Search

Modern metallurgical and smelting activities discharge the lead-containing wastewater, causing serious threats to human health. Bacteria and urease applied to microbial-induced carbonate precipitation (MICP) and enzyme-induced carbonate precipitation (EICP) are denatured under high Pb 2+ concentration. The nano-hydroxyapatite (nHAP)-assisted biomineralization technology was applied in this study for Pb immobilization. Results showed that the extracellular polymers and cell membranes failed to secure the urease activity when subjected to 60 mM Pb 2+ . The immobilization efficiency dropped to below 50% under MICP, whereas it due to a lack of extracellular polymers and cell membranes dropped to below 30% under EICP. nHAP prevented the attachment of Pb 2+ either through competing with bacteria and urease or promoting Ca 2+ /Pb 2+ ion exchange. Furthermore, CO 3 2- from ureolysis replaced the hydroxyl (-OH) in hydroxylpyromorphite to encourage the formation of carbonate-bearing hydroxylpyromorphite of higher stability (Pb 10 (PO 4 ) 6 CO 3 ). Moreover, nHAP application overcame an inability to provide nucleation sites by urease. As a result, the immobilization efficiency, when subjected to 60 mM Pb 2+ , elevated to above 80% under MICP-nHAP and to some 70% under EICP-nHAP. The findings highlight the potential of applying the nHAP-assisted biomineralization technology to Pb-containing water bodies remediation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hydroxyapatite (HA) is a major component of the inorganic minerals in the hard tissues of humans and has been widely used as a biomedical ceramic material in orthopedic and dentistry applications. Because human bone contains several impurities, including carbonates, chlorides, fluorides, magnesium, and strontium, human bone minerals differ from stoichiometric HA. Additionally, natural bone is composed of nano-sized HA, and the nanoscale particles exhibit a high level of biological activity. In this paper, HA is prepared via the hydrothermal process because its reaction conditions are easy to control and it has been shown to be quite feasible for large-scale production. Therefore, the hydrothermal process is an effective and convenient method for the preparation of HA. Furthermore, eggshell is adopted as a source of calcium, and mulberry leaf extract is selectively added to synthesize HA. The eggshell accounts for 11% of the total weight of a whole egg, and it consists of calcium carbonate, calcium phosphate, magnesium carbonate, and organic matter. Eggshell contains a variety of trace elements, such as magnesium and strontium, making the composition of the synthesized HA similar to that of the human skeleton. These trace elements exert considerable benefits for bone growth. Moreover, the use of eggshell as a raw material can permit the recycling of biowaste and a reduction in process costs. The purpose of this study is to prepare HA powder via the hydrothermal method and to explore the effects of hydrothermal conditions on the structure and properties of the synthesized HA. The room-temperature precipitation method is used for the control group. Furthermore, the results of an immersion test in simulated body fluid confirm that the as-prepared HA exhibits good apatite-forming bioactivity, which is an essential requirement for artificial materials to bond to living bones in the living body and promote bone regeneration. In particular, it is confirmed that the HA synthesized with the addition of the mulberry leaf extract exhibits good in vitro biocompatibility. The morphology, crystallite size, and composition of the carbonated nano-HA obtained herein are similar to those of natural bones. The carbonated nano-HA appears to be an excellent material for bioresorbable bone substitutes or drug delivery. Therefore, the nano-HA powder prepared in this study has great potential in biomedical applications.

Highly nanocrystalline carbonated hydroxyapatite (CHAp) is synthesized by hydrothermal technique with four different stoichiometric compositions for microstructural and mechanical analysis. HAp is one of the most biocompatible material and addition of carbonate ions lead to increase in fracture toughness highly required in biomedical applications. The structural properties and its purity as single phase is confirmed by X-ray diffraction. Lattice imperfections and structural defects is investigated using XRD pattern model simulation, i.e. Rietveld's analysis. The substitution of CO 3 2- in HAp structure leads to a decrease in crystallinity which ultimately lessens crystallite size of sample as verified by XRD analysis. FE-SEM micrographs confirms the formation of nanorods with cuboidal morphology and porous structure of HAp and CHAp samples. The particle size distribution histogram validates the constant decrease in size due to carbonate addition. The mechanical testing of prepared samples revealed the increase in mechanical strength from 6.12 MPa to 11.52 MPa due to the addition of carbonate content, which leads to a rise in fracture toughness, a significant property of an implant material from 2.93 kN to 4.22 kN. The cumulative effect of CO 3 2- substitution on HAp structure and mechanical properties has been generalized for the application as biomedical implant material or biomedical smart materials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hydroxyapatite (HA) has been commonly used as an alternative bone substitute. But it has drawbacks, such as poor degradation and limited osteogenesis. Low-crystalline carbonated hydroxyapatite (L-CHA), which has greater biodegradability than HA, is suggested as one of the main components of bone minerals, but the exact mechanism behind the roles of carbonate substituted in biological behaviors of low-crystalline HA is still a mystery. In this study, L-CHAs with different carbonate contents were prepared, and the effects of the content on the physicochemical properties, in vitro cytological responses, and in vivo bone defects repair effects of L-CHAs were investigated. The results demonstrated that CO 3 2- had successfully entered the lattice structure of L-CHAs with a maximum content of 9.2 wt %. Both low-crystalline undoped HA (L-HA) and L-CHAs were nanocrystalline (20-30 nm) with significantly higher specific surface areas, protein adsorption capacities, and biodegradability compared to high-crystalline HA (H-HA) with submicron crystalline size (200-400 nm). Besides, the amounts of the adsorbed protein and released Ca 2+ ions increased in a carbonate-content-dependent manner. Compared to L-HA and H-HA, L-CHAs promoted the adhesion and proliferation of bone marrow mesenchymal stem cells and significantly upregulated the levels of alkaline phosphatase (ALP) activity and the expression of osteogenesis-related genes. In addition, L-CHA-9 not only showed a faster biodegradation rate but also effectively promoted bone regeneration when implanted in the critical-sized bone defects of rabbit femora. This study provided evidence for the development of L-CHA as a promising biodegradable and bioactive material with great osteoconductivity and osteogenic capability with respect to conventional HA.

Hydroxyapatite (HA) is a complex material, which is often nanocrystalline when found within a biological setting. This work has directly compared the structural characteristics derived from data collected using a conventional laboratory-based X-ray diffractometer with those collected from a dedicated pair distribution function (PDF) beamline at Diamond Light Source. In particular, the application of PDF analysis methods to carbonated HA is evaluated. 20 synthetic samples were measured using both X-ray diffraction (XRD) and PDFs. Both Rietveld refinement (of laboratory XRD data) and real-space refinement (of PDF data) were used to analyse all samples. The results of Rietveld and real-space refinements were compared to evaluate their application to crystalline and nanocrystalline hydroxyapatite. Significant relationships were observed between real-space refinement parameters and increasing carbonate substitution. Understanding the local order of synthetic hydroxyapatite can benefit several fields, including both biomedical and clinical settings., (open access.)

