Ivan Panayotov

Functional mapping of human sound and carious enamel and dentin with Raman spectroscopy.

The goals of this trial were, first, to produce a Raman mapping of decay and sound dentin samples, through accurate analysis of the Raman band spectra variations of mineral and organic components. The second goal was to confirm the correlation between the Raman signal and the signal of a fluorescent camera, by assaying the concentration of pentosidine and natural collagen fluorescent crosslink using reverse phase high-pressure liquid chromatography. The first correlation assumed a possible relationship between the signal observed with the camera and Raman spectroscopy. The second correlation assumed an association with the Maillard reaction. Absence of a correlation for this trial was that no association could be found between Raman spectra characteristics, fluorescence variation and the HPLC assay. Our results void this absence.

Polyetheretherketone (PEEK) for medical applications

Polyetheretherketone (PEEK) is a polyaromatic semi-crystalline thermoplastic polymer with mechanical properties favorable for bio-medical applications. Polyetheretherketone forms: PEEK-LT1, PEEK-LT2, and PEEK-LT3 have already been applied in different surgical fields: spine surgery, orthopedic surgery, maxillo-facial surgery etc. Synthesis of PEEK composites broadens the physicochemical and mechanical properties of PEEK materials. To improve their osteoinductive and antimicrobial capabilities, different types of functionalization of PEEK surfaces and changes in PEEK structure were proposed. PEEK based materials are becoming an important group of biomaterials used for bone and cartilage replacement as well as in a large number of diverse medical fields. The current paper describes the structural changes and the surface functionalization of PEEK materials and their most common biomedical applications. The possibility to use these materials in 3D printing process could increase the scientific interest and their future development as well.

In vitro investigation of fluorescence of carious dentin observed with a Soprolife® camera

ObjectivesOur aim was to determine the origin of the red fluorescence of carious dentine observed with the Soprolife® camera.MethodsWe conducted in vitro studies to evaluate the origin of the red fluorescence using acids and matrix metalloproteinase (MMP) to mimic caries and methylglycoxal (MGO) to evaluate the effect of glycation reactions on the red fluorescence. In every step of these models, we detected the changes of dentin photonic response with Soprolife® in daylight mode and in treatment mode. A Raman spectroscopy analysis was performed to determine the variations of the dentin organic during the in vitro caries processes. Raman microscopy was performed to identify change in the collagen matrix of dentine.ResultsThe red fluorescence observed in carious dentine using a Soprolife® camera corresponds to the brownish color observed using daylight. Demineralization using nitric acid induces a loss of the green fluorescence of dentine. The red fluorescence of carious dentine is resistant to acid treatment. Immersion of demineralized dentine in MGO induces a change of color from white to orange-red. This indicates that the Maillard reaction contributes to lesion coloration. Immersion of demineralized dentine in an MMP-1 solution followed by MGO treatment results in a similar red fluorescence. Raman microspectroscopy analysis reveals accumulation of AGEs product in red-colored dentine.ConclusionsOur results provide important information on the origin of the fluorescence variation of dentine observed with the Soprolife® camera. We demonstrate that the red fluorescence of carious dentine is linked to the accumulation of Advanced Glycation End products (AGE).Clinical relevanceThe study provides a new biological basis for the red fluorescence of carious dentine and reinforces the importance of the Soprolife® camera in caries diagnostics.

Initial stem cell adhesion on porous silicon surface: molecular architecture of actin cytoskeleton and filopodial growth

The way cells explore their surrounding extracellular matrix (ECM) during development and migration is mediated by lamellipodia at their leading edge, acting as an actual motor pulling the cell forward. Lamellipodia are the primary area within the cell of actin microfilaments (filopodia) formation. In this work, we report on the use of porous silicon (pSi) scaffolds to mimic the ECM of mesenchymal stem cells from the dental pulp (DPSC) and breast cancer (MCF-7) cells. Our atomic force microscopy (AFM), fluorescence microscopy, and scanning electron microscopy (SEM) results show that pSi promoted the appearance of lateral filopodia protruding from the DPSC cell body and not only in the lamellipodia area. The formation of elongated lateral actin filaments suggests that pores provided the necessary anchorage points for protrusion growth. Although MCF-7 cells displayed a lower presence of organized actin network on both pSi and nonporous silicon, pSi stimulated the formation of extended cell protrusions.

Confocal Raman data analysis enables identifying apoptosis of MCF-7 cells caused by anticancer drug paclitaxel

Abstract. Confocal Raman microscopy is a noninvasive, label-free imaging technique used to study apoptosis of live MCF-7 cells. The images are based on Raman spectra of cells components, and their apoptosis is monitored through diffusion of cytochrome c in cytoplasm. K-mean clustering is used to identify mitochondria in cells, and correlation analysis provides the cytochrome c distribution inside the cells. Our results demonstrate that incubation of cells for 3 h with 10 μM of paclitaxel does not induce apoptosis in MCF-7 cells. On the contrary, incubation for 30 min at a higher concentration (100 μM) of paclitaxel induces gradual release of the cytochrome c into the cytoplasm, indicating cell apoptosis via a caspase independent pathway.

Multiphoton imaging of the dentine-enamel junction.

