Experimental Correction of Homeostasis Changes during Alloxan-Induced Diabetes by Implantation of Islet Cells Cultured in Fibrous TiNi-Based Scaffold.

The use of fibrous scaffolds made of titanium nickelide and carrying islet cells (IC) after a course of alloxan administration was studied in vivo. Scanning electron microscopy showed good adhesion of IC in the pore space of the scaffolds. In 28 days, mature tissue comprising both cellular and fibrous components filled the inner space of the scaffold by 90%. The advantage of intraperitoneal implantation of IC in vitro seeded in fibrous titanium nickelide scaffolds in comparison with injection of IC (6×105 cells) to Wistar rats was demonstrated in the dynamics of pancreatic function recovery (normalization of blood glucose and glycosylated hemoglobin concentrations by day 21 of the experiment), regeneration of the white hematopoietic lineage cells, and lengthening (by 2 times) of the lifespan of animals with alloxan-induced diabetes. Injection of IC improved rat survival by only 40%. Thus, the hybrid tissue-engineered construct (fibrous titanium nickelide scaffold+IC) is biocompatible when administered intraperitoneally, stimulates regeneration in rats with alloxan-induced diabetes, improves blood glucose utilization processes, and restores suppressed leukopoiesis. It is assumed that hepatocytes can be the main target cells of the hybrid tissue-engineering construct.

Imbalance of morphofunctional responses of Jurkat T lymphoblasts at short-term culturing with relief zinc- or copper-containing calcium phosphate coating on titanium.

Morphofunctional response of Jurkat T cells that were cultured for 24 h on substrates prepared from commercially pure titanium with relief microarc bilateral calcium phosphate coating containing copper or zinc was studied. Changes in the concentration of essential trace elements contained in this coating can cause significant imbalance of molecular processes of differentiation, secretion, apoptosis, and necrosis and reduce tumor cell survival.

Cell-IQ visualization of motility, cell mass, and osteogenic differentiation of multipotent mesenchymal stromal cells cultured with relief calcium phosphate coating.

The Cell-IQ continuous surveillance system allowed us to establish the following changes in a 14- day culture in vitro: a twofold suppression of the directional migration of multipotent mesenchymal stromal cells of human adipose tissue (MMSC-AT) towards the samples with a microarc calcium phosphate (CP) coating from synthetic hydroxyapatite; a tenfold decrease in the cell mass on the interphase with the samples, which was accompanied by a slight reduction in the expression of membrane determinants of stromal stem cells; and an enhancement of their osteogenic differentiation (osteocalcin secretion and mineralized matrix formation) on the 21st day of the study. Calcium phosphate particles, but not the calcium and phosphorus ions, may trigger the phenotypic transformation of the MMSC-AT behavior in vitro.

MORPHOFUNCTIONAL CHANGES OF JURKAT LINE T-LYMPHOBLASTS AT SHORT-TERM CONTACT WITH RELIEF CALCIUM PHOSPHATE SURFACE.

