Supersaturated Calcification Solution Research Articles

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.

Structural characterization of hydroxyapatite layer coatings on titanium supports

This paper presents a study on an alternative coating method based on biomimetic techniques which are designed to form a crystalline HA layer in a manner similar to the process of natural bone formation. The HA formation on the surface of titanium alloy pretreated with NaOH solution is investigated. Two types of solutions such as supersaturated calcification solution (SCS) and modified SCS (M-SCS) were used to investigate bone-like apatite formation on alkali-treated titanium. The HA deposits were investigated by means of scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results obtained indicate that HA layers with preferred orientation and composition similar to that found in bone can be biomimetically synthesized depending on the ion composition and concentration of the solution. The data suggest that the method utilized in this work can be successfully applied to obtain deposition of uniform coatings of crystalline hydroxyapatite on titanium substrates.

SEM and EDX studies of bioactive hydroxyapatite coatings on titanium implants

This work presents a study on an alternative coating method based on biomimetic techniques which are designed to form a crystalline hydroxyapatite layer very similar to the process corresponding to the formation of natural bone. The HA formation on the surface of titanium alloy pretreated with NaOH solution is investigated. Two types of solutions such as supersaturated calcification solution (SCS) and modified SCS (M-SCS) were used to investigate bone-like apatite formation on alkali-treated titanium. The hydroxyapatite deposits are investigated by means of scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX). The data suggest that the method utilized in this work can be successfully applied to obtain deposition of uniform coatings of crystalline hydroxyapatite on titanium substrates.

A Resorbable Surface Formed on Ti Alloy Material

This paper performs a new way of a resorbable surface on Ti alloy preparation. Precalcification of a chemically treated Ti or Ti alloy surface by a supersaturated calcification solution - SCS2 (high content of Ca2+ as well as (PO4)3- ions) allows to short of an induction period in SBF solution (practically immediate reaction stats a hydroxyapatite precipitation). Soluble octacalcium phosphate (Ca8H2(PO4)6. 5H2O was detected by RTG on the surface after calcification. Presumption is: a healing time of the calcified Ti alloy could be shortened.

Fast deposition of hydroxyapatite coating on titanium to modify cell affinity of corneal fibroblast in vitro

By two step acid-alkali pretreatment and immersing into supersaturated calcification solution, hydroxyapatite (HA) coating was deposited on titanium (Ti) discs. The composition, surface morphology and cross-section of the coating were analyzed by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Fibroblasts of rabbit cornea were seeded on HA coated Ti disc, pure Ti disc and glass. Cell adhesion, proliferation and morphology were detected at 24, 48 and 72 h, respectively. It is shown for the first time that HA coating can significantly enhance the adhesion and proliferation of rabbit corneal fibroblast in comparison with that of pure Ti.

Biomimetic Fabrication and Characterization of BG/COL/HCA Scaffolds for Bone Tissue Engineering

The bone tissue engineering scaffold was developed by compounded the type I collagen with the porous scaffold of the sol-gel derived bioactive glass (BG) in the system CaO-P2O5-SiO2. The resultant porous scaffold was treated in supersaturated calcification solution (SCS) to form the surface layer of hydroxyl-carbonate-apatite (HCA) since the type I collagen possessed good biocompatibility and bio-absorbability, and also, the ability of inducting calcium phosphates to precipitated inside and outside the collagen fibers where the collagen fibers acted as bio-macromolecules template for formation of bone-like inorganic minerals in nature bone such as: octo-calcium phosphate (OCP), tri-calcium phosphate (TCP) and hydroxyl-carbonate-apatite (HCA). On the other hand, the sol-gel derived bioactive glass also played an important role in formation of the above bio-minerals owing to its serial chemical reactions with the body fluid. The in vitro study in supersaturated calcification solution SCS indicated that the surface of the porous scaffold was able to induce formation of bone-like HCA crystals on the pore walls of the scaffold which possessed satisfactory cells biocompatibility.

