I. A. Khlusov

A hybrid PHB–hydroxyapatite composite for biomedical application: production, in vitro and in vivo investigation

Samples of a hybrid composite of polyhydroxybutyrate (PHB), a biodegradable polyester, and hydroxyapatite (HA), with different PHB/HA ratios, have been prepared using mechanical-physical method. Electron microscopy, X-ray structure analysis and differential thermal analysis have been used to investigate the structure and physicochemical properties of the composite, depending on the PHB/HA ratio. The properties of the surface of the HA-loaded composite are significantly different from those of the pure polymer. As the HA percentage in the composite increases, free interface energy, the cohesive force, i.e., the strength of the adhesive bond between the composite surface and the water phase, and surface wettability increase. The HA percentage of the composite does not influence its melting temperature, but affects the temperature for the onset of decomposition: as the HA content increases from 0 to 10% (w/w), T d decreases from 260°C to 225°C. The degree of crystallinity of PHB/HA increases from 77% to 89% with an increase in the HA fraction from 10% to 50%. Functional properties of the composites have been investigated in vitro and in vivo. The best parameters of growth and differentiation of murine marrow osteoblasts are registered on PHB/HA samples containing 10% and 20% HA. In ectopic bone formation assay it has been proven that the hybrid PHB/HA composites can function as scaffolds and that bone tissue develops on their surface and in pores.

The Structure and Physical and Mechanical Properties of a Novel Biocomposite Material, Nanostructured Titanium–Calcium-Phosphate Coating

The paper presents the results of a complex study of the structure, phase composition and physical and mechanical properties of a new biocomposite material, bulk nanostructured titanium–calcium-phosphate coating, as well as its biological testing. The high-strength nanostructured titanium was obtained by multiple uniaxial pressing using an original press-mold within the temperature interval from 1023 to 623 K and the additional rolling deformation in combination with prior-recrystalline annealing. Such a treatment produces improvement in the mechanical properties of titanium up to the level of high-strength titanium alloys, for example, Ti-6Al-4V. It was found that the micro-arc technique of formation of calcium phosphate (Ca-P) coating in aqueous solutions of phosphoric acid, hydroxylapatite and calcium carbonate powders provides the generation of β-tricalcium phosphate that points to high biocompatibility. Adhesion strength of Ca-P coating to nanostructured titanium is no less than 25 MPa. Biological tests in vivo showed that the novel coating promotes implant integration into bone marrow cells and provides the growth of bone tissue on an implant surface. The novel calcium phosphate coating is highly biocompatible, nontoxic and can be used for osteosynthesis.

Osteogenic potential of mesenchymal stem cells from bone marrow in situ: role of physicochemical properties of artificial surfaces.

Correlation analysis demonstrated the role of inorganic parameters of the surfaces of calcium phosphate materials in the regulation of osteogenic differentiation of mesenchymal precursors. The progenitor stromal cells were isolated from syngeneic bone marrow immobilized in vitro on calcium phosphate surfaces with different structure, phasic, and elemental composition. After 45 days of subcutaneous ectopic osteogenesis in BALB/c mice, the tissues grown on these matrixes were characterized histologically. It was found that adhesion of bone marrow cells is the initial stage determining their future proliferation (conduction) over the artificial surface and the area of formed tissue plate. The success of histogenesis depends on surface roughness. The optimal roughness class was 4–5 (Russian State Standards), which enables differentiation of progenitor stromal cells under the specific microenvironmental conditions into the connective and adipose tissue cells. Differentiation of the progenitor cells into the stromal cells producing the hemopoiesis-inducing microenvironment also takes place in the foci of active hemopoiesis. Induction of osteogenic potential of the stromal precursors (osteoinduction) is determined by the ratio between calcium and phosphate atoms in surface coatings. In our experimental system, osteogenic differentiation of stromal mechanocytes was blocked only at Ca/P<0.5.

Study of physicochemical and biological properties of calcium phosphate coatings prepared by RF magnetron sputtering of silicon-substituted hydroxyapatite

Coatings based on pure silicon and silicon-substituted hydroxyapatite were grown by RF magnetron sputtering. The coating surface morphology, phase and elemental composition were studied by scanning electron microscopy, energy-dispersive X-ray analysis, and infrared spectroscopy. It was found that coatings are X-ray-amorphous, their elemental composition being controlled by the sputtered target composition. The distribution of elements over the coating surface is homogeneous. Medical and biological properties of coatings were studied in vivo and in vitro. Osteogenic properties of coatings were studied. Coatings grown by sputtering of a stoichiometric hydroxyapatite target are biocompatible without osteoinductive activity. The introduction of silicate ions into the hydroxyapatite structure that forms an electrode target significantly enhances the in vivo effect of CaP magnetron coatings on the osteogenic activity and stromal bone-marrow stem cells.

