Hydroxyapatite Crystal Structure Research Articles

Cu/Gd co-doped hydroxyapatite/poly lactic-co-glycolic acid composites enhance MRI imaging and bone defect regeneration.

Background: The hydroxyapatite (HA)/poly(lactide-co-glycolide) acid (PLGA) composite material is a widely used orthopedic implant due to its excellent biocompatibility and plasticity. Recent advancements in cation doping have expanded its potential biological applications. However, conventional HA/PLGA composites are not visible under X-rays post-implantation and have limited osteogenic induction capabilities. Copper (Cu) is known to regulate osteoblast proliferation and differentiation, while gadolinium (Gd) can significantly enhance the magnetic resonance imaging (MRI) capabilities of materials. Methods: This study aimed to investigate whether incorporating Cu and Gd into an HA/PLGA composite could enhance the osteogenic properties, in vivo bone defect repair, and MRI characteristics. We prepared a Cu/Gd@HA/PLGA composite and assessed its performance. Results: Material characterization confirmed that Cu/Gd@HA retained the morphology and crystal structure of HA. The Cu/Gd@HA/PLGA composite exhibited excellent nuclear magnetic imaging capabilities, porosity, and hydrophilicity, which are conducive to cell adhesion and implant detection. In vitro experiments demonstrated that the Cu/Gd@HA/PLGA composite enhanced the proliferation, differentiation, and adhesion of MC3T3-E1 cells, and upregulated COL-1 and BMP-2 expression at both gene and protein levels. In vivo studies showed that the Cu/Gd@HA/PLGA composite maintained strong T1-weighted MRI signals and significantly improved the bone defect healing rate in rats. Conclusion: These findings indicate that the Cu/Gd@HA/PLGA composites significantly enhance T1-weighted MRI capabilities, promote osteoblast proliferation and differentiation in vitro, and accelerate bone defect healing in vivo.

Nickel-ion substituted hydroxyapatite matrices for metal-affinity chromatographic purification of recombinant proteins.

Hydroxyapatite (HAp) is a calcium phosphate ceramic, widely used as a matrix for protein chromatography. The crystal structure of HAp is amenable to a wide range of substitutions, thus allowing for the alteration of its properties. In this study, nickel-ion substituted HAp (NiSHAp) was synthesized using a wet-precipitation method, followed by spray drying. This resulted in the structural incorporation of nickel ions within well-defined microspheres, which were suitable for chromatographic applications. The chromatographic experiments were conducted with NiSHAp and compared with spray-dried hydroxyapatite (SHAp) matrices. Protein purification experiments were conducted using refolded recombinant L-asparaginase (L-Asp), which was produced as inclusion bodies in Escherichia coli. The results showed that NiSHAp effectively adsorbed L-Asp, which was selectively eluted using a phosphate buffer, surpassing the efficiency of imidazole-based elution. In contrast, SHAp showed weaker binding and lower selectivity. The significance of this study lies in developing a scalable NiSHAp matrix for protein purification, especially for large-scale applications. The NiSHAp matrix offers a cost-effective alternative to commercial immobilized metal affinity chromatography (IMAC) adsorbents, especially for purifying His-tagged proteins. This innovative approach exhibits the advantages of mixed-mode chromatography by combining the properties of hydroxyapatite and IMAC in a single matrix, with the potential of improved industrial-scale protein purification.

Specific Features of the Crystal Structure of Calcium Hydroxyapatite in Native Bone Tissue

Specific Features of the Crystal Structure of Calcium Hydroxyapatite in Native Bone Tissue

Hormonal and non-hormonal oral contraceptives given long-term to pubertal rats differently affect bone mass, quality and metabolism.

