Modified-simulated Body Fluid Research Articles

Integration of hydroxyapatite on polydopamine-coated zinc oxide nanoparticles: Physicochemical, antibacterial and cytocompatibility study

Ensuring the effective antibacterial with enhanced cytocompatibility of zinc oxide (ZnO) nanomaterials is still a challenging task. Here, we initially coated ZnO NPs with polydopamine (PDA), then integrated hydroxyapatite (HAp), and eventually obtained HAp@ZnO/PDA composite NPs to investigate the cytocompatible and antibacterial properties of the NPs in terms of different concentrations (200 to 6.25 µg/mL). The morphological analysis confirmed that the PDA-coating assisted the facile integration of HAp on the surface of ZnO/PDA NPs, and successfully synthesized the HAp@ZnO/PDA-06, HAp@ZnO/PDA-12, and HAp@ZnO/PDA-24 NPs by varying the modified-simulated body fluid (m-SBF) incubation time. Moreover, the morphological analysis also acknowledged that the prolonged incubation period (i.e., more than 6 h) is not recommendable for better HAp integration. Similarly, the antibacterial test confirmed that all three types of HAp@ZnO/PDA composite NPs including pure ZnO and ZnO/PDA NPs exhibited dose-dependent bactericidal action, and they were more efficient against Gram-positive Staphylococcus aureus compared to Gram-negative Escherichia coli. According to the results, the average E. coli survival percentage on pure ZnO, ZnO/PDA, HAp@ZnO/PDA-06, HAp@ZnO/PDA-12, and HAp@ZnO/PDA-24 NPs at the concentration range from 200 to 6.25 µg/mL was around 52%, 59%, 59%, 57%, and 55% respectively while the average S. aureus survival percentage on those NPs at the concentration range from 200 µ to 6.25 µg/mL was around 37%, 45%, 47%, 45%, and 41% respectively. In terms of cytocompatibility, the HAp@ZnO/PDA NPs treated samples showed enhanced MC3T3-E1 cell adhesion and proliferation when compared to pure ZnO NPs. The result explained that in all the tested NPs above 50 µg/mL, the cell viability is less than 50% while the concentration of NPs below 50 µg/mL showed more than 100% of cell viability, and at 50 µg/mL, the cell viability exhibited by pure ZnO, ZnO/PDA, HAp@ZnO/PDA-06, HAp@ZnO/PDA-12, and HAp@ZnO/PDA-24 composite NPs was 42%, 75%, 83%, 78%, and 70% respectively in average. Moreover, the cell viability reached above 350% in the 6.25 µg/mL of NPs treated samples. Hence, the study concluded that preparing HAp@ZnO/PDA composite NPs following mussel-inspired PDA coating and simulated body fluid (SBF) incubation techniques provided a facile way for the enhancement of the cytocompatibility of ZnO NPs without losing its potential antibacterial properties.

The promoting effect of the TiO2 nano-porous arrays after polydopamine-based modification and Arg-Gly-Asp peptide immobilization on biomineralization and osteogenesis

TiO2 nano-porous arrays with appropriate structural parameters, roughness and wettability have application potential in implantable titanium-based (Ti-based) alloys. In this research, the polydopamine (PDA) and Arg-Gly-Asp (RGD) peptide are adopted to construct a favorable osteogenic microenvironment on the anodized TiO2 nano-porous arrays before and after heat treatment. The characterization of surface morphologies and chemical compositions has been conducted to confirm the feasibility of the PDA-based modification and RGD peptide immobilization. Via immersion in the modified-simulated body fluid (m-SBF) for 7 days, the PDA-based modified samples have represented enhanced biomineralization performance. Moreover, the cell adhesion and proliferation assay, alkaline phosphatase (ALP) activity assay and quantitative real-time polymerase chain reaction (qRT-PCR) assay in vitro have indicated that the heat-treated TiO2 nano-porous arrays modified with PDA and RGD peptide have endowed the seeded MC3T3-E1 mouse pre-osteoblasts with excellent osteogenic performance. Such modified TiO2 nano-porous arrays have great latent capacity in implantable materials for biomineralization and osteogenesis.