Multiphoton microscopy has been used to reveal structural details of dentine and enamel at the dentin-enamel junction (DEJ) based on their 2-photon excited fluorescence (2PEF) emission and second harmonic generation (SHG). In dentine tubule 2PEF intensity varies due to protein content variation. Intertubular dentin produces both SHG and 2PEF signals. Tubules are surrounded by a thin circular zone with a lower SHG signal than the bulk dentine and the presence of collagen fibers perpendicular to the tubule longitudinal axis is indicated by strong SHG responses. The DEJ appears as a low intensity line on the 2PEF images and this was never previously reported. The SHG signal is completely absent for enamel and aprismatic enamel shows a homogeneous low 2PEF signal contrary to prismatic enamel. The SHG intensity of mantle dentine is increasing from the dentine-enamel junction in the first 12 μm indicating a progressive presence of fibrillar collagen and corresponding to the more external part of mantle dentine where matrix metallo-proteases accumulate. The high information content of multiphoton images confirms the huge potential of this method to investigate tooth structures in physiological and pathological conditions.

Investigation of the in vitro photocatalytic antibacterial activity of nanocrystalline TiO2 and coupled TiO2/Ag containing copolymer on the surface of medical grade titanium

Antibacterial surfaces have been in the focus of research for years, driven by an unmet clinical need to manage an increasing incidence of implant-associated infections. The use of silver has become a topic of interest because of its proven broad-spectrum antibacterial activity and track record as a coating agent of soft tissue implants and catheters. However, for the time being, the translation of these technological achievements for the improvement of the antibacterial property of hard tissue titanium (Ti) implants remains unsolved. In our study, we focused on the investigation of the photocatalysis mediated antibacterial activity of silver (Ag), and Ti nanoparticles instead of their pharmacological effects. We found that the photosensitisation of commercially pure titanium discs by coating them with an acrylate-based copolymer that embeds coupled Ag/Ti nanoparticles can initiate the photocatalytic decomposition of adsorbed S. salivarius after the irradiation with an ordinary visible light source. The clinical isolate of S. salivarius was characterised with MALDI-TOF mass spectrometer, while the multiplication of the bacteria on the surface of the discs was followed-up by MTT assay. Concerning practical relevance, the infected implant surfaces can be made accessible and irradiated by dental curing units with LED and plasma arc light sources, our research suggests that photocatalytic copolymer coating films may offer a promising solution for the improvement of the antibacterial properties of dental implants.

SVSVGMKPSPRP: a broad range adhesion peptide

Abstract Background: A combinatorial phage display approach was previously used to evolve a 12-mer peptide (SVSVGMKPSPRP) with the highest affinity for different semiconductor surfaces. The discovery of the multiple occurrences of the SVSVGMKPSPRP sequence in an all-against-all basic local alignment search tool search of PepBank sequences was unexpected, and a Google search using the peptide sequence recovered 58 results concerning 12 patents and 16 scientific publications. The number of patent and articles indicates that the peptide is perhaps a broad range adhesion peptide. Methods: To evaluate peptide properties, we conducted a study to investigate peptide adhesion on different inorganic substrates by mass spectrometry and atomic force microscopy for gold, carbon nanotubes, cobalt, chrome alloy, titanium, and titanium alloy substrates. Results: Our results showed that the peptide has a great potential as a linker to functionalize metallic surfaces if specificity is not a key factor. This peptide is not specific to a particular metal surface, but it is a good linker for the functionalization of a wide range of metallic materials. Conclusion: The fact that this peptide has the potential to adsorb on a large set of inorganic surfaces suggests novel promising directions for further investigation. Affinity determination of SVSVGMKPSPRP peptide would be an important issue for eventual commercial uses.

A New Rat Model for Translational Research in Bone Regeneration.

The European Union (EU) directive 2010/63/EU on the protection of animals used for scientific purpose focused on reducing the number of animals and refinement of breeding. Animal studies are necessary to protect human health. Lots of animal models exist to study bone regeneration, but a reliable, well reproducible, and relatively inexpensive model with the possibility for multiple testing in the same animal is still missing. Rats may serve as good models for this due to the small animal size and good cost/benefit ratio. The present study aimed to develop a novel rat caudal vertebrae critical size defect model for bone regeneration and implant osseointegration studies The study was performed using Wistar rats with weight from 380 to 450 g. An incision was made on the dorsal side of the tail. After skin and muscles retractions, the vertebrae were exposed. Critical size defects for bone tissue engineering or implant placements for titanium body experiments were possible in each of the first four caudal vertebrae. Micro-computed tomography (CT) and histology were used to detect bone growth. There was no bone formation in the defects after 1 or 2 months of healing. When a calcium phosphate biomaterial was used (Bio-Oss®; Geistlich Pharma AG, Wolhusen, Switzerland), a good stability of the material in the defect was noted and bone growth was visible after 1 or 2 months. Results based on implant placement showed good primary stability after 3 months of healing. MicroCT showed integrated implant position through the vertebra. These results suggest that the rat caudal vertebrae may serve as a good new model for studying bone regeneration and implant osseointegration with the possibility of multiple testing within the same experimental animal and the potential to decrease number of experimental animals.

Sprayed Cells and Polyelectrolyte Films for Biomaterial Functionalization: The Influence of Physical PLL–PGA Film Treatments on Dental Pulp Cell Behavior

Further development of biomaterials is expected as advanced therapeutic products must be compliant to good manufacturing practice regulations. A spraying method for building-up polyelectrolyte films followed by the deposition of dental pulp cells by spraying is presented. Physical treatments of UV irradiation and a drying/wetting process are applied to the system. Structural changes and elasticity modifications of the obtained coatings are revealed by atomic force microscopy and by Raman spectroscopy. This procedure results in thicker, rougher and stiffer film. The initially ordered structure composed of mainly α helices is transformed into random/β-structures. The treatment enhanced dental pulp cell adhesion and proliferation, suggesting that this system is promising for medical applications.