In vitro bioactivity of sol–gel-derived hydroxyapatite particulate nanofiber modified titanium

A chemically-etched titanium surface was modified by electrospinning a sol–gel-derived hydroxyapatite (HAp) that was subjected to calcination within the temperature range of 200–1400°C in the normative atmospheric condition. After heat treatment, crystal structures of the filmed titanium oxide and HAp on the titanium’s surface were identified using wide-angle X-ray diffraction. A highly porous layer of HAp was found to have formed on the oxidized titanium surfaces. The surfaces of three different samples; (1) electrospun HAp, (2) HAp calcined at 600°C, and (3) HAp calcined at 800°C, were investigated for their ability to foster promotion, proliferation, and differentiation of human osteoblasts (HOB) (in the 9th passage) in vitro up to 6 days. Among the three samples, cells cultured on the HAp calcined at 800°C titanium surfaces displayed the best results with regard to adhesion, growth, and proliferation of HOB. This novel method for fabrication of titanium substrates would provide a promising improvement for titanium-based medical devices over the current standards, which lack such substrates. These titanium substrates explicitly provide enhanced HOB proliferation in terms of both desired surface properties and their produced bulk quantity.     

In vitro bioactivity and osteoblast-like cell test of zinc containing fluoridated hydroxyapatite films

Zinc containing fluoridated hydroxyapatite (ZnFHA) films on Ti6Al4V substrates was prepared using sol–gel dip-coating method. The release of zinc ions from ZnFHA film was controlled mainly by the zinc content in the film. The release behavior showed an initial rapid increase release followed by a tapering-off and directed to a constant value at longer time. After soaking in SBF for 8 days, a layer was deposited and completely covered the original surface of the ZnFHA film, indicating good in vitro “bioactivity.” The osteoblast-like MG63 cells were seeded on the ZnFHA films; FHA film and Ti6Al4V substrate were used as control. The cell culture result showed that cell adhesion and proliferation on ZnFHA films were significantly increased compared with the controls. The results in this work suggest that ZnFHA films on Ti6Al4V substrates can function as an implant with good bioactivity and cytocompatibility.     

Influence of thermal treatment on the “in vitro” bioactivity of wollastonite materials

The aim of this work was to study the influence of the composition and thermal treatment of the in vitro bioactivity of wollastonite materials obtained by sol–gel method. For this purpose, gels in the system SiO₂–CaO were obtained applying calcium nitrate and tetraethoxysilicate as precursors. The gels were heated to 700°C and then sintered up to 1400°C. The bioactivity of the gel-derived materials in simulated body fluid (SBF) was investigated and characterized. Additional changes in ionic concentration, using inductively couple plasma atomic emission spectroscopy (ICP-AES), were determined. The results showed that all materials obtained were bioactive and indicate that the absence of phosphorous in the material composition is not an essential requirement for the development of a Hydroxyapatite layer. The bioactivity was influenced by the thermal treatment, the different phases (glass-phase, wollastonite and pseudowollastonite) as well as the porous size. On the gel-derived materials the bioactivity decreased with the sintering temperature.     

Effect of glass–ceramic microstructure on its in vitro bioactivity

Two routes were used to obtain a glass–ceramic composed of 43.5 wt % SiO₂ – 43.5 wt % CaO – 13 wt % ZrO₂. Heat treatment of a glass monolith produced a glass–ceramic (WZ1) containing wollastonite-2M and tetragonal zirconia as crystalline phases. The WZ1 did not display bioactivity in vitro. Ceramizing the glass via powder technology routes formed a bioactive glass–ceramic (WZ2). The two glass–ceramics, WZ1 and WZ2, were composed of the same crystalline phases, but differed in microstructure. The in vitro studies carried out on WZ2 showed the formation of an apatite-like layer on its surface during exposure to a simulated body fluid. This paper examined the influence of both chemical and morphological factors on the in vitro bioactivitity. The interfacial reaction product was examined by scanning and transmission electron microscopy. Both instruments were fitted with energy-dispersive X-ray analyzers. Measurements of the pH made directly at the interface of the two glass–ceramics were important in understanding their different behavior during exposure to the same physiological environment.     