Human leukemic T-lymphoblastoid cells (hereinafter Jurkat T-cells) were used to model T-lymphocytes morphofunctional reaction to 24-h in vitro contact with relief (roughness index Ra = 2.2--2.7 mm) pure titanium substrates (12_12_1 mm3) covered by calcium phosphate (CP) bilateral coating that was prepared by micro-arc method. Jurkat T-cell culture on plastic surface of well plate (2D control of culture growth), as well as the cells that contacted for 24 h with oxide (TiO2) micro-arc coating on pure titanium substrate (3D control) served as comparison tests. 27--98 % of immortalized cells in 2D control culture had CD3+CD4+CD71+CD45RA+ immunophenotype and secreted IL-2, IL-4, IL-8, IL-10 and TNFa, but not IL-1b and IL-6. Other markers of cell activation, differentiation, maturation and death (CD8, CD16, CD56, CD25, CD95) were found at 0­2.5% of the cell population. Microtextured CP surface elevated statistically IL-8 outcome to 183 and 160 % of corresponding control values in 2D- and 3D-cultures of Jurkat T-cells. CD4/CD8 ratio fell to 9 : 1 (at 13 : 1 and 82 : 1 in 2D- and 3D-controls, respectively) both by CD4+ depletion and by CD8+ cell percent raise. Total amount of cells (TAC) in Jurkat T-cell culture after 24-h contact with CP coating was decreased to 88 % 2D control level (P < 0.04) that could favor to a suppression of cell division. TAC reduction in Jurkat T-cell culture was accompanied by accelerated IL-8 spontaneous secretion (r = ­0.97, P < 0.00009). For all this, IL-8 induced apoptosis (r = 0.94, P < 0.0001) in low concentrations (pg/ml). The obtained result is the feature of Jurkat T-cells reaction on CP but not TiO2 coatings, and it may be used in case of materials selection for endoprosthesis replacement and fracture osteosynthesis in patients suffering by hematological and bone malignancies.

IMMUNOPHENOTYPICAL CHARACTERISTICS OF CELLULAR COMPOSITION IN BREAST MILK.

The morphology and immunophenotype of female colostrum adherent cells with the help of CD3, CD31, CD34, CD45, CD68, vimentin, and osteocalcin antibodies panel was studied in short-term (6­7 days) culture in vitro. Approximately equal (1 : 1) ratio of fibroblast-like and rounded cells was observed in 20 % of cultural flasks. The cells with regular shape mixed with single fibroblasts were noted in 80 % of cultural flasks. The diameter of spreaded cells varied within 10­100 mm. All cells adhered to plastics did not express CD3 and interacted slightly (sl) with antibodies to CD31, CD34, and CD45. At the same time, adherent cells with intensive CD68, vimentin and osteocalcin staining have been revealed. Literature data allows to interpret CD68+CD3­CD31slCD34slCD45sl immunophenotype of significant part of mother colostrum adherent cells as belonging to monocyte-macrophage lineage. Marked expression of stromal antigens (vimentin, osteocalcin) in 40­45 % adherent cells in cultural medium without osteogenic supplements (beta-glycerophosphate, ascorbic acid, dexamethasone) proposes an existence of osteoblasts fraction differentiated in colostrum from mesenchymal stem cells under an action of breast milk humoral factors.

Modulating effect of matrices with calcium phosphate coating on cytotoxicity of strontium ranelate and ibandronic acid in vitro.

Strontium ranelate (29 µg/ml) and ibandronic acid (50 µM) produced a cytotoxic effect on rat bone marrow myelokaryocytes in vitro. Strontium ranelate increased the number of myelokaryocytes with signs of necrosis, ibandronic acid increased the number of apoptotic and necrotic cells in the 9-day 2D culture of bone marrow cells on the plastic surface of the wells of culture plates. Co-culturing of the bone marrow with 3D matrices with microarc calcium phosphate coating that simulated bone mineral matrix increased intracellular ROS concentration, but abolished the cytotoxic effect of these drugs.

[The combined use of bisphosphonates and strontium ranelate with osseosubstituting materials].

In review the possibility of biomaterials osseointegration improvement with help of bisphosphonates or strontium ranelate is discussed. For this purpose, they are added to hydroxyapatite used for implants coating, or are included as a component of bulk calcium phosphate materials. Strontium is employed as a compound of biodegradable metal alloys, also. Combined use of carrier (implant) with bisphosphonates or strontium ranelate promotes controlling local delivery of pharmaceutical molecules into lesion, enhances the therapy efficiency, and decreases a dose and systemic toxicity of the drugs. Bisphosphonates and strontium ranelate increase the mass, a count and thickness of bone trabeculas, improve the bone biomechanical properties in the place of implants fixation, and diminish the bone fracture risk. Main studies are devoted to pharmacologic mechanisms of implants osseointegration improvement. Bisphosphonates as isoprenoid lipids chemical analogues diminish by concurrent principle the osteoclastsfarnesylpyrophosphate synthase activity and inhibit the prenylation. Unprenylated small GTPases don't fasten onto osteoclasts membrane that weakens cellular resorptive activity and accelerates their apoptosis. Strontium ranelate enhances osteoblasts replicative activity and suppresses their apoptosis, also retards osteoclasts resorptive function and accelerates their apoptosis. Its effects are conditioned by activating Wnt-signaling pathway by means of calcium-sensing receptor and by changing the RANKL/RANK/OPG system.