Hydroxyapatite forming ability, ion release and antibacterial activity of the melt-derived SiO2–P2O5–Na2O–CaO–F glasses modified by replacing CaO with SrO and ZnO

Since the discovery of 1970s, bioactive glass has been a hot topic of research because of its excellent biological activity, which makes it a material that can repair and replace human bone tissue organs. In this work, the bioactive glasses in the system SiO2–P2O5–Na2O–CaO–F with different amounts of strontium oxide (SrO) and zinc oxide (ZnO) were prepared by the conventional melt quenching technology. The hydroxyapatite (HA) forming ability, ion release and antibacterial activity of these prepared glasses were investigated and the obtained results illustrated that SrO-doped samples had a better ability to form HA in modified simulated body fluid (MSBF) than ZnO-doped samples. As the immersion time of the sample in MSBF increased, the content of HA phase gradually increased. In the same immersion time, the formation ability of HA and the variation of SrO substitution amount showed a non-linear trend, which is mainly related to the influence of SrO content on the glass network structure. The results of ion concentration showed that the formation of HA was the result of the comprehensive action of various ions in the solution, especially the release rate of Si4+ ions, which had a direct impact on the formation ability of HA. The antibacterial test illustrated that the difference in antibacterial activity of bacteria solution at different sample concentrations may be related to the high pH environment and the osmotic effects caused by the non-physiological concentration of ions in the solution. The glass sample contained 4 wt% SrO showed the minimum bactericidal concentration at 64 mg/mL. The glass samples prepared in this experiment had good biological activity and antibacterial effect, making them suitable for using in dentistry and orthopedic applications, as well as providing a valuable composition reference for the preparation of bioactive glass with excellent comprehensive properties.

Monitoring and Assessing the Degradation Rate of Magnesium-Based Artificial Bone In Vitro Using a Wireless Magnetoelastic Sensor.

A magnetoelastic-based (MB) sensor was employed as a novel method to monitor and assess the degradation rate of magnesium-based artificial bone (MBAB) in vitro, which can be used as an implant to repair a bone defect, providing a quantitative method to depict the degradation rate of MBAB. MBABs were fabricated by the Pro/Engineering software and a precision machine tool using high-purity (HP) magnesium. The MB sensor was embedded in the neutral surface of MBAB by an unharmful quick adhesive, forming the MB sensor-embedded MBAB (EMBAB). The modified simulated body fluid (MSBF) media (PH = 7.4), mimicking the human internal environment, and the NaOH media (PH = 12), accelerating EMBAB’s degradation, were used to immerse the EMBAB for 15 days at 37 °C. The EMBAB was then tested daily on a self-developed experimental platform to monitor the relative output power under a 100 N external force. The results showed that the relative output power of the sensing coil gradually increased with the EMBAB’s degradation. The degradation rate of the EMBAB could be calculated on the basis of the changes of the relative output power caused by the MB sensor and of the degradation time. With the EMBAB’s degradation, an increasing strain directly worked on the MB sensor, significantly changing the value of the relative output power, which means that the EMBAB was characterized by a quick degradation rate. During the 15 days of the experiment, the degradation rates on the 7th and 15th days were 0.005 dbm/day and 0.02 dbm/day, and 0.02 dbm/day and 0.04 dbm/day in MSBF and alkaline media, respectively. Therefore, the MB sensor provides a wireless and passive method to monitor and assess the degradation rate of bone implants in vitro.

