Mechanism of bonelike apatite formation on bioactive tantalum metal in a simulated body fluid.

Development of tantalum metal with bone-bonding ability is paid much attention because of its attractive features such as high fracture toughness, high workability and its achievement on clinical usage. Formation of bonelike apatite is an essential prerequisite for artificial materials to make direct bond to living bone. The apatite formation can be assessed in vitro using a simulated body fluid (SBF) that has almost equal compositions of inorganic ions to human blood plasma. The present authors previously showed that the apatite formation on tantalum metal in SBF was remarkably accelerated by treatment with NaOH aqueous solution and subsequent firing at 300 degrees C, while untreated tantalum metal spontaneously forms the apatite after a long soaking period. The purpose of the present study is to clarify the reason why the NaOH and heat treatments accelerate the apatite formation on tantalum metal. X-ray photoelectron spectroscopy was used to analyze changes in surface structure of the tantalum metal at an initial stage after immersion in SBF. Untreated tantalum metal had tantalum oxide passive layer on its surface, while amorphous sodium tantalate was formed on the surface of the tantalum metal by the NaOH and heat treatments. After soaking in SBF, the untreated tantalum metal sluggishly formed small amount of Ta-OH groups by a hydration of the tantalum oxide passive layer on its surface. In contrast, the treated tantalum metal rapidly formed Ta-OH groups by exchange of Na+ ion in the amorphous sodium tantalate on its surface with H3O+ ion in SBF. Both the formed Ta-OH groups combined with Ca2+ ion to form a kind of calcium tantalate, and then with phosphate ion, followed by combination with large amount of Ca2+ ions and phosphate ions to build up apatite layer. The formation rate of Ta-OH groups on the treated tantalum metal predominates the following process including adsorption of Ca2+ ion and phosphate ion on the surface. It is concluded that the acceleration of the apatite nucleation on the tantalum metal in SBF by the NaOH and heat treatments was attributed to the fast formation of Ta-OH group, followed by combination of the Ta-OH groups with Ca2+ and phosphate ions.     

An X-ray photoelectron spectroscopy study of the process of apatite formation on bioactive titanium metal

Bioactive titanium metal, prepared by treatment with NaOH followed by an annealing stage to form a sodium titanate layer with a graded structure on its surface, forms a biologically active bone-like apatite layer on its surface in the body, and bonds to bone through this apatite layer. In this study, process of apatite formation on the bioactive titanium metal in a simulated body fluid was investigated using X-ray photoelectron spectroscopy. The bioactive titanium metal formed Ti-OH groups soon after soaking in the simulated body fluid, via the exchange of the Na(+) ions in the sodium titanate on its surface with H(3)O(+) ions in the fluid. The Ti-OH groups on the metal combined with the calcium ions in the fluid immediately to form a calcium titanate. After a long period, the calcium titanate on the metal took the phosphate ions as well as the calcium ions in the fluid to form the apatite nuclei. The apatite nuclei then proceeded to grow by consuming the calcium and phosphate ions in the fluid. These results indicate that the Ti-OH groups formed on the metal induce the apatite nucleation indirectly, by forming a calcium titanate. The initial formation mechanism of the calcium titanate may be attributable to the electrostatic interaction of the negatively charged Ti-OH groups with the positively charged calcium ions.     

Apatite formation on zirconium metal treated with aqueous NaOH.

Previous studies by the authors have shown that titanium metal, titanium alloys and tantalum metal which were subjected to aqueous NaOH solution and subsequent heat treatments form an apatite surface layer upon immersion in a simulated body fluid (SBF) with ion concentrations nearly equal to those in human blood plasma. These metals form the apatite surface layer even in living body, and bond to living bone through the apatite layer. In the present study, the apatite-forming ability of NaOH-treated zirconium metal in SBF has been investigated. A hydrated zirconia gel layer was formed on the surface of the zirconium metal on exposure to 1-15 M NaOH aqueous solutions at 95 degrees C for 24h. It was observed that the metals treated in NaOH aqueous solutions with concentrations above 5 M form an apatite layer on their surface in SBF. This indicates that the Zr-OH group of the zirconia gel induces apatite nucleation. The present study points to the possibility of obtaining bioactive zirconium after treatment by NaOH.     