[Molecular mechanisms of action of bisphosphonates and strontium ranelate].

Bisphosphonates are chemical analogs of isoprene lipids, which competitively decrease the activity of farnesyl diphosphate synthase in osteoclasts and thus retard prenylation. The non-prenylated small GTPases cannot attach to the membrane of osteoclasts, which decreases their resorptive function and accelerates apoptosis. Strontium ranelate activates the Wnt signal pathway (with participation of calcium-sensitive receptor), increases the replication activity (by changing the function of RANKL/RANK/OPG system) thus suppressing the apoptosis of osteoblasts, and retards the resorptive function by accelerating the apoptosis of osteoclasts.

[Effects of strontium ranelate on mononuclear leukocyte culture].

Strontium ranelate (SR) effect on the functional parameters of human blood mononuclear leukocytes culture has been investigated. SR in concentrations of 2.9, 29, and 290 mg/ml produces a dose-dependent decrease in the activity of alkaline phosphatase and the content of calcium and potassium ions in supernatants. In concentrations of 2.9 and 29 mg/ml, SR decreases, while in a concentration of 290 mg/ml it increases the intercellular phosphate ion content. Fibroblast- and osteoblast-like cells, which are capable of regulating mineral compound of intercellular liquid, are SR cellular targets in human blood mononuclear leukocytes culture.

Human Mesenchymal Stem Cells as a Carrier for a Cell-Mediated Drug Delivery.

A number of preclinical and clinical studies have demonstrated the efficiency of mesenchymal stromal cells to serve as an excellent base for a cell-mediated drug delivery system. Cell-based targeted drug delivery has received much attention as a system to facilitate the uptake a nd transfer of active substances to specific organs and tissues with high efficiency. Human mesenchymal stem cells (MSCs) are attracting increased interest as a promising tool for cell-based therapy due to their high proliferative capacity, multi-potency, and anti-inflammatory and immunomodulatory properties. In particular, these cells are potentially suitable for use as encapsulated drug transporters to sites of inflammation. Here, we studied the in vitro effects of incorporating synthetic polymer microcapsules at various microcapsule-to-cell ratios on the morphology, ultrastructure, cytokine profile, and migration ability of human adipose-derived MSCs at various time points post-phagocytosis. The data show that under appropriate conditions, human MSCs can be efficiently loaded with synthesized microcapsules without damaging the cell's structural integrity with unexpressed cytokine secretion, retained motility, and ability to migrate through 8 µm pores. Thus, the strategy of using human MSCs as a delivery vehicle for transferring microcapsules, containing bioactive material, across the tissue-blood or tumor-blood barriers to facilitate the treatment of stroke, cancer, or inflammatory diseases may open a new therapeutic perspective.

Structural and functional state of the bone marrow during its in vitro interaction with ferromagnetic nanoparticles.

Hemopoietic islets, associations of stromal (macrophages, fibroblasts) and blood (including stem) cells, are structural and functional units of the bone marrow. We studied cellular and molecular processes developing following short-term (1 h) contact of hemopoietic islets with ferromagnetic nanoparticles in a multicellular system of the bone marrow in vitro. It was established that nanodispersions of magnetite (Fe(3)O(4), mean particle diameter 18 nm) and iron coated with carbon (Fe(C), particle diameter 5-10 nm) in a dose of 3 mg/liter had a minor effect on processes of necrotic and apoptotic cell death. Nanodispersion of carbon-coated iron (Fe(C)) most mildly stimulated oxidizing processes recorded by intracellular levels of reactive oxygen species. These nanoparticles, in contrast to magnetite, did not reduce the amount of hemopoietic islets in the bone marrow cell suspension.

