring for Fabrication of Artificial Polymeric Scaffolds for Regenerative Medicine

We present a femtosecond Laser Two‐Photon Polymerization (LTPP) system of large scale three‐dimensional structuring for applications in tissue engineering. The direct laser writing system enables fabrication of artificial polymeric scaffolds over a large area (up to cm in lateral size) with sub‐micrometer resolution which could find practical applications in biomedicine and surgery. Yb:KGW femtosecond laser oscillator (Pharos, Light Conversion. Co. Ltd.) is used as an irradiation source (75 fs, 515 nm (frequency doubled), 80 MHz). The sample is mounted on wide range linear motor driven stages having 10 nm sample positioning resolution (XY—ALS130‐100, Z—ALS130‐50, Aerotech, Inc.). These stages guarantee an overall travelling range of 100 mm into X and Y directions and 50 mm in Z direction and support the linear scanning speed up to 300 mm/s. By moving the sample three‐dimensionally the position of laser focus in the photopolymer is changed and one is able to write complex 3D (three‐dimensional) structures. An illumination system and CMOS camera enables online process monitoring. Control of all equipment is automated via custom made computer software “3D‐Poli” specially designed for LTPP applications. Structures can be imported from computer aided design STereoLihography (stl) files or programmed directly. It can be used for rapid LTPP structuring in various photopolymers (SZ2080, AKRE19, PEG‐DA‐258) which are known to be suitable for bio‐applications. Microstructured scaffolds can be produced on different substrates like glass, plastic and metal. In this paper, we present microfabricated polymeric scaffolds over a large area and growing of adult rabbit myogenic stem cells on them. Obtained results show the polymeric scaffolds to be applicable for cell growth practice. It exhibit potential to use it for artificial pericardium in the experimental model in the future.We present a femtosecond Laser Two‐Photon Polymerization (LTPP) system of large scale three‐dimensional structuring for applications in tissue engineering. The direct laser writing system enables fabrication of artificial polymeric scaffolds over a large area (up to cm in lateral size) with sub‐micrometer resolution which could find practical applications in biomedicine and surgery. Yb:KGW femtosecond laser oscillator (Pharos, Light Conversion. Co. Ltd.) is used as an irradiation source (75 fs, 515 nm (frequency doubled), 80 MHz). The sample is mounted on wide range linear motor driven stages having 10 nm sample positioning resolution (XY—ALS130‐100, Z—ALS130‐50, Aerotech, Inc.). These stages guarantee an overall travelling range of 100 mm into X and Y directions and 50 mm in Z direction and support the linear scanning speed up to 300 mm/s. By moving the sample three‐dimensionally the position of laser focus in the photopolymer is changed and one is able to write complex 3D (three‐dimensional) structures....

Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy.

Xenograft models are suitable for in vivo study of leukemias pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.

Combination of thermal extrusion printing and ultrafast laser fabrication for the manufacturing of 3D composite scaffolds

We present a novel approach to manufacturing 3D microstructured composite scaffolds for tissue engineering applications. A thermal extrusion 3D printer – a simple, low-cost tabletop device enabling rapid materialization of CAD models in plastics – was used to produce cm-scale microporous scaffolds out of polylactic acid (PLA). The fabricated objects were subsequently immersed in a photosensitive monomer solution and direct laser writing technique (DLW) was used to refine its inner structure by fabricating a fine mesh inside the previously produced scaffold. In addition, a composite material structure out of four different materials fabricated via DLW is presented. This technique, empowered by ultrafast lasers allows 3D structuring with high spatial resolution in a great variety of photosensitive materials. A composite scaffold made of distinct materials and periodicities is acquired after the development process used to wash out non-linked monomers. Another way to modify the 3D printed PLA surfaces was also demonstrated - ablation with femtosecond laser beam. Structure geometry on macro- to micro- scales could be finely tuned by combining these fabrication techniques. Such artificial 3D substrates could be used for cell growth or as biocompatible-biodegradable implants. To our best knowledge, this is the first experimental demonstration showing the creation of composite 3D scaffolds using convenient 3D printing combined with DLW. This combination of distinct material processing techniques enables rapid fabrication of diverse functional micro-featured and integrated devices. Hopefully, the proposed approach will find numerous applications in the field of tissue engineering, as well as in microelectromechanical systems, microfluidics, microoptics and others.