Biomimetic apatite formation on chemically treated titanium

Titanium treated in NaOH can form hydroxycarbonated apatite (HCA) after exposition in simulated body fluid (SBF). Generally, titanium is covered with a passive oxide layer. In NaOH this passive film dissolves and an amorphous layer containing alkali ions is formed on the surface. When exposed to SBF, the alkali ions are released from the amorphous layer and hydronium ions enter into the surface layer, resulting in the formation of Ti-OH groups in the surface. The released Na(+) ions increase the degree of supersaturation of the soaking solution with respect to apatite by increasing pH, and Ti-OH groups induce apatite nucleation on the titanium surface. The acid etching of titanium in HCl under inert atmosphere was examined as a pretreatment to obtain a uniform initial titanium surface before alkali treatment. Acid etching in HCl leads to the formation of a micro-roughened surface, which remains after alkali treatment in NaOH. It was shown by SEM, gravimetric and solution analysis that the apatite nucleation was uniform and the thickness of precipitated HCA layer increased continuously with time. The treatment of titanium by acid etching in HCl and subsequently in NaOH is a suitable method for providing the metal implant with bone-bonding ability.     

TEM-EDX study of mechanism of bonelike apatite formation on bioactive titanium metal in simulated body fluid

Bioactive titanium metal, which forms a bonelike apatite layer on its surface in the body and bonds to the bone through the apatite layer, can be prepared by NaOH and heat treatments to form an amorphous sodium titanate layer on the metal. In the present study, the mechanism of apatite formation on the bioactive titanium metal has been investigated in vitro. The metal surface was examined using transmission electron microscopy and energy dispersive X-ray spectrometry as a function of the soaking time in a simulated body fluid (SBF) and complemented with atomic emission spectroscopy analysis of the fluid. It was found that, immediately after immersion in the SBF, the metal exchanged Na(+) ions from the surface sodium titanate with H(3)O(+) ions in the fluid to form Ti-OH groups on its surface. The Ti-OH groups, immediately after they were formed, incorporated the calcium ions in the fluid to form an amorphous calcium titanate. After a long soaking time, the amorphous calcium titanate incorporated the phosphate ions in the fluid to form an amorphous calcium phosphate with a low Ca/P atomic ratio of 1.40. The amorphous calcium phosphate thereafter converted into bonelike crystalline apatite with a Ca/P ratio of 1.65, which is equal to the value of bone mineral. The initial formation of the amorphous calcium titanate is proposed to be a consequence of the electrostatic interaction of negatively charged units of titania, which are dissociated from the Ti-OH groups, with the positively charged calcium ions in the fluid. The amorphous calcium titanate is speculated to gain a positive charge and to interact with the negatively charged phosphate ions in the fluid to form the amorphous calcium phosphate, which eventually stabilizes into bonelike crystalline apatite.     

Bioactive macroporous titanium surface layer on titanium substrate

A macroporous titanium surface layer is often formed on titanium and titanium alloy implants for morphological fixation of the implants to bone via bony ingrowth into the porous structure. The surface of titanium metal was recently shown to become highly bioactive by being subjected to 5.0 M-NaOH treatment at 60 degrees C for 24 h and subsequent heat treatment at 600 degrees C for 1 h. In the present study, the NaOH and heat treatments were applied to a macroporous titanium surface layer formed on titanium substrate by a plasma spraying method. The NaOH and heat treatments produced an uniform amorphous sodium titanate layer on the surface of the porous titanium. The sodium titanate induced a bonelike apatite formation in simulated body fluid at an early soaking period, whereby the apatite layer grew uniformly along the surface and cross-sectional macrotextures of the porous titanium. This indicates that the NaOH and heat treatments lead to a bioactive macroporous titanium surface layer on titanium substrate. Such a bioactive macroporous layer on an implant is expected not only to enhance bony ingrowth into the porous structure, but also to provide a chemical integration with bone via apatite formation on its surface in the body.     

Apatite-forming ability of CaO-containing titania.