Bioactive borosilicate glass scaffolds: in vitro degradation and bioactivity behaviors

Bioactive borosilicate glass scaffolds with the pores of several hundred micrometers and a competent compressive strength were prepared through replication method. The in vitro degradation and bioactivity behaviors of the scaffolds have been investigated by immersing the scaffolds statically in diluted phosphate solution at 37°C, up to 360 h. To monitor the degradation progress of the scaffolds, the amount of leaching elements from the scaffolds were determined by ICP-AES. The XRD and SEM results reveal that, during the degradation of scaffolds, the borosilicate scaffolds converted to hydroxyapatite. The compressive strength of the scaffolds decreased during degradation, in the way that can be well predicted by the degradation products, or the leachates, from the scaffolds. MTT assay results demonstrate that the degradation products have little, if any, inhibition effect on the cell proliferation, when diluted to a certain concentration ([B] <2.690 and pH value at neutral level). The study shows that borosilicate glass scaffold could be a promising candidate for bone tissue engineering material.     

In vitro study on the influence of strontium-doped calcium polyphosphate on the angiogenesis-related behaviors of HUVECs

Angiogenesis is of great importance in bone tissue engineering, and has gained large attention in the past decade. Strontium-doped calcium polyphosphate (SCPP) is a novel biodegradable material which has been proved to be able to promote in vivo angiogenesis during bone regeneration. An in vitro culture system was developed in the present work to examine its influence on angiogenesis-related behaviors of human umbilical vein endothelial cells (HUVECs), including cell adhesion, spreading, proliferation and migration. The effects of microtopography, chemical property and the ingredients in the degradation fluid (DF) on cell behaviors were discussed. The results showed that cells attached and spread better on SCPP scaffold than on calcium polyphosphate (CPP), which might partially result from the less rough surface of SCPP scaffold and the less hydrogel formed on the surface. In addition, cell proliferation was significantly improved when treated with SCPP DF compared with the treatment with CPP DF. Statistical analysis indicated that Sr(2+) in SCPP DF might be the main reason for the improved cell proliferation. Moreover, cell migration, another important step during angiogenesis, was evidently stimulated by SCPP DF. The improved in vivo angiogenesis by SCPP might be assigned to its better surface properties and strontium in the DF. This work also provides a new method for in vitro evaluation of biodegradable materials' potential effects on angiogenesis.     

The influence of lithium fluoride on in vitro biocompatibility and bioactivity of calcium aluminate–PMMA composite cement

The objective of this study is to assess the influence of lithium fluoride on in vitro biocompatibility and bioactivity of calcium aluminate (CA)-polymethylmethacrylate (PMMA) composite cement exhibiting quick setting time ( < 15 min), low exothermic temperature (< 47 degrees C), and high compressive strength (> 100 MPa). The biocompatibility was measured by examining cytotoxicity tests such as the agar diffusion test with L929 cell line and the hemolysis test with fresh rabbit blood. To estimate the bioactivity of CA-PMMA composite cement, we determined hydroxyapatite (HAp) formation on the surface of composite cement in the simulated body (SBF) solution by using thin-film XRD, XPS, SEM, EPMA and ICP-AES. The results of biocompatibility tests indicated that all experimental compositions of this study had no cytotoxicity and no hemolysis so that there was no cytotoxicity with regard to non-reacted monomers (MMA and TEGDMA) and lithium fluoride. The results of bioactivity tests revealed that CA-PMMA composite cement without lithium fluoride did not form HAp on its surface after 60 days of soaking in the SBF. On the other hand, LiAl2(OH)7 . 2H2O and HAp were formed on the surface of CA-PMMA composite cement including 1.0% by weight of lithium fluoride after 7 and 15 days of soaking in the SBF, respectively. The 5 microm of LiAl2(OH)7 . 2H2O and HAp mixed layers were formed on the surface of specimen after 60 days of soaking in the SBF.     

Effect of particle size on in vitro cytotoxicity of titania and alumina nanoparticles

Aluminium oxide (Al₂O₃) and titanium dioxide (TiO₂) nanoparticles (NPs) have been widely used in nanotechnology-based products. Recently, researchers and the public have raised concerns about the adverse effects of these NPs in biological systems, particularly in humans. The aim of this study was to investigate the possible adverse effects of these two common metal oxide NPs on human lung epithelium cells (A549) and to investigate NP size-dependent effects on these cells, considering both the primary and hydrodynamic particle size. NPs were found to inhibit cell viability and proliferation at the highest concentration level (10?mg/mL) included in this study, as measured by a clonogenic assay. Moreover, cell viability, proliferation and metabolism were impaired to a greater extent by the smaller NPs (5?nm TiO₂ and 10?nm Al₂O₃) relative to the larger particles (200?nm TiO₂ and 50?nm Al₂O₃) included in this study, as measured by cell proliferation and metabolism. Notably, the observed cytotoxic effects correlated to the primary size, rather than the hydrodynamic size. Similarly, NP cytotoxicity was found to be correlated with the NP surface area. These findings highlight the importance of including primary size and surface area information in NP characterisation in cytotoxicity studies.     

