Functional evaluation of mineral trioxide aggregate cement with choline dihydrogen phosphate.

To improve the cytocompatibility of mineral trioxide aggregate (MTA) cement and its ability for reparative dentin formation, the effect of adding choline dihydrogen phosphate (CDHP), which is reported to be biocompatible, to MTA cement was investigated. The L929 cell proliferation showed that the addition of CDHP improved cell viability. The addition of CDHP shortened the setting time of MTA cement, with a significant decrease in consistency above 0.4 g/mL. Diametral tensile strength of the set cement was improved by the addition of 0.4 g/mL CDHP. Solubility was judged to be within the range of clinical application. The spontaneous precipitation of low crystalline hydroxyapatite was examined by immersing the set cement in phosphate buffer saline, and it was found that the ability of the cement with 0.4 g/mL of CDHP was significantly improved compared with that of the cement without CDHP.

Mechanical and Biocompatibility Properties of Calcium Phosphate Bioceramics Derived from Salmon Fish Bone Wastes.

Natural calcium phosphates derived from fish wastes are a promising material for biomedical application. However, their sintered ceramics are not fully characterized in terms of mechanical and biological properties. In this study, natural calcium phosphate was synthesized through a thermal calcination process from salmon fish bone wastes. The salmon-derived calcium phosphates (sCaP) were sintered at different temperatures to obtain natural calcium phosphate bioceramics and then were investigated in terms of their microstructure, mechanical properties and biocompatibility. In particular, this work is concerned with the effects of grain size on the relative density and microhardness of the sCaP bioceramics. Ca/P ratio of the sintered sCaP ranged from 1.73 to 1.52 when the sintering temperature was raised from 1000 to 1300 °C. The crystal phase of all the sCaP bioceramics obtained was biphasic and composed of hydroxyapatite (HA) and tricalcium phosphate (TCP). The density and microhardness of the sCaP bioceramics increased in the temperature interval 1000-1100 °C, while at temperatures higher than 1100 °C, these properties were not significantly altered. The highest compressive strength of 116 MPa was recorded for the samples sintered at 1100 °C. In vitro biocompatibility was also examined in the behavior of osteosarcoma (Saos-2) cells, indicating that the sCaP bioceramics had no cytotoxicity effect. Salmon-derived biphasic calcium phosphates (BCP) have the potential to contribute to the development of bone substituted materials.

Preparation of chitosan-hydroxyapatite composite mono-fiber using coagulation method and their mechanical properties.

Autograft has been carried out for anterior cruciate ligament (ACL) reconstruction surgery. However, it has negative aspect because patients lose their healthy ligaments from other part. We focus on a chitosan-hydroxyapatite (HAp) composite fiber as a scaffold of ligament regeneration. Chitosan- HAp composite fiber was made by using coagulation method. Chitosan-NaH2PO4 solution was coagulated with coagulation bath including calcium ion to get the mono-fiber and then treated with sodium hydroxide solution to form HAp in fiber matrix. The mechanical property of the fiber was improved by the stretching of the wet one because of the orientation of chitosan molecule and the interaction between chitosan and HAp. Maximum stress was improved with increasing of sodium dihydrogen phosphate until 0.03M. The swelling ratio of the fiber was inhibited by composited with HAp. Additionally, bone-bonding ability was confirmed by SBF soaking tests.

Alternating Current Electrophoretic Deposition for the Immobilization of Antimicrobial Agents on Titanium Implant Surfaces.

One prominent cause of implant failure is infection; therefore, research is focusing on developing surface coatings that render the surface resistant to colonization by micro-organisms. Permanently attached coatings of antimicrobial molecules are of particular interest because of the reduced cytoxicity and lower risk of developing resistance compared to controlled release coatings. In this study, we focus on the chemical grafting of bioactive molecules on titanium. To concentrate the molecules at the metallic implant surface, we propose electrophoretic deposition (EPD) applying alternating current (AC) signals with an asymmetrical wave shape. We show that for the model molecule bovine serum albumin (BSA), as well as for the clinically relevant antifungal lipopeptide caspofungin (CASP), the deposition yield is drastically improved by superimposing a DC offset in the direction of the high-amplitude peak of the AC signal. Additionally, in order to produce immobilized CASP coatings, this experimental AC/DC-EPD method is combined with an established surface activation protocol. Principle component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data confirm the immobilization of CASP with higher yield as compared to a diffusion-controlled process, and higher purity than the clinical CASP starting suspensions. Scratch testing data indicate good coating adhesion. Importantly, the coatings remain active against the fungal pathogen C. albicans as shown by in vitro biofilm experiments. In summary, this paper delivers a proof-of-concept for the application of AC-EPD as a fast grafting tool for antimicrobial molecules without compromising their activities.