Experimental Study of Biomimetic Mineralization on the Surface of Piezoelectric Pulp-Cap Film

To study the biomimetic mineralization behaviour of piezoelectric pulp-cap films, bioelectret chitosan films were prepared by polarization in an electric field and soaked in PBS with lysozyme for piezoelectricity attenuation testing. The results showed that comparing these with samples stored in an exsiccator, the films soaked in PBS had accelerated d33 loss. Calcium phosphate crystals nucleated and grew on the surfaces of samples soaked in supersaturated calcification solution at 37 for 1, 6, 12 and 24 h. OCP and HA were found to co-precipitate on the surfaces within 24 h of immersion. This novel piezoelectric inorganic-organic pulp-cap has the potential to be applied in dental pulp capping.

Biomineralization of Electrospun Nano-HA/PHB GTR Membrane

Poly 3-hydroxybutyrate (PHB) as a kind of polysaccharides has been proved promising for tissue engineering because of its biocompatibility and biodegradability. But its poor mechanical properties and hydrophilicity limit its application. In order to explore a new useful porch to improve the performance of PHB-based GTR membrane, membrane composed of nano-HA / PHB composite was manufactured through the air/jet electrospinning process which can potentially generate nanometer scale diameter fibers and enlarge surface area of materials while maintaining high porosity. Successively, the biomineralization behavior of the membrane in supersaturated calcification solution (SCS) was studied. The Results of this investigation show that the successfully manufactured porous nano-HA/PHB membrane has high activity in SCS and its ability of inducing the formation of mineral crystal in vitro than that of the unfilled PHB membrane. It can be concluded that the addition of nano-HA and the novel technology could improve the performance of the PHB-based GTR membrane.

Growth of Calcium Phosphate on Poled Piezoelectric Poly-L-lactic Acid Membrane by a Biomimetic Method

The bioactivity of poled piezoelectric PLLA membrane was investigated by studying the calcium phosphate formation in vitro using a biomimetic method. Samples (φ10mm) were poled under DC electric field of 8~l0kV/cm at 70°C for 30 min followed by cooling under the electric field. Surface chemistry of the samples before and after poling treatment was studied by X-ray photoelectron spectroscopy (XPS). Poled/unpoled samples were immersed in supersaturated calcification solution (SCS) for periods up to 24 h (36.5°C). The surface morphology and composition of the soaked samples were evaluated by using scanning electron microscope (SEM) and X-ray diffraction analysis (XRD). Poled samples showed two different charged surfaces, negatively-charged surface (N-PLLA) and positively-charged surface (P-PLLA). On the N-PLLA surfaces, SEM together with XRD showed a gradually formed calcium phosphate (Ca-P), while no obvious Ca-P on either P-PLLA or unpoled samples was observed. This study demonstrated that poled piezoelectric PLLA substrates induce substantially higher level of Ca-P formation than electrically neutral substrates and only N-PLLA, however, can improve Ca-P formation after immersion in SCS.

Studies on induction of l-aspartic acid modified chitosan to crystal growth of the calcium phosphate in supersaturated calcification solution by quartz crystal microbalance

Acidic amino acids, such as aspartic acid (L-Asp) and glutamic acid, are the primary bioactive molecules of the glycoprotein on the organic/inorganic interface of biomineralized tissues. In this study, the induction of chitosan film modified with L-Asp on the crystal growth of hydroxyapatite (HAP) was investigated by a novel in situ analysis approach, quartz crystal microbalance (QCM), associated with the dynamically structural and morphological characterization of precipitation products on various phases by X-ray diffraction (XRD), Fourier-transformed infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). The natural chitosan exhibited no inducing ability on the crystal growth of HAP. However, the growth rate of induced HAP was dramatically accelerated by the L-Asp modification of chitosan film and increased with the increase of the concentration of L-Asp in the chitosan substrate. It was shown that the chelation of calcium ion with L-Asp provided a nucleation centre and the cluster nuclei was formed by adsorbing further PO(4)(3-), Ca(2+), and then HAP deposited on the original HAP coating in the supersaturated calcification solution (SCS). The developed method allows a kinetic evaluation of the induction of organic film on crystal nucleation and the growth of HAP in vitro.