Evaluation of in vitro bioactivity and biocompatibility of Bioglass®-reinforced polyethylene composite

The bioactivity and biocompatibility of Bioglass®-reinforced high-density polyethylene composite (Bioglass®/HDPE) have been evaluated in simulated body fluid (SBF) and by in vitro cell culture, respectively. The formation of a biologically active hydroxy-carbonate apatite (HCA) layer on the composite surface after immersion in SBF was demonstrated by thin-film X-ray diffraction, infrared spectroscopy and scanning electron microscopy, indicating the in vitro bioactivity of Bioglass®/HDPE composites. The HCA layer was formed on the 40 vol% composite surface within 3 days immersion in SBF at a formation rate comparable to those on bioactive glass-ceramics, showing that in vitro bioactivity could be obtained in a composite. Furthermore, the composite was biocompatible to primary human osteoblast-like cells. In comparison with unfilled HDPE and tissue culture plastic control, a significant increase in cellular metabolic activity was found on the composite. Therefore, Bioglass®/HDPE composites have a promising biological response as a potential implant material.     

In vitro study of the SBF and osteoblast-like cells on hydroxyapatite/chitosan–silica nanocomposite

Hydroxyapatite/chitosan–silica (HApCSi) nanocomposites were synthesized by co-precipitated method and their potential application as filler materials for bone regeneration were investigated in simulated body fluid (SBF). To study their biocompatibility, they were cultured with rat osteoblast-like UMR-106 cells for 3, 7, 14, and 21 days. Studies of the silica contents in chitosan matrix showed the presence of silinol (Si–OH) groups in CSi hybrid and their decrease after being composited with calcium phosphate (CaP) which is desirable for the formation of the apatite. XRD and TEM studies showed that the HAp formed in the CSi matrix were nanometer (20–40 nm) in size. Nanocomposites of HApCSi20 processed with 20%v/v silica whisker showed a micro hardness of 84.7 ± 3.3 MPa. Mineralization study in SBF showed the formation of apatite crystals on the HApCSi surface after being incubated for 7 days. In vitro biocompatibility, cell morphology, proliferation, and cell adhesion tests confirmed the osteoblast attachment and growth on the HApCSi20 surface. The density of cells and the production of calcium nodules on the substrate were seen to increase with increasing cultured time. The mechanical evaluation and in vitro experiment suggested that the use of HApCSi composite will lead to the formation of new apatite particles and thus be a potential implant material.     

Antibacterial effect of bioactive glasses on clinically important anaerobic bacteria in vitro

Bioactive glasses (BAGs) of different compositions have been studied for decades for clinical use and they have found many dental and orthopaedic applications. Particulate BAGs have also been shown to have antibacterial properties. This large-scale study shows that two bioactive glass powders (S53P4 and 13–93) and a sol–gel derived material (CaPSiO II) have an antibacterial effect on 17 clinically important anaerobic bacterial species. All the materials tested demonstrated growth inhibition, although the concentration and time needed for the effect varied depending on the BAG. Glass S53P4 had a strong growth-inhibitory effect on all pathogens tested. Glass 13–93 and sol–gel derived material CaPSiO II showed moderate antibacterial properties.     

Evaluation of polyethersulfone highflux hemodialysis membrane in vitro and in vivo

A new hemodialysis membrane manufactured by a blend of polyethersulfone (PES) and polyvinylpyrrolidone (PVP) was evaluated in vitro and in vivo. Goat was selected as the experimental animal. The clearance and the reduction ratio after the hemodialysis of small molecules (urea, creatinine, phosphate) for the PES membrane were higher in vitro than that in vivo. The reduction ratio of β₂-microglobulin was about 50% after the treatment for 4 h. The biocompatibility profiles of the membranes indicated slight neutropenia and platelet adhesion at the initial stage of the hemodialysis. Electrolyte, blood gas, and blood biochemistry were also analyzed before and after the treatment. The results indicated that PES hollow fiber membrane had a potential widely use for hemodialysis.     

Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation

Microspheres have been prepared from the resorbable linear polyester of β-hydroxybutyric acid (polyhydroxybutyrate, PHB) by the solvent evaporation technique and investigated in vitro and in vivo. Biocompatibility of the microspheres has been proved in tests in the culture of mouse fibroblast cell line NIH 3T3 and in experiments on intramuscular implantation of the microspheres to Wistar rats for 3 months. Tissue response to the implantation of polymeric microspheres has been found to consist in a mild inflammatory reaction, pronounced macrophage infiltration that increases over time, involving mono- and poly-nuclear foreign body giant cells that resorb the polymeric matrix. No fibrous capsules were formed around polymeric microparticles; neither necrosis nor any other adverse morphological changes and tissue transformation in response to the implantation of the PHB microparticles were recorded. The results of the study suggest that polyhydroxybutyrate is a good candidate for fabricating prolonged-action drugs in the form of microparticles intended for intramuscular injection.     

Study on physicochemical properties and in vitro bioactivity of tricalcium silicate–calcium carbonate composite bone cement

In this article, a novel bone cement composed of tricalcium silicate (Ca(3)SiO(5); C(3)S) and calcium carbonate (CaCO(3)) was prepared with the weight percent of CaCO(3) in the range of 0, 10, 20, 30, and 40%. The initial setting time was dramatically reduced from 90 to 45 min as the content of CaCO(3) increased from 0 to 40%, and the workable paste with a liquid/powder (L/P) ratio of 0.8 ml/g could be injected between 2 and 20 min (nozzle diameter 2.0 mm). The composite cement showed higher mechanical strength (24-27 MPa) than that of the pure Ca(3)SiO(5) paste (14-16 MPa). Furthermore, the composite cement could induce apatite formation and degrade in the phosphate buffered saline. The results indicated that the Ca(3)SiO(5)-CaCO(3) paste had better hydraulic properties than pure Ca(3)SiO(5) paste, and also the composite cement was bioactive and degradable. The novel bone cement could be a potential candidate as a bone substitute.     

Human tissue allograft processing: impact on in vitro and in vivo biocompatibility

This work investigates the impact of chemical and physical treatments on biocompatibility for human bone/tendon tissues. Nontreated and treated tissues were compared. In vitro testing assessed indirect and direct cytotoxicity. Tissues were subcutaneously implanted in rats to assess the immunological, recolonization, and revascularization processes at 2–4 weeks postimplantation. No significant cytotoxicity was found for freeze-dried treated bones and tendons in comparison to control. The cellular adhesion was significantly reduced for cells seeded on these treated tissues after 24 h of direct contact. A significant cytotoxicity was found for frozen treated bones in comparison to freeze-dried treated bones. Tissue remodeling with graft stability, no harmful inflammation, and neo-vascularization was observed for freeze-dried chemically treated bones and tendons. Frozen-treated bones were characterized by a lack of matrix recolonization at 4 weeks postimplantation. In conclusion, chemical processing with freeze-drying of human tissues maintains in vitro biocompatibility and in vivo tissue remodeling for clinical application.     

Effect of polymerization degree of calcium polyphosphate on its microstructure and in vitro degradation performance

Preparation, characterization and in vitro study of a series of calcium polyphosphate (CPP) with different polymerization degree were reported. A series of CPP with different polymerization degree were prepared by controlling calcining time. Average polymerization degree was analyzed by liquid state ³¹P nuclear magnetic resonance (NMR). The microstructure was observed by scanning electric microscope (SEM). X-ray diffraction (XRD) analysis was used to demonstrate that polymerization degree would not affect the crystal system and space group of CPP. The results showed that polymerization degree increased with the increase of calcining time. Degradation studies were performed during 32 days in physiological saline solution (aqueous solution, 0.9 wt.%NaCl) to assess the effect of polymerization degree on the degradation velocity of the samples. It was also shown that the degradation velocity of CPP (polymerization degree = 13) doubles than another two samples (polymerization degree = 9,19). The results in the present study may be able to provide some fundamental data for controlling CPP degradation.     