In vitro degradation and cytocompatibility of a silane/Mg(OH)2 composite coating on AZ31 alloy by spin coating

A new organic-inorganic composite coating was generated on AZ31 alloy surface with sol-gel method to improve the anti-corrosion performance and biocompatibility of AZ31 alloy in the physiological environment. The results of electrochemical impedance spectroscopy (EIS), potentiodynamic polarization, pH variations in modified-simulated body fluid (m-SBF) demonstrated that the silane/Mg(OH)2 composite coating significantly enhanced the anti-corrosion ability and slowed down the degradation of AZ31 alloy under in vitro condition. Fourier-transform infrared spectroscopy (FTIR) showed that the major composition of in-situ formation layer was Mg(OH)2. The surface morphology, chemical and phase constitutions of the composite coating were detected using scanning electron microscopy (SEM). The results demonstrated that the composite coating was uniform and defect-free. X-ray diffraction analysis (XRD) was also employed to characterize the corrosion products of all specimens immersed in m-SBF after 30 days. MTT and ALP tests indicated that the composite coating greatly enhanced the cell proliferation and osteogenic differentiation of osteoblasts on the AZ31 alloy surface.

In-vitro biodegradation and corrosion-assisted cracking of a coated magnesium alloy in modified-simulated body fluid

A calcium phosphate coating was directly synthesized on AZ91D magnesium (Mg) alloy. Resistance of this coating to corrosion in a modified-simulated body fluid (m-SBF) was investigated by potentiodynamic polarization and electrochemical impedance spectroscopy (EIS). Mechanical properties of the bare and coated alloy were investigated using slow strain rate tensile (SSRT) and fatigue testing in air and m-SBF. Very little is reported in the literature on human-body-fluid-assisted cracking of Mg alloys, viz., resistance to corrosion fatigue (CF) and stress corrosion cracking (SCC). This study has a particular emphasis on the effect of bio-compatible coatings on mechanical and electrochemical degradations of Mg alloys for their applications as implants. The results suggest the coating to improve the general as well as pitting corrosion resistance of the alloy. The coating also provides visible improvement in resistance to SCC, but little improvement in CF resistance. This is explained on the basis of pitting behaviour in the presence and absence of the coating.

In vitro investigation of biodegradable polymeric coating for corrosion resistance of Mg-6Zn-Ca alloy in simulated body fluid

A silane-based biodegradable coating was developed and investigated to improve corrosion resistance of an Mg-6Zn-Ca magnesium alloy to delay the biodegradation of the alloy in the physiological environment. Conditions were optimized to develop a stable and uniform hydroxide layer on the alloys surface—known to facilitate silane-substrate adhesion. A composite coating of two silanes, namely, diethylphosphatoethyltriethoxysilane (DEPETES) and bis-[3-(triethoxysilyl) propyl] tetrasulfide (BTESPT), was developed, by the sol-gel route. Corrosion resistance of the coated alloy was characterized in a modified-simulated body fluid (m-SBF), using potentiodynamic polarization and electrochemical impedance spectroscopy (EIS). The silane coating provided significant and durable corrosion resistance. During the course of this, hydrogen evolution and pH variation, if any, were monitored for both bare and coated alloys. The coating morphology was characterized using scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX) and the cross-linking in the coating was studied using Fourier transform infrared spectroscopy (FTIR). As indicated by X-ray diffraction (XRD) results, an important finding was the presence of hydrated magnesium phosphate on the sample that was subjected to immersion in m-SBF for 216h. Magnesium phosphate is reported to support osteoblast formation and tissue healing.

Evaluation of alkaline pre-treatment of PLLA fibers for biomimetic hydroxyapatite coating

A poly(l-lactic acid) (PLLA) and hydroxyapatite (HA) composite was formed via a biomimetic coating process. Before treatment in a modified-simulated body fluid (m-SBF), the surfaces of the PLLA fibers were etched with alkaline reagents to make the polymer surface easier for apatite to nucleate and grow. Several elements of this etching procedure (including the etching reagent, concentration, and length of pre-treatment) were varied to determine the optimal combination for producing a predictable HA coating with the desired properties of consistent coating distribution and thickness. The alkaline etching reagents used in this study were sodium hydroxide (NaOH), sodium hypochlorite (NaOCl), calcium hydroxide (Ca(OH)2), and calcium hypochlorite (Ca(OCl)2). The weight gained through m-SBF coating, changes in the mechanical properties of the PLLA, and HA coating morphology were observed to determine the effects of different pre-treatment strategies on the biomimetic coating process. It was confirmed that the most effective etching reagents are Ca(OH)2 and Ca(OCl)2. It was also found that decreased etching reagent concentration leads to a decrease in HA coating and increased etching time increases HA deposition.