It was recently shown that titanium metal and its alloys spontaneously form a bonelike apatite layer on their surfaces in the living body and bond to the bone through the apatite layer, when the sodium ions are incorporated into titanium oxide layer of their surfaces by chemical and heat treatments. It is expected that their apatite-forming ability, and hence their bone-bonding ability, could be enhanced, if the calcium ions are incorporated into their surface titanium oxide layers instead of the sodium ions, because the calcium ions released from their surface layers can increase the ionic activity product of the apatite of the surrounding fluid more effectively than the sodium ions. In the present study, in order to investigate the effect of incorporation of the calcium ions into the titanium oxide layer on its apatite-forming ability, apatite-forming abilities of titania gels which have different CaO contents and subjected to different heat treatments were examined in a simulated body fluid with ion concentrations nearly equal to those of the human blood plasma. It was found that CaO-containing gels do not form the apatite on their surfaces as far as they take an amorphous phase in spite of the fact that they release larger amounts of the calcium ions with increasing CaO contents of the gels. They form the apatite when they take an anatase-like structure even though they do not contain CaO. These results indicate that a specific structure of the titanium oxide is more important for the apatite nucleation than the magnitude of the ionic activity products of the apatite in the surrounding fluid.     

Preparation of bioactive titanium metal via anodic oxidation treatment

Titania with specific structures of anatase and rutile was found to induce apatite formation in vitro. In this study, anodic oxidation in H(2)SO(4) solution, which could form anatase and rutile on titanium metal surface by conditioning the process, was employed to modify the structure and bioactivity of biomedical titanium. After the titanium metal was subjected to anodic oxidation treatment, thin film X-ray diffraction and scanning electron microscopy results showed the titanium metals surfaces were covered by porous titania of anatase and/or rutile. In simulated body fluid (SBF), the titanium anodically oxidized under the conditions with spark-discharge could induce apatite formation on its surface. The induction period of apatite formation was decreased with increasing amount of either anatase or rutile by conditioning the anodic oxidation. After the titanium metal, anodically oxidized under the conditions without spark-discharge, was subjected to heat treatment at 600 degrees C for 1 h, it could also induce apatite formation in SBF because the amount of anatase and/or rutile was increased by the heat treatment. Our results showed that induction of apatite-forming ability on titanium metal could be attained by anodic oxidation conjoined with heat treatment. So it was believed that anodic oxidation in H(2)SO(4) solution was an effective way to prepare bioactive titanium.     

Surface potential change in bioactive titanium metal during the process of apatite formation in simulated body fluid

Bioactive titanium metal can be prepared by NaOH and heat treatments that present the metal with a graded bioactive surface layer of amorphous sodium titanate. This study used laser electrophoresis together with transmission electron microscopy (TEM) and energy-dispersive X-ray microanalysis (EDX) to relate the surface potential change of the bioactive titanium metal with its surface structural change in simulated body fluid (SBF). The surface potential of the metal was highly negative immediately after immersion in SBF. With increasing soaking time, the surface potential increased, revealing a maximum positive value, and then decreased to a constant negative value. TEM-EDX showed that immediately after immersion in SBF, the metal surface formed Ti-OH groups by exchanging Na(+) ions in the surface sodium titanate with H3O(+) ions in the fluid. Thereafter, with increasing soaking time the metal surface formed an amorphous calcium titanate, then an amorphous calcium phosphate, and, finally, apatite with bone-like composition and structure. These results indicate that the process of apatite formation on bioactive titanium metal is initiated by the formation of Ti-OH groups with negative charges that interact with calcium ions with positive charges to form calcium titanate. The calcium titanate gains a positive charge and later interacts with phosphate ions with negative charges, forming amorphous calcium phosphate. The amorphous calcium phosphate eventually transforms and stabilizes into bone-like crystalline apatite with a negative charge.     

Apatite-forming ability of a zirconia/alumina nano-composite induced by chemical treatment

Induction of an apatite-forming ability on a nano-composite of a ceria-stabilized tetragonal zirconia polycrystals (Ce-TZP) and alumina (Al2O3) polycrystals via chemical treatment with aqueous solutions of H3PO4, H2SO4, HCl, or NaOH has been investigated. The Ce-TZP/Al2O3 composite is attractive as a load-bearing bone substitute because of its mechanical properties. The chemical treatments produced Zr-OH surface functional groups, which are known to be effective for apatite nucleation in a body environment. The composite, after chemical treatment, was shown to form a bonelike apatite layer when immersed in a simulated body fluid containing ion concentrations nearly equal to those in human blood plasma. This implies that it may form apatite in the living body and bond to living bone through the apatite layer. This type of bioactive Ce-TZP/Al2O3 composite is therefore expected to be useful as a bone substitute, even under load-bearing conditions.     