We investigated the effects of hormonal and non-hormonal oral contraceptives (OCs) on bone mass, mineralization, composition, mechanical properties, and metabolites in pubertal female SD rats. OCs were given for 3-, and 7 months at human equivalent doses. The combined hormonal contraceptive (CHC) was ethinyl estradiol and progestin, whereas the non-hormonal contraceptive (NHC) was ormeloxifene. MicroCT was used to assess bone microarchitecture and BMD. Bone formation and mineralization were assessed by static and dynamic histomorphometry. The 3-point bending test, nanoindentation, FTIR, and cyclic reference point indentation (cRPI) measured the changes in bone strength and material composition. Bone and serum metabolomes were studied to identify potential biomarkers of drug efficacy and safety and gain insight into the underlying mechanisms of action of the OCs. NHC increased bone mass in the femur metaphysis after 3 months, but the gain was lost after 7 months. After 7 months, both OCs decreased bone mass and deteriorated trabecular microarchitecture in the femur metaphysis and lumbar spine. Also, both OCs decreased the mineral: matrix ratio and increased the unmineralized matrix after 7 months. After 3 months, the OCs increased carbonate: phosphate and carbonate: amide I ratios, indicating a disordered hydroxyapatite crystal structure susceptible to resorption, but these changes mostly reversed after 7 months, indicating that the early changes contributed to demineralization at the later time. In the femur 3-point bending test, CHC reduced energy storage, resilience, and ultimate stress, indicating increased susceptibility to micro-damage and fracture, while NHC only decreased energy storage. In the cyclic loading test, both OCs decreased creep indentation distance, but CHC increased the average unloading slope, implying decreased microdamage risk and improved deformation resistance by the OCs. Thus, reduced bone mineralization by the OCs appears to affect bone mechanical properties under static loading, but not its cyclic loading ability. When compared to an age-matched control, after 7 months, CHC affected 24 metabolic pathways in bone and 9 in serum, whereas NHC altered 17 in bone and none in serum. 6 metabolites were common between the serum and bone of CHC rats, suggesting their potential as biomarkers of bone health in women taking CHC. Both OCs have adverse effects on various skeletal parameters, with CHC having a greater negative impact on bone strength.

Structural and biological evaluation of novel vanadium/Yttrium co-doped hydroxyapatite for bone tissue engineering applications

Hydroxyapatite (HA) is widely used in various medical fields due to its exceptional bioactivity, bio-affinity, and biocompatibility. However, the potential of biomaterials co-doped with metal ions for specific biomedical applications remains underexplored. In this study, we modified the HA structure by introducing vanadium (V) and yttrium (Y) to enhance its physicochemical and biological properties. V-Y co-doped HA (V-Y:HA) samples were prepared by the wet precipitation method using a reflux condensation setup with three different concentrations (0.005 M, 0.01 M, and 0.02 M) of V and Y. The addition of V and Y significantly influenced the biochemical properties and crystal structure of HA. X-ray diffraction (XRD) analysis revealed a modified crystal structure and the presence of the β-tricalcium phosphate (β -TCP) phase in the sample with a higher V concentration. V and Y incorporation resulted in smaller crystallite sizes and noticeable changes in lattice parameters. Scanning electron microscopy (SEM) images displayed sphere-like morphology with extensive aggregation. Fourier-transform infrared spectroscopy (FT-IR) and Raman analysis confirmed the presence of functional groups and phosphate vibrational modes in V-Y:HA samples. X-ray photoelectron spectroscopy (XPS) revealed the ionic and oxide forms of vanadium and the electronic states of yttrium. The as-prepared samples exhibited excellent mechanical strength, reduced porosity, and improved hemocompatibility compared to pure HA. The V-Y:HA sample with a higher V concentration demonstrated enhanced antibacterial activity against P. aeruginosa and S. aureus. In-vitro bioactivity assessment after 21 days revealed enhanced apatite formation on the surface of HA and V-Y:HA materials. These findings suggest that the novel V-Y:HA nanomaterials have great potential as biomaterials for bone tissue engineering and related applications.

Mn導入骨類似組成リン酸カルシウムセメントの創製

Amorphous calcium phosphate (ACP) is known as a precursor of hydroxyapatite (HAp) which is an inorganic main component of bone. The ACP gradually crystallizes to low crystalline apatite near human body temperature. If we introduce an active ingredient at the time of synthesis of ACP and we can introduce an ingredient effective for bone repair or an ingredient having functionality into the crystal structure of HAp, an active ingredient-releasing material responsive to bone metabolism would obtaine, and the protein might produce. Mn is an essential trace element in the living body and is present in the body as an active metal such as Mn-SOD (Mn-superoxmutase) having an antioxidant activity and enhancing the activity of proteoglycan synthetase during bone formation. In this research, we try to introduce Mn into ACP, prepare cement using the obtained powder. In the synthetic powder, Mn was introduced at the same rate as the preparation. The obtained cement had crystallized and low-crytalline bone-like apatite composition. The porosity changed with the amount of Mn added. The cements exhibited protein adsorption properties regardress of the addition of Mn. It is expected that proteins involved in bone metabolism adsorb to the cement.