Surface characteristics of hydroxyapatite films deposited on anodized titanium by an electrochemical method

The biocompatibility of anodized titanium (Ti) was improved by an electrochemically deposited calcium phosphate (CaP) layer. The CaP layer was grown on the anodized Ti surface in modified simulated body fluid (M-SBF) at 85°C. The phases and morphologies for the CaP layers were influenced by the electrolyte concentration. Nano flake-like precipitates that formed under low M-SBF concentrations were identified as hydroxyapatite (HAp) crystals orientated in the c-axis direction. In high M-SBF concentrations, the CaP layer formed micro plate-like precipitates on anodized Ti, and micropores were covered with HAp. Proliferation of murine preosteoblast cell (MC3T3-E1) on the HAp/anodized Ti surfaces was significantly higher than for untreated Ti and anodized Ti surfaces.

Detection of apatite formation on titania by modified-simulated body fluid using surface plasmon resonance

Detection of apatite formation on titania by modified-simulated body fluid using surface plasmon resonance

Structural and in vitro adhesion analysis of a novel covalently coupled bioactive composite

The interfacial adhesion between a restorative composite and tooth is one of the major factors that determine the ultimate performance of composite restoration. A novel polyurethane (PU) composite material was prepared by chemically binding the nano-hydroxyapatite (nHA) to the diisocyanate component in the PU backbone by utilizing solvent polymerization. The procedure involved stepwise addition of monomeric units of the PU and optimizing the reagent concentrations. The resultant materials were characterized structurally (Raman Spectroscopy) and in vitro bioactive analysis was conducted in modified-simulated body fluid for periodical time intervals. The in vitro study evaluated the push-out bond strength of existing obturating material and novel covalently linked PU/nHA composites to dentin after long-term storage in deionized water and artificial saliva. Human extracted molar roots were filled with experimental samples and analyzed at predetermined time intervals. The shear bond strength of samples was measured and surface morphologies were evaluated. Covalent bond formation was achieved between PU and nHA without intermediate coupling agent. With the increase in concentration of nHA, the composite showed more bioactivity and adhesion toward tooth structure. Bond strength of this new composite were in accordance with obutrating material, therefore, the material can be used as an obturating material because of its direct adhesion with tooth structure.

The cytocompatibility and osseointegration of the Ti implants with XPEED® surfaces

This study evaluated cytocompatibility and osseointegration of the titanium (Ti) implants with resorbable blast media (RBM) surfaces produced by grit-blasting or XPEED(®) surfaces by coating of the nanostructured calcium. Ti implants with XPEED(®) surfaces were hydrothermally prepared from Ti implants with RBM surfaces in a solution containing alkaline calcium. The surface characteristics were evaluated by using a scanning electron microscope (SEM) and surface roughness measuring system. Apatite formation was measured with SEM after immersion in modified-simulated body fluid and the amount of calcium released was measured by inductively coupled plasma optical emission. The cell proliferation was investigated by MTT assay and the cell attachment was evaluated by SEM in MC3T3-E1 pre-osteoblast cells. Thirty implants with RBM surfaces and 30 implants with XPEED(®) surfaces were placed in the proximal tibiae and in the femoral condyles of 10 New Zealand White rabbits. The osseointegration was evaluated by a removal torque test in the proximal tibiae and by histomorphometric analysis in the femoral condyles 4 weeks after implantation. The Ti implants with XPEED(®) surfaces showed a similar surface morphology and surface roughness to those of the Ti implants with RBM surfaces. The amount of calcium ions released from the surface of the Ti implants with XPEED(®) surfaces was much more than the Ti implants with RBM surfaces (P < 0.05). The cell proliferation and cell attachment of the Ti implants showed a similar pattern to those of the Ti implants with RBM surfaces (P > 0.1). Apatite deposition significantly increased in all surfaces of the Ti implants with XPEED(®) surfaces. The removable torque value (P = 0.038) and percentage of bone-to-implant contact (BIC%) (P = 0.03) was enhanced in the Ti implants with XPEED(®) surfaces. The Ti implants with XPEED(®) surfaces significantly enhanced apatite formation, removal torque value, and the BIC%. The Ti implants with XPEED(®) surfaces may induce strong bone integration by improving osseointegration of grit-blasted Ti implants in areas of poor quality bone.