Effect of the content of hydroxyapatite nanoparticles on the properties and bioactivity of poly(l-lactide) – Hybrid membranes

Poly(l-lactide)/hydroxyapatite, PLLA/HA, composite membranes for bone regeneration with different concentrations of nanoparticles have been prepared and their physicochemical properties and bioactivity have been determined. Hydroxyapatite nanoparticles act as nucleating agent of the poly(l-lactide) crystals, as detected by DSC, and as reinforcing filler, as proven by the monotonous increase of the elastic modulus of the microporous membranes with increasing nano-filler content. The bioactivity, which regards to the use of these materials in bone regeneration, was tested by immersing the samples in a simulated body fluid, SBF. A faster deposition of a biomimetic apatite layer was observed as increases the content of hydroxyapatite nanoparticles, thus membranes with a 15% (w/w) of hydroxyapatite particles (relative to PLLA weight) present a homogeneous layer of hydroxyapatite on the surface of their pores after 7 days of immersion in SBF. An especial emphasis has been made on the influence of a plasma treatment on the bioactivity of the membranes. With this aim, the membranes were submitted to a plasma treatment previously to their immersion in a simulated body fluid. It has been observed that the surface of a PLLA membrane after 21 days of immersion in SBF is still not completely covered by hydroxyapatite whereas the same sample treated with plasma show a smooth layer of biomimetic hydroxyapatite. The increase of bioactivity achieved with this treatment was less important in high hydroxyapatite content composites.     

Preparation and in vitro evaluation of mesoporous hydroxyapatite coated β-TCP porous scaffolds

A mesoporous hydroxyapatite (HA) coating was prepared on a β-tricalcium phosphate (β-TCP) porous scaffold by a sol-gel dip-coating method using the block copolymer Pluronic F127 (EO₁₀₆PO₇₀EO₁₀₆) as the template. For application as a bone graft, in vitro cell response and bone-related protein expression of mesoporous HA coated β-TCP scaffold were investigated, using the non-mesoporous HA coated scaffold as the control group, to evaluate the influence of the mesoporous structure on the biological properties of HA coating. It was found that the increased surface area of the mesoporous HA coating greatly affected the response of MC3T3-E1 osteoblasts and the expression of proteins. An enzyme-linked immunosorbent assay recorded a significantly higher expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) in the mesoporous group than those in the control group (*p < 0.05) after different incubation periods. The introduction of mesopores enhanced the expression of ALP and BSP in the cells grown on the mesoporous HA coatings, on the premise of maintaining the protein expression in a sequence to ensure the correct temporo-spatial expression in osteogenesis. These results indicated that the mesoporous HA coating would provide a good environment for cell growth, suggesting that it could be used as the coating material for the surface modification of the tissue engineering scaffolds.     

In vitro and in vivo biocompatibility of graded hydroxyapatite–zirconia composite bioceramic

To obtain bioceramics with good osteoinductive ability and mechanical strength, graded hydroxyapatite–zirconia (HA–ZrO₂) composite bioceramics were prepared in this work. The biocompatibility of the bioceramics was investigated in vitro based on acute toxicity and cytotoxicity tests and hemolysis assay. Results showed the studied graded HA–ZrO₂ had little toxicity to mouse and L929 mouse fibroblasts. Also, hemolysis assay indicated a good blood compatibility of the bioceramics. Based on the results of in vitro tests, animal experiments were performed on white New Zealand rabbits by implantation into hip muscles and femur. It was found that the graded HA–ZrO₂ composite bioceramics exhibited superior osteoinductive ability, which may be a promising bioceramics implant.     

Effect of Boron Substitution for Bi on Superconductivity of the Bi-2212 Phase in the Bi-Sr-Ca-Cu-O System

Possibility of boron substitution for Bi and the substitution effect on superconductivity is investigated for the Bi-2212 phase of Bi-Sr-Ca-Cu-O. From X-ray diffraction study, it is found that samples in the (Bi_2?x B _x )Sr₂CaCu₂O _z system are mainly of the single 2212 phase in a composition range of 0.0≤x≤0.6, and both of the lattice parameters a and c change with increasing x up to 0.6. From measurements of the magnetic susceptibility and the electrical resistivity, the superconducting transition temperature is found to increase up to 0.6 with increasing x. These results are considered to show that boron is substitutable for Bi up to x=0.6 in the (Bi_2?x B _x )Sr₂CaCu₂O _z system and that the boron substitution causes the number of hole-carriers to decrease in this system.