Growth of hydroxyapatite plate-like nanoparticles by additive free precipitation for the deposition of aligned coatings

In recent years, nanoparticles' morphology has been tailored to tune nanoparticle specific properties for several applications. In particular, plate-like particles have been synthesized to create an analog of nacre microstructure exhibiting outstanding mechanical performance attributed to nacre's micro and nano hierarchy. However, the synthesis of equiaxed hydroxyapatite (HA) plates for constructing an oriented architecture has remained as an astounding challenge. In this study, we designed an additive-free chemical precipitation route in which the pH was increased stepwise to maintain the particles' equiaxed 2d shape. Our results showed that the produced particles exhibited HA's crystal structure and highly sustained the equiaxed/rectangular plate morphology of the octacalcium phosphate (OCP) phase, formed as an interphase during the precipitation. The reaction time, reactant concentration, and heat treatment were found essential in controlling the particles' morphology, size, and crystal symmetry. The crystal features of the particles were dissected via high-resolution scanning and transmission electron microscopes (HRSEM and HRTEM) and X-ray diffraction (XRD) analysis. Moreover, the Williamson-Hall plot and Rietveld Size-strain algorithm were implemented to calculate the crystallite size and strain of the particles. HRSEM and HRTEM micrographs showed that ultrathin (10–20 nm) 2d HA particles with mean lengths of 140–150 nm grew through the c-axis. In the last stage, we attempted to use the particles to deposit oriented coatings by electrophoretic deposition (EPD).

Bactericidal and in vitro osteogenic activity of nano sized cobalt-doped silicate hydroxyapatite

Hydroxyapatite (HA) particles with enhanced antibacterial properties can be prepared by integrating metal ions into the crystal structure of the nanoparticles. Cobalt and silicate ions containing HA (Co/Si-HA) with the formula Ca10-xCox(PO4)6-y(SiO4)y(OH)2 (x = 0.2, 0.6, and 1.0 and y = 0.5) was successfully synthesised by using microwave-assisted wet precipitation method. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and inductively coupled plasma mass spectrometry (ICP-MS) techniques were used to characterise the synthesised nanoparticles. The results revealed that the incorporation of SiO44− ions increased the lattice parameters and decreased the crystallite size of HA. However, the incorporation of Co2+ions led to the reduction of lattice parameters and the particle size of the SiHA nanoparticles. In vitro antibacterial activity of materials was evaluated using disk diffusion and minimum inhibitory concentration (MIC) protocols. The findings indicated that incorporating Co2+ ions into SiHA inhibited the growth of Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus). The cytotoxicity of materials evaluated using the Sarcoma osteogenic (Saos-2) cell line revealed that they were cytocompatible and exhibited no adverse side effects. The osteogenic differentiation of cells was confirmed by the significant increase in the alkaline phosphatase (ALP) activity by incorporating Co2+/SiO44− ions into the HA crystal structure. Our results show that the nanoparticles prepared in this study have a promising future in biomaterial-tissue engineering applications.

Surface and structural characterization of Cu-exchanged hydroxyapatites and their application in H2O2 electrocatalytic reduction

We report on the preparation of hydroxyapatite (HA) nanoparticles with high specific surface area, terminated with calcium-rich {010} facets, and their subsequent surface functionalization with copper by cationic exchange with copper nitrate solutions at different concentrations. Elemental analysis highlighted a progressive increase of the amount of copper incorporated into the HA, reaching a maximum of ∼ 7 wt%. A combined X-ray diffraction and solid-state NMR investigation showed no significant structural differences after the Cu functionalization, confirming that the copper exchange occurs mainly at the surface until saturation and, for the higher Cu concentrations, also in the sub-surface/bulk layers of the material, without altering the HA crystal structure. The gradual substitution of surface Ca2+ by Cu2+ was studied also by IR spectroscopy using carbon monoxide as probe molecule. Finally, we assessed the catalytic activity of the materials testing the electrochemical reduction of H2O2 by cyclic voltammetry. We observed a progressive increase in catalytic activity correlated with the amount of Cu, suggesting the possible application of copper-exchanged HA as electrochemical H2O2 sensors.

