L. Pramatarova

Comparative study of cytotoxicity of detonation nanodiamond particles with an osteosarcoma cell line and primary mesenchymal stem cells.

Recently, nanodiamonds (NDs) have attracted great interest due to their unique physical and chemical properties that could be used in various biological applications. However, depending on the origin, NDs often contain different impurities which may affect cellular functions and viability. Therefore, before their biomedical application, the cytotoxicity of newly produced NDs should be assessed. In the present study, we have evaluated cytotoxicity of four types of ND particles with two cell models: a human osteosarcoma cell line, MG-63, and primary rat mesenchymal stem cells (rMSCs). Detonation-generated nanodiamond (DND) particles were purified with different acid oxidizers and impurities’ content was determined by elemental analysis. The particles size distribution was measured revealing that the DND particles have an average size in the range of 51–233 nm. Cytotoxicity was assessed by optical microscopy and proliferation assay after 72 hours exposure of the cells to nanoparticles. We observed cell-specific and material-specific toxicity for all tested particles. Primary stem cells demonstrated higher sensitivity to DND particles than osteosarcoma cells. The most toxic were the DND particles with the smallest grain size and slight content of non-diamond carbon, while DNDs with higher grain size and free from impurities had no significant influence on cell proliferation and morphology. In addition, the smaller DND particles were found to form large aggregates mainly during incubation with rMSCs. These results demonstrate the role of the purification method on the properties of DND particles and their cytotoxicity as well as the importance of cell types used for evaluation of the nanomaterials.

Peculiarities of hydroxyapatite/nanodiamond composites as novel implants

Hydroxyapatite/detonation nanodiamond composites are created on silica glass and cover glass by simple soaking process in an open deposition type set-up. The supersaturated solution (simulated body fluid, SBF) is prepared in a way to resemble the composition of human blood plasma. The composite growth is carried out through the addition of detonation nanodiamond particles to the SBF. Scanning electron microscopy, X-ray diffraction and FTIR spectroscopy are used to determine the surface morphology and the structure of the hydroxyapatite /detonation nanodiamond composite layers. The applied methods provide evidence that the nanodiamond surface functional groups interact strongly with the biological solution. The detonation nanodiamond surface is chemically multifunctional (surface OH, C-O-H, C = C, C-O-C and C = O groups exist), so that the hydroxyapatite is grown both by physical adsorption and chemical interaction. The OH- groups are regarded to play an important role in the hydroxyapatite growth on a diamonds surface from SBF, as they charge it negatively and attract Ca2+ ions, which in turn attract PO43- ions, thus forming apatite nuclei.

Initial biocompatibility of plasma polymerized hexamethyldisiloxane films with different wettability

Understanding the relationships between material surface properties, behaviour of adsorbed proteins and cellular responses is essential to design optimal material surfaces for tissue engineering. In this study we modify thin layers of plasma polymerized hexamethyldisiloxane (PPHMDS) by ammonia treatment in order to increase surface wettability and the corresponding biological response. The physico-chemical properties of the polymer films were characterized by contact angle (CA) measurements and Fourier Transform Infrared Spectroscopy (FTIR) analysis.Human umbilical vein endothelial cells (HUVEC) were used as model system for the initial biocompatibility studies following their behavior upon preadsorption of polymer films with three adhesive proteins: fibronectin (FN), fibrinogen (FG) and vitronectin (VN). Adhesive interaction of HUVEC was evaluated after 2 hours by analyzing the overall cell morphology, and the organization of focal adhesion contacts and actin cytoskeleton. We have found similar good cellular response on FN and FG coated polymer films, with better pronounced vinculin expression on FN samples while. Conversely, on VN coated surfaces the wettability influenced significantly initial celular interaction spreading. The results obtained suggested that ammonia plasma treatment can modulate the biological activity of the adsorbed protein s on PPHMDS surfaces and thus to influence the interaction with endothelial cells.

Towards real time 3D quantitative characterisation of in situ layer growth using white light interference microscopy

Quantitative 3D characterisation of layer growth or modification in situ in liquid systems is a challenge because of the changing morphology of the layer and the presence of the growth liquid. Because of the limited bandwidth of many surface profiling techniques, measurement of microscopic surface roughness is generally limited to that of static surfaces. The aim of the present work is to develop a new technique using high speed scanning interference microscopy combined with an adapted immersion head for use in liquid growth systems. For several years we have been developing a real time 3D surface analysis system based on a high speed camera and cabled logic processing, combined with continuous scanning white light interferometry. An optical measurement head is also being developed for use in liquid immersion conditions, with the view of measuring layer growth or modification in biomaterials. In this paper we report on the present status of the development of our second prototype 4D system and also of the optical immersion head for in situ measurements, describing the achievements made and the difficulties still to be overcome.