A simple biomimetic method for calcium phosphate coating

A biomimetic calcium phosphate coating is expected to enhance the bioactivity and bone inductivity of human implants. This study presents a very simple, highly effective biomimetic method to obtain a calcium phosphate coating on a titanium substrate. Using NaH 2PO 4, a stable solution was prepared with high calcium and phosphate ion concentrations. This solution turned to a supersaturated calcification solution (SCS) when NaHCO 3 was added. The addition of NaHCO 3 elevated the pH value of the solution gradually and steadily. A uniform coating approximately 40 μm thick was found on the substrates after 24 h immersion. The compositions of the coatings were adjustable from hydroxyapatite (HA) to HA/dicalcium phosphate dihydrate (DCPD). The calcium phosphate deposits were investigated by means of scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transformed infrared (FT-IR) spectroscopy.

Hydroxyapatite nucleation on Na ion implanted Ti surfaces

Hydroxyapatite (HA) coating is the widely common approach to improve the biocompatibility of orthopaedic and dental titanium-based implants. Low-temperature processses are of current interest. In these processes, biologically active HA having the chemistry and structure of mineralized tissue may be formed either in vivo or under in vivo simulated conditions. Ti surfaces treated with NaOH have been shown to induce HA formation upon exposure to simulated body fluid [1– 3]. The effect can be related to: (i) generation of hydroxylated surface Ti–OH groups acting as nucleating sites for HA. The process occurs due to Ti reacting with NaOH and by a subsequent hydrolysis of the reaction product Na2TiO3; (ii) increased supersaturation of HA due to increased surface pH resulting from Na2TiO3 hydrolysis; and (iii) etching-generated rugged surface morphology providing additionally mechanical anchorage structures. The procedure of Kim et al. [1] involves treatment of Ti in NaOH (>0.5 M) at 60 ◦C for >24 h followed by 1 h dry-heating at 600 ◦C and then incubation in simulated body fluid (SBF). Two modifications have been reported by Wen et al. The first refers to treatment of Ti6Al4V in diluted NaOH (≤0.4 M) at 140 ◦C for 5 h (without post-heating) before incubating in a supersaturated calcification solution, SCS [2]. The second uses an additional step of etching Ti6Al4V in HCl+H2SO4 before reacting with NaOH at 140 ◦C and precipitation from SCS [3]. This paper presents the surface treatment technique involving ion implantation of Na into Ti to show that Na reacts with Ti to form surface-incorporated Na2TiO3 and a microporous ragged surface topography, and such surfaces are reactive to induce HA nucleation upon exposing to SBF. The substrates used were plates (10× 10× 1 mm3) of commercially pure Ti (Goodfellow). Standard procedures of polishing and cleaning were applied before use. Na ions were implanted at doses ranging from 8×1016 to 4×1017 ions cm−2. The ion energies were set at 18 to 22 keV to deposit Na within a theoretical surface depth of (22–28)± 14 nm. Exposure experiments in simulated body fluid (SBF [4]) were conducted at 37 ◦C in polystyrene vials. The surfaces were characterized using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and light microscopy (LM). The XRD analysis of the as-implanted samples shows Na2TiO3 to be a new major phase introduced into the surface, Fig. 1a. The signal intensity increases

Modulation of apatite crystal growth on Bioglass by recombinant amelogenin.