Dental caries diagnosis using terahertz spectroscopy and birefringence.

Dental caries is a widespread chronic infectious disease which may induce a series of oral and general problems if untreated. As a result, early diagnosis and follow-up following radiation-free dental caries therapy are critical. Terahertz (THz) waves with highly penetrating and non-ionizing properties are ideally suited for dental caries diagnosis, however related research in this area is still in its infancy. Here, we successfully observe the existence of THz birefringence phenomenon in enamel and demonstrate the feasibility of utilizing THz spectroscopy and birefringence to realize caries diagnosis. By comparing THz responses between healthy teeth and caries, the transmitted THz signals in caries are evidently reduced. Concomitantly, the THz birefringence is also unambiguously inhibited when caries occurs due to the destruction of the internal hydroxyapatite crystal structure. This THz anisotropic activity is position-dependent, which can be qualitatively understood by optical microscopic imaging of dental structures. To increase the accuracy of THz technology in detecting dental caries and stimulate the development of THz caries instruments, the presence of significant THz birefringence effect induced anisotropy in enamel, in combination with the strong THz attenuation at the caries, may be used as a new tool for caries diagnosis.

In vitro bioactivity of 3D microstructure hydroxyapatite/collagen based-egg white as an antibacterial agent.

The present study aims to design 3D scaffold hydroxyapatite (HA)/collagen (Coll) based egg-white (EW) as antibacterial properties. The calcium source in HA synthesis derived from the Pinctada maxima shell cultivated on Bali Island has proven biocompatibility, and the compressive strength exceeded human bone. HA synthesis by precipitation with heat treatment in oven-dried at 80°C (HA-80) and annealed at 900°C (HA-900), has crystallinity 48% and 85%, respectively, were used for scaffold design. The physicochemical properties of X-ray diffractometer spectra showed that increasing temperature affected the crystallinity and HA phase formed. Furthermore, the crystal structure of HA changed in nanocomposite due to the substitution of Coll and EW, and the Fourier transform infrared spectroscopy spectra confirmed that the absorption peak of the phosphate group (1027-1029 cm-1 ) decreased intensity, presumably by protein binding of EW and Coll. The cell viability of HA/Coll/EW in 24, 48, and 72 h incubation period was 112.34 ± 4.36, 104.89 ± 3.41, 72.88 ± 6.85, respectively. The decreases of cell viability due to high cell density and reduced nutrients in wells. Antibacterial activity of HA/Col/EW exhibited a strong zone of inhibition against bacteria causing periodontitis; Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Staphylococcus aureus.

Anisotropic expansion effect of Sr doping on the crystal structure of hydroxyapatite

The anisotropic expansion effect of Sr on the HA crystal structure is proposed where the relative expansion rate in the c-axis direction is about 2.22 times that in the a-axis direction.

Complete chemical and structural characterization of selenium-incorporated hydroxyapatite

Hydroxyapatite (HAp) has long been used as synthetic bone tissue replacement material. Recent advances in this area have led to development of dual-functional bioceramics exhibiting high biocompability/osteoconductivity together with the therapeutic effect. Selenium, in that respect, is an effective therapeutic agent with promising antioxidant activity and anticancer effects. In this study, selenium-incorporated hydroxyapatite (HAp:Se) particles have been synthesized by modified aqueous precipitation method using calcium (Ca(NO3)2·4H2O) and phosphate ((NH4)2HPO4) salts and sodium selenite (Na2SeO3). The effects of selenium incorporation and post-synthesis calcination treatment (900–1100 °C) on physical, chemical properties and crystal structure of resultant HAp powders have been investigated. Complete chemical identification was performed with spectroscopical analyses including Fourier transform infrared and x-ray photoelectron spectroscopy to elucidate the mechanism and chemical nature of selenium incorporation in HAp. Meanwhile, detailed x-ray diffraction studies by Rietveld refinement have conducted to explain changes in the HAp crystal structure upon selenium incorporation.

