Chondrogeneic Potential of MSC from Different Sources in Spheroid Culture.

We performed a comparative study of the proliferative potential of human mesenchymal stromal cells (MSC) from three sources (tooth pulp, adipose tissue, and Wharton's jelly) in spheroid culture; human chondroblasts served as the positive control. Histological examination revealed signs of chondrogenic differentiation in all studied cell cultures and the differences in the volume and composition of the extracellular matrix. Spheroids formed by MSC from the tooth pulp and Wharton's jelly were characterized by low content of extracellular matrix and glycosaminoglycans. Spheroids from adipose tissue MSC contained maximum amount of the extracellular matrix and high content of glycosaminoglycans. Chondrocytes produced glycosaminoglycan-enriched matrix. Type II collagen was produced by chondrocytes (to a greater extent) and adipose tissue MSC (to a lesser extent). The results of our study demonstrate that MSC from the adipose tissue under conditions of spheroid culturing exhibited maximum chondrogenic potential.

Formation of Tissue-Engineered Construct of Human Cartilage Tissue in a Flow-Through Bioreactor.

We performed culturing of a cell-engineered construct of human cartilage tissue consisting of biopolymer microstructured collagen-containing hydrogel, human adipose tissue mesenchymal stromal cells, and induction chondrogenic culture medium in a specially designed flow-through bioreactor. On day 16 of the experiment, human adipose tissue mesenchymal stromal cells acquired flattened shape typical for chondroblasts, demonstrated high proliferative activity, and formed extracellular matrix. The observed histological changes in the cultured system attested to the beginning of the formation of a tissue-engineered construct of human cartilage tissue.

In vitro and in vivo biocompatibility studies of a recombinant analogue of spidroin 1 scaffolds.

The goal of this study was to generate porous scaffolds from the genetically engineered protein, an analogue of Nephila clavipes spidroin 1 (rS1/9) and to assess the properties of new rS1/9 scaffolds essential for bioengineering. The salt leaching technique was used to make the rS1/9 scaffolds of interconnected macroporous structure with spontaneously formed micropores. The tensile strength of scaffolds was 18 ± 5 N/cm(2). Scaffolds were relatively stable in a phosphate buffer but degraded in oxidizing environment after 11 weeks of incubation. Applicability of the recombinant spidroin 1 as a substrate for cell culture was demonstrated by successful 3T3 cells growth on the surface of rS1/9 films (270 ± 20 cells/mm(2) vs. 97 ± 8 cells/mm(2) on the glass surface, p < 0.01). The 3T3 fibroblasts readily proliferated within the rS1/9 scaffold (from initially plated 19 ± 2 cells/mm(3) to 3800 ± 304 cells/mm(3) after 2 weeks). By this time, cells were uniformly distributed between the surface and deeper layers (27% ± 8% and 33% ± 4%, respectively; p > 0.05), whereas the initial distribution was 58% ± 7% and 11% ± 8%, respectively; p < 0.05). The rS1/9 scaffolds implanted subcutaneously into Balb/c mice were well tolerated. Over a 2-month period, the scaffolds promoted an ingrowth of de novo formed vascularized connective tissue elements and nerve fibers. Thus, scaffolds made of the novel recombinant spidroin 1 analogue are potentially applicable in tissue engineering.

Micro- and nanostructural characteristics of 3D porous carriers ElastoPHB-3D.

Microstructure of the surface and micro- and nanostructure of the internal surface of 3D porous carrier ElastoPHB-3D were studied by methods of electron microscopy and atomic force microscopy. Biological properties of ElastoPHB-3D samples were evaluated using culture of L929 mouse fibroblasts. High porosity and pore size of biodegradable matrixes ElastoPHB-3D and their good biofunctional properties as the substrate for cell culturing allow us to recommend ElastoPH-3D as a promising carrier for cell transplantation and creation of artificial organs.

Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood.

Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated. PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction. To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted. Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs. Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method. C16:0 constituted the largest proportion, up to 70%. Of the long-chain hydroxy acids, only beta-OH-C14:0 was detected and it did not exceed 0.06 mol%. The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood. It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems. Thus, the technology of PHA purification must satisfy rather stringent specific requirements.

Comparative analysis of human serum albumin adsorption and complement activation for intraocular lenses.