From superresolution to nanodetection: overview of far field optical nanoscopy techniques for nanostructures

Far field optical nanoscopy has been brought to the forefront with the 2014 Nobel Prize for chemistry in fluorescent nanoscopy for revealing intra-cellular details of tens of nm. In this review, we present an improved classification scheme that summarizes the many optical nanoscopy techniques that exist. We place particular emphasis on unlabelledsuperresolution techniques that provide real improved resolving power and unlabellednanodetection techniques for characterizing unresolved nanostructures. Superresolution is illustrated with sub-100 nm imaging of diatoms with tomographic diffractive microscopyand adenoviruseswith submerged microsphere optical nanoscopy. Three sub-categories of nanodetectionare then presented. Contrast enhancement is illustrated with surface enhanced ellipsometric contrast microscopy for the study of bacterial motility and strobed phase contrast microscopy for measuring the mechanical properties of vesicle membranes. High sensitivity phase measurement using interference microscopy demonstrates how nanostructured surfaces and structures can be characterized in biomaterials, laser textured stainless steel and defects within thin polymer films. Finally, deconvolution is illustrated with the use of through-focus scanning optical microscopy in critical dimension measurement and characterization of 40 nm linewidths in microelectronic devices. In this way we show how new far field optical nanoscopy techniques are being developed for unlabelled characterization of nano and biomaterials.

Study of Organosilicon Plasma Polymer Used in Composite Layers with Biomedical Application

In this work we study the ability of plasma polymer (PP) films obtained from hexamethyldisiloxane (HMDS) on silica glass (SG) to induce hydroxyapatite (HA)‐based composite layers from a mixture of simulated body fluid (SBF) and clear solution of detonation nanodiamond (DND) by a biomimetic process. The grown composites (PPHMDS/HADND) were studied by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and Rutherford backscattering (RBS) techniques. FTIR spectra of the PPHMDS indicated diminishing of the polymer characteristic bands when the polymer is immersed in DND clear solution. Furthermore, after sample immersion in the SBF‐DND mixture, the FTIR spectra showed the presence of carbonate‐containing HA through the characteristic vibration modes of P‐O in the phosphate group and C‐O in the carbonate group. The formation of HA layers, rich in silica and/or carbon was confirmed by RBS and SEM. The cell viability measured after 7 days on the polymer surface is more then 95% for...

Natural Opal as a Model System for Studying the Process of Biomineralization

Opal is a natural nanostructure model. It possesses nanosized close packed silica spheres and a regular sublattice of voids, filled with molecular water and accessible for filling by other substances. Using natural opal as a substrate, no complimentary technique is needed to produce a patterned surface as it is present naturally. Thus, the possession of nano-dimensions for efficient influencing of different biological events can be used in the laboratory and biologically integrated multifunctional devices (biomaterials, sensors) could be created. Additionally, biomineralization mechanisms may be studied using model systems. The main purpose of the work is to use nanostructured or other functionalized materials as models to contribute to the study of biomineralization. Particularly this paper reports on the ability of natural opal from Bulgarias Eastern Rhodopes mountain to induce the deposition of a surface layer of calcium phosphate from simulated body fluid. Raman, infrared spectroscopy and XRD were used to show that opal consists of two main phases: microcrystalline quartz and cristobalite and that the observed with optical microscopy layer deposited from the simulated body fluid on both phases was calcium phosphate.

Study of the calcium phosphate layer grown on AISI 316 stainless steel from simulated body fluid

A calcium phosphate layer was deposited on the surface of AISI 316 stainless steel by immersion in a solution supersaturated with calcium and phosphorus ions. The substrates were pre-modified by ion implantation of Ca and P, in order to induce nuclei for calcium phosphate growth on the surface. The reactivity of these surfaces towards crystal growth in aqueous solution was examined by Fourier transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. This showed that the deposited layer was a mixture of hydroxyapatite and other calcium phosphates.

Makyoh-topography studies of the morphology of the cross sections of renal stones

Makyoh topography is applied to study the morphology of mechanically polished cross sections of renal stones. The results are compared to roughness and height profile data obtained by white-light interferometry, and to microstructure studies using optical and scanning electron microscopy. It is shown that Makyoh topography is able to furnish qualitative information on the surface roughness and height profile. Correlation with compositional data obtained by X-ray diffraction are established and discussed in terms of mechanical properties. Potential application for the study of the inner structure of the renal stones and implications for shock wave lithotripsy are discussed.

Effect of nanodiamond modification of siloxane surfaces on stem cell behaviour

Mesenchymal stem cells (MSCs) hold a great promise for use in many cell therapies and tissue engineering due to their remarkable potential to replicate indefinitely and differentiate into various cell types. Many efforts have been put to study the factors controlling stem cell differentiation. However, still little knowledge has been gained to what extent biomaterials properties influence stem cell adhesion, growth and differentiation. Research utilizing bone marrow-derived MSCs has concentrated on development of specific materials which can enhance specific differentiation of stem cells e.g. osteogenic and chondrogenic. In the present work we have modified an organosilane, hexamethyldisiloxane (HMDS) with detonation nanodiamond (DND) particles aiming to improve adhesion, growth and osteodifferentiation of rat mesenchymal stem cells. HMDS/DND films were deposited on cover glass using two approaches: premixing of both compounds, followed by plasma polymerization (PP) and PP of HMDS followed by plasma deposition of DND particles. We did not observe however an increase in rMSCs adhesion and growth on DND-modified PPHMDS surfaces compared to unmodified PPHMDS. When we studied alkaline phosphatase (ALP) activity, which is a major sign for early osteodifferentiation, we found the highest ALP activity on the PPHMDS/DND material, prepared by consequent deposition while on the other composite material ALP activity was the lowest. These results suggested that DND-modified materials were able to control osteodifferention in MSCs depending on the deposition approach. Modification of HMDS with DND particles by consequent plasma deposition seems to be a promising approach to produce biomaterials capable to guide stem cell differentiation toward osteoblasts and thus to be used in bone tissue engineering.