Investigation on structural properties and bioactivity of nanosized biphasic calcium phosphate

Synthetic biphasic bioceramics composed of hydroxyapatite (HA) and other calcium phosphates (CPs) can provide promising efficiency in the treatment of bone defects based on the rapid dissolution of the CPs, to allow its replacement by freshly formed bone, along with the slow resorption of the HA, to preserve the volume of the grafted area. For this purpose, the present study was conducted to prepare nanosized biphasic calcium phosphate (n-BCP) using a facile mechanochemical process. The experimental outputs were also compared to the commercial-grade HA in terms of physicochemical features. The Rietveld refinement was utilized to calculate the phase contents and crystal structures of the composites, including crystallite size, lattice parameters, and unit cell volume. Besides, the atomic arrangement of Ca1, Ca2, PO43−, and OH− groups in the hexagonal crystal structure of HA and triclinic structure of anhydrous dicalcium phosphate (DCPA) was determined. The phase fraction, crystallite size, and the powder density of the biphasic structures, which were derived by Rietveld refinement, were found to be affected by ball-milling. In addition to biphasic structures, a monolithic hexagonal HA with an isotropic crystal growth was formed after 7 h of ball-milling under an argon atmosphere. The in vitro test in a simulated body fluid (SBF) confirmed the bioactivity of the biphasic structure through the formation of a bone-like apatite layer after one week.

Adsorption of the Tartrate Ions in the Hydroxyapatite/Aqueous Solution of NaCl System

The research on the interaction of tartrate ions with the surface of hydroskyapatite was presented, including the measurements of the kinetics of tartrate ion adsorption and tartrate ion adsorption as a function of pH. The adsorption of tartrate ions was calculated from the loss of tartrate concentration in the solution as measured by a radioisotope method using C-14 labeled tartaric acid. In order to explain the mechanism of interaction of tartrate ions with hydroxyapatite, supplementary measurements were carried out, i.e., potentiometric measurements of the balance of released/consumed ions in the hydroxyapatite/electrolyte solution system, zeta potential measurements, FTIR spectrophotometric measurements and the hydroxyapatite crystal structure and particle size distribution were characterized. It was found that the adsorption of tartrate ions occurs as a result of the exchange of these ions with hydroxyl, phosphate and carbonate ions. Replacing the ions with the abovementioned tartrate ions leads to the appearance of a negative charge on the surface of the hydroxapatite. On the basis of XRD study and particle size distribution, a decrease in the size of crystallites and the diameter of hydroxyapatite particles in contact with a solution of 0.001 mol/dm3 of tartaric acid was found.

Effect of Carbonate to Phosphate Molar Ratios on the Physico-Chemical Properties of Carbonated Hydroxyapatite Nanopowder

The aim of this study was to incorporate carbonate ions (CO3 2–) into the hydroxyapatite (HA) crystal structure followed by investigation on the effect of different carbonate to phosphate (CO3 2–/PO4 3–) ratios on the phase purity, crystal structure as well as CO3 2– content present in the apatite structure. CO3 2– substitution has been proposed to enhance the performance of HA-based material, particularly on the physico-chemical properties. Three different compositions of carbonated hydroxyapatite (CHA) powder with different CO3 2–/ PO4 3– ratios (namely, CHA 1:1, CHA 2:1 and CHA 4:1) were chemically synthesised by nanoemulsion method at 37°C and characterised for their physico-chemical properties. Results demonstrated that all as-synthesised powders formed single phase B-type CHA without any additional phases. Interestingly, an increasing amount of CO3 2– substituted into the apatite structure gives rise to the formation of CHA structure with a variation on their cell parameters and the degree of crystallinity. An increase in the CO3 2–/ PO4 3– ratio was also found to lead a higher amount of CO3 2– content present in the as-synthesised powder (in a range of 4 wt % to 10 wt %), which is comparable to the CO3 2– content found in the human bone mineral.

Strontium and silicate co-substituted hydroxyapatite: Mechanochemical synthesis and structural characterization

Synthetic hydroxyapatite is a bioceramic material promising for medical applications. The introduction of substituents into the crystal structure of hydroxyapatite allows tailoring its properties and obtaining materials with improved implantation characteristics. In the present work, it was shown for the first time that silicate and strontium co-substituted hydroxyapatite can be synthesized via the “soft” mechanochemical method. The synthesis was carried out by subjecting the reaction mixture to ball milling in the absence of any organic surfactant or solvent. The product of the mechanochemical synthesis was a single-phase material with a nanocrystalline structure. The crystal structure of co-substituted hydroxyapatite Ca9.5Sr0.5(PO4)5.5(SiO4)0.5(OH)1.5 was analyzed for the first time. The synthesized co-substituted hydroxyapatite does not require any further processing or treatment and can be used as a biomaterial in the as-synthesized state.