Synthetic biomimetic carbonated hydroxyapatite (CHA) has shown significant promise in bone tissue engineering for its mechanical and chemical biocompatibility and osteogenic potential. Variations in the size of hydroxyapatite particles have also been shown to contribute to the hydroxyapatite's osteogenic success. However, synthesizing biomimetic CHA with optimal osteogenic properties using a simple synthesis methodology to make highly reproducible, biomimetic, and osteogenic CHA has not been evaluated fully. The objective of this study was to synthesize submicron CHA particles using a nanoemulsion method. We hypothesized that by varying the synthesis technique we could control particle size while still creating highly biomimetic CHA typically produced during nanoemulsion synthesis. Furthermore, we hypothesized that 500 nm CHA particles would induce greater osteoblastic differentiation compared to larger or smaller CHA particles. X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and dynamic light scattering were used to characterize the chemical composition, shape, and size of CHA synthesized through variations in pH, temperature and stirring speed during synthesis. Manipulation of pH showed the ability to selectively tailor CHA particle size from 200-900 nm in a reproducible manner while maintaining the chemical composition. In addition, 500 nm particles elicited the most rapid increase in osteoblastic differentiation and did not decrease cell viability compared to 200 and 900 nm particles., (© 2021 Wiley Periodicals LLC.)

In daily clinical practice, there is a notable variety of synthetic bone substitute, with various resorption rates, different chemical and structural characteristics that influence on bone regeneration and are not suitable for every clinical use. New biomaterials based on nanotechnology have been developed to be bioabsorbable as new bone is formed. This study intends to evaluate THP-1 cell viability when exposed to extracts of unsintered nanostructured carbonated hydroxyapatite (cHA) microspheres processed at 5 and 37°C compared to sintered hydroxyapatite processed at 90°C. cHA shows, in previous studies, biocompatibility, and better bioabsorption rates, consequently, improve the deposition of new bone and tissue repair. The results demonstrated that the tested biomaterials did not activate inflammatory role through THP-1 cells and did not affect activated macrophages independently of their crystallinities, suggesting their safety and biocompatibility. These results are of fundamental importance for the advancement of research on smart materials, especially in what controls the effect of nanostructured cHA microspheres in the biological environment, seems to be a promising biomaterial in clinical application on regenerative medicine., (© 2020 Wiley Periodicals LLC.)

A scaffold that mimics physicochemical structure of nerve and supplies calcium ions in axonal environment is an attractive alternative for nerve regeneration, especially when applied in critical nerve defect. Various scaffold material, design, including their combination with several growth-induced substances and cells application have been being investigated and used in the area of nerve tissue engineering. However, the development remains challenges today because they are still far from ideal concerning their stability, reproducibility, including complicated handling related to the poor mechanical strength. In view of the current basis, in this study, the introduction of carbonated hydroxyapatite (CHA) as promising candidate to increase mechanical properties of nerve scaffold is reported. The incorporation of CHA was not only expected to provide better mechanical properties of the scaffold. Under physiological condition, CHA is known to be the most stable phases of calcium phosphate compound. Therefore, CHA was expected to provide controlled release calcium for better axonal environment and promote fasten nerve regeneration. This study shows that CHA incorporated gelatin membrane has ideal microstructure to prevent fibrous tissue ingrowth into the injury site, while retaining its capability to survive nerve tissue by allowing adequate glucose and specific proteins diffusion. The provided Ca 2+ release to the environment promoted neuronal growth, without suppressing acetylcholine esterase release activity. Neurite elongation was dramatically higher in the gelatin membrane incorporated with CHA. Introduction of CHA into gelatin membrane represents a new generation medical device for nerve reconstruction, with CHA was considered as a promising factor., (© 2020 Wiley Periodicals, Inc.)

In this present study, in vitro characterization on bioactivity and biocompatibility of the copper-doped hydroxyapatite (Cu-HA) coatings deposited onto Ti6Al4V alloy with increasing copper content obtained using solution precursor plasma spray process were evaluated under simulated body fluid (SBF) and cytotoxicity tests. The growth of flake-like structures was observed at the coatings' surface after 7 days of immersion in SBF. Elemental composition analysis showed a calcium-deficient carbonate-containing apatite for the grown layer. Broadening of the HA peaks were also observed in the X-ray diffraction patterns and infrared absorption spectra of the immersed coatings associated with the possible formation of an amorphous layer at the surface of the coatings. Copper incorporation does not alter the bioactivity of HA but only slowed down for 10 mol% copper content. Cytotoxicity evaluation using MTT assays showed that 3 mol% Cu-HA coating was not toxic under in vitro characterizations., (© 2020 Wiley Periodicals, Inc.)

The necessity for finding a compromise between mechanical and biological properties of biomaterials spurs the investigation of the new methods to control and optimize scaffold processing for tissue engineering applications. A scaffold composed of ε-polycaprolactone fibers reinforced with carbonated hydroxyapatite (CHAP) dually doped with selenite oxyanions (Se) and cationic gold (Au) was synthesized using the electrospinning technique and studied at different contents of Au. Despite the fact that the amount of the Au dopant was relatively low, variations to it induced significant microstructural changes, affecting the cell response and mechanical properties in return. Au nanoparticles segregated as a separate, ternary phase at the highest Au content, corresponding to x = 0.8 in the Au x Ca 10-1.5x (PO 4 ) 5.8 (SeO 2 ) 0.2-x (CO 3 ) x (OH) 2 stoichiometric formula of Au/Se-CHAP. Their appearance coincided with a rapid degeneration in the density and adhesion of osteoblastic cells grown on the scaffolds. In spite of this adverse effect, the cell spreading and proliferation improved with increasing the amount of the Au dopant in the Au/Se-CHAP particles of the scaffold in the x = 0.0-0.6 range, suggesting that the biological effects of Au in the ionic and in the nanoparticulate form on the implant integration process may be diametrically opposite. The addition of Au had a dramatic effect on some mechanical properties, such as toughness and strain at break, which were both reduced twice upon the introduction of Au into Se-CHAP at the lowest amount (x = 0.2) compared to the Au-free composite. The significant variation of physical and biological properties of these composite scaffolds with trace changes in the content of the Au dopant inside the ceramic filler particles is promising, as it provides a new, relatively subtle avenue for tailoring the properties of tissue engineering scaffolds for their intended biomedical applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Synthesis of hydroxyapatite coated activated carbon nanocomposite was carried out by in-situ chemical precipitation method. Different characterizations confirm that, hydroxyapatite successfully coated over activated carbon powder. Extensive sorption studies of U(VI) on the nanocomposite were conducted to know the effect of contact time, humic acid, carbonate, ionic strength and pH. The study revealed that, the composite material is a more efficient sorbent for U(VI) compared to precursors, which removes U(VI) ion without altering physicochemical properties of water. Sorption exhibits multilayer adsorption on heterogeneous surface and follows chemisorptions. Practical applicability of the material was demonsteted by spiking tap water with U(VI) ion at three different initial concentrations (50, 100 and 150 µg L -1 ) and the tap water was allowed to passed through a cartridge packed with composite. It was observed that, the concentration of U(VI) ion in eluted water reduced to 98.28%, 96.20% and 97.40%, respectively. This revealed that, the material possesses a huge potential for sequestrating dissolved U(VI) ion and can be used as alternate filtering material for dissolved U(VI) in complex natural water system.