Intraocular liquid, in contrast to blood, has no cellular components; therefore, proteins (human serum albumin [HSA], and [alpha, beta, gamma] globulins) are the major components that determine patients' response to the intraocular lens (IOL) surface. In addition to the amount of adsorbed proteins, the possibility of its conformational changes, including conformational changes of globulins C1 and C3 that respond for the activation of the complements system by the classical and alternative pathways, cannot be excluded. The interaction between IOLs and protein components of intraocular liquid directly influences the ocular exudative reaction in the early postoperational period, the intensity of cellular and pigmental scurf on the surface of the IOLs, and the state of endothelial cells of the cornea in the distant postoperational period. Our goal was to compare the interaction of commercial IOLs made from polymethylmethacrylate, silicone, poly-2-hydroxyethyl methacrylate (p-HEMA), and copolymer p-HEMA with collagen with HSA and the complement system. The total internal reflection fluorescence (TIRF) method and hemolytic assay were used for this task, respectively. It has been demonstrated that the probability of biocompatibility of commercially produced IOLs on the stage of protein adsorption can be evaluated using the kinetic of HSA-fluorescein isothiocyanate adsorption onto the IOL surface by the TIRF METHOD: In the case of IOLs from p-HEMA, a negative correlation was shown between the degree of irreversible adsorption of HSA and the minimum relative rate constant of the surface-induced complement activation. We did not find any correlation between hydrophilicity/hydrophobicity of lenses and their adsorptional properties including complement activation. From suggested adsorptional criteria in vitro for biocompatible surfaces, the hydrogel lens from p-HEMA has a lower probability of biocompatibility in comparison with other IOLs.

Carbon coated polyethylene: effect of surface energetics and topography on human platelet adhesion.

The influence of surface energy and structural properties of carbon coated polyethylene (PE) on the human platelet adhesion was studied. Three types of amorphous carbon coating were obtained by plasma pulse discharge, with the number of pulses grading as 10, 50, 100. Human serum albumin adsorption experiments have been carried out with all samples in vitro. Platelet adhesion analysis by SEM included determination of total quantity of adherent platelets, and respective quantities of platelets at different stages of activation (single, spread, aggregates). Surface topographies ranged from bare PE and such (10 pulses), to globular 0.5 microm in size (50 pulses), and complex fibrillar 3-4 microm structures (100 pulses). Surface free energy varies from 31.7 +/- 0.6 to 40.4 +/- 0.6 mN/m for uncoated PE and 10 pulse coatings, respectively, as determined by contact angle techniques. All studied coatings demonstrate weaker platelet activation properties in comparison with untreated PE. Among all studied coatings, the 50 pulse coated surface seems to be the least suitable for contact with platelets, mainly due to its structural rather than to its energy properties. These data are related to a sharp decrease in the adsorbed protein level for the samples with 50 pulse coatings. The applied analysis of platelet activation enables more accurate characterization of platelet-biomaterial interaction.

Competitive adsorption of human serum albumin and gamma-globulin from a binary protein mixture onto hexadecyltrichlorosilane coated glass.

The kinetics of competitive adsorption of proteins onto hexadecyltrichlorosilane coated glass (HTS-glass) from model solutions containing fluorescein isothiocyanate (FITC)-labeled human serum albumin (HSA-FITC) and gamma-globulin (HGG-FITC) were studied by total internal reflection fluorescence (TIRF) spectroscopy. The processes of displacement of HSA-FITC by HGG are independent of the conformational state of HSA adsorbed onto glass. On HTS-glass, displacement of protein is hindered by the presence of large numbers of CH3-terminated alkyl tails which induce conformational (reorientational) changes in HSA-FITC and HGG-FITC adsorbed from simple solutions. In contrast to HSA, adsorption of HGG onto HTS-glass from a simple solution is characterized by the absence of irreversible adsorption in the initial portion of the kinetic curve. Competition between HSA and HGG-FITC induces replacement of end-on adsorbed HGG-FITC on HTS-glass surface with subsequent desorption of the HGG-FITC into solution. Upon further increase in the HSA concentration in solution the competition of HSA for adsorption sites prevails, which leads to a decrease in the amount of adsorbed HGG-FITC and, consequently, to a decrease in the rate of its displacement.

The role of proteins in the nucleation and formation of calcium-containing deposits on biomaterial surfaces.