Influence of dicalcium phosphate dihydrate coating on the in vitro degradation of Mg-Zn alloy

To reduce the degradation rate and further to improve the biocompatibility of magnesium alloy, dicalcium phosphate dihydrate (CaHPO4·2H2O, DCPD) has been fabricated on a kind of magnesium-zinc alloy by way of electrodeposition method. The experimental results of XRD, SEM and EDS showed that the electrodeposited coating on the Mg-Zn alloy mainly contains the flake-like DCPD, along with some octacalcium phosphate (Ca8(HPO4)2(PO4)4·4H2O, OCP). After the in vitro degradation of the coated alloy in modified-simulated body fluid (m-SBF), it was proved that the coating could reduce the degradation rate effectively, and the samples were covered by calcium phosphate salts with a higher Ca/P ratio. Therefore, it indicates that compared with the bare alloy, the DCPD coating rendered a more biocompatible surface, and is a promising coating candidate for biomedical magnesium materials.

Effect of laser surface melting on corrosion behaviour of AZ91D Mg alloy in simulated-modified body fluid

High corrosion rate in physiological environment of the body is the major drawback of magnesium alloys for their successful applications as biodegradable orthopaedic implants. In the present study, corrosion behaviour of AZ91D magnesium alloy after laser surface melting (LSM) was studied in modified-simulated body fluid at 37 °C. The improved corrosion resistance of AZ91D alloy using LSM was found to depend on the solidification microstructure in the laser-melted zone. The general and pitting corrosion resistance of laser-treated surface was significantly enhanced due to the refined continuous network of β-Mg17Al12 phases and the increased Al concentration in the laser-melted zone.

Evaluation of Biomimetic Solution for Coating Powder Metallurgy Porous Titanium Samples

In order to improve implant-bone attachment, porous titanium has been used to achieve the ingrowth of bone tissue within the porous structure. Although this biomaterial has shown efficient bone adhesion for orthopedic and dental implants, the ideal surface must have chemical bonds at the implant-bone interface. In this work, samples of pure porous titanium were produced by powder metallurgy technique and submitted to biomimetic process in order to evaluate the material’s bioactivity and to enhance its osteoconductivity. The samples were immersed in modified simulated body fluid (mSBF) which induces the nucleation and growth of a calcium phosphate bioactive film and a chemical bond with titanium. SEM, EDX and FTIR analyses showed that a calcium phosphate deposition occurred without the need of pre-treatments to increase the surface bioactivity. As a result, this research revealed the potential for obtaining a bonelike apatite film on this porous titanium by biomimetic method.

Evaluating the stress corrosion cracking susceptibility of Mg–Al–Zn alloy in modified-simulated body fluid for orthopaedic implant application

Applications of magnesium alloys as biodegradable orthopaedic implants are critically dependent on the mechanical integrity of the implant during service. In this study, the stress corrosion cracking susceptibility of sand-cast Mg–Al–Zn alloy in modified-simulated body fluid was evaluated using the slow strain rate test method. The study suggests that the stress corrosion cracking susceptibility of the sand-cast magnesium alloy is not substantial and this aspect should not be a concern for its implant applications.