Bone-like apatite layer formation on hydroxyapatite prepared by spark plasma sintering (SPS)

Hydroxyapatite (HA) compacts with high density and superior mechanical properties were fabricated by spark plasma sintering (SPS) using spray-dried HA powders as feedstock. The formation of bone-like apatite layer on SPS consolidated HA compacts were investigated by soaking the HA compacts in simulated body fluid (SBF) for various periods (maximum of 28 days). The structural changes in HA post-SBF were analyzed with scanning electron microscopy, grazing incidence X-ray diffraction and X-ray photoelectron spectroscopy. It was found that a layer consisting microcrystalline carbonate-containing hydroxyapatite was formed on the surface of HA compacts after soaking for 24h. The formation mechanism of apatite on the surface of HA compacts after soaking in SBF was attributed to the ion exchange between HA compacts and the SBF solution. The increase in ionic concentration of calcium and phosphorus as well as the increase in pH after SBF immersion resulted in an increase in ionic activity product of apatite in the solution, and provided a specific surface with a low interface energy that is conducive to the nucleation of apatite on the surface of HA compacts.     

Calcium phosphate formation at the surface of bioactive glass in vitro

The calcium phosphate formation at the surface of bioactive glass was studied in vitro. Glass rods and grains were immersed in different aqueous solutions and studied by means of scanning electron microscopy and energy dispersive x-ray analysis. Surface morphological changes and weight loss of corroded grains were monitored. In-depth compositional profiles were determined for rods immersed in the different solutions. The solutions used were tris-buffer (tris-hydroxymethylaminomethane + HCl), tris-buffer prepared using citric acid (tris-hydroxymethylaminomethane + C6H8O7.H2O), and a simulated body fluid, SBF, containing inorganic ions close in concentration to those in human blood plasma. It was found that the calcium phosphate formation at the surface of bioactive glass in vitro proceeds in two stages. When immersing the glass in tris or in SBF a Ca,P-rich surface layer forms. This accumulation takes place within the silica structure. Later, apatite crystals forming spherulites appear on the surface. The Ca/P-ratio of initially formed calcium phosphate was found to be about unity. It is proposed that this is due to bonding of phosphate to a silica gel. The surface is stabilized, i.e., leaching is retarded, by the rapid Ca,P-accumulation within the silica structure before apatite crystals are observed on the surface. It is proposed that the initially formed calcium phosphate is initiated within the silica gel. The crystallizing surface provides nucleation sites for extensive apatite formation on the glass surface. In the presence of citrate no Ca,P-accumulation occur at the glass surface, but soluble Ca-citrate complexes form. By comparing the weight loss during corrosion in tris with that in the calcium and phosphate containing SBF, it is possible to establish whether the glass can induce apatite formation at its surface or not.     

Novel silicon-doped hydroxyapatite (Si-HA) for biomedical coatings: an in vitro study using acellular simulated body fluid

Magnetron co-sputtering was used to produce silicon-doped hydroxyapatite (Si-HA) as coatings intended for potential applications such as orthopedic and dental implants. It was found that the crystallinity of the as-sputtered coatings increased after annealing, resulting in a nanocrystalline apatite structure. Subsequently, the bioactivity of the coatings was evaluated in an acellular simulated body fluid (SBF). Physicochemical evaluation demonstrated that a carbonate-containing apatite layer, which is essential for bonding at the bone/implant interface, was formed on the coating surfaces after immersion in SBF between 4 and 7 days. The annealed coatings exhibited enhanced bioactivity and chemical stability under physiological conditions, as compared with the as-sputtered coatings. It is proposed that the rate at which the carbonate-containing apatite layer forms is dependent on the scale factor of the structure. A nanocrystalline structure can provide a higher number of nucleation sites for the formation of apatite crystallites, leading to a more rapid precipitation of carbonate-containing apatite layer. This work shows that Si-HA coatings offer considerable potential for applications in hard tissue replacement, owing to their ability to form a carbonate-containing apatite layer rapidly.     