Background and objective: Tetracycline and its derivatives, combined with calcium phosphates, have been proposed as a delivery system to control inflammatory processes and chronic infections. The objective of this study was to evaluate the microspheres of alginate encapsulated minocycline-loaded nanocrystalline carbonated hydroxyapatite (CHAMINO) as a biomimetic device to carry out target-controlled drug delivery for alveolar bone repair. Methods: CHAMINO microspheres were implanted in a rat central incisor socket after 7 and 42 days. New bone was formed in both groups between 7 and 42 days of implantation. However, the bone growth was significantly higher for the CHAMINO microspheres. Results: The minocycline (MINO) loading capacity of the nanocrystaline carbonated hydroxyapatite (CHA) nanoparticles was 25.1±2.2 µg MINO/mg CHA for adsorption over 24 hrs. The alginate microspheres containing minocycline-loaded CHA were biologically active and inhibited the Enterococcus faecalis culture growth for up to seven days of the MINO release. An osteoblastic cell viability assay based on the resazurin reduction was conducted after the cells were exposed to the CHAMINO powder and CHAMINO microspheres. Thus, it was found that the alginate extracts encapsulated the minocycline-loaded CHA microspheres and did not affect the osteoblastic cell viability, while the minocycline-doped CHA powder reduced the cell viability by 90%. Conclusion: This study concluded that the alginate microspheres encapsulating the minocycline-loaded nanocrystalline carbonated hydroxyapatite exhibited combined antibacterial activity against Enterococcus faecalis with cytocompatibility and osteoconduction properties. The significant improvement in the new bone formation after 42 days of implantation suggests that the CHAMINO microsphere has potential in clinical applications of bone regeneration., Competing Interests: The authors report no conflicts of interest in this work.

Background: Zinc-doped hydroxyapatite has been proposed as a graft biomaterial for bone regeneration. However, the effect of zinc on osteoconductivity is still controversial, since the release and resorption of calcium, phosphorus, and zinc in graft-implanted defects have rarely been studied. Methods: Microspheres containing alginate and either non-doped carbonated hydroxyapatite (cHA) or nanocrystalline 3.2 wt% zinc-doped cHA (Zn-cHA) were implanted in critical-sized calvarial defects in Wistar rats for 1, 3, and 6 months. Histological and histomorphometric analyses were performed to evaluate the volume density of newly formed bone, residual biomaterial, and connective tissue formation. Biomaterial degradation was characterized by transmission electron microscopy (TEM) and synchrotron radiation-based X-ray microfluorescence (SR-µXRF), which enabled the elemental mapping of calcium, phosphorus, and zinc on the microsphere-implanted defects at 6 months post-implantation. Results: The bone repair was limited to regions close to the preexistent bone, whereas connective tissue occupied the major part of the defect. Moreover, no significant difference in the amount of new bone formed was found between the two microsphere groups. TEM analysis revealed the degradation of the outer microsphere surface with detachment of the nanoparticle aggregates. According to SR-µXRF, both types of microspheres released high amounts of calcium, phosphorus, and zinc, distributed throughout the defective region. The cHA microsphere surface strongly adsorbed the zinc from organic constituents of the biological fluid, and phosphorus was resorbed more quickly than calcium. In the Zn-cHA group, zinc and calcium had similar release profiles, indicating a stoichiometric dissolution of these elements and non-preferential zinc resorption. Conclusions: The nanometric size of cHA and Zn-cHA was a decisive factor in accelerating the in vivo availability of calcium and zinc. The high calcium and zinc accumulation in the defect, which was not cleared by the biological medium, played a critical role in inhibiting osteoconduction and thus impairing bone repair., Competing Interests: The authors report no conflicts of interest in this work.

In this study, iron oxide nanoparticles (IO-NPs) with a mean diameter of 102.85 nm were firstly synthesized via a facile green route using Ulva spp. aqueous extract as a bioreductant agent. Then, IO-NPs were loaded into carbonated hydroxyapatite (c-Hap) and the final product was named as the iron oxide nanoparticles loaded carbonated hydroxyapatite (IO-NPs-Lc-Hap). Subsequently, IO-NPs-Lc-Hap was characterized by FT-IR, SEM, XRD and EDX analysis methods. MG colour removal efficiencies of Ulva spp., Hap, IO-NPs and IO-NPs-Lc-Hap materials were also evaluated by adsorption and/or Fenton-like reaction methods. IO-NPs-Lc-Hap with the highest decolourization capacity was chosen as a heterogeneous Fenton-like catalyst for Malachite Green (MG). For Fenton-like decolourization of MG, the optimum H 2 O 2 concentration, initial dye concentration and catalyst concentration were determined to be 30 mM, 100 mg/L and 1.0 g/L, respectively. At these optimum conditions, 100% decolourization efficiency and 33.3% COD removal were obtained. On the other hand, 94% decolourization efficiency and 42% COD removal were achieved for the real textile wastewater at the obtained optimum conditions. The experimental decolourization reaction rate for MG was determined as - r d = 0.0779 [(mg dye0 .3 ) (g cat -0.3 ) (min -1 )] × q t 0 .7 . Also, the catalyst had high decolourization efficiencies at the end of six sequence usages.

In this work, we evaluate the tissue response and tolerance to a designed 3D porous scaffold composed of nanocrystalline carbonate-hydroxyapatite and agarose as a preliminary step in bone repair and regeneration. These scaffolds were subcutaneously implanted into rats, which were sacrificed at different times. CD4+, CD8+ and ED1+ cells were evaluated as measurements of inflammatory reaction and tolerance. We observed some inflammatory response early after subcutaneous implantation. The 3D interconnected porosity increased scaffold integration via the formation of granulation tissue and the generation of a fibrous capsule around the scaffold. The capsule is initially formed by collagen which progressively invades the scaffold, creating a network that supports the settlement of connective tissue and generating a compact structure. The timing of the appearance of CD4+ and CD8+ cell populations is in agreement with the resolved inflammatory response. The appearance of macrophage activity evidences a slow and gradual degradation activity. Degradation started with the agarose component of the scaffold, but the nano-apatite was kept intact for up to 30 days. Therefore, this apatite/agarose scaffold showed a high capacity for integration by a connective network that stabilizes the scaffold and results in slow nano-apatite degradation. The fundamental properties of the scaffold would provide mechanical support and facilitate bone mobilization, which is of great importance in the masticatory system or large bones.

Background: Optimal osseointegration has been recognized as a pivotal factor in determining the long-term success of biomedical implants., Materials and Methods: In the current study, highly carbonated hydroxyapatite (CHA) with carbonate contents of 8, 12 and 16 wt% and pure hydroxyapatite (HA) were fabricated via a novel hydrothermal method and deposited on the titanium substrates to generate corresponding CHA bioceramic coatings (designated as C8, C12 and C16, respectively) and HA bioceramic coatings (designated as C0)., Results: C8, C12 and C16 were endowed with nanoscale, hierarchical hybrid micro-/nanoscale and microscale surface topographies with rod-like superstructures, respectively. Compared with C0, the micro-/nanotextured CHA bioceramic coatings (C8, C12 and C16) possessed excellent surface bioactivity and biocompatibility, as well as better wettability, which mediated improved protein adsorption, giving rise to simultaneous enhancement of a biological cascade of events of rat bone-marrow-derived mesenchymal stem cells including cell adhesion, proliferation, osteogenic differentiation and, notably, the production of the pro-angiogenic growth factor, vascular endothelial growth factor-A. In particular, C12 with biomimetic hierarchical hybrid micro-/nanorod topography exhibited superior fractal property and predominant performance of protein adsorption, cell adhesion, proliferation and osteogenesis concomitant with angiogenesis., Conclusion: All these results suggest that the 12 wt% CHA bioceramic coating with synergistic modification of surface chemistry and topography has great prospect for future use as implant coating to achieve optimum osseointegration for orthopedic and dental applications., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The development of new dosimeters with good dosimetric properties is important for quality control in radiation applications. A new practical electron spin resonance (ESR) dosimeter based on carbonated hydroxyapatite that simulated the composition and structure of tooth enamel was specially synthesized. The synthesized material was investigated by transmission electron microscope, X-ray diffraction, fourier transform infrared spectroscopy and X-ray photo electron spectroscopy to confirm to the main composition of carbonated hydroxyapatite with CO32- successfully doped into the crystal lattice through optimizing the synthesis process of C/P molar ratio, pH value dynamical adjustment, annealing temperature and time. The dosimetric properties were systematically investigated by ESR spectroscopy. The results indicated that the radiation induced signal had a good dose response within a relatively wide dose range. The dose response was linear in the dose range of 0-400 Gy with a correlation coefficient of 0.9999 and had dose accumulative effect in the experimental dose range of 0-100 Gy. In a wider dose range up to 30 kGy, the dose response also presented linear feature in double-logarithmic coordinate system with a correlation coefficient of 0.9970. The dose detection limit was about 0.34Gy with a given probability of 95% confidence level depending upon a rigid calculation algorithm. The signal was extremely stable in the observation time of 360 days with a variation coefficient of 3.8%. The radiation sensitivity of the material showed no remarkable variation against photon energy from 662 KeV to 1.25 MeV and dose rate from 0.86 Gy/min to 12.17 Gy/min. The material showed more sensitive in lower photon energy range below 662 keV, which hint additional calibration may need when using in special photon energy condition. The preliminary results suggested that this newly developed dosimeter was potential to become a practical dosimeter that would expand the application fields of ESR dosimetry., Competing Interests: The authors have declared that no competing interests exist.