Evaluation of calcium alginate gel as electrode material for alternating current iontophoresis of lidocaine using excised rat skin.

Iontophoresis (IOP) is a noninvasive method of delivering medication transcutaneously through the skin. The electrodes used in this method should tightly fit to rough and irregular surfaces and be biologically safe, easy to handle and prepare, and cost-effective. To satisfy these requirements, calcium alginate gel can be a candidate electrode for IOP. Using calcium alginate gel electrodes, we examined whether lidocaine can be effectively transported across an excised rat skin by squarewave alternating current (AC) application. A squarewave AC with either a 70% or 80% duty cycle was continuously applied to 0.5% calcium alginate gel electrodes containing 10% lidocaine at 10 V and 1 kHz for 60 min. Lidocaine concentration was measured using a spectrophotometer and the temperature of the gel was determined. The lidocaine concentrations for AC-IOP at the 70% and 80% duty cycles were significantly higher than that without AC-IOP. Furthermore, the group with the 80% duty cycle showed higher lidocaine concentrations than the group with the 70% duty cycle. The temperatures of all the groups were lower than 28 °C throughout the procedure. In conclusion, the calcium alginate gel can be used as a possible matrix for IOP electrodes.

Photoluminescence and doping mechanism of theranostic Eu3+/Fe3+ dual-doped hydroxyapatite nanoparticles.

Theranostic nanoparticles currently have been regarded as an emerging concept of 'personalized medicine' with diagnostic and therapeutic dual-functions. Eu3+ doped hydroxyapatite (HAp) has been regarded as a promising fluorescent probe for in vivo imaging applications. Additionally, substitution of Ca2+ with Fe3+ in HAp crystal may endow the capability of producing heat upon exposure to a magnetic field. Here we report a preliminary study of doping mechanism and photoluminescence of Eu3+ and Fe3+ doped HAp nanoparticles (Eu/Fe:HAp). HAp with varied concentration of Eu3+ and Fe3+ doping are presented as Eu(10 mol%):HAp, Eu(7 mol%)-Fe(3 mol%):HAp, Eu(5 mol%)-Fe(5 mol%):HAp, Eu(3 mol%)-Fe(7 mol%):HAp, and Fe(10 mol%):HAp in the study. The results showed that the HAp particles, in nano-size with rod-like morphology, were successfully doped with Eu3+ and Fe3+, and the particles can be well suspended in cell culture medium. Photoluminescence analysis revealed that particles have prominent emissions at 536 nm, 590 nm, 615 nm, 650 nm and 695 nm upon excitation at a wavelength of 397 nm. Moreover, these Eu/Fe:HAp nanoparticles belonged to B-type carbonated HAp, which has been considered an effective biodegradable and biocompatible drug/gene carrier in biological applications.

Collagen type IV-specific tripeptides for selective adhesion of endothelial and smooth muscle cells.

Controlling the balance of endothelial cells (ECs) and smooth muscle cells (SMCs) in blood vessels is critically important to minimize the risk associated with vascular implants. Extracellular matrix (ECM) plays a key role in controlling the cellular balance, suggesting a promising source of cell-selective peptides. To obtain EC- or SMC-selective peptides, we start by highlighting sequence differences found among ECM molecules as enriched targets for cell-selective peptides. We explored the EC- or SMC-selective performance of tripeptides that are specifically enriched only in collagen type IV, but not in types I, II, III, and V. Collagen type IV was chosen since it is the major ECM in the basement membrane of blood vessels, which separates ECs and SMCs. Among 114 collagen type IV-derived tripeptides pre-screened from in silico analysis, 22 peptides (19%) were found to promote cell-selective adhesion, as determined by peptide array. One of the best performing EC-selective peptides (Cys-Ala-Gly (CAG)) was mixed into an electrospun fine-fiber, a vascular graft material, for practical application. Compared to unmodified fiber, the CAG containing fiber surface was found to enhance adhesion of ECs (+190%) while limiting SMCs (-20%). These results are not only consistent with the hypothesis of ECM as a source of cell selective peptides, but also suggest a new genre of EC- or SMC-selective peptides for tissue engineering applications. Collectively, these findings favorably support the screening approach used to discover new peptides for these purposes.