Pilot in vitro study of the parameters of artificial niche for osteogenic differentiation of human stromal stem cell pool.

The aim of this research is experimental investigation of the topography and evaluation of some parameters of artificial microterritories promoting osteogenic differentiation of stromal stem cells. A technique of short-term culturing of prenatal human lung stromal cells with fibroblastoid morphology on calcium phosphate substrates with known topography was used. Judging from secretory activity of the cell culture (osteocalcin, alkaline phosphatase), stromal stem cells directly interacting with calcium phosphate discs have advantage in manifestation of osteoblast-like functional activity in comparison with cells cultured on plastic. Rough surfaces of calcium phosphate discs stimulate the formation of spatial human fibroblastoid cell culture. The cells with positive reaction to acid phosphatase are located on spheroliths forming the relief of calcium phosphate coatings. The cells with positive reaction to alkaline phosphatase (marker of osteoblasts) populate hollows (niches) of the artificial surface. The niche for induction of osteogenic differentiation of human multipotent mesenchymal stem cells is apparently a structural and functional formation. It can be characterized by an index calculated as the ratio of the total area occupied by alkaline phosphatase-positive cells to the area of artificial surface occupied by one stained cell.

Osteogenic and Angiogenic Properties of Heparin as a System for Delivery of Biomolecules for Bone Bioengineering: a Brief Critical Review.

The review considers complex, controversial, and individual effects of heparin and its derivatives on the bone and circulatory systems in dependence of the dose, the state of the cells and tissues of the recipient. General data on the anticoagulant activity of heparin and its derivatives are presented; special attention is paid to the effect of heparin on mesenchymal cells and tissues and its role in angiogenesis. We also discuss the ability of heparin to bind osteogenic and angiogenic biomolecules in the context of the development of systems for their delivery and sustained controlled release and propose a schematic representation of the positive and side effects of heparin as a delivery system for biomolecules in tissue engineering.

Surface characterization and biological assessment of corrosion-resistant a-C:H:SiOx PACVD coating for Ti-6Al-4V alloy.

The paper focuses on the SiOx-doped amorphous hydrocarbon (a-C:H:SiOx) coating on the titanium (Ti-6Al-4V) alloy substrate obtained by plasma-assisted chemical vapor deposition (PACVD) in a mixture of argon gas and polyphenylmethylsiloxane vapor using a bipolar substrate bias. It is shown that the a-C:H:SiOx coating deposition results in the formation of a negative surface potential important for application of this coating for medical implants. The a-C:H:SiOx coatings improve the corrosion resistance of Ti alloy to 0.5 M NaCl solution and phosphate-buffered saline. In particular, the corrosion current density of the a-C:H:SiOx-coated sample in a 0.5 M NaCl solution at 22 °C decreases from 1∙10-8 to 1.7∙10-10 A/cm2, that reduces the corrosion rate from 9∙10-5 to 15∙10-7 mm/year. The a-C:H:SiOx coating facilitates the surface endothelization of an implant located in the thoracic aorta of a mini pig, and reduces the risk of thrombosis and implant failure. This effect can be explained by the ability of the a-C:H:SiOx coating ability to reduce in vitro a 24-hour secretion of pro-inflammatory interleukins (IL-6, IL-12(p70), IL-15, and IL-17) and cytokines (IFN-g and TNF-a) by blood mononuclear cells (MNCs) and elevates the concentration of anti-inflammatory interleukin IL-1Ra. In vitro analysis shows no cytotoxicity of the a-C:H:SiOx coating for the human blood MNCs, suggesting a promising PACVD on Ti alloys for cardiovascular implants, including pumps for mechanical heart support systems.