Formation and properties of bioactive surface layers on titanium

The plasma electrolytic oxidation technique was used to form a calcium phosphate coating containing hydroxyapatite on a titanium implant surface. The composition, morphology, and anticorrosion and mechanical properties of the obtained layers were studied. The optimal conditions of the polarization mode and electrolyte composition were found. Experiments performed in vivo and in vitro showed that the bioactivity of deposited films depends on the chemical composition (concentrations and Ca/P ratio) and the surface roughness of calcium phosphate layers.

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 artifi cial 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 artifi cial surface occupied by one stained cell.

Application of High-Frequency Magnetron Sputtering to Deposit Thin Calcium-Phosphate Biocompatible Coatings on a Titanium Surface

Thin calcium-phosphate coatings were deposited on titanium substrates by high-frequency magnetron sputtering. The elemental composition of coatings and types of chemical bonds were studied by Rutherford backscattering (RBS) and Fourier transform infrared spectroscopy (FTIR), respectively. An analysis of the IR spectra detected absorption bands caused by vibrations of phosphate PO43− groups and pyrophosphate H2PO4− anions, which are typical of apatites. The RBS results showed that the coating contains elements typical of calcium phosphates, i.e., Ca, P, and O; 45.4 ± 1.1, 3.6 ± 0.5, and 41.1 ± 0.7 at %, respectively. The Ca/P atomic ratio depends on sputtering conditions and varies in the range 1.7–4.0. The physicomechanical characteristics of the coatings and their solubility in a biological liquid were studied. The grown coatings can significantly reduce dissolution of substrates and extraction of dopants into the surrounding solution.

Relationship between osteogenic characteristics of bone marrow cells and calcium phosphate surface relief and solubility.

The capacity of mouse bone marrow cells to adhere to calcium phosphate surfaces and form tissue plates depending on the surface relief and solubility was studied in ectopic bone formation test. Calcium phosphate coating of titanium disks, made by the anodic spark (microarch) oxidation in 10% orthophosphoric acid with hydroxyl apatite particles, differed by the structure (thickness of coating, size of pores, and roughness) and solubility (level of in vitro oxidation of 1-week extracts of implants). Chemical (phasic and element) composition of the studied calcium phosphate coatings was virtually the same. The findings indicate that histogenesis is regulated by physicochemical characteristics of the implant surface. It seems that the osteogenic potential of calcium phosphate surfaces is largely determined by their relief, but not by pH of degradation products.

Ferroelectric polymer scaffolds based on a copolymer of tetrafluoroethylene with vinylidene fluoride: Fabrication and properties

A solution blow spinning technique is a method developed recently for making nonwoven webs of micro- and nanofibres. The principal advantage of this method compared to a more traditional electrospinning process is its significantly higher production rate. In this work, the solution blow spinning method was further developed to produce nonwoven polymeric scaffolds based on a copolymer of tetrafluoroethylene with vinylidene fluoride solution in acetone. A crucial feature of the proposed method is that high-voltage equipment is not required, which further improves the methods economics. Scanning electron microscopy analysis of the samples demonstrated that the surface morphology of the nonwoven materials is dependent on the polymer concentration in the spinning solution. It was concluded that an optimum morphology of the nonwoven scaffolds for medical applications is achieved by using a 5% solution of the copolymer. It was established that the scaffolds produced from the 5% solution have a fractal structure and anisotropic mechanical properties. X-ray diffraction, infrared spectroscopy, Raman spectroscopy and differential scanning calorimetry demonstrated that the fabricated nonwoven materials have crystal structures that exhibit ferroelectric properties. Gas chromatography has shown that the amount of acetone in the nonwoven material does not exceed the maximum allowable concentration of 0.5%. In vitro analysis, using the culture of motile cells, confirmed that the nonwoven material is non-toxic and does not alter the morpho-functional status of stem cells for short-term cultivation, and therefore can potentially be used in medical applications.

Adhesion of Staphylococcus Aureus to Implants with Different Physicochemical Characteristics

Adhesion of pathogenic Staphylococcus aureus (strain 209) to BT1-0 titanium disks (12 mm in diameter) with different coatings and noncoated was studied in vitro by photocolorimetry. Transparency of bacterial suspension in normal saline was evaluated after 2-h culturing with the implants at 37°C. The decrease of S. aureus content in the suspension due to its adsorption on implants was negligible and increased by 0.9-5.5% in comparison with the control (adhesion to glass). When the specimens were placed into bacterial suspension, the density of staphylococcal adsorption on the surface considerably increased (by 9-53%) in comparison with the control, which attested to active participation of the implants in bacterial adsorption. The degree of bacterial adhesion to the implants decreased in the following order: disk with calcium phosphate ceramic coating-disk with calcium phosphate X-ray amorphous coating-disk without coating-disk with cermet coating. The adhesion of Staphylococcus is a stochastic process depending on the sum of implant characteristics, in particular, on the phase composition of the coating, electric conductivity, and Ca/P ionic ratio. The authors conclude that the formation of antibacterial properties of coating by saturating them with antibiotics or impregnation with metals, specifically silver ion implantation, is justified, because it reduces the postimplantation infection risk.