In vitro evaluation of calcium alginate gels as matrix for iontophoresis electrodes.

Calcium alginate gel has some unique properties, such as the capability to keep the drugs, bioadhesiveness, safety, and low cost. The purpose of this study is to determine whether calcium alginate gel can be used as a matrix of electrodes for iontophoresis (IOP). We measured the concentration of lidocaine transported from calcium alginate gels with various concentrations of alginic acid using an in vitro experimental cell with square-wave alternating current (AC) application. Temperature and pH changes were also determined during AC-IOP. The results revealed that lidocaine was released from calcium alginate gels at concentrations nearly 1.71-fold larger at 5 V, 60 min after AC application than in the case of passive diffusion. Lidocaine transport depended on the alginic acid concentration in the gels. Although there were slight increases in temperature and pH, chemical and thermal burns were not severe enough to be a concern. In conclusion, the calcium alginate gel can be used as a possible matrix for IOP electrodes.

Surface plasmon resonance biosensor with high anti-fouling ability for the detection of cardiac marker troponin T.

Designing a surface recognition layer with high anti-fouling ability, high affinity, and high specificity is an important issue to produce high sensitivity biosensing transducers. In this study, a self-assembled monolayer (SAM) consisting of a homogeneous mixture of oligo(ethylene glycol) (OEG)-terminated alkanethiolate and mercaptohexadecanoic acid (MHDA) on Au was employed for immobilizing troponin T antibody and applied in detecting cardiac troponin T by using surface plasmon resonance (SPR). The mixed SAM showed no phase segregation and exhibited human serum albumin resistance, particularly with an antibody-immobilized surface. X-ray photoemission spectra revealed that the chemical composition ratio of OEG to the mixed SAM was 69% and the OEG packing density was 82%. The specific binding of troponin T on the designed surface indicated a good linear correlation (R=0.991, P<0.0009) at concentrations lower than 50 µgmL(-1) with the limit of detection of 100 ngmL(-1) using a SPR measuring instrument. It is concluded that the mixed SAM functions as designed since it has high detection capability, high accuracy and reproducibility, as well as shows strong potential to be applied in rapid clinical diagnosis for label-free detection within 2 min.

Synthesis and luminescence properties of Eu(III)-doped nanoporous silica spheres.

Europium (III) (Eu(3+))-doped nanoporous silica spheres were synthesized, and the states of Eu(3+) ions in the silica framework structure were investigated. The ordered nanopores were preserved with the doping at the Eu(3+) molar concentration to Si up to 10 mol%, and the O-Si-O and Si-OH groups in the structures were clearly rearranged with the doping, indicating the interaction of Eu(3+) with the O atoms. The significant morphological changes in the spheres were observed with the doping. The photoluminescence spectral shapes due to the transitions of (5)D(0)-(7)F(1) and (5)D(0)-(7)F(2) were indicative of the presence of the Eu(3+) in an environment of a low symmetry. It was found that the Eu(3+) was located inside the silica framework to electrostatically interact with the environmental O atoms, which would prevent the aggregation among Eu(3+) ions to show the efficient luminescence. Therefore, the interactions between the Eu(3+) ions and silica framework structures in the spheres were successfully clarified.

Immobilization of folic acid on Eu3+-doped nanoporous silica spheres.

Folic acid (FA) was immobilized on Eu(3+)-doped nanoporous silica spheres (Eu:NPSs) through mediation of the 3-aminopropyltriethoxysilane adlayer. The ordered nanopores of Eu:NPS were preserved by the immobilization. The FA-immobilized Eu:NPSs showed the characteristic photoluminescence peak due to interactions between the FA molecules and Eu(3+) ions, and highly dispersed stability in phosphate buffered saline.

Detection of interfacial phenomena with osteoblast-like cell adhesion on hydroxyapatite and oxidized polystyrene by the quartz crystal microbalance with dissipation.