Controlling Enamel Remineralization by Amyloid-Like Amelogenin Mimics.

In situ regeneration of the enamel-like structure of hydroxyapatite (HAp) crystals under oral conditions is significant for dental caries treatment. However, it is still a challenge for dentists to duplicate the elegant and well-aligned apatite structure bonding to the surface of demineralized enamel. A biocompatible amelogenin-inspired matrix, a phase-transited lysozyme (PTL) film mimicking an N-terminal amelogenin with central domain (N-Ame) combined with synthetic peptide (C-AMG) based on the functional domains of C-terminal telopeptide (C-Ame) is shown here, which is formed by amyloid-like lysozyme aggregation at the enamel interface through a rapid one-step aqueous coating process. In the PTL/C-AMG matrix, C-AMG facilitated the oriented arrangement of amorphous calcium phosphate (ACP) nanoparticles and their transformation to ordered enamel-like HAp crystals, while PTL served as a strong interfacial anchor to immobilize the C-AMG peptide and PTL/C-AMG matrix on versatile substrate surfaces. PTL/C-AMG film-coated enamel induced both of the in vivo and in vitro synthesis of HAp crystals, facilitated epitaxial growth of HAp crystals and recovered the highly oriented structure and mechanical properties to levels nearly identical to those of natural enamel. This work underlines the importance of amyloid-like protein aggregates in the biomineralization of enamel, providing a promising strategy for treating dental caries.

Characterization of Nano-Scale Hydroxyapatite Coating Synthesized from Eggshells Through Hydrothermal Reaction on Commercially Pure Titanium

Commercially pure titanium (c.p. Ti) is often used in biomedical implants, but its surface cannot usually combine with the living bone. A coating of hydroxyapatite (HA) on the surface of titanium implants provides excellent mechanical properties and has good biological activity and biocompatibility. For optimal osteocompatibility, the structure, size, and composition of HA crystals should be closer to those of biological apatite. Our results show that the surface of c.p. Ti was entirely covered by rod-like HA nanoparticles after alkali treatment and subsequent hydrothermal treatment at 150 °C for 48 h. Nano-sized apatite aggregates began to nucleate on HA-coated c.p. Ti surfaces after immersion in simulated body fluid (SBF) for 6 h, while no obvious precipitation was found on the uncoated sample. Higher apatite-forming ability (bioactivity) could be acquired by the samples after HA coating. The HA coating featured bone-like nanostructure, high crystallinity, and carbonate substitution. It can be expected that HA coatings synthesized from eggshells on c.p. Ti through a hydrothermal reaction could be used in dental implant applications in the future.

Development Carbonated Hydroxyapatite Powders from Oyster Shells (<i>Crassostrea gigas</i>) by Carbonate Content Variations

Carbonated hydroxyapatite (CHAp) is an inorganic mineral that more closely resembles the main component of composing human hard tissue in 2-8 wt% carbonate content. CHAp powders have been synthesized from oyster shells using the precipitation method. Oyster shells are one type of shellfish from the bivalve class which is rich in calcium carbonate content. In this research, CaO from oyster shells obtained from the decomposition process of CaCO3 was used as a source of calcium and diammonium hydrogen phosphate and ammonium bicarbonate as well as a precursor of phosphate and carbonate, respectively. In addition, carbonate content variations were x = 0, 0.3, 0.8 and 1.2 which were characterized by fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), and scanning electron microscope-energy dispersive X-Ray (SEM-EDX) to determine the functional groups, crystallographic properties, morphology and Ca/P molar ratio, respectively. Carbonate ion substitution in the hydroxyapatite crystal structure is known to decrease crystallinity and crystallite size. The theory is in accordance with the results obtained in this study with the crystallite size is 74.322, 46.933, 37.727, and 31.499 nm for 0.95, 2.7, 5.7, and 9.35 wt.% carbonate content, respectively.