This study aimed to evaluate bone repair in rat dental sockets after implanting nanostructured carbonated hydroxyapatite/sodium alginate (CHA) and nanostructured carbonated hydroxyapatite/sodium alginate containing 5% strontium microspheres (SrCHA) as bone substitute materials. Twenty male Wistar rats were randomly divided into two experimental groups: CHA and SrCHA (n=5/period/group). After one and 6 weeks of extraction of the right maxillary central incisor and biomaterial implantation, 5 μm bone blocks were obtained for histomorphometric evaluation. The parameters evaluated were remaining biomaterial, loose connective tissue and newly formed bone in a standard area. Statistical analysis was performed by Mann-Withney and and Wilcoxon tests at 95% level of significance. The histomorphometric results showed that the microspheres showed similar fragmentation and bio-absorbation (p>0.05). We observed the formation of new bones in both groups during the same experimental periods; however, the new bone formation differed significantly between the weeks 1 and 6 (p=0.0039) in both groups. The CHA and SrCHA biomaterials were biocompatible, osteoconductive and bioabsorbable, indicating their great potential for clinical use as bone substitutes.

Ionic substitutions have been proposed as a tool to control the functional behavior of synthetic hydroxyapatite (HA), particularly for Bone Tissue Engineering applications. The effect of simultaneous substitution of different levels of carbonate (CO 3 ) and silicon (Si) ions in the HA lattice was investigated. Furthermore, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured on multi-substituted HA (SiCHA) to determine if biomimetic chemical compositions were osteoconductive. Of the four different compositions investigates, SiCHA-1 (0.58 wt % Si) and SiCHA-2 (0.45 wt % Si) showed missing bands for CO 3 and Si using FTIR analysis, indicating competition for occupation of the phosphate site in the HA lattice; 500°C was considered the most favorable calcination temperature as: (i) the powders produced possessed a similar amount of CO 3 (2-8 wt %) and Si (<1.0 wt %) as present in native bone; and (ii) there was a minimal loss of CO 3 and Si from the HA structure to the surroundings during calcination. Higher Si content in SiCHA-1 led to lower cell viability and at most hindered proliferation, but no toxicity effect occurred. While, lower Si content in SiCHA-2 showed the highest ALP/DNA ratio after 21 days culture with hMSCs, indicating that the powder may stimulate osteogenic behavior to a greater extent than other powders. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1775-1785, 2017., (© 2017 Wiley Periodicals, Inc.)

Design and synthesis of materials with better properties and performance are essential requirements in the field of biomaterials science that would directly improve patient quality of life. For this purpose, in situ silica-coated silicate and carbonate co-doped hydroxyapatite (Sc/S.C.HA) nanopowder was synthesized via the sol-gel method. Characterisation of the prepared nanopowder was carried out by XRD, FTIR, TEM, SEM, EDX, ICP, zeta potential, acid dissolution test, and cell culture test. The substitution of the silicate and carbonate ions into hydroxyapatite structure was confirmed by FTIR analysis. XRD analysis showed that silica is an amorphous phase, which played a role in covering the surface of the S.C.HA nanoparticles as confirmed by acid dissolution test. Low thickness and low integrity of the amorphous silica surface layer facilitated ions release from S.C.HA nanoparticles into physiological saline solution. Zeta potential of the prepared nanopowder suspended in physiological saline solution was -27.3±0.2mV at pH7.4. This negatively charged surface, due to the presence of amorphous silica layer upon the S.C.HA nanoparticles, not only had an accelerating effect on in vitro biomineralization of apatite, but also had a positive effect on cell attachment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Simulated body fluid (SBF) is widely used as part of an in vitro method to evaluate implant materials such as their apatite forming ability (AFA), a typical indication of potential bone-bonding ability in vivo. We report the use of carbonate-buffered SBFs as potential solutions for implant evaluation and the effect of proteins, represented by bovine serum albumin (BSA) in SBFs on the formation of hydroxyapatite (HA). These solutions are buffered by the thermodynamic equilibrium with 5% CO 2 in an incubator, and result in a deposition of carbonated HA. Using several titanium-based surfaces, these solutions were studied in comparison with the widely-used SBF (ISO 23317). The presence of BSA strongly inhibited the formation of HA in traditional SBF, while HA can still be observed in carbonate-buffered SBFs. A kinetic study reveals that the inhibitory effect is concentration dependent with 0.1g/L and 1g/L of BSA having little effect on HA growth but a complete inhibition of HA formation at 5g/L of BSA, as tested using NaOH treated titanium with a known positive AFA. The decrease in solution pH and free calcium concentrations in SBFs due to the addition of BSA is not significant, suggesting other causes for the strong inhibitory effect., Statement of Significance: The successful use of simulated body fluids (SBFs) to evaluate potential bioactive implants relies on the better understanding of the heterogeneous nucleation and growth of hydroxyapatite in solution. Although a standardized recipe for SBF was developed over a decade ago, a few key issues remain to be understood, i.e. the behavior of carbonate-buffered SBFs having similar buffering mechanism as human blood, and the effect of proteins on hydroxyapatite formation on bioactive materials. This paper addresses these two issues and would help the reader better understand the subtleties in this domain and better interpret the results generated using SBFs., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Synthetic hydroxyapatite (HAp) is used clinically as a material for bone prostheses owing to its good bone-bonding ability; however, it does not contribute to bone remodeling. Carbonate-substituted hydroxyapatite (CAp) has greater bioresorption capacity than HAp while having similar bone-bonding potential, and is therefore considered as a next promising material for bone prostheses. However, the effects of the CAp instability on inflammatory and immune responses are unknown in detail. Here, we show that the surface layer of CAp is more hydrated than that of HAp and induces changes in the shape and function of macrophage-like cells. HAp and CAp were synthesized by wet method and molded into disks. The carbonate content of CAp disks was 6.2% as determined by Fourier transform (FT) infrared spectral analysis. Diffuse reflectance infrared FT analysis confirmed that physisorbed water and surface hydroxyl groups (OH - ) were increased whereas structural OH - was decreased on the CAp as compared to the HAp surface. The degree of hydroxylation in CAp was comparable to that in bone-apatite structures, and the CAp surface exhibited greater hydrophilicity and solubility than HAp. We investigated immune responses to these materials by culturing RAW264 cells (macrophage precursors) on their surfaces. Cell spreading on the CAp disk was suppressed and the secretion level of inflammatory cytokines was reduced as compared to cells grown on HAp. These results indicate that the greater surface hydration of CAp surface can attenuate adverse inflammatory responses to implanted bone prostheses composed of this material. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1063-1070, 2017., (© 2017 Wiley Periodicals, Inc.)

