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.     

Processing and bioactivity of 45S5 Bioglass®-graphene nanoplatelets composites

Well dispersed 45S5 Bioglass^® (BG)-graphene nanoplatelets (GNP) composites were prepared after optimising the processing conditions. Fully dense BG nanocomposites with GNP loading of 1, 3 and 5 vol% were consolidated using Spark plasma sintering (SPS). SPS avoided any structural damage of GNP as confirmed using Raman spectroscopy. GNP increased the viscosity of BG-GNP composites resulting in an increase in the sintering temperature by ~50 °C compared to pure BG. Electrical conductivity of BG-GNP composites increased with increasing concentration of GNP. The highest conductivity of 13 S/m was observed for BG-GNP (5 vol%) composite which is ~9 orders of magnitude higher compared to pure BG. For both BG and BG-GNP composites, in vitro bioactivity testing was done using simulated body fluid for 1 and 3 days. XRD confirmed the formation of hydroxyapatite for BG and BG-GNP composites with cauliflower structures forming on top of the nano-composites surface. GNP increased the electrical conductivity of BG-GNP composites without affecting the bioactivity thus opening the possibility to fabricate bioactive and electrically conductive scaffolds for bone tissue engineering.     

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.     

A microstructural examination of apatite induced by Bioglass® in vitro

Bioglass®, a clinically used bone graft material, has been tested in vitro in a simulated body fluid (SBF) up to four weeks. Apatite crystals were not only found to form on its surface but also in the reaction solutions. The apatite crystals have been examined by high-resolution transmission electron microscopy (TEM). The crystals formed in the solutions appear identical in morphology and structure with those formed on the Bioglass® surface. It may be that the soluble Si in the solution serves as the nucleating site for the apatite crystal or that apatite nuclei are released from the Bioglass® surface to the solution resulting in crystal growth.     

Biocompatibility and bioactivity of PDLLA/TiO2 and PDLLA/TiO2/Bioglass® nanocomposites

Biocompatibility and bioactivity of polymer matrix composites containing titanium dioxide (TiO₂) nanoparticles were investigated. The solvent casting method was used to prepare poly (d,l-lactic acid) (PDLLA) films with 0 and 20 wt.% TiO₂ nanoparticles and with 20 wt.% TiO₂ mixed with 5 wt.% micrometre-sized (< 5 μm) Bioglass® particles. The samples were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy Dispersive X-ray (EDX) analyses. A Zygo® light interferometer was used to examine the surface roughness of the samples. The bioactivity and the surface reactivity of the materials were determined by investigating the formation of hydroxyapatite (HA) on the surface of samples upon immersion in simulated body fluid (SBF) for up to 28 days. Heterogeneous distributed HA crystals were found on composite films containing TiO₂ after 21 days exposure to SBF. Cell cytotoxicity and viability were determined by using live/dead and MTS assay on osteoblast-like MG-63 cells. The PDLLA films containing different concentrations of TiO₂ and Bioglass® particulate inclusions showed no effect on cell viability in live/dead assay after incubation period of 7 days. All three groups of samples demonstrated significant increase in relative metabolic activity in MTS assay after 7 days incubation (while a slower proliferation rate was obtained for cells on the PDLLA film containing both TiO₂ and Bioglass® compared to the Thermanox® control). The bioactive behaviour of the nanocomposites may make them attractive materials for fabrication of tissue engineering scaffolds.     

The story of Bioglass®

Historically the function of biomaterials has been to replace diseased or damaged tissues. First generation biomaterials were selected to be as bio-inert as possible and thereby minimize formation of scar tissue at the interface with host tissues. Bioactive glasses were discovered in 1969 and provided for the first time an alternative; second generation, interfacial bonding of an implant with host tissues. Tissue regeneration and repair using the gene activation properties of Bioglass provide a third generation of biomaterials. This article reviews the 40 year history of the development of bioactive glasses, with emphasis on the first composition, 45S5 Bioglass, that has been in clinical use since 1985. The steps of discovery, characterization, in vivo and in vitro evaluation, clinical studies and product development are summarized along with the technology transfer processes.     

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.     