The effects of a recombinant mouse amelogenin (rM179) on the growth of apatite crystals nucleated on a bioactive glass (45S5 type Bioglass) surface were investigated with a view to gaining a better understanding of the role of amelogenin protein in tooth enamel formation and of its potential application in the design of novel enamel-like biomaterials. Bioglass discs were incubated in phosphate-buffered saline (PBS) to preform a calcium phosphate surface layer and subsequently immersed in blank, bovine serum albumin (BSA)- and rM179-containing supersaturated calcification solutions (SCS(B), SCS(BSA) and SCSrM179), respectively. Calcium phosphate layers formed on all the treated samples and were characterized to be apatite by X-ray diffraction and Fourier transmission infrared spectrophotometry. Under scanning electron microscopy, plate-shaped crystals (approximately 50 nm thick and 300-600 nm across) were observed on the samples after PBS incubation. The crystals grown from SCS(B) were of the typical plate shape except for an increased thickness, while needle-shaped crystals (200-300 nm long and 50-70 nm thick) were precipitated on the SCS(BSA)-immersed samples. Interestingly, it was found that the crystals deposited on the SCSrM179-immersed samples adopted an elongated, curved shape (approximately 500 nm long and approximately 120 nm thick). Further TEM observations showed that the crystals generated by the SCSrM179 immersion appeared to be composed of bundles of lengthwise crystals (15-20 nm thick) orientated parallel to one another, much alike the long and thin crystals observed in the very early stage of enamel formation. The significant modulation by the rM179 protein of apatite crystal growth is quite different from the overall inhibition observed by BSA and most likely is relevant to the specific function of the amelogenin matrix in controlling enamel crystal growth in vivo.

Controlled crystal growth of calcium phosphate on titanium surface by NaOH-treatment

A simple supersaturated calcification solution (SCS) was used to deposit calcium phosphate (Ca-P) on the surface of NaOH-treated titanium (NaOH-Ti), in order to study the corresponding deposition mechanism of calcium phosphate (Ca-P). No Ca-P coating from SCS was found on titanium without NaOH-treatment, while NaOH-Ti possessed the capability to induce a Ca-P coating on the titanium surface. Octacalcium phosphate (OCP) crystals were first grown on an NaOH-Ti surface, followed by hydroxyapatite (HA) with a [0 0 1] preferential orientation on OCP. It is found that two factors controlled the growth of Ca-P crystals on NaOH-Ti from SCS. First, the surface morphology of NaOH-Ti characterized with crevices seems to be beneficial for inducing a Ca-P coating from SCS; second, the basic hydroxyl, Ti-OH, radical has increased in NaOH-Ti with the increase of treating time and concentration, which facilitate the nucleation of Ca-P crystals.

Crystal growth of calcium phosphate on chemically treated titanium

Calcium phosphate (Ca–P) phases were grown on titanium which was treated with H 3PO 4 and diluted alkali solution by immersing in supersaturated calcification solutions (SCSs) at 37°C, i.e. fast calcification solution (FCS) and Hanks'-balanced salt solution (HBSS). A significant difference between the sizes of the Ca–P crystals precipitated from FCS and from HBSS was observed and ascribed to the fact that the ion concentrations of Ca 2+ and HPO 2− 4 in HBSS are much lower than those in FCS and some inhibitors of Ca–P growth such as Mg 2+ and CO 2− 3 ions are present in the former. Micron-scaled thin crystal plates composed of hydroxyapatite and octacalcium phosphate grew directly on the treated surfaces from FCS whereas nano-sized, bone-like apatite particles deposited from HBSS. It was found that the longer the H 3PO 4 etching was, the more easier Ca–P phases were deposited on the treated surface when the alkali ageing time was fixed. No Ca–P deposition was observed on any untreated or single-step treated metals after 2 weeks of immersion in either HBSS or FCS. The present study makes it possible to prepare Ca–P coatings on titanium implants by simple chemical method, which might enhance their activity of bone bonding.