Hydroxyapatite formation on alkali-treated titanium with different content of Na+ in the surface layer

Titanium can form a bone-like apatite layer on its surface in SBF when it is treated in NaOH. When pre-treated titanium is exposed to SBF, the alkali ions are released from the surface into the surrounding fluid. The Na+ ions increase the degree of supersaturation of the soaking solution with respect to apatite by increasing pH. On the other hand, the released Na+ cause an increase in external alkalinity that triggers an inflammatory response and leads to cell death. Therefore, it would be beneficial to decrease the release of Na+ into the surrounding tissue. The purpose of this study was to evaluate the hydroxyapatite formation on alkali-treated titanium with different content of Na+ in the surface layer. Using SEM, gravimetric analysis and measurement of calcium and phosphate concentration, it was found that the rate of apatite formation was not significantly influenced by a lower amount of Na+ in the surface layer. Titanium with the lowest content of Na+ could be more suitable for implantation in the human body. The amount of alkali ions released in the surrounding tissue is lower and the rate of apatite formation is identical to titanium with the highest content of Na+ in the surface layer.     

Apatite formation on the surface of Ceravital-type glass-ceramic in the body.

Previous studies on surface structural changes in vitro as well as in vivo of bioactive A-W-type glass-ceramics and Bio-glass-type glasses showed that the essential condition for glasses and glass-ceramics to bond to living bone is formation of a bonelike apatite layer on their surfaces in the body. Gross et al., however, had explained the bone-bonding mechanism of Ceravital-type apatite-containing glass-ceramic without mentioning formation of the surface apatite layer. In the present study, apatite formation on the surface of one of Ceravital-type glass-ceramics was investigated in vitro as well as in vivo. An apatite-containing glass-ceramic of the composition Na2O 5, CaO 33, SiO2 46, Ca(PO3)2 16 wt%, which was named KGS by Gross et al., was soaked in an acellular simulated body fluid which had ion concentrations almost equal to those of the human blood plasma. The same kind of glass-ceramic was implanted into a rabbit tibia. Thin-film x-ray diffraction, Fourier transform infrared reflection spectroscopy, and scanning electron microscopic observation of the surfaces of the specimens soaked in the simulated body fluid showed that Ceravital-type glass-ceramic also forms a layer of carbonate-containing hydroxyapatite of small crystallites and/or a defective structure on its surface in the fluid. Electron probe x-ray microanalysis of the interface between the glass-ceramic and the surrounding bone showed that a thin layer rich in Ca and P is present at the interface. These findings indicated that Ceravital-type glass-ceramics also form the bonelike apatite layer on its surface in the body and bond to living bone through the apatite layer.     

Experimental and clinical performance of porous tantalum in orthopedic surgery

Porous tantalum, a new low modulus metal with a characteristic appearance similar to cancellous bone, is currently available for use in several orthopedic applications (hip and knee arthroplasty, spine surgery, and bone graft substitute). The open-cell structure of repeating dodecahedrons is produced via carbon vapor deposition/infiltration of commercially pure tantalum onto a vitreous carbon scaffolding. This transition metal maintains several interesting biomaterial properties, including: a high volumetric porosity (70-80%), low modulus of elasticity (3MPa), and high frictional characteristics. Tantalum has excellent biocompatibility and is safe to use in vivo as evidenced by its historical and current use in pacemaker electrodes, cranioplasty plates and as radiopaque markers. The bioactivity and biocompatibility of porous tantalum stems from its ability to form a self-passivating surface oxide layer. This surface layer leads to the formation of a bone-like apatite coating in vivo and affords excellent bone and fibrous in-growth properties allowing for rapid and substantial bone and soft tissue attachment. Tantalum-chondrocyte composites have yielded successful early results in vitro and may afford an option for joint resurfacing in the future. The development of porous tantalum is in its early stages of evolution and the following represents a review of its biomaterial properties and applications in orthopedic surgery.     

Simple surface modification of poly(epsilon-caprolactone) to induce its apatite-forming ability

A biodegradable polymer coated with a bone-like apatite layer on its surface is useful as a scaffold for bone tissue regeneration. In this work, a poly(epsilon-caprolactone) (PCL) surface was modified by an O2 plasma surface treatment to form oxygen-containing functional groups. The plasma-treated samples were subsequently dipped alternately in an alcoholic solution containing calcium ions and one containing phosphate ions to deposit apatite precursors on the surface. The surface-modified PCL samples formed a dense and uniform surface bone-like apatite layer after immersion for 24 h in a simulated body fluid with ion concentrations approximately equal to those of human blood plasma. This surface-modification process is applicable to two-dimensional PCL plates and three-dimensional PCL meshes. In the resulting apatite-PCL composite, the apatite layer strongly adhered to the PCL surface and remained intact after a tape-detachment test. Therefore, this type of composite material will be a useful scaffold for bone tissue engineering.     