The surface functionalization of 45S5 Bioglass®-based glass-ceramic scaffolds and its impact on bioactivity

The first and foremost function of a tissue engineering scaffold is its role as a substrate for cell attachment, and their subsequent growth and proliferation. However, cells do not attach directly to the culture substrate; rather they bind to proteins that are adsorbed to the scaffold's surface. Like standard tissue culture plates, tissue engineering scaffolds can be chemically treated to couple proteins without losing the conformational functionality; a process called surface functionalization. In this work, novel highly porous 45S5 Bioglass-based scaffolds have been functionalized applying 3-AminoPropyl-TriethoxySilane (APTS) and glutaraldehyde (GA) without the use of organic solvents. The efficiency and stability of the surface modification was assessed by X-ray photoemission spectroscopy (XPS). The bioactivity of the functionalized scaffolds was investigated using simulated body fluid (SBF) and characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). It was found that the aqueous heat-treatment applied at 80 degrees C for 4 hrs during the surface functionalization procedure accelerated the structural transition of the crystalline Na2Ca2Si3O9 phase, present in the original scaffold structure as a result of the sintering process used for fabrication, to an amorphous phase during SBF immersion. The surface functionalized scaffolds exhibited an accelerated crystalline hydroxyapatite layer formation upon immersion in SBF caused by ion leaching and the increased surface roughness induced during the heat treatment step. The possible mechanisms behind this phenomenon are discussed.     

Initial in vitro biocompatibility of a bone cement composite containing a poly-ε-caprolactone microspheres

The biocompatibility of a reinforced calcium phosphate injectable bone substitute (CPC-IBS) containing 30% poly-ε-caprolactone (PCL) microspheres was evaluated. The IBS consisted of a solution of chitosan and citric acid as the liquid phase and tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powder as the solid phase with 30% PCL microspheres. The surface of the CPC-IBS was observed by SEM, and analyzed by EDX profiles. The initial setting of the sample was lower in the IBS containing 0% citric acid than in the IBS containing 10 or 20% citric acid. The compressive strength of the PCL-incorporated CPC-IBS was measured using a Universal Testing Machine. The 20% citric acid samples had the highest mechanical strength at day 12, which was dependent on both time and the citric acid concentration. The in vitro bioactivity experiments with simulated body fluid (SBF) confirmed the formation of apatite on the sample surfaces after 2, 7, and 14 days of incubation in SBF. Ca and P ion release profile by ICP method also confirmed apatite nucleation on the CPC-IBS surfaces. The in vitro biocompatibility of the CPC-IBS was evaluated by using MTT, cellular adhesion, and spreading studies. In vitro cytotoxicity tests by MTT assay showed that the 0 and 10% CPC-IBS was cytocompatible for fibroblast L-929 cells. The SEM micrograph confirmed that MG-63 cells maintained their phenotype on all of the CPC-IBS surfaces although cellular attachment was better in 0 and 10% CPC-IBS than 20% samples.     

Calcium–phosphate–silicate composite bone cement: self-setting properties and in vitro bioactivity

In this study, a novel low temperature setting calcium phosphate–silicate cement was obtained by mixing CaHPO₄ · 2H₂O (DCPD) and Ca₃SiO₅ (C₃S) with 0.75 M sodium phosphate buffers (pH = 7.0) as liquid phase. The self-setting properties of the obtained DCPD/C₃S paste with liquid to powder ratio (L/P) of 0.6 ml/g, such as setting times, injectability, degradability and compressive strength were investigated and compared with that of DCPD/CaO cement system. The results indicated that, with the weight ratio of C₃S varied from 20% to 40%, the workable DCPD/C₃S pastes could set within 20 min, and the hydrated cement showed significantly higher compressive strength (around 34.0 MPa after 24 h) than that of the DCPD/CaO cement system (approximately 10.0 MPa). Furthermore, the in vitro pH value of the cements was investigated by soaking in simulated body fluid (SBF) for 12 h, and the result indicated that the DCPD/C₃S did not induce significant increase or decrease of pH value in SBF. Additionally, the composite cement possesses better ability to support and stimulate cell proliferation than the DCPD/CaO cement. With good hydraulic properties, improved biocompatibility and moderate degradability, the novel DCPD/C₃S bone cement may be a potential candidate as bone substitute.     