Solution blow spinning of PLLA/hydroxyapatite composite scaffolds for bone tissue engineering.

Composite poly-L-lactide acid-based scaffolds with hydroxyapatite (HAp) content up to 75 wt.% were fabricated via solution blow spinning. The influence of HAp concentration on structure, wettability, mechanical properties and chemical and phase composition of the produced materials was examined. It was found that with an increase of HAp content the average fiber diameter was increased, the uniaxial strength and relative elongation were reduced, while the phase composition and surface wettability did not change. The performance of the scaffolds during implantation in the parietal bone of a rat skull for a period from 15 to 90 days was studied. The materials have shown high ability to integrate with both soft and hard tissues. It was found that scaffolds with 25 wt.% HAp content significantly enhance osteogenesis during scarification (damage) of the periosteum. Overall, the fabricated scaffolds proved to be highly efficient for replacing bone defects in long tubular bones.

[Osteogenic and angiogenic properties of heparin as a system of biomolecule delivery for bone bioengineering: a brief critical review]. / Osteogennye i angiogennye svoistva geparina kak sistemy dostavki biomolekul pri bioinzhenerii kosti: kratkii kriticheskii obzor.

The review discusses the complex, ambiguous and individual effects of heparin and its derivatives on the bone and circulatory systems, in dependence of the dosage, the state of the cells and tissues of recipients. General data on the anticoagulant activity of heparin and its derivatives are presented; aspects of the effect of heparin on mesenchymal cells and tissues and its role in angiogenesis are considered in details. Particular attention is paid to the ability of heparin to bind osteogenic and angiogenic biomolecules: thus us especially important for the development of systems for their delivery and sustained controlled release. A schematic representation of the positive and side effects of heparin as a delivery system for biomolecules in tissue engineering is proposed.

Flexible intramedullary nails for limb lengthening: a comprehensive comparative study of three nails types.

This study aims to investigate the comparative study of calcium phosphate coatings for flexible intramedullary nails (FINs) used to lengthen long tubular bones. The presence of a calcium phosphate coating deposited by micro-arc oxidation (MAO) or a composite coating based on a co-polymer of vinylidene fluoride with tetrafluoroethylene (VDF-TeFE) and hydroxyapatite (HA) on the surface of the FIN significantly enhanced the regeneration of bone in the area of osteotomy during limb lengthening by combined osteosynthesis. The investigation of the physico-chemical properties of the FIN coated with calcium phosphate via MAO demonstrated that the improved bone tissue formation resulted from favourable conditions for adhesion, proliferation and differentiation of multipotent stem cells into osteoblasts on the coating surface. The composite coatings only stimulated the formation of bone tissue in vivo, primarily because of the piezoelectric properties of the VDF-TeFE co-polymer.

[Secretion of niche signal molecules in conditions of osteogenic differentiation of multipotent mesenchymal stromal cells induced by textured calcium phosphate coating]. / Sekretsiia signal'nykh molekul krovetvornykh nish v usloviiakh osteogennoi differentsirovki mul'tipotentnykh mezenkhimnykh stromal'nykh kletok, indutsirovannoi rel'efnym kal'tsii-fosfatnym pokrytiem.