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 °C for 2 h in order to investigate the products' high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to β-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

Hydroxyapatite and carbonate-substituted hydroxyapatite are widely used in bone tissue engineering and regenerative medicine. Both apatite materials were embedded into recently developed ceramic/polymer composites, subjected to Simulated Body Fluid (SBF) for 30days and characterized using ATR-FTIR spectroscopic imaging to assess their behaviour and structures. The specific aim was to detect the transition phases between both types of hydroxyapatite during the test and to analyze the surface modification caused by SBF. ATR-FTIR spectroscopic imaging was successfully applied to characterise changes in the hydroxyapatite lattice due to the elastic properties of the scaffolds. It was observed that SBF treatment caused a replacement of phosphates in the lattice of non-substituted hydroxyapatite by carbonate ions. A detailed study excluded the formation of pure A type carbonate apatite. In turn, CO 3 2- content in synthetic carbonate-substituted hydroxyapatite decreased. The usefulness of ATR-FTIR spectroscopic imaging studies in the evaluation of elastic and porous β-glucan hydroxyapatite composites has been demonstrated., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The powder composites are predominantly used for filling of voids in bone and as drug delivery carrier to prevent the infection or inflammatory reaction in the damaged tissues. The objective of this work was to study the synthesis of agarose encapsulation on carbonated hydroxyapatite powder and their biological and drug delivery properties. Mesoporous, nanosized carbonated hydroxyapatite/agarose (CHAp/agarose) powder composites were prepared by solvothermal method and subsequently calcined to study the physico-chemical changes, if it subjected to thermal exposure. The phase of the as-synthesized powder was CHAp/agarose whereas the calcinated samples were non-stoichiometric HAp. The CHAp/agarose nanorods were of length 10-80nm and width 40-190nm for the samples synthesized at temperatures 120°C (ST120) and 150°C (ST150). The calcination process produced spheres (10-50nm) and rods with reduced size (40-120nm length and 20-30nm width). Composites were partially dissolved in SBF solution followed by exhibited better bioactivity than non-stoichiometric HAp confirmed by gravimetric method. Hemo and biocompatibility remained unaffected by presence of agarose or carbonate in the HAp. Specific surface area of the composites was high and exhibited an enhanced amoxicillin and 5-fluorouracil release than the calcined samples. The composites demonstrated a strong antimicrobial activity against E. coli, S. aureus and S. epidermidis. The ST120 showed prolonged drug (AMX and 5-Fcil) release and antimicrobial efficacy than ST150 and calcined samples. This technique would be simple and rapid for composites preparation, to produce high quality crystalline, resorbable, mesoporous and bioactive nanocomposite (CHAp/agarose) powders. This work provides new insight into the role of agarose coated on bioceramics by solvothermal technique and suggests that CHAp/agarose composites powders are promising materials for filling of void in bone and drug delivery applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Apatite forming ability of hydroxyapatite (HAP) and carbonate hydroxyapatite (CHAP) containing composites was compared. Two composite materials, intended for filling bone defects, were made of polysaccharide polymer and one of two types of hydroxyapatite. The bioactivity of the composites was evaluated in vitro by soaking in a simulated body fluid (SBF), and the formation of the apatite layer was determined by scanning electron microscopy with energy-dispersive spectrometer and Raman spectroscopy. The results showed that both the composites induced the formation of apatite layer on their surface after soaking in SBF. In addition, the sample weight changes and the ion concentration of the SBF were scrutinized. The results showed the weight increase for both materials after SBF treatment, higher weight gain and higher uptake of calcium ions by HAP containing scaffolds. SBF solution analysis indicated loss of calcium and phosphorus ions during experiment. All these results indicate apatite forming ability of both biomaterials and suggest comparable bioactive properties of composite containing pure hydroxyapatite and carbonate-substituted one., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hollow hydroxyapatite (HA) microspheres, with a high-surface-area mesoporous shell, can provide a unique bioactive and osteoconductive carrier for proteins to stimulate bone regeneration. However, synthetic HA has a slow resorption rate and a limited ability to remodel into bone. In the present study, hollow HA microspheres with controllable amounts of carbonate substitution (0-12 wt.%) were created using a novel glass conversion route and evaluated in vitro and in vivo. Hollow HA microspheres with ~12 wt.% of carbonate (designated CHA12) showed a higher surface area (236 m(2) g(-1)) than conventional hollow HA microspheres (179 m(2)g(-1)) and a faster degradation rate in a potassium acetate buffer solution. When implanted for 12 weeks in rat calvarial defects, the CHA12 and HA microspheres showed a limited capacity to regenerate bone but the CHA12 microspheres resorbed faster than the HA microspheres. Loading the microspheres with bone morphogenetic protein-2 (BMP2) (1 μg per defect) stimulated bone regeneration and accelerated resorption of the CHA12 microspheres. At 12 weeks, the amount of new bone in the defects implanted with the CHA12 microspheres (73±8%) was significantly higher than the HA microspheres (59±2%) while the amount of residual CHA12 microspheres (7±2% of the total defect area) was significantly lower than the HA microspheres (21±3%). The combination of these carbonate-substituted HA microspheres with clinically safe doses of BMP2 could provide promising implants for healing non-loaded bone defects., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nanocrystalline biocompatible single-phase Mn-doped A-type carbonated hydroxyapatite (A-cHAp) powder has been synthesized by mechanical alloying of a stoichiometric mixture of CaCO3, CaHPO4·2H2O and MnO powder for 10 h at room temperature under open air. The A-type carbonation in HAp (substitution of CO3(2-) for OH(-)) is confirmed by FTIR analysis. Microstructure characterization in terms of lattice imperfections and phase quantification of ball milled samples are made by analyzing XRD patterns employing the Rietveld structure refinement method. Rietveld analysis of XRD patterns recorded from Mn-doped HAp samples has been used to locate Mn(2+) cations in HAp. The Ca2 vacancy site is found to be more favorable for Mn substitution. Microstructure characterization by HRTEM corroborates the findings of the X-ray analysis where the presence of a significant amount of amorphous phase of HAp analogous to indigenous bone mineral is clearly found. MTT assay shows sufficiently high percentage cell viability confirming the cytocompatibility of the sample.

Microwave-assisted methods have been frequently used in many processes owing to their numerous advantages such as performing fast, efficient and homogenous processes and reducing side reactions. In view of these benefits, in this study it was purposed to produce bone-like hydroxyapatite (HA) by inducing biomimetic process with microwave-irradiation. This is why, concentrated body fluid (SBF) i.e. 10×SBF-like solution was used and it was precipitated in different microwave powers i.e. 90W, 360W, 600W, and 1200W and in different exposure times. For comparison, precipitation process was also carried out at room temperature for 6h and at 80°C for 1h. The obtained HA structures were characterized by appropriate instrumental techniques. As a result, microwave-induced precipitation at 600W for 9 times 30s was determined as the optimum condition for the production of HA which has similar properties to the cortical bone. At this condition, B-type HA with 9.22% (wt.) carbonate content, 1.61 Ca/P molar ratio and amorphous structure was obtained easily, rapidly and efficiently. So, this is the first time microwave technology has been used to precipitate HA from SBF solution., (Copyright © 2015 Elsevier B.V. All rights reserved.)

