Supersaturated Calcification Solution Research Articles

Biomimetic hydroxyapatite (HAp) coatings on pure Mg and their physiological corrosion behavior

The deposition of bioactive hydroxyapatite (HAp) coatings on pure magnesium (Mg) surfaces was studied. A modification of the biomimetic deposition method was used, which consisted in the incorporation of a supersaturated calcification solution (SCS). Different preparation parameters were modulated to obtain HAp and/or doped apatite coatings with different characteristics. The influence of the surface pre-treatments and soaking time in SCS on the physicochemical properties and the corrosion performance of the biomimetic HAp coatings were evaluated. Two different pre-treatments were explored to condition the sample’s surface for the biomimetic HAp deposition. After the surface pre-treatments, the Mg samples were covered with HAp coatings by soaking in SCS during two immersion periods specifically chosen for this study: 2 h and 6 h, respectively. Scanning electron microscopy, coupled with energy-dispersive X-ray spectroscopy (SEM/EDS), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Raman Spectroscopy, and Fourier transform infrared (FTIR), were applied for the chemical, morphological, and structural characterization of the resulting biomimetic HAp-coatings. The in vitro corrosion behavior was studied by open circuit potential (OCP) measurements and electrochemical impedance spectroscopy (EIS) during immersion tests in Ringer’s physiological solution for up to 14 days. The results showed that HAp coatings were successfully deposited on the pure Mg substrates. Furthermore, the corrosion studies in Ringer’s solution showed that HAp coating behavior depends not only on the pre-treatment type but also on the immersion time in SCS. These results open the door to propose different strategies to control—on demand—the corrosion rate of Mg coated with biomimetic HAp. These systems could be a promising alternative for designing and developing new temporary implants for the human body.

In Vitro Study of Octacalcium Phosphate Behavior in Different Model Solutions

Octacalcium phosphate (OCP), a new-generation bone substitute material, is a considered precursor of the biological bone apatite. The two-layered structure of OCP contains the apatitic and hydrated layers and is intensively involved in ion-exchange surface reactions, which results in OCP hydrolysis to hydroxyapatite and adsorption of ions or molecular groups presented in the environment. During various in vitro procedures, such as biomaterial solubility, additive release studies, or the functionalization technique, several model solutions are applied. The composition of the environmental solution affects the degree and rate of OCP hydrolysis, its surface reactivity, and further in vitro and in vivo properties. The performed study was aimed to track the structural changes of OCP-based materials while treating in the most popular model solutions of pH values 7.2–7.4: simulated body fluid (SBF), Dulbecco’s phosphate-buffered saline (DPBS), supersaturated calcification solution (SCS), normal saline (NS), and Dulbecco’s modified Eagle’s medium (DMEM). Various degrees of OCP hydrolysis and/or precipitate formation were achieved through soaking initial OCP granules in the model solutions. Detailed data of X-ray diffraction, Fourier-transform infrared spectroscopy, atomic emission spectrometry with inductively coupled plasma, and scanning electron microscopy are presented. Cultivation of osteosarcoma cells was implemented on OCP pre-treated in DMEM for 1–28 days. It was shown that NS mostly degraded the OCP structure. DPBS slightly changed the OCP structure during the first treatment term, and during further terms, the crystals got thinner and OCP hydrolysis took place. Treatment in SBF and SCS caused the precipitate formation along with OCP hydrolysis, with a larger contribution of SCS solution to precipitation. Pre-treating in DMEM enhanced the cytocompatibility of materials. As a result, on performing the in vitro procedures, careful selection of the contact solution should be made to avoid the changes in materials structure and properties and get adequate results.

Apatite coatings on chemically modified titanium using a new accelerated biomimetic route

To improve the osseointegration and biological performance of biomedical titanium, hydroxyapatite (HA) was coated onto chemically modified titanium (Ti) using a novel and fast biomimetic route which incorporated supersaturated calcification solutions (SCS). As a result, it is possible to obtain HA coatings on both acid and base chemically-modified titanium surfaces within 2 h using SCS. After coating, acid etched and heat treated Ti exhibited a fine and homogenous coating of hydroxyapatite crystals that did not change morphology after 4 h. Alkaline-modified Ti showed a denser and thicker nano-sized hydroxyapatite layer (particle size about 100 nm) at 2 h due to the presence of hydrogen sodium titanate. Therefore, no heat treatment was necessary to induce HA precipitation.

