Biocompatibility of biphasic α,ß-tricalcium phosphate ceramics in vitro.

The biocompatibility of biphasic α,ß-tricalcium phosphate ceramics, obtained by annealing a compact preform based on ß-tricalcium phosphate powder, was studied in vitro. It was found that within 10-30 days the adhesion of primary dental pulp stem cells located on the surface of biphasic α,ß-tricalcium phosphate ceramics is suppressed. Decrease of the cell number on the surface of biphasic α,ß-tricalcium phosphate ceramics, most likely, can be associated with both the pH level (acidic) as a result of hydrolysis of the more soluble phase of α-tricalcium phosphate and with the nature of surface that changes as a result of the formation and growth of hydroxyapatite crystals.

Antiproliferative Effects of Mesenchymal Stem Cells and Epithelial Cells on Lymphocytes.

We analyzed the interactions between peripheral blood lymphocytes from heterologous donors with mesenchymal stem cells obtained from the tooth pulp and trophoblast. In mixed cultures, proliferation of both lymphocytes and mesenchymal stem cells was suppressed. Similar suppressive effects were observed in lymphocyte cultures mixed with epithelial cells (hepatocytes HeG2 and renal epithelial cells HEK293). This suppression can be determined by impairment of normal adhesion contacts between cells of different origin.

Interaction of Lymphocytes with Mesenchymal Stem Cells.

We studied the interaction of neural stem cells and dental pulp-derived mesenchymal stem cells with lymphocytes from autologous and heterologous donors. Flow cytometry analysis with the use of CFSE-labeled lymphocytes demonstrated an increase in the content of proliferating CD8, CD16 and CD56 cells, but not CD4 cells in cultures of HLA-DR-negative mesenchymal stromal cells from the dental pulp co-cultured with lymphocytes. In neural cultures expressing HLA-DR, all subpopulations of T cells and NK cells were activated. No differences between the autologous and heterologous cultures were revealed.

Effect of sunlight transformed by luminophore-containing materials on cell functions in vitro and in vivo.

Effect of sunlight transformed by luminophore-containing materials on cell viability and functional state of the retina was assessed using the photodamage model. Exposure to the luminescent component of light improved viability of NIH 3T3 cells and promoted recovery of electric activity in rabbit retina after photodamage.

Mesenchymal stem cells from human dental pulp: isolation, characteristics, and potencies of targeted differentiation.

We studied cell cultures isolated from the pulp of third molar germ of an adult human and from the skin of a human fetus on gestation day 10. Both cultures expressed similar repertoire of surface markers typical of multipotent mesenchymal cells (CD44, CD90, and CD105). Under in vitro conditions, dental pulp cells were more susceptible to factors inducing their differentiation into adipogenic, chondrogenic, and osteogenic lineage cells.

[New type titan alloy with shape memory for use in dental implantology].

The paper summarizes the results of in vitro and in vivo studies that have proved biocompatibility and medical safety of Ta and Ti-Nb-Ta-bases alloys. According to some in vitro data Ti-Nb-Ta-based alloy possesses certain advantages when comparing to Ta-based. In particular, it contributes to elevation of viability of cellular elements and to definite increase of their adhesive potential.

[Perspectives of use of polytetrafluoroethylene with nanostructured surface in dentistry].

The perspectives of use of porous polytetrafluorethilen (PTPE) with modified surface combined with mesenchymal stem cells for tissue-engineering constructions were studied. The paper also describes the mode of PTPE surface modification consisting in nanostructured metallic or ceramic layer application resulting in biocompatibility and surface adhesion rates increase. The magnetic atomizing of Ti and Ti-Ca-P-C-O-N nanolayers enhances the material integration potential as well as adhesion rates thus making it perspective when combined with mesenchymal stem cells for bone defects plasty.

Evaluation of collagen gel microstructure by scanning electron microscopy.

We performed qualitative comparison of freeze drying and chemical drying as methods of preparing 3D wet specimens for scanning electron microscopy. Human fibroblasts immobilized in collagen gel were used as a model system. Specimens fixed with glutaraldehyde were frozen in liquid nitrogen and freeze-dried at low temperature in high vacuum. In parallel experiments, glutaraldehyde-fixed samples were dehydrated in ascending ethanol solutions, absolute ethanol, and 100% hexamethyldisilazane and then dried at room temperature. Scanning electron microscopy microphotographs of collagen fibers and cells were characterized by high resolution and the absence of collapsed or deformed structures even at high magnification (×50,000) for both chemical drying and high-vacuum freeze drying. However, high-vacuum freeze drying is superior to chemical drying for the investigation of the internal space of 3D scaffolds, because sample fracture can be prepared directly in liquid nitrogen. These techniques are a part of the sample preparation process for scanning electron microscopy and can also be used for studies of cell adhesion, morphology, and arrangement in wet specimens (3D gels and flexible tissue engineering scaffolds).