CO3(2-) doping is an effective method to increase the biological activity of nano-hydroxyapatite (n-HA). In the present study, calcium nitrate and trisodium phosphate were chosen as raw materials, with a certain amount of Na2CO3 as a source of CO-3(2-) ions, to synthesize nano-carbonate hydroxyapatite (n-CHA) slurry by solution precipitation method. The structure and micro-morphology of n-CHA were characterized by transmission electron microscope (TEM), X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FTIR) and Raman spectroscopy (RS). The results revealed that the synthetic n-HA crystals are acicular in nanometer scale and have a crystal size of 20-30 nm in diameter and 60-80 nm in length, which are similar to natural bone apatite. And the crystallinity of n-CHA crystals decreases to the increment of CO3(2-). Samples with more CO3(2) have composition and structure more similar to the bone apatite. The value of lattice parameters a decreases, value of c increases, and c/a value increases with the increase in the amount of CO3(2-), in accordance with crystal cell parameters change rule of type B replacement. In the AB mixed type (substitution OH- and PO4(3-)) CHA, IR characteristic peak of CO3(2-) out-of-plane bending vibration appears at 872 cm(-1), meanwhile, the asymmetry flexible vibration band is split into band at 1 454 cm(-1) and band at 1 420 cm(-1), while weak CO3(2)-peak appears at 1 540 cm(-1). CO3(2-) Raman peak of symmetric stretching vibration appears at 1 122 cm(-1). CO3(2-) B-type (substitution PO4(3-)) peak appeared at 1 071 cm(-1). Through the calculation of integral area ratio of PO4(3-)/ CO3(2-), OH-/CO3(2-), and PO4(3-)/OH-, low quantity CO3(2-) is B-type and high quantity CO3(2-) is A-type (substitution OH-). The results show that the synthesized apatite crystals are AB hybrid substitued nano-carbonate hydroxyapatite, however B-type replacement is the main substitute mode. Due to similarity inthe shape, size, crystal structure and growth mode, the synthesized apatite crystals can be called a kind of bone-like apatite.

Single phase nanocrystalline biocompatible A-type carbonated hydroxyapatite (A-cHAp) powder has been synthesized by mechanical alloying the stoichiometric mixture of CaCO3 and CaHPO4.2H2O powders in open air at room temperature within 2h of milling. The A-type carbonation in HAp is confirmed by FTIR analysis. Structural and microstructure parameters of as-milled powders are obtained from both Rietveld's powder structure refinement analysis and transmission electron microscopy. Size and lattice strain of nanocrystalline HAp particles are found to be anisotropic in nature. Mechanical alloying causes amorphization of a part of crystalline A-cHAp which is analogous to native bone mineral. Some primary bond lengths of as-milled samples are critically measured. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay test reveals high percentage of cell viability and hence confirms the biocompatibility of the sample. The overall results indicate that the processed A-cHAp has a chemical composition very close to that of biological apatite., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In order to improve the biocompatibility of metallic implants, bioactive components are often used as coatings so that a real bond with the surrounding bone tissue can be formed. We prepared ethyl cellulose/carbonated hydroxyapatite composite coatings (ECHCs) on Ti6Al4V substrates with carbonated hydroxyapatite coatings (CHACs) without ethyl cellulose as controls. The inorganic constituent on the CHACs and ECHCs is calcium-deficient carbonated hydroxyapatite with a flaky texture and a low degree of crystallinity. The flaky carbonated hydroxyapatite plates aggregate to form macropores with an aperture size of around 0.5-2.0 μm. The presence of ethyl cellulose provides superior morphology, contact angle, and biocompatibility characteristics. In comparison to CHACs, ECHCs exhibit a smoother, crack-free surface because the cracks are filled by ethyl cellulose. Moreover, the contact angle of ECHCs is 37.3°, greater than that of CHACs (13.0°). Surface biocompatibility was investigated by using human bone mesenchymal stem cells (hBMSCs). The attachment, spreadability, viability and proliferation of hBMSCs on ECHCs are superior to those on CHACs. Thus, the crack-free ECHCs have excellent biocompatibility and are appropriate for use as biological implants.

Normal bone mineral is a carbonated-apatite, but there are limited data on the effect of mineral containing carbonate on cell response. We characterized surface chemical compositions of three experimental carbonated hydroxyapatite (CO3(2-) HA) substrates and investigated their effect on osteoblast differentiation. Carbonate was incorporated into the hydroxyapatite powders while phosphate and hydroxyl groups were shown to be reduced by analyzing the chemical composition of the substrate surfaces. CO3(2-) HA powders with increasing carbonate concentrations designated as C1 (3.88%), C2 (4.85%), and C3 (5.82%) were molded, pressed, and fired into 14 mm discs. We observed that calcium phosphate ratios increased monotonically with increasing carbonate content, whereas differentiation of MG63 cells decreased. CO3(2-) HA surfaces also affected factor production. Addition of carbonate caused a 70% reduction in osteoprotegerin (OPG) compared to cultures on pure HA, but the effect of carbonate was not dose-dependent. Low carbonate content reduced VEGF-A by 80%, but higher levels of carbonate reversed this effect in a concentration dependent manner, with the C3 VEFG-A levels approximately twice that of C1 levels. These observations collectively indicate that bone cells are sensitive to carbonate content in bone mineral and the effects of carbonate substitution vary with the outcome being measured. Overall, this study provides a preliminary understanding of how carbonate substitution within hydroxyapatite modulates cellular behavior., (© 2013 Wiley Periodicals, Inc.)

First-principles calculations were performed for CO3(2-) ions in hydroxyapatite in order to investigate the atomic structures and thermodynamic stability of CO3(2-) and its related defects. Two different chemical equilibrium conditions in high-temperature and aqueous-solution environments were considered, and atomic and ionic chemical potentials for the individual chemical equilibrium conditions were evaluated to calculate defect formation energies. It was found that A-type CO3(2-) (substituting OH(-)) is energetically more favorable than B-type CO3(2-) (substituting PO4(3-)) in the high-temperature environment, whereas B-type is preferred to A-type in the aqueous solution environment. This result successfully reproduces experimentally observed trends. In the formation of A-type and B-type CO3(2-), OH(-) vacancies or protons (interstitial or substitutional) act as charge-compensating defects., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Thin carbonate-containing hydroxyapatite (CA) films coating partially stabilized zirconia (Y-TZP) were prepared (CA-Y-TZP) to establish a metal-free implant system. CA was coated using a molecular precursor method. The CA film was deposited on the surface of Y-TZP using a precursor solution, which was a mixture of a calcium-ethylenediaminetetraacetic acid (EDTA) complex and phosphate compounds. The deposited CA film was characterized by X-ray diffraction, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy measurements. A focus ion beam system technique revealed that the thickness of the CA film was less than 1.0 µm. Biological evaluations of CA-Y-TZP were performed by immersion experiments in simulated body fluid (SBF) and implantation experiments in the tibiae and femoral condyles of rabbits. In the SBF immersion experiment, apatite deposition progressed more on CA-Y-TZP at the early stage of immersion than on Y-TZP without the CA coating. Animal experiments revealed that bone formation on CA-Y-TZP was similar with than on Y-TZP. Histomorphometrical evaluations showed a significantly higher bone-to-implant contact ratio and bone mass on CA-Y-TZP after implantation into the femoral trabecular bone of rabbits. Therefore, CA-Y-TZP appears to be applicable as a metal-free implant., (© 2014 Wiley Periodicals, Inc.)