The adhesion process of osteoblast-like cells on hydroxyapatite (HAp) and oxidized polystyrene (PSox) was investigated using a quartz crystal microbalance with dissipation (QCM-D), confocal laser scanning microscope (CLSM), and atomic force microscope (AFM) techniques in order to clarify the interfacial phenomena between the surfaces and cells. The interfacial viscoelastic properties (shear viscosity (η(ad)), elastic shear modulus (µ(ad)), and tan δ) of the preadsorbed protein layer and the interface layer between the surfaces and cells were estimated using a Voigt-based viscoelastic model from the measured frequency (Δf) and dissipation shift (ΔD) curves. In the ΔD-Δf plots, the cell adhesion process on HAp was classified as (1) a mass increase only, (2) increases in both mass and ΔD, and (3) slight decreases in mass and ΔD. On PSox, only ΔD increases were observed, indicating that the adhesion behavior depended on the surface properties. The interfacial µ(ad) value between the material surfaces and cells increased with the number of adherent cells, whereas η(ad) and tanδ decreased slightly, irrespective of the surface. Thus, the interfacial layer changed the elasticity to viscosity with an increase in the number. The tan δ values on HAp were higher than those on PSox and exceeded 1.0. Furthermore, the pseudopod-like structures of the cells on HAp had periodic stripe patterns stained with a type I collagen antibody, whereas those on PSox had cell-membrane-like structures unstained with type I collagen. These results indicate that the interfacial layers on PSox and HAp exhibit elasticity and viscosity, respectively, indicating that the rearrangements of the extracellular matrix and cytoskeleton changes cause different cell-surface interactions. Therefore, the different cell adhesion process, interfacial viscoelasticity, and morphology depending on the surfaces were successfully monitored in situ and evaluated by the QCM-D technique combined with other techniques.

Effect of interfacial proteins on osteoblast-like cell adhesion to hydroxyapatite nanocrystals.

A quartz crystal microbalance with dissipation (QCM-D) technique was employed to detecting the protein adsorption and subsequent osteoblast-like cell adhesion to hydroxyapatite (HAp) nanocrystals. The interfacial phenomena with the preadsorption of three proteins (albumin (BSA), fibronectin (Fn), and collagen (Col)), the subsequent adsorption of fetal bovine serum (FBS), and the adhesion of the cells were investigated. The QCM-D measured the frequency shift (Δf) and dissipation energy shift (ΔD), and the viscoelastic properties of the adlayers were evaluated using ΔD-Δf plot and Voigt-based viscoelastic model. The Col adsorption significantly showed higher Δf, ΔD, elasticity, and viscosity values as compared to the BSA and Fn adsorption, and the subsequent FBS adsorption depended on the preadsorbed proteins. The ΔD-Δf plot of the cell adhesion also showed a different behavior depending on the surfaces, and the Fn- and Col-modified surfaces showed the rapid mass and ΔD changes by forming the viscous interfacial layers with cell adhesion, indicating that the processes were affected by the cellular reaction through the extracellular matrix (ECM) proteins. The confocal laser scanning microscope images of adherent cells showed a different morphology and pseudopod on the surfaces. The cells adhered to the surfaces modified with the Fn and Col had significantly uniaxially expanded shapes and fibrous pseudopods, and those modified with the BSA had a round shape. Therefore, the different cell-protein interactions would cause the arrangement of the ECM and the cytoskeleton changes at the interfaces, and these phenomena were successfully detected by the QCM-D and Voigt-based model.

Competitive adsorption of fibronectin and albumin on hydroxyapatite nanocrystals.

Competitive adsorption of two-component solutions containing fibronectin (Fn) and albumin (Ab) on hydroxyapatite (HAp) nanocrystals was analyzed in situ using the quartz crystal microbalance with dissipation (QCM-D) technique. Adsorption of the one-component protein (Fn or Ab) and the two-component proteins adjusted to different molar ratios of Fn to Ab at a fixed Fn concentration was investigated. The frequency shift (Δf; Hz) and the dissipation energy shift (ΔD) were measured with the QCM-D technique, and the viscoelastic changes of adlayers were evaluated by the saturated ΔD/Δf value and the Voigt-based viscoelastic model. For the adsorption of the one-component protein, the Fn adlayer showed a larger mass and higher viscoelasticity than the Ab adlayer, indicating the higher affinity of Fn on HAp. For the adsorption of the two-component proteins, the viscoelastic properties of the adlayers became elastic with increase in Ab concentration, whereas the adsorption mass was similar to that of Fn in the one-component solution regardless of the Ab concentration. The specific binding mass of the Ab antibody to the adlayers increased with increase in Ab concentration, whereas that of the Fn antibody decreased. Therefore, Fn preferentially adsorbs on HAp and Ab subsequently interacts with the adlayers, indicating that the interfacial viscoelasticity of the adlayers was dominated by the interaction between Fn and Ab.