In vitro degradation and mechanical behaviour of calcium phosphate coated Mg-Ca alloy

ABSTRACT The mechanical and physical properties of Mg-Ca alloys make them suitable for temporary orthopaedic implant applications. However, the rapid degradation of these alloys limits their use in the physiological environment. The effect of calcium phosphate (CaP) coating on biomineralization, biodegradation, and mechanical behaviour of the alloy in physiological environment is investigated in this work. Supersaturated calcification solution was used for developing CaP layer on the HNO3 pretreated substrate. The surface morphology and composition of phases present in the samples after immersion in supersaturated simulated body fluid were analysed using a scanning electron microscope fitted with energy dispersive spectroscopy. The results show that the CaP coating improved biomineralization and reduced the degradation rate. The CaP coating also helped in controlling the reduction in mechanical properties in the physiological environment. Hence, the CaP coating can be used as a surface modification technique to tailor the degradation rate of Mg-Ca alloys.

Effect of ions concentration in buffer solutions on the nucleation of hydroxyapatite surface on the octacalcium phosphate granules

Octacalcium phosphate granules were subsequently immersed for different lengths of time from 1 day to 44 days at constant temperature 37±0.4°C in solutions, simulating the extracellular body fluids: Supersaturated Calcification Solution (SCS), Simulated Body Fluid (SBF), based on the ISO-10993 “Biological Evaluation of Medical Devices” standard. The role of buffer`s systems pH-value, time of the material exposure and calcium and phosphorus ionic concentrations were analysed with the main focus on kinetics of degradation, changes in the morphological characteristics and phase composition of octacalcium phosphate samples.

Bismuth-Doped Nanohydroxyapatite Coatings on Titanium Implants for Improved Radiopacity and Antimicrobial Activity.

This study aims to present the possibility to obtain bismuth-doped nanohydroxyapatite coatings on the surface of the titanium implants by using a solution-derived process according to an established biomimetic methodology. The bioactivity of the titanium surface was increased by an alkali-thermal treatment. Then, under biomimetic conditions, the titanium surface was coated with a Bi-doped nanohydroxyapatite layer by using a modified supersaturated calcification solution (SCS) containing a bismuth salt. The apatite deposits were analyzed by scanning electron microscopy coupled with X-ray analysis (SEM-EDX), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and digital X-rays radiography method. The results indicate that the Bi-doped nanohydroxyapatite coatings on titanium surface were produced. These coatings exhibit a good radiopacity, thus enhancing their applications in dental and orthopedic fields. Additionally, the Bi-doped nanohydroxyapatite coatings show significant antimicrobial activity against Escherichia coli and Staphylococcus aureus bacteria.

Cerium-doped hydroxyapatite/collagen coatings on titanium for bone implants

The novelty of the present research consists in the possibility of obtaining cerium-doped hydroxyapatite/collagen coatings on the titanium support, to improve the performance of the bone implants. These coatings were deposited on the titanium surface by biomimetic method using a modified supersaturated calcification solution (SCS) additionally containing a cerium source and collagen. Prior to the deposition of the apatite layer, an alkali ÷ thermal oxidation pretreatment has been applied to ensure an increase in the bioactivity of the titanium surface. The coatings were examined by scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy. The EDX and XRD investigations of the coatings indicated that cerium was incorporated in the hydroxyapatite lattice. The collagen presence in the coatings was confirmed by FTIR analysis. The cerium-doped hydroxyapatite/collagen coatings showed good antibacterial efficacy against Escherichia coli and Staphylococcus aureus bacteria, being more effective against Escherichia coli. These coatings have a significant potential to be used in the dental and orthopedic implants, as the osseointegration depends on much more factors than simple formation of hydroxyapatite.

Biomineralization of electrospun polycaprolactone-guided bone regeneration membrane

To evaluate the biomineralization of the tissue-engineering electrospun polycaprolactone (PCL) scaffold and its potential use for guided bone regeneration (GBR) membranes. PCL ultrafinefiber scaffolds were fabricated by electrospinning and then immersed in supersaturated calcification solution (SCS) for biomineralization investigation. The electrospun PCL scaffolds and the calcium phosphate coating were identified by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Water-contact angles were also measured to evaluate the hydrophilicity of the modified surface. The biocompatibility of the composite was investigated by culturing osteoblasts on the scaffolds. Cell behavior was observed by SEM. The electrospun PCL scaffold was composed of ultrafine fibers and well-interconnected pores. The deposits on the fibers grew in number and size as the biomineralization time increased. Then, the covering of the whole PCL film was identified as dicalcium phosphate dehydrate and apatite. Good cell attachment and proliferation behavior were observed on the membranes. The quick apatite formation on the surface of the PCL electrospun scaffold indicated that SCS biomineralization may be an effective approach for obtaining PCL/calcium phosphate composites. The cellular biocompatibility of the composite scaffold was preliminarily confirmed by the in vitro culture of osteoblasts on the scaffold. As such, the composite scaffold is a promising biomimetic extracellular matrix biomaterial for bone tissue engineering and GBR membranes.