A bio-degradable scaffold incorporating osteoinductive factors is one of the alternative methods for achieving the regeneration of a mandibular bone defect. The current pilot study addressed such a bone reconstruction in a non-human primate model, Macaca fascicularis monkeys, with an engineered poly(ɛ-caprolactone) (PCL) scaffold, provided with a carbonate-substituted hydroxyapatite coating. The scaffolds were implanted into unilaterally created mandibular segmental defects in 24 monkeys. Three experimental groups were formed: (1) scaffolds with rhBMP-2 (n = 8), (2) scaffolds with autologous mixed bone marrow cells (n = 8), and (3) empty scaffolds as a control group (n = 8). Evaluation was based on clinical observation as well as micro-CT, mechanical, and histological analyses. Despite a high infection rate, the overall results showed that the currently designed PCL scaffolds had insufficient load-bearing capability, and complete bone union was not achieved after 6 months of implantation. Nevertheless, the group of PCL scaffolds loaded with rhBMP-2 showed evidence of bone-regenerative potential, in contrast to PCL with autologous mixed bone marrow cells and the control group., (© 2013 Wiley Periodicals, Inc.)

Hydroxyapatite serves as a bioactive material for biomedical purposes, because it shares similarities with the inorganic part of the bone. However, how this material deposits on metallic surfaces using biomimetic matrices remains unclear. In this study, we deposited dihexadecyl phosphate, a phospholipid that bears a simple chemical structure, on stainless steel and titanium surfaces using the Langmuir-Blodgett (LB) technique; we employed the resulting matrix to grow carbonated hydroxyapatite. We obtained the calcium phosphate coating via a two-step process: we immersed the surfaces modified with the LB films into phosphate buffer, and then, we exposed the metal to a solution that simulated the concentration of ions in the human plasma. The latter step generated carbonated hydroxyapatite, the same mineral existing in the bone. The free energy related to the surface roughness and composition increased after we modified the supports. We investigated the film morphology by scanning electron and atomic force microscopies and determined surface composition by infrared spectroscopy and energy dispersive X-ray. We also studied the role of the surface roughness and the surface chemistry on cell viability. The surface-modified Ti significantly increased osteoblastic cells proliferation, supporting the potential use of these surfaces as osteogenic materials., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A new injectable in situ forming hydroxyapatite and thermosensitive chitosan gel (chitosan/HA/Na2CO3 gel) promoted by Na2CO3 was preliminarily synthesized. This study was the first to use Na2CO3 as coagulant to construct the chitosan thermosensitive gel. The sol–gel phase transition, degradation, and morphology of the gel were examined. We found that chitosan/HA/Na2CO3 sol with 1.4% Na2CO3 has a suitable gelation time (9 min) and degradation rate. SEM images of the dried gel show a porous netlike framework. TEM, EDS, and XRD were combined to confirm the presence of hydroxyapatite. In vitro cell culture was performed by using rat bone mesenchymal stem cells (rBMSCs). rBMSCs survived well on the chitosan gel scaffold that formed in vitro and in vivo, indicating that the chitosan gel was a suitable substrate for the attachment and proliferation of rBMSCs. Subcutaneous implantation of the chitosan gel formed in situ into a nude mouse revealed that the chitosan gel loaded with rBMSCs could lead to angiogenesis.

We demonstrate the application of the combined experimental-computational approach for studying the anionic impurities in hydroxyapatite (HAp). Influence of the carbonation level (x) on the concentration of the NO3(2-) radicals in the HAp nanocrystals of Ca10-xNax(PO4)6-x(CO3)x(OH)2 with x in the range 0 < x < 2 and average sizes of 30 nm is investigated by different analytical methods including electron paramagnetic resonance (EPR). Stable NO3(2-) radicals are formed under X-ray irradiation of nano-HAp samples from NO3(-) ions incorporated in trace amounts during the wet synthesis process. Density functional theory (DFT) based calculations show energetic preference for the PO4 group substitution by NO3(-) ions. Comparison of the calculated and experimental spectroscopic parameters (g and hyperfine tensors) reveals that EPR detects the NO3(2-) radicals located in the positions of the PO4 group only. It is shown that with the increase in x, the carbonate ions substitute the NO3(2-)/NO3(-) ions. DFT calculations confirm that carbonate incorporation in HAp structure is energetically more favorable than the formation of the nitrate defect.

The aim of this study was to evaluate the soft tissue response to poly(ε-caprolactone) (PCL) implants with and without carbonate-substituted hydroxyapatite (CHA) coating compared to the commonly used titanium alloy (Ti-6Al-4V)-machined surface. Experimental materials were implanted subcutaneously in New Zealand white rabbits for 5 weeks. The tissue attachment strength, as evaluated by a tissue peel test, histological and histomorphology analysis, as well as scanning electron microscopy were compared between groups. The peel test result revealed no statistically significant difference between groups. Histological analysis found fibrous capsule formation around all implant materials. The fibrous capsule around PCL implants with and without CHA coating was significantly thinner compared with the capsule thickness around the titanium implants. However, the inflammatory cells, as present at the fibrous capsule-implant interface, were found to be significantly lower in the Ti-group. In conclusion, the current data do not prove that PCL or PCL with a CHA coating results in a superior soft tissue response compared with a machined titanium implant., (Copyright © 2013 Wiley Periodicals, Inc.)

Water is well recognized as an important component in bone, typically regarded as a constituent of collagen, a pore-filling fluid in bone, and an adsorbed species on the surface of bone crystallites. The possible siting and role of water within the structure of the apatite crystallites have not been fully explored. In our experiments, carbonated hydroxyl- and fluorapatites were prepared in D(2)O and characterized by elemental analysis, thermal gravimetric analysis, powder X-ray diffraction, and infrared and Raman spectroscopy. Two hydroxylapatites and two fluorapatites, with widely different amounts of carbonate were analyzed by solid state (2)H NMR spectroscopy using the quadrupole echo pulse sequence, and each spectrum showed one single line as well as a low-intensity powder pattern. The relaxation time of 7.1 ms for 5.9 wt% carbonated hydroxylapatite indicates that the single line is likely due to rapid, high-symmetry jumps in translationally rigid D(2)O molecules, indicative of structural incorporation within the lattice. Discrimination between structurally incorporated and adsorbed water is enhanced by the rapid exchange of surface D(2)O with atmospheric H(2)O. Moreover, a (2)H resonance was observed for samples dried under a variety of conditions, including in vacuo heating to 150°C. In contrast, a sample heated to 500°C produced no deuterium resonance, indicating that structural water had been released by that temperature. We propose that water is located in the c-axis channels. Because structural water is observed even for apatites with very low carbonate content, some of the water molecules must lie between the monovalent ions.