A collagen sponge incorporating a hydroxyapatite/chondroitinsulfate composite as a scaffold for cartilage tissue engineering.

Because cartilage has limited potential for self-repair, tissue engineering is expected to replace the present therapies for damaged cartilage, such as total knee arthroplasty. However, scaffolds suitable for cartilage tissue engineering have not been established. We synthesized a novel porous scaffold, a collagen sponge incorporating a hydroxyapatite/chondroitinsulfate composite (pCol-HAp/ChS), containing materials which resemble extracellular matrices in bone and cartilage tissues. In this report, the physical, mechanical and biological properties of the scaffold are compared with those of a collagen sponge (pCol) and pCol incorporating a hydroxyapatite composite (pCol-HAp). HAp/ChS had smaller crystals and a larger total surface area than HAp. SEM images of the three materials showed pCol-HAp/ChS to have the roughest surface. The mechanical properties suggest that pCol-HAp/ChS and pCol/HAp are similar, and superior to pCol. Seeding experiments showed a uniform distribution of mesenchymal stem cells (MSCs) in pCol-HAp/ChS and pCol/HAp. Histochemical staining after 2 weeks of culture revealed pCol-HAp/ChS to be the most chondrogenic. From these results, pCol-HAp/ChS is expected to be a candidate for a scaffold for cartilage tissue engineering in place of collagen sponge.

Rapid magnetic catch-and-release purification by hydrophobic interactions.

A reversible, conventional, and rapid purification method of hydrophobically tagged products using hydrophobic magnetic nanoparticles was developed. The reversible purification system entails simply controlling the polarity of solvents. First, for the catching procedure, poor solvents were added into a well-dispersed system of magnetic nanoparticles and tagged products. Once the poor solvents were added to the system, the products were recrystallized among the nanoparticles and the aggregation of magnetic nanoparticles occurred due to hydrophobic interactions. These aggregates with the products contained within them were able to be collected rapidly by magnets. Then, the releasing procedure can be easily performed by redispersing the collected aggregates into good solvents. The availability of this purification protocol was confirmed by using a hydrophobically tagged fluorescent model product. Furthermore, this rapid purification method was successfully applied to a peptide elongation reaction system which enabled the synthesis of peptides such as Leu-Enkephalin in high purity, in high yield, and in a short time.

Fabrication of hydroxyapatite ultra-thin layer on gold surface and its application for quartz crystal microbalance technique.

We present a method for coating gold quartz crystal microbalance with dissipation (QCM-D) sensor with ultra-thin layer of hydroxyapatite nanocrystals evenly covering and tightly bound to the surface. The hydroxyapatite layer shows a plate-like morphology and less than 20 nm in thickness. The hydroxyapatite sensor operated in liquid with high stability and sensitivity. The in-situ adsorption mechanism and conformational change of fibrinogen on gold, titanium and hydroxyapatite surfaces were investigated by QCM-D technique and Fourier-transform infrared spectroscopy. The change of secondary structures of fibrinogen adsorbed on the surfaces depended on the adsorbed amounts of protein. The secondary structure of fibrinogen adsorbed on the surfaces changes with increasing coverage. This is explained by repulsion among fibrinogens, affecting water structure and thus the strength of fibrinogen interactions on the surface. The study indicates that the hydroxyapatite sensor is applicable for qualitative and conformational analysis of protein adsorption.

Preparation of alginic acid layers on stainless-steel substrates for biomedical applications.

This study is concerned with the blood compatibility of alginic acid layers immobilized on gamma-aminopropyltriethoxysilane (gamma-APS)-grafted stainless-steel (SUS316L). The surfaces were characterized with contact angle measurement and X-ray photoelectron spectroscopy (XPS). The blood compatibility was evaluated in terms of platelet adhesion and blood clotting time. An in vitro platelet adhesion assay indicated that only a small number of platelets adhered to substrate surfaces modified with gamma-APS and subsequently with alginic acid. Moreover, alginic-acid-immobilized SUS316L substrates had little effect on the blood clotting time. This indicated that alginic-acid-immobilized SUS316L substrates do not adsorb some blood-clotting proteins or factors, or stimulate them.