In experiments in vivo using diffusion chambers, the morphology and composition of calcium-containing deposits on natural and artificial biomaterials that had no direct contact with cells were studied using scanning electron microscopy with energy dispersion X-ray microanalysis. It was revealed that the formation of a protein layer containing protein-calcium complexes is the key event in biomaterial calcification. A mechanism of formation of a calcium-containing protein matrix that creates the conditions for supersaturation of the crystal-forming medium over critical value has been proposed. The formation of nuclei of insoluble calcium phosphate starts predominantly deep in an adsorbed protein layer enriched by calcium ions.

[Interaction of heparinized polymer materials with plasma proteins and thrombocytes]. / Vzaimodeistvie geparinizirovannykh polimernykh materialov s belkami plazmy krovi i trombotsitami.

The interaction of heparinized polymer materials with human plasma proteins (serum albumin (SA), fibrinogen (F), antithrombin III (AT-III)) and platelets was studied. The level of SA and F adsorption irreversibility was demonstrated to depend on the mode of heparin immobilization. There was a negative correlation between the parameters of protein adsorption and the level of adsorbed AT-III. The relationship of the count of platelets adhered to the surface of heparinized polymer materials, to the time of contact was shown to be extreme. There was a correlation between the count of adhesive platelets and the parameters of plasma protein and AT-III adsorption. Based on experimental evidence, a scheme was proposed, which describes the impact of protein adsorption processes on the anticoagulative activity of surface-bound heparin, It is concluded that AT-III plays a particular role as the major component of the adsorption layer that is responsible for the nature of interactions of polymer materials with blood at the protein and cellular levels.

Analysis of albumin deposits on hydroxylated siloxane films. Implications for surface treatment of medical devices.

The authors have developed methods to enhance albumin binding to modified silicone rubber (SR) films. An intermediate bifunctional coupling agent, polyvinylmethyl siloxane-comethyl-1-ethanol siloxane (PVMS-CO-MES), is prepared from a cyclic tetramer, vinyl-methyl siloxane, by an oxymercuration-demercuration reaction, and cross-linked to silicone rubber under mild peroxide catalytic conditions. Free mercury on the surface was obtained under many reaction conditions and is shown to materially enhance 125I-labeled albumin binding. The mechanism most likely occurs via disulfide bond breakage, protein denaturation, and aggregation. The possible role of iodine-mercury bonds, an artefactual source, is ruled out with the aid of total internal reflectance-fluorescence measurements of the albumin adsorption rate constant. Although in situ albumin aggregation via disulfide bond breakage is a potentially attractive method for biocompatible protein gel formation, the toxicity of mercury makes the current method unfit for clinical practice.

An N-substituted polyurea coating with high affinity for heparin.

A new N-substituted polyurea with tertiary amino groups in the polycarbamidic chain (NPUTA) has been synthesized. The polymer is soluble in C1-C4 alcohols, has high adhesion to polar molds, and has high H2O uptake (130-150%). The material can be coated on many biomaterials (polyurethanes, charcoal hemosorbents, cellulosic hemodialysis membranes), and high amounts of heparin can be adsorbed onto treated surfaces. NPUTA cast from 0.5-3.5% ethanol solutions can absorb large amounts of heparin from anti-coagulant solution (40-60 micrograms/cm2) and heparinized plasma. Heparin release into phosphate buffered saline (PBS) solution or plasma is minimal. The influence of NPUTA solution concentration and pre-absorbed heparin on the protein adsorption, platelet adhesion, surface induced hemolysis, and complement activation of these films has been investigated. Radiolabeled protein assays, radiolabeled platelet assays, and other methods were used. It was shown that modified surfaces for the listed materials, with heparinization, demonstrate improved in vitro blood compatibility without any changes in functional properties. For example, treatment with NPUTA/heparin does not reduce sorption of middle molecules by activated charcoal hemosorbent, while markedly and significantly decreasing platelet adhesion and complement activation. NPUTA/heparin modified, glutaraldehyde treated bovine pericardium exhibited significantly reduced calcification in a rat subcutaneous implant model. Other ex vivo circulation experiments also confirm the blood compatibility of different NPUTA treated surfaces.

The heterogeneity of protein/surface interactions and structural alterations of adsorbed albumin and immunoglobulin G.