Porous materials from titanium-cobalt alloys for hybrid implants.

We proposed a new method to increase the biocompatibility of porous materials that were synthesized from titanium and cobalt allows by the method of self-propagating high-temperature synthesis. This method suggested the introduction of calcium hydroxyapatite into the reaction mixture. Administration of calcium hydroxyapatite into the reaction mixture had a modifying effect on the structure and surface of the pore space and biocompatibility of composite materials. Administration of calcium hydroxyapatite crystals was followed by a significant decrease in the size of pores and appearance of water-soluble fractions, which inhibited the activity of cells. However, treatment with amorphous nanodispersed calcium hydroxyapatite increased the biocompatibility and adhesiveness of materials for mesenchymal stem cells. The pore space and mechanical characteristics of materials obtained with amorphous nanodispersed calcium hydroxyapatite were similar to the properties of natural bone. Moreover, these materials surpassed titanium-cobalt allows in biocompatibility. Our results indicate that the introduction of amorphous nanodispersed calcium hydroxyapatite into the reaction mixture during self-propagating high-temperature synthesis has a modifying effect on the pore space of composite materials and increases their biocompatibility and adhesiveness for cells. We conclude that these materials may be used as a carrier of stem cells and progenitor cells in hybrid implants.

Localization of green fluorescent protein in mouse preimplantation embryos.

The localization of enhanced green fluorescent protein (EGFP) was studied in preimplantation embryos obtained from reciprocal mating of hemizygous C57Bl/6-Tgn (ACTbEGFP)1Osb/J mice with C57Bl/6 mice. Specific fluorescence of EGFP was observed in all oocytes and embryos obtained from transgenic females during all preimplantation stages and in embryos inheriting the EGFP gene from transgenic males starting from the 8 blastomere stage during the compactization period. EGFP mRNA or EGFP synthesized during oogenesis can be retained in embryos during the entire preimplantation period, while expression of EGFP gene transferred from the father coincides with the onset of compactization. The possibility of using these embryos in experimental mammalian embryology is discussed.

Biosynthetic wound coatings as susbtrates for cell growth.

Experimental studies of composite materials formed on the basis of fluorine-containing latex and bioactive polysaccharides showed that physicochemical properties of composite materials and their adhesion characteristics can be modulated by variations of polysaccharide-latex ratio and the nature of polysaccharides. The ratio of components ensuring the formation of biosynthetic films that meet the standards for modern wound coating and maintain adhesion and growth of substrate-dependent mammalian cells was determined. These materials can considerably increase the efficiency of treatment of extensive and deep skin wounds in cases when application of cell cultures is indicated.

Acceleration of fibril formation and thermal stabilization of collagen fibrils in the presence of taxifolin (dihydroquercetin).

We studied the effect of flavonoid taxifolin (dihydriquercetin) on the structure and thermal stability of collagen I fibrils. Taxifolin accelerated fibril formation with reconstruction of periodical cross-striation characteristic of these fibrils. Differential scanning calorimetry showed elevation of melting temperature of collagen fibrils formed in neutral or weakly alkaline media, but not of individual tropocollagen molecules in acid medium. Taxifolin capacity to stimulate fibril formation and promote stabilization of fibrillar forms of collagen can be used in medicine.

Use of thermosensitive polymer material on the basis of N-isopropylacrylamide and N-tert-butylacrylamide copolymer in cell technologies.

We developed thermosensitive polymer substrates on the basis of N-isopropylacrylamide and N-tert-butylacrylamide co-polymer and studied their interaction with cultured substrate-dependent mammalian cells. It was shown that these polymers promote cell adhesion and proliferation at a level comparable to polystyrene treated for cell culturing and provide effective cell detachment after lowering culturing temperature below a critical level determined by phase transition temperature in aqueous solutions of polymers. A dependence of phase transition temperature on the ratio between N-isopropylacrylamide and N-tert-butylacrylamide was demonstrated. Differences in the dynamics of cell detachment from the surface of polymer substrates with various proportions between the components were shown.

Immobilization and long-term culturing of mouse embryonic stem cells in collagen-chitosan gel matrix.

We propose a method of creation of a 3D matrix consisting of native collagen fibers and natural polysaccharide chitosan. The collagen-chitosan hydrogels maintain viability and prolipherative activity of embryonic stem cells obtained from internal cells of mouse blastocyst. The proposed system forming hydrogels in situ can be used in cell therapy for immobilization and targeted delivery of stem cells.

Factors affecting survival of reconstructed mouse embryos after nuclear transfer.