Controlled release of strontium ions from a bioactive Ti metal with a Ca-enriched surface layer

A nanostructured sodium hydrogen titanate layer ∼1 μm in thickness was initially produced on the surface of titanium metal (Ti) by soaking in NaOH solution. When the metal was subsequently soaked in a mixed solution of CaCl₂ and SrCl₂, its Na ions were replaced with Ca and Sr ions in an Sr/Ca ratio in the range 0.18–1.62. The metal was then heat-treated at 600 °C to form strontium-containing calcium titanate (SrCT) and rutile on its surface. The treated metal did not form apatite in a simulated body fluid (SBF) even after 7 days. When the metal formed with SrCT was subsequently soaked in water at 80 °C, the treated metal formed bone-like apatite on its surface within 1 day in SBF since the Ca ions were partially replaced with H₃O⁺ ions. However, it released only 0.06 ppm of Sr ions even after 7 days in phosphate-buffered saline. When the metal was soaked after the heat treatment in 1 M SrCl₂ solution instead of water, the treated metal released 0.92 ppm of Sr ions within 7 days while maintaining its apatite-forming ability. The Ti formed with this kind of bioactive SrCT layer on its surface is expected to be highly useful for orthopedic and dental implants, since it should be able to promote bone growth by releasing Sr ions and tightly bond to the bone through the apatite formed on its surface.     

Acceleration of apatite nucleation on microrough bioactive titanium for bone-replacing implants

The viability of a new two-step method for obtaining bioactive microrough titanium surfaces for bone replacing implants has been evaluated. The method consists of (1) Grit blasting on titanium surface to roughen it; and (2) Thermo-chemical treating to obtain a bioactive surface with bone-bonding ability by means of nucleating and growing an apatite layer on the treated surface of the metal. The aim of this work is to evaluate the effect of surface roughness and chemical composition of the grit-blasting particles on the ability of the surfaces of nucleating and growing a homogeneous apatite layer. The determination and kinetics of the nucleation and growing of the apatite layer on the surfaces has mainly been studied with environmental scanning electron microscopy (ESEM) and grazing-incidence X-ray diffractometry. The results show that Al(2)O(3)-blasted and thermochemically-treated titanium surfaces accelerates nucleation of the apatite, whereas SiC-blasted and thermochemically-treated titanium surfaces inhibits apatite nucleation, compared with the well studied polished and thermochemically-treated titanium surfaces. The acceleration of the apatite nucleation on the Al(2)O(3)-blasted microrough titanium surfaces is because concave parts of the microroughness that are obtained during grit blasting provides to the rough and bioactive surfaces with a chemical- and electrostatic-favored situation for apatite nucleation. This consists of a high density of surface negative charges (also assisted by the nanoroughness of the surface obtained after the thermochemical treatment) and an increased concentration of the Ca(2+)-ions of the fluid, which have a limited mobility at the bottom of the concave parts.     

Effect of calcium salt content in the poly(epsilon-caprolactone)/silica nanocomposite on the nucleation and growth behavior of apatite layer

The effect of calcium salt content in the poly(epsilon-caprolactone) (PCL)/silica nanocomposite on the nucleation and growth behavior of apatite layer in simulated body fluid (SBF) was investigated. The specimens were prepared with low (L) and high (H) concentrations of calcium nitrate tetrahydrate through a sol-gel method. After soaking in the SBF at 36.5 degrees C for 1 week, a densely packed apatite layer that had a smooth surface and a Ca/P ratio similar to bone was formed on specimens containing a low concentration of calcium salt while a loosely packed apatite layer with a rugged surface and a higher Ca/P ratio than that of bone occurred on specimens containing a high concentration of calcium salt. The results are explained in terms of the degree of supersaturation of apatite in the SBF, as determined by the concentrations of constituent ions of apatite and pH. The practical implication of the results is that a dense and bone-like apatite layer on the PCL/silica nanocomposite in vitro, and perhaps in vivo, can be achieved by adopting an appropriate calcium salt content.