In vitro degradation and mechanical integrity of calcium-containing magnesium alloys in modified-simulated body fluid

The successful applications of magnesium-based alloys as degradable orthopaedic implants are mainly inhibited due to their high degradation rates in physiological environment and consequent loss in the mechanical integrity. This study examines the degradation behaviour and the mechanical integrity of calcium-containing magnesium alloys using electrochemical techniques and slow strain rate test (SSRT) method, respectively, in modified-simulated body fluid (m-SBF). Potentiodynamic polarisation and electrochemical impedance spectroscopy (EIS) results showed that calcium addition enhances the general and pitting corrosion resistances of magnesium alloys significantly. The corrosion current was significantly lower in AZ91Ca alloy than that in AZ91 alloy. Furthermore, AZ91Ca alloy exhibited a five-fold increase in the surface film resistance than AZ91 alloy. The SSRT results showed that the ultimate tensile strength and elongation to fracture of AZ91Ca alloy in m-SBF decreased only marginally (∼15% and 20%, respectively) in comparison with these properties in air. The fracture morphologies of the failed samples are discussed in the paper. The in vitro study suggests that calcium-containing magnesium alloys to be a promising candidate for their applications in degradable orthopaedic implants, and it is worthwhile to further investigate the in vivo corrosion behaviour of these alloys.

HRTEM Analysis of Apatite Formed on Bioactive Titanium in Modified-SBF

Process of the hydroxyapapite(HA) precipitation on bioactive titanium metal prepared by NaOH in a modified-simulated body fluid(mSBF) was investigated by high resolution transmission electron microscope (HRTEM) attached with energy dispersive X-ray spectrometer(EDX). The amorphous titanate phase on titanium surface is form by NaOH treatment and an amorphous titanate incorporated calcium and phosphate ions in the liquid to form an amorphous calcium phosphate. With increasing of soaking time in the liquid, the HA particles are observed in amorphous calcium phosphate phase with a Ca/P atomic ratio of I.30. The octacalcium phosphate (OCP) structure is not detected in HRTEM image and electron diffraction pattern. After a long soaking time, the HA particles grow as needle-like shape on titanium surface and a large particle-like aggregates of needle-like substance were observed to form on titanium surface within needle-like shape. A long axis of needle parallels to c-direction of the hexagonal HA structure.

Comparison of physical characteristics and cell culture test of hydroxyapatite/collagen composite coating on NiTi SMA: electrochemical deposition and chemically biomimetic growth

A hydroxyapatite (HA)/collagen (COL) composite coating on NiTi shape memory alloy (SMA) was prepared by eletrochemical deposition (ELD) in modified simulated body fluid (MSBF). To draw comparisons of physical characteristics and bioactivity of the composite coating, the HA/COL composite coating was also prepared by chemically biomimetic growth (BG) and the ELD coating was re-soaked in MSBF again for further biomimetic growth (called EBG method in this paper). It was indicated that the c-axis of HA crystals was oriented parallel to the longitudinal direction of the COL fibril in BG and EBG coating, which could not found in ELD coating. The EBG method could induce a denser, thicker and better crystallized HA/COL coating. The cell culture test indicated that the BG coating presented better cell biocompatibility.

Preparation of bone-like composite coating using a modified simulated body fluid with high Ca and P concentrations

A carbonate hydroxyapatite (HA)–collagen (Col) composite coating on NiTi shape memory alloy (SMA) was synthesized by biomimetic growth in modified simulated body fluid (MSBF), which containing high Ca and P ions concentrations. The morphology of composite coating was uniform and porous. The major phase of coating, identified by Infrared Spectroscopy (IR) and X-ray diffraction (XRD), was HA with B-type carbonate substitution. It was also proved by IR results that the collagen was present in the coating. Transmission Electron Microscopy (TEM) and high resolution TEM analysis showed that the c-axis of HA crystals aligned parallel to the longitudinal direction of the collagen fibrils. The relative orientation maintained in the composite coating may benefit the NiTi SMA to achieve better properties in hard tissue replacement.