The theoretical model is developed for the reversible and irreversible protein adsorption in kinetic regime by assuming the continuous energetical heterogeneity for protein/surface interaction and the possibility of structural alterations of adsorbed molecules. The simplest rectangular distributions of adsorption centers in energy of activation are used to explain the logarithmic kinetics of IgG and human serum albumin (HSA) adsorption on a quartz surface. To explain the Freindlich character of HSA adsorption onto a precoated surface, the exponential distributions of adsorption centers in energy of activation are used. A competitive analysis of some of the approaches allowed for the energetical heterogeneity of protein/surface interaction is made. The possibility of lateral electrostatic repulsion to form the logarithmic kinetics of HSA adsorption is checked experimentally. The influence of the temperature on HSA adsorption onto quartz is discussed also.

The interaction of heparinized biomaterials with human serum, albumin, fibrinogen, antithrombin III, and platelets.

The influence of the method of heparin (HEP) immobilization on human serum albumin (HSA), fibrinogen (FG), and antithrombin III (AT-III) adsorption, platelet adhesion, and activation on the surface of polyvinylchloride, polyurethane Vitur, and a copolymer of styrene and divinylbenzene was measured. The negative correlation between the degree of irreversibility of plasma protein adsorption and the amount of adsorbed AT-III for HEP, immobilized onto the polymer surface passivated with HSA, FG, and plasma was found. The same negative correlation was observed between the amount of AT-III adsorbed on these systems and the number of adhered platelets. Schemes of the interaction of surface bound-HEP with AT-III, including the influence of an irreversibly adsorbed protein layer and adhered platelets, have been proposed. The essential role of AT-III in heparinized biomaterials/platelet interaction has been shown. A new method of combined immobilization of HEP and platelet adhesion inhibitor has been elaborated on.

The effect of cells on biomaterial calcification: experiments with in vivo diffusion chambers.

The results of these in vivo experiments show that bovine pericardium can undergo calcification without direct contact with tissue. It is clear that the direct interaction of cells with implanted samples both promotes and accelerates the process of calcification. Moreover, dietary calcium supplements, calcium chloride and vitamin D can intensify the rate and extent of this process.

Biomaterial calcification without direct material-cell interaction.

This report summarizes 1) features of the local redistribution of calcium ions and formation of complexes with calcium (Ca) in the presence of polymer samples; 2) the adsorption of Ca ions and Ca containing complexes onto biomaterial surfaces; 3) the character and composition of Ca containing deposits; and 4) the role of cellular and humoral factors in calcification. All experiments were done with three types of medical grade polymer material (from USSR): silicone rubber (SR), polyurethane "Vitur" (PU), and polyethylene (PE). Biochemical, radioisotopic, SEM, and EDAX methods were used in in vitro and in vivo experiments. The diffusion chamber model was used in animal experiments. SR was shown to induce greater changes in complex formation processes and to adsorb more Ca containing complexes than PE or PU. In addition to the degree of SR calcification seen after 21 days, implantation accentuated these findings. The possibility of calcification of polymer materials without direct contact of material and cells was observed. Combining the in vitro and in vivo experimental data, the authors propose a hypothetical scheme of biomaterial calcification.

Influence of mold properties on surface structure of a polyurethane-siloxane block co-polymer. Implications for blood compatibility.

The authors studied the surface structure of organosiloxane-polyether urethane co-polymer films cast against low and high surface free energy materials (glass/ceramic and metal). A novel surface interaction parameter, the gold nucleus density distribution (GND), determined by partial gold decoration transmission electron microscopy, was used to indirectly assess the dispersive contribution to interfacial surface free energy. A water wetting assay was used to characterize the hydrophobic/hydrophilic balance of the polymer film surfaces. Two biological interaction parameters, one involving the kinetics of albumin and fibrinogen adsorption from plasma and the other a platelet adhesion index (RIPA), were used to estimate blood compatibility. Results indicate that mold properties influence the surface structure of this block co-polymer, sequestering polysiloxane groups at mold, and possibly air interfacial regions of the film. However, as shown by ESCA and FTIR analysis, specific chemical groups did not correlate with the blood compatibility indices. The GND correlate with initial albumin and fibrinogen adsorption rates. Initial protein adsorption was less well predicted by surface water wettability. Neither surface structure nor surface interaction data predicted later (60-120 min) protein sorption or platelet adhesion. Subsequent events may be influenced by protein turnover and cell-surface interactions, and are less influenced by polymer surface properties.