Manufacture of nanosized apatite coatings on titanium with different surface treatments using a supersaturated calcification solution

The biomimetic method is used for the deposition of calcium phosphate coatings (Ca - P) on the surface of different biomaterials. However, the application of this method requires long exposure times in order to obtain a suitable layer thickness for its use in medical devices. In this paper, we present a fast approach to obtain apatite coatings on titanium, using a combination of supersaturated calcification solution (SCS) with chemical modification of the titanium surface. Also, it was evaluated the effect of four different surface treatments on the apatite deposition rate. Commercially pure titanium plates were activated by chemical or thermochemical treatments. Then, the activated samples were immersed in a solution with high content of calcium and phosphate ions at 37 oC for 24 h, mimicking the physiological conditions. The coatings were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The use of SCS solutions allowed the formation of crystalline hydroxyapatite coatings within a period of 24 h with a thickness between 1 and 5.3 µm. Besides, precipitates of hydroxyapatite nanoparticles with a globular configuration, forming aggregates with submicrometer size, were found in SCS solutions.

Hydroxyapatite-silver nanoparticles coatings on porous polyurethane scaffold

The present paper is focused on a study regarding the possibility of obtaining hydroxyapatite-silver nanoparticle coatings on porous polyurethane scaffold. The method applied is based on a combined strategy involving hydroxyapatite biomimetic deposition on polyurethane surface using a Supersaturated Calcification Solution (SCS), combined with silver ions reduction and in-situ crystallization processes on hydroxyapatite-polyurethane surface by sample immersing in AgNO3 solution. The morphology, composition and phase structure of the prepared samples were characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX), X-ray diffraction (XRD), UV-Vis spectroscopy and X-ray photoelectron spectroscopy (XPS) measurements. The data obtained show that a layer of hydroxyapatite was deposited on porous polyurethane support and the silver nanoparticles (average size 34.71 nm) were dispersed among and even on the hydroxyapatite crystals. Hydroxyapatite/polyurethane surface acts as a reducer and a stabilizing agent for silver ions. The surface plasmon resonance peak in UV-Vis absorption spectra showed an absorption maximum at 415 nm, indicating formation of silver nanoparticles. The hydroxyapatite-silver polyurethane scaffolds were tested against Staphylococcus aureus and Escherichia coli and the obtained data were indicative of good antibacterial properties of the materials.

Investigation on the effect of collagen and vitamins on biomimetic hydroxyapatite coating formation on titanium surfaces

This study uses an in vitro experimental approach to investigate the roles of collagen and vitamins in regulating the deposition of hydroxyapatite layer on the pure titanium surface. Titanium implants were coated with a hydroxyapatite layer under biomimetic conditions by using a supersaturated calcification solution (SCS), modified by adding vitamins A and D3, and collagen. The hydroxyapatite deposits on titanium were investigated by means of scanning electron microscopy (SEM) coupled with X-ray analysis (EDX), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy. The results obtained have shown that hydroxyapatite coatings were produced in vitro under vitamins and collagen influence.

Fabrication of mineralized electrospun PLGA and PLGA/gelatin nanofibers and their potential in bone tissue engineering

Surface mineralization is an effective method to produce calcium phosphate apatite coating on the surface of bone tissue scaffold which could create an osteophilic environment similar to the natural extracellular matrix for bone cells. In this study, we prepared mineralized poly(d,l-lactide-co-glycolide) (PLGA) and PLGA/gelatin electrospun nanofibers via depositing calcium phosphate apatite coating on the surface of these nanofibers to fabricate bone tissue engineering scaffolds by concentrated simulated body fluid method, supersaturated calcification solution method and alternate soaking method. The apatite products were characterized by the scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), and X-ray diffractometry (XRD) methods. A large amount of calcium phosphate apatite composed of dicalcium phosphate dihydrate (DCPD), hydroxyapatite (HA) and octacalcium phosphate (OCP) was deposited on the surface of resulting nanofibers in short times via three mineralizing methods. A larger amount of calcium phosphate was deposited on the surface of PLGA/gelatin nanofibers rather than PLGA nanofibers because gelatin acted as nucleation center for the formation of calcium phosphate. The cell culture experiments revealed that the difference of morphology and components of calcium phosphate apatite did not show much influence on the cell adhesion, proliferation and activity.