Novel starch thermoplastic/Bioglass® composites: Mechanical properties, degradation behavior and in-vitro bioactivity

The present research aims to evaluate the possibility of creating new degradable, stiff and highly bioactive composites based on a biodegradable thermoplastic starch-based polymeric blend and a Bioglass® filler. Such combination should allow for the development of bioactive and degradable composites with a great potential for a range of temporary applications. A blend of starch with ethylene–vinyl alcohol copolymer (SEVA-C) was reinforced with a 45S5 Bioglass® powder presenting a granulometric distribution between 38 and 53 m. Composites with 10 and 40 wt % of 45S5 Bioglass® were compounded by twin-screw extrusion (TSE) and subsequently injection molded under optimized conditions. The mechanical properties of the composites were evaluated by tensile testing, and their bioactivity assessed by immersion in a simulated body fluid (SBF) for different periods of time. The biodegradability of these composites was also monitored after several immersion periods in an isotonic saline solution. The tensile tests results obtained indicated that SEVA-C/Bioglass® composites present a slightly higher stiffness and strength (a modulus of 3.8 GPa and UTS of 38.6 MPa) than previously developed SEVA-C/Hydroxylapatite (HA) composites. The bioactivity of SEVA-C composites becomes relevant for 45S5 amounts of only 10 wt %. This was observed by scanning electron microscopy (SEM) and confirmed for immersion periods up to 30 days by both thin-film X-ray diffraction (TF-XRD) (where HA typical peaks are clearly observed) and induced coupled plasma emission (ICP) spectroscopy used to follow the elemental composition of the SBF as function of time. Additionally, it was observed that the composites are biodegradable being the results correlated with the correspondent materials composition.     

The effect of loading icariin on biocompatibility and bioactivity of porous β-TCP ceramic

In order to enhance the ability of calcium phosphate-based biomaterials for bone defect repair, icariin (Ica), one natural product with ability of promoting osteoblasts differentiation in vitro and enhancing bone formation in vivo, was loaded into porous β-tricalcium phosphate ceramic (β-TCP) disks. The obtained Ica-loaded porous β-TCP ceramic (Ica/β-TCP) disks were characterized by SEM. The SEM photos indicated that the disks had porous structure and the surface morphology of the porous β-TCP ceramic (β-PTCP) disks had no obvious difference from the Ica/β-TCP disks. The Ica release curve of Ica/β-TCP disks showed a burst release during the first 1 day and the concentration of released Ica during the first 3 days had low cytotoxicity. The loading Ica in Ica/β-TCP disks hardly affected the attachment and morphology of Ros17/28 cells, however, the Ica/β-TCP disks were favorable to supporting the proliferation and differentiation of Ros17/28 cells better compared with the β-PTCP disks. There was plenty of bone-like apatite formed on the surface of Ica/β-TCP disks soaked in SBF solution for three days. After back intramuscular implantation of rats for three months, no obvious osteogenic evidence was detected in β-PTCP disks, but new bone formation was observed in Ica/β-TCP disks. Fibrous tissues and slight inflammatory reaction was also found in the Ica/β-TCP disks and β-TCP disks. Therefore, the loading Ica did not change the biocompatibility of β-TCP ceramic, but enhanced the bioactivity of β-TCP ceramic in vivo. The Ica/β-TCP ceramic had potential to be used for bone defect repair.     

Microstructure,mechanical properties,and in vitro biocompatibility of spark plasma sintered hydroxyapatite–aluminum oxide–carbon nanotube composite