Preparation of bioactive Ti6Al4V surfaces by a simple method

Boiling diluted alkali incubation was found to be an effective way to prepare bioactive Ti6Al4V surfaces, whether polished or not, as indicated in vitro after immersion in two different supersaturated calcification solutions (SCSs). The induction of calcium phosphate (Ca-P) precipitation from the SCSs is most probably made possible by the formation of a new TiO 2 surface layer and a large number of submicron-scaled etched pits therein. The morphologies and composition of the Ca-P deposited from different SCSs are entirely different from each other. The processes on Ti6Al4V surfaces during treatment and immersion were investigated in detail by means of scanning electron microscopy combined with energy dispersive X-ray analysis, X-ray photoelectron spectroscopy and X-ray diffraction.

Preparation of bioactive microporous titanium surface by a new two-step chemical treatment.

Microporous oxide layers allowing fast deposition of calcium phosphate layers (CPLs) were formed on commercially pure titanium (c.p.Ti) after the application of a newly developed two-step chemical treatment. The micropores were of submicrometre size. The two-step treatment was carried out by etching c.p.Ti samples with HCl and H2SO4 first and then treating them in boiling 0.2 N NaOH solution at 140 degrees C for 5 h. Conformal CPLs, about 20 microm thick, were deposited on the two-step treated c.p.Ti surface by means of a two-day immersion in an in vitro supersaturated calcification solution. The CPL was characterized to be mainly composed of two sublayers, i.e. an outside loose octacalcium phosphate crystal sublayer and an inside dense carbonated apatite sublayer. A scratching test indicated that the apatite sublayer was strongly bonded to the c.p.Ti substrate. Moreover, it was observed that the untreated or single-step treated c.p.Ti surfaces are not only morphologically different from one another but significantly different from the two-step treated one, in that no precipitation was observed on them up to 14 d immersion in the same calcification solution. It is indicated that the two-step chemical treatment is a simple and easily controllable method to prepare bioactive titanium surfaces and subsequently to induce the rapid precipitation of conformal and adherent CPL from in vitro supersaturated calcification solutions.

Fast precipitation of calcium phosphate layers on titanium induced by simple chemical treatments

A simple two-step chemical treatment i.e. etching with HCl and H 2SO 4 followed by immersion in boiling dilute NaOH solution, has been developed by our group to prepare bioactive microporous titanium surfaces allowing fast deposition of a calcium phosphate layer (CPL) from an in vitro supersaturated calcification solution (SCS). In this work, a precalcification (Pre-Ca) procedure was applied by soaking the two-step treated titanium in Na 2HPO 4 and then saturated Ca(OH) 2 solution before immersion in SCS to accelerate further the CPL precipitation. The treated titanium surfaces with Pre-Ca were characterized after 1, 2, 4, 8 and 16 h of immersion in SCS by means of scanning electron microscopy together with energy dispersive X-ray analysis, X-ray diffraction and infrared absorption analysis. It was observed that the CPL precipitation rate with Pre-Ca averaged 1 μm h −1, twice as fast as without Pre-Ca. No precipitation was observed on untreated titanium with Pre-Ca up to day 14 of immersion in the SCS.

A simple method to prepare calcium phosphate coatings on Ti6Al4V.

A two-step chemical treatment followed by immersion in a supersaturated calcification solution (SCS) was found to be a simple way to prepare calcium phosphate (Ca-P) coatings on Ti6Al4V. The Ca-P deposition on the treated metallic surfaces could be accelerated by employing a pre-calcification (Pre-Ca) procedure prior to immersion in SCS. The two-step treatment was performed by etching the metallic plates with a mixture of HCl and H2SO4 followed by ageing in boiling diluted NaOH solution at 140 degrees C. Pre-Ca was carried out by incubating the two-step treated plates in Na2HPO4 solution and then in saturated Ca(OH)2 solution. The formation of a bioactive microporous surface oxide layer on Ti6Al4V by the two-step treatment was most probably responsible for the induction of Ca-P precipitation. The deposition rates and compositions of Ca-P coatings in two different SCSs were investigated by means of scanning electron microscopy, X-ray diffraction and infrared spectrophotometry.