Biomimetic synthesis of poly(lactic-co-glycolic acid)/multi-walled carbon nanotubes/apatite composite membranes

Bioactive guided tissue regeneration (GTR) membrane has had some success for periodontal therapy. In this study, poly(lactic-co-glycolic acid) (PLGA)/multi-walled carbon nanotubes (MWNTs) composite membranes were incu- bated in three supersaturated calcification solutions (SCS) of different pH values for 21 days to prepare a PLGA/ MWNTs/apatite composite. Scanning electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), energy dispersive spectroscopy (EDS), water contact angle measurement and mechanical testing were used for characterization. It was found that after 21 days incubation, apatite with low crystallite size and crystallinity was formed on the PLGA/MWNTs composite membranes. The Ca-poor carbapatite was similar in morphology and compo- sition to that of natural bone. The size and shape of the apatite crystals immersed in three SCS were different from each other. The hydrophilicity and mechanical properties of the PLGA/MWNTs composite membranes were significantly enhanced after mineralization. This indicated that biomimetic mineralization may be an effective method to improve the biocompatibility and bone inductivity of certain materials. The PLGA/MWNTs/apatite composites may be potentially use- ful in GTR applications, particularly as GTR membranes for periodontal tissue regeneration.

The effect of vitamins to hydroxyapatite growth on porous polyurethane substrate

This work presents a study on the possibility of obtaining hydroxyapatite thin layers on porous polyurethane support using a modified coating method, which is based on a biomimetic technique. To investigate bone-like apatite coatings formation on polyurethane surface, two types of solutions such as supersaturated calcification solution (SCS) and modified SCS (M-SCS) were used. The modified supersaturated calcification solution was prepared with added at original SCS appropriate quantities of vitamin A and vitamin D2. The hydroxyapatite deposits are investigated by scanning electron microscopy (SEM) coupled with energy dispersive X-ray analysis (EDX) and ATR-IR spectroscopy. The presence of vitamins (A and D2) in M-SCS solution favours the formation of hydroxyapatite layer on polymeric substrate. The results obtained from SEM images have showed that porous polyurethane/hydroxyapatite scaffolds with estimated porosity of over 60% in an interconnected network, were produced under vitamins influence.

Preparation of hydroxyapatite coating on CoCrMo implant using an effective electrochemically-assisted deposition pretreatment

Abstract A hydroxyapatite (HA) coating is efficiently generated on CoCrMo implant alloys by employing an electrochemically-assisted deposition (ECAD) pretreatment followed by chemical immersion in a supersaturated calcification solution. CoCrMo was electrochemically treated in a calcium- and phosphate-containing solution for short time. A thin layer about 200 nm thicknesses, consisting mainly of amorphous calcium phosphate, was formed on the surface of the CoCrMo after the ECAD pretreatment. After the chemical immersion for 2 h, a layer of octacalcium phosphate was formed on the surfaces. A layer of HA was formed after 72 h immersion. The effectiveness of these treatments was demonstrated by comparison with the alkaline treated CoCrMo. Results showed that the ECAD pretreatment highly enhanced the capability of formation of HA on the CoCrMo surface, while only a small amount of CaP islands were formed on CoCrMo through traditional chemical treatment. A tensile test showed that the coating tightly bonded to the substrate. This work has explored an effective ECAD pretreatment to activate CoCrMo implant surface for generating HA coating.

Development of a New Combined Test Setup for Accelerated Dynamic pH-Controlled in vitro Calcification of Porcine Heart Valves

Fifty years after their first implantation, bioprosthetic heart valves still suffer from tissue rupture and calcification. Since new bioprostheses exhibit a lower risk of calcification, fast and reliable in vitro methods need to be evaluated for testing the application of new anti-calcification techniques. This report describes a modification of the well-known in vitro dynamic calcification test method (Glasmacher et al, Leibniz University Hannover (LUH)), combined with the pH-controlled, constant solution supersaturation (CSS) method (University of Patras (UP)). The CSS method is based on monitoring the pH of the solution and the addition of calcium and phosphate ion solutions through the implementation of two syringe pumps. The pH and the activities of all ions in the solutions are thus kept constant, resulting in higher calcification rates compared to conventional in vitro methods in which solution supersaturation is allowed to decrease without any further control. To verify this hypothesis, five glutaraldehyde preserved porcine aortic valves were tested. Three of the valves were tested according to a free-drift methodology: the valves were immersed in a supersaturated calcification solution, with an initial total calcium times total phosphate product of (CaxP)=10.5 (mmol/L)2, renewed weekly. Two valves were tested by the new pH-controlled loop system, implementing the CSS methodology. All valves were tested for a 4-week period, loaded at 300 cycles per minute, resulting in a total of 12 million cycles at the end of the testing period. The degree of calcification was determined weekly by means of mux-ray, and by conventional, clinical and micro-computer tomography (CT, muCT). The results showed that the valves mineralizing at constant solution supersaturation in vitro yielded higher rates of calcification compared to the valves tested at conditions of decreasing solution supersaturation without any control, indicating the development of a new, accelerated, controllable in vitro calcification method.