Reconstruction of mouse embryos was performed by injection of donor genetic material from differentiated cells of various types (cumulus cells, cardiomyocytes, and epithelial cells) into recipient cells (mature oocytes and zygotes). A medium for microsurgery was selected, which enhanced survival of both embryonic and somatic cells during the reconstructive manipulations. Special preparation of somatic cells to transplantation was carried out, which employed factors synchronizing the cells in a certain phase of the cell cycle in order to enhance their capacity to maintain the development of reconstructed embryos. The processes of nucleus reprogramming in specialized cells under the action of cytoplasmic factors of oocytes and zygotes were examined. During in vitro culturing of reconstructed embryos, the most successful development was observed in embryos implanted with donor material from cumulus cells. Mouse embryos reconstructed with a certain genome and subsequent production and use of stem cells are considered as the model system for developing the basic principles of replacement therapy.

Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells.

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of vascular endothelial growth factor (VEGF165) to human aortic endothelial cells (HAEC) over an extended time period. Films were prepared using a 50:50 (w/w) mixture of non-crosslinkable and crosslinkable copolymers of the following monomer compositions (w/w): 85:15, N-isopropylacrylamide (NiPAAm):N-tert-butylacrylamide (NtBAAm); and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (ABzPh, crosslinking agent), respectively. After crosslinking by UV irradiation, the ability of films to incorporate a fluorescently labeled carrier protein (FITC-labeled BSA, 1 mg loaded per film), at 4 degrees C, was first established. Incorporation into the matrix was confirmed by the observation that increasing film thickness from 5 to 10 microm increased release from collapsed films at 37 degrees C (1.76 +/- 0.15 and 10.98 +/- 3.38 microg/mL, respectively, at 24 h postloading) and that this difference was maintained at 5 days postloading (1.81 +/- 0.25 and 13.8 +/- 2.3 microg/mL, respectively). Incorporation was also confirmed by visualization using confocal microscopy. When 10-microm films were loaded with a BSA solution (1 mg/mL) containing VEGF165 (3 microg/mL), sustained release of VEGF165 was observed (10.75 +/- 3.11 ng at 24 h; a total of 31.32 +/- 8.50 ng over 7 days). Furthermore, eluted VEGF165 increased HAEC proliferation by 18.2% over control. The absence of cytotoxic species in medium released from the copolymer films was confirmed by the lack of effect of medium (incubated with copolymer films for 3 days) on HAEC viability. In conclusion this study has shown that NiPAAm:NtBAAm copolymers can be loaded with a therapeutic protein and can deliver bioactive concentrations to human vascular endothelial cells over an extended time period.

[A study of the effect of irradiation hardness on the properties of the fluoropolymeric matrix modified by polyanionic biologically active substances]. / Izuchenie vliianiia zhestkosti oblucheniia na svoistva ftropolimernoi matritsy, modifitsirovannoi polianionnymi biologicheski aktivnymi veshchestvami.

The properties of a synthetic substrate responsible for the behavior of substrate-dependent cells in the culture were studied. The effect of the composition of a system water-soluble biopolymer (sodium alginate or methyl cellulose)-synthetic latex SKF-26 and the effect of various types of radiations on its biophysical properties were studied. The results obtained indicate that the addition of water-soluble biopolymeric additives to synthetic polymeric films improves the adhesion of cells to the substrate, the adhesion being closely related to the concentration of additives. It was found that the modification methods that determine changes in the charge of the substrate affect the capacity of different cell types for adhesion and proliferation. It was also found that the hardness of irradiation does not affect the vapor permeability and the extent of film swelling.

[Self-assembly of collagen type I molecules without telopeptides]. / Izuchenie samosborki molekul kollagena tipa I s udalennymi telopeptidami.

The effect of temperature on the kinetics of formation of fibrils from rat tail collagen molecules devoid of telopeptides was studied. It was shown that the rats of fibril formation at 30 and 35 degrees C increases five- and eightfold, respectively, as compared with that at 25 degrees C. It was found that enthalpy of fibril denaturation at 30 degrees C is maximal for the collagen both with intact telopeptides and devoid of telopeptides. It was found that essential for the fibrilogenesis of type I collagen devoid of telopeptides are temperatures of 30 and 35 degrees C.

[The use of 1H-NMR spectroscopy for the study of cell cultures]. / Primenenie 1H-IaMR spektroskopii dlia issledovaniia kul'tur kletok.

Production of lactate by the HSR-1, HSR-8, HET-SR fibroblasts have been investigated by 1H-NMR method. Were investigated both monolayer cell cultures and cells immobilized in collagen lattice. Represented data demonstrate the possibility of the NMR-spectroscopy to investigate growth's processes in the cell cultures.

[Use of polarized thermomicroscopy for recording formation and degrading processes of collagen fibrils]. / Primenenie poliarizatsionnoi termomikroskopii dlia registratsii protsessov formirovaniia i degradatsii kollagenovykh fibrill.

Polarized thermomicroscopic method were used for registration of collagen fibril formation and thermal degradation processes. It was compared with differential scanning microcalorimetry and optical density measurement methods and recommended as a fast method for registration of collagen fibril formation and degradation processes.