Secretion of 21 cytokines, chemokines and growth factors (LIF, SCF, SDF-1a, SCGF-b, M-CSF, MCP-3, MIF, MIG, TRAIL, GRO-a; IL-1a, IL-2ra, IL-3, IL-12(p40), IL-16, IL-18, HGF, TNF-b, b-NGF, IFN-a2, CTACK) has been studied in vitro in the culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) in conditions of its osteogenic differentiation caused by 14-day contact with calcium phosphate (CP) surface with different roughness. Bilateral X-ray amorphous CP coatings were prepared on the samples of commercially pure titanium in the anodal regime using a micro-arc method. An aqueous solution prepared from 20 wt% phosphoric acid, 6 wt% dissolved hydrohyapatite nanopowder (particle diameter 10-30 nm with single agglomerates up to 100 nm), and 9 wt% dissolved calcium carbonate was used to obtain CP coating. hAMMSCs isolated from lipoaspirate were co-cultured after 4 passages with the CP-coated samples at final concentration of 1.5´105 viable karyocytes per 1.5 mL of standard nutrition medium (without osteogenic stimulators) for 14 days (a determination of [CD45,34,14,20], CD73, CD90 и CD105 cell immunophenotype; an analysis of secretory activity) and 21 days (alizarin red S staining of culture) with medium replacement every 3-4 days. Under conditions of in vitro contact with rough CP coating hAMMSCs differentiated into osteoblasts synthesizing the mineralized bone matrix; this was accompanied by 2-3-fold increasing ratio of [CD45,34,14,20]+ hemopoietic cells. The following humoral factors of hemopoietic niches acted as the signal molecules escalating in vitro the hemopoietic base in 14 days of differentiating three-dimensional culture of hAMMSCs: either leukemia inhibitory factor (LIF) and stem cell factor (SCF) cytokines under mean index of CP roughness Ra=2.4-2.6 mm or stromal derived factor-1 (SDF-1a, CXCL12 chemokine) under Ra=3.1-4.4 mm.

Voltammetric Behavior, Identifying and Quantitatively Determining Iron-Based Nanoparticles, and Evaluating Their Stability in Simulated Solutions of Gastric Juice.

The electrochemical behavior of Fe3O4 nanoparticles, iron nanoparticles coated with carbon, and diazonium salts by voltammetry methods using a carbon paste electrode (CPE) has been studied. There has been developed a voltammetric method for identifying and quantifying solid-phase iron-based nanoparticles in the background electrolyte of 0.02 mole/dm3 Trilon B (pH 3.5) with the use of the CPE. Investigations of nanoparticles' stability with various coatings in a simulated solution of gastric juice have been carried out. Nanoparticles stability has been evaluated on the basis of determining Fe(III) ions in a simulated solution after contacting with nanoparticles within different periods of time using the method of inversion voltammetry. It has been shown that nanoparticles coated with carbon and salts of arendiazonium are the most resistant to aggressive media.

Comparative investigations of structure and properties of micro-arc wollastonite-calcium phosphate coatings on titanium and zirconium-niobium alloy.

Investigation results of micro-arc wollastonite-calcium phosphate (W-CaP) biocoatings on the pure titanium (Ti) and Zr-1wt.%Nb (Zr-1Nb) alloy were presented. The voltages of 150-300 V generate the micro-arc oxidation (MAO) process with the initial amplitude current of 150-550 A and 100-350 A for Ti and Zr-1Nb substrates, respectively. The identical dependencies of changes of the coating thickness, surface roughness and adhesion strength on the process voltage were revealed for the both substrates. The W-CaP coatings with the thickness of 10-11 µm were formed on Ti and Zr-1Nb under the low process voltage of 130-150 V. Elongated wollastonite particles with the size in the range of 40-100 µm were observed in such coatings. The structure of the coatings on Ti was presented by the X-ray amorphous and crystalline phases. The X-ray reflexes relating to the crystalline phases of Ti and wollastonite were observed only in XRD patterns of the coatings deposited under 130-200 V on Ti. While, the crystalline structure with phases of CaZr4(PO4)6, ß-ZrP2O7, ZrO2, and Zr was detected in the coatings on Zr-1Nb. FT-IRS, XRD, SEM, and TEM data confirmed that the increase of the process voltage to 300 V leads to the dissociation of the wollastonite. No toxic effect of specimens on a viability, morphology and motility of human adipose-derived multipotent mesenchymal stem cells was revealed in vitro.