Characterization and Degradation Study of Calcium Phosphate Coating on Magnesium Alloy Bone Implant In Vitro

Magnesium alloys have similar mechanical properties with natural bone, and they degrade within a certain time span. Therefore, magnesium alloys are suitable to be made as bone screws or plates. However, high susceptibility to corrosion has limited their applications in the orthopedic field. They would possess great medical functions if the degradation rates of magnesium alloys could be reduced. This paper describes approaches to form calcium phosphate coating on magnesium alloy (AZ31) to control the degradation rate. Samples of AZ31 were placed in the supersaturated calcification solution prepared with Ca (NO3)2 , NaH2PO4, and NaHCO3, and then, the calcium phosphate coating was formed. The composition, phase structure, and morphology of the coatings were investigated. The degradation behaviors of the naked and coated magnesium (Mg) alloys were studied in simulated body fluid. The results of this paper have shown that the coatings significantly decrease the degradation rate of the original Mg alloy, indicating that the Mg alloy with calcium phosphate coating is a promising degradable bone material.

Calcium phosphate coating on magnesium alloy by biomimetic method: Investigation of morphology, composition and formation process

Magnesium alloy has similar mechanical properties with natural bone and can degrade via corrosion in the electrolytic environment of the human body. Calcium phosphate has been proven to possess bioactivity and bone inductivity. In order to integrate both advantages, calcium phosphate coating was fabricated on magnesium alloy by a biomimetic method. Supersaturated calcification solutions (SCSs) with different Ca/P ratio and Cl− concentration were used as mimetic solutions. The morphology, composition and formation process of the coating were studied with scanning electron microscopy (SEM), energy dispersive spectrometer (EDS), Fourier transformed infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The results show that a uniform calcium phosphate coating was observed on magnesium alloy, the properties of which could be adjusted by the SCSs with different Ca/P ratio. The formation process of the coating was explored by immersing magnesium alloy in SCSs with different Cl− concentration which could adjust the hydrogen production. According to SEM results, the hydrogen bubbles were associated with the formation of grass-like and flower-like coating morphologies. In conclusion, the biomimetic method was effective to form calcium phosphate coating on magnesium alloy and the morphology and composition of the coating could be accommodated by the Ca/P ratio and Cl− concentration in SCSs.

Calcium phosphate coating on magnesium alloy for modification of degradation behavior

Magnesium alloy has similar mechanical properties with natural bone, but its high susceptibility to corrosion has limited its application in orthopedics. In this study, a calcium phosphate coating is formed on magnesium alloy (AZ31) to control its degradation rate and enhance its bioactivity and bone inductivity. Samples of AZ31 plate were placed in the supersaturated calcification solution prepared with Ca(NO3)2, NaH2PO4 and NaHCO3, then the calcium phosphate coating formed. Through adjusting the immersion time, the thickness of uniform coatings can be changed from 10 to 20 μm. The composition, phase structure and morphology of the coatings were investigated. Bonding strength of the coatings and substrate was 2–4 MPa in this study. The coatings significantly decrease degradation rate of the original Mg alloy, indicating that the Mg alloy with calcium phosphate coating is a promising degradable bone material.

In vitro transformation of OCP into carbonated HA under physiological conditions

Octacalcium phosphate (OCP) is a precursor phase during the in vivo mineralization of biological tissues. OCP can easily convert into hydroxyapatite (HA), which is the main inorganic constituent of human bone and teeth. In this study titanium alloys (Ti6Al4V) were coated with a crystalline OCP layer at 37 °C by using a supersaturated calcification solution (SCS). The in vitro bioactivity and mineralization behaviour was investigated by soaking the OCP coatings into simulated body fluid (SBF) for various time periods between 10 and 168 h. Samples were characterized by SEM-EDX, FT-IR- and XRD-analyses. In the early mineralization stages of OCP hydrolysis and partial dissolution of the crystals results in the formation of carbonated hydroxyapatite (CHA). The plate-shaped morphology of the OCP crystals remains unchanged and the thickness of the crystals increases with soaking time. After 100 h in SBF a continuous CHA layer grew upon the OCP coating proving the in vitro bioactivity of thus processed coatings. Thus, OCP coatings might enhance the osteoconductivity of orthopedic titanium based implants.