In the present work, HA reinforced with Al₂O₃ and multiwalled carbon nanotubes (CNTs) is processed using spark plasma sintering (SPS). Vickers micro indentation and nanoindentation of the samples revealed contrary mechanical properties (hardness of 4.0, 6.1, and 4.4 GPa of HA, HA–Al₂O₃ and HA–Al₂O₃–CNT samples at bulk scale, while that of 8.0, 9.0, and 7.0 GPa respectively at nanoscale), owing to the difference in the interaction of the indenter with the material at two different length scales. The addition of Al₂O₃ reinforcement has been shown to enhance the indentation fracture toughness of HA matrix from 1.18 MPa m^1/2 to 2.07 MPa m^1/2. Further CNT reinforcement has increased the fracture toughness to 2.3 times (2.72 MPa m^1/2). In vitro biocompatibility of CNT reinforced HA–Al₂O₃ composite has been evaluated using MTT assay on mouse fibroblast L929 cell line. Cell adhesion and proliferation have been characterized using scanning electron microscopy (SEM), and have been quantified using UV spectrophotometer. The combination of cell viability data as well as microscopic observations of cultured surfaces suggests that SPS sintered HA–Al₂O₃–CNT composites exhibit the ability to promote cell adhesion and proliferation on their surface and prove to be promising new biocompatible materials.     

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.     

Gallium-containing phospho‐silicate glasses: Synthesis and in vitro bioactivity

A series of Ga-containing phospho-silicate glasses based on Bioglass 45S5, having molar formula 46.2SiO₂·24.3Na₂O·26.9CaO·2.6P₂O₅·xGa₂O₃ (x = 1.0, 1.6, 3.5), were prepared by fusion method. The reference Bioglass 45S5 without gallium was also prepared. The synthesized glasses were immersed in simulated body fluid (SBF) for 30 days in order to observe ion release and hydroxyapatite (HA) formation. All Ga-containing glasses maintain the ability of HA formation as indicated by main X-ray diffractometric peaks and/or electronic scanning microscopy results. HA layer was formed after 1 day of SBF soaking in 45S5 glass containing up to 1.6% Ga₂O₃ content. Moreover, gallium released by the glasses was found to be partially precipitated on the glass surface as gallium phosphate. Further increase in gallium content reduced the ion release in SBF. The maximum of Ga³⁺ concentration measured in solution is ~ 6 ppm determined for 3.5% Ga₂O₃ content. This amount is about half of the toxic level (14 ppm) of gallium and the glasses release gallium till 30 days of immersion in SBF. Considering the above results, the studied materials can be proposed as bioactive glasses with additional antimicrobial effect of gallium having no toxic outcome.     

In vitro and in vivo assessment of the biocompatibility of an Mg–6Zn alloy in the bile

There is a great clinical need for biodegradable bile duct stents. Biodegradable stents made of an Mg–6Zn alloy were investigated in both vivo animal experiment and in vitro cell experiments. During the in vivo experiments, blood biochemical tests were performed to determine serum magnesium, serum creatinine (CREA), blood urea nitro-gen (BUN), serum lipase (LPS), total bilirubin (TB) and glutamic-pyruvic transaminase (GPT) levels. Moreover, tissue samples of common bile duct (CBD), liver and kidney were taken for histological evaluation. In the in vitro experiments, primary mouse extrahepatic bile duct epithelial cells (MEBDECs) were isolated and cultured. Cytotoxicity testing was carried out using the MTT method. Flow cytometry analyses with propidium iodide staining were performed to evaluate the effect of Mg–6Zn alloy extracts on cell cycle. The in vivo experiments revealed no significant differences (P > 0.05) in serum magnesium, CREA, BUN, LPS, TB or GPT before and after the operation. Based on the HE results, hepatocytes, bile duct epithelial cells, renal glomerulus and renal tubule tissues did not present significant necrosis. In the in vitro experiments, the cell relative growth rate curve did not change significantly from 20 to 40 % extracts. In vitro experiments showed that 20–40 % Mg–6Zn extracts are bio-safe for MEBDECs. In vivo experiments showed that Mg–6Zn stents did not affect several important bio-chemical parameters or, harm the function or morphology of the CBD, kidney, pancreas and liver. Our data suggested that this Mg–6Zn alloy is a safe biocompatible material for CBD.     

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.     

Evaluation of emcocel® 50 and emcocel® 90, a new excipient in direct compression

Abstract

The tabletting properties of a new microcrystalline cellulose product, Emcocel® 50 and Emcocel® 90 were evaluated and compared with the tabletting properties of Avicel® PH 101. The evaluation of placebo tablets, of the dilution potential, of formulations with active compounds as Aspirin, Phenobarbital and a spraydried extract in high concentrations showed that Emcocel® has comparative tabletting properties in regard of Avicel® PH 101.     

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.