Combining gelatins functionalized with the tyrosine-derived groups desaminotyrosine or desaminotyrosyl tyrosine with hydroxyapatite (HAp) led to the formation of composite materials with much lower swelling ratios than those of the pure matrices. Shifts of the infra-red (IR) bands related to the free carboxyl groups could be observed in the presence of HAp, which suggested a direct interaction of matrix and filler that formed additional physical cross-links in the material. In tensile tests and rheological measurements the composites equilibrated in water had increased Young's moduli (from 200 kPa up to 2 MPa) and tensile strengths (from 57 kPa up to 1.1 MPa) compared with the matrix polymers without affecting the elongation at break. Furthermore, an increased thermal stability of the networks from 40 to 85°C could be demonstrated. The differences in the behaviour of the functionalized gelatins compared with pure gelatin as a matrix suggested an additional stabilizing bond between the incorporated aromatic groups and the HAp as supported by the IR results. The composites can potentially be applied as bone fillers., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The influence of ionic substituents in calcium phosphates intended for bone and tooth replacement biomedical applications is an important research topic, owing to the essential roles played by trace elements in biological processes. The present study investigates the mechanical and biological evaluation of ionic doped hydroxyapatite/β-tricalcium phosphate mixtures which have been prepared by a simple aqueous precipitation method. Heat treating the resultant calcium phosphates in a carbonated atmosphere led to the formation of ionic doped carbonated hydroxyapatite/β-tricalcium phosphate mixtures containing the essential ions of biological apatite. The structural analysis determined by Rietveld refinement confirmed the presence of hydroxyapatite as the main phase, together with a considerable amount of β-tricalcium phosphate. Such phase assemblage is essentially due to the influence of substituted ions during synthesis. The results from mechanical tests proved that carbonate substitutions are detrimental for the mechanical properties of apatite-based ceramics. In vitro proliferation assays of osteoblastic-like cells (MC3T3-E1 cell line) to powders revealed that carbonate incorporation can either delay or accelerate MC3T3 proliferation, although reaching the same proliferation levels as control cells after 2 weeks in culture. Further, the powders enable pre-osteoblastic differentiation in a similar manner to control cells, as indirectly measured by ALP activity and Type-I collagen medium secretion., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Double-step hydrothermal processing is a process where powder compacts of calcium phosphates are exposed to vapor of solvent solution, followed by being immersed in the solution. In the present study, we investigated the effects of ammonium carbonate on formation of calcium-deficient hydroxyapatite (CDHA) through double-step hydrothermal processing. The synthesized CDHA has high crystallinity when the solution has relatively low concentration of the ammonium carbonate ranging from 0.01 to 0.25 mol dm(-3). Carbonate content in the prepared samples were distinctly increased with increasing the concentration of ammonium carbonate to indicate formation of carbonate-containing calcium-deficient hydroxyapatite (CHAp) with low crystallinity. Morphology of the CHAp formed on the compacts varied progressively from rods and rosette-like shape to irregular shape with increase in the initial concentration of the ammonium carbonate in the solution. Application of ammonium carbonate in the double-step hydrothermal processing allows fabrication of irregular-shaped CDHA containing carbonate ions in both phosphate and hydroxide site, with low crystallinity, when the initial concentration of ammonium carbonate was 0.5 mol dm(-3) and more.

Three dimensional interconnected macroporous (pore diameter: 600-800 μm) hydroxyapatite/agarose disks have been evaluated in this study as potential bone regeneration scaffolds. With this purpose, the adhesion and proliferation of human Saos-2 osteoblasts on this biomaterial were analyzed. As an index of cell function, the following parameters were measured: cell morphology, viability, cell size/complexity, cell cycle, reactive oxygen species (ROS) content, and lactate dehydrogenase (LDH) release. The existence of anoikis induced by inappropriate contacts between the cell and the scaffold has been detected by scanning electron microscopy, confocal microscopy, and flow cytometry. The intracellular nitric oxide content has been also measured as potential inducer of anoikis. The positive effects of previous scaffold coating with type I collagen on osteoblast adhesion as well as the collagen protection against anoikis have been demonstrated in this study., (© 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.)

Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) fibers containing carbonated hydroxyapatite (CHA) nanoparticles with different CHA amounts (5, 10 and 15 wt%) were electrospun with the aid of ultrasonic power for dispersing the nanoparticles. Scanning electron microscopy and energy-dispersive x-ray spectroscopy results showed that the distribution of CHA within the CHA/PHBV nanocomposite fibers was homogeneous when the CHA content was 10 wt%. Slight particle agglomeration occurred when the CHA content was 15 wt%. The diameters of the electrospun CHA/PHBV nanocomposite fibers and PHBV polymer fibers were around 3 µm. Fourier transform infrared spectroscopic analysis further confirmed the presence of CHA in CHA/PHBV nanocomposite fibers. Both PHBV and CHA/PHBV fibrous membranes exhibited similar tensile properties. Compared with PHBV solvent-cast film, the PHBV fibrous membrane was hydrophobic but the incorporation of CHA nanoparticles dramatically enhanced its wettability. In vitro studies revealed that both types of electrospun fibrous membranes (PHBV and CHA/PHBV) supported the proliferation of human osteoblastic cells (SaOS-2). The alkaline phosphatase activity of SaOS-2 cells seeded on the CHA/PHBV fibrous membranes was higher than that of the cells seeded on the PHBV fibrous membranes after 14 days of cell culture. The electrospun CHA/PHBV nanocomposite fibrous membranes show promises for bone tissue engineering applications.

The crystal structures of the ferric carbonate-hydroxyapatite (Fe-CHAp) and oxyapatite (Fe-OAp) pseudomorphs were investigated by powder neutron diffraction and Fourier transform infrared spectroscopy. At low iron loadings, Fe-CHAp (x = 0.1) is A-B type carbonate apatite-2H, where atmospheric CO(2) displaces tunnel hydroxyl and framework phosphate (Ca(2+) + 2PO(4)(3-) --> square(Ca) + 2CO(3)(2-) and Ca(2+) + OH(-) --> Fe(3+) + CO(3)(2-)), while Fe-CHAp (x = 0.2) is A type carbonate apatite-2M. For high iron loadings (x = 0.5), near the solubility limit, Fe(3+) incorporation includes concomitant oxidation of hydroxyl groups (Ca(2+) + OH(-) --> Fe(3+) + O(2-)). The discontinuity in the lattice metric at x approximately 0.2 together with a progressive reduction of OH(-) and CO(3)(2-), substantiates these incorporation mechanisms. The general formula of Fe-CHAp is [Ca(4-x)(F)Fe(x)][Ca(6-y)(T) square(y)][(PO(4))(6-y)(CO(3))(y)][(OH(4))(2-x)(CO(3))(x)] (0 < or = x < or = 0.2, 0 < or = y < or = 0.2) and Fe-OAp is [Ca(3.8)(F)Fe(0.2)][Ca(6-x)(T)Fe(x)][(PO(4))][O(0.2+x)(OH)(1.8-x)] (0 < x < or = 0.3). These new data are placed in the context of earlier crystallographic studies, with the Ca(I)O(6) metaprism twist angle (phi) shown to be a reliable indicator of symmetry, such that for phi(max) > 27.(0) degrees, monoclinic and triclinic structures are preferable.

Human osteoclasts derived from CD14+ve precursors were cultured on discs of stoichiometric hydroxyapatite (HA) and carbonate-substituted hydroxyapatite (CHA) of varying carbonate contents. The development of osteoclasts was qualitatively different on ceramics compared to dentine, occurring in discrete, confluent subpopulations, which suggests local cell signaling may be important in the process. Resorption was quantified by scanning electron microscopy, surface profilometry, and by calcium release into the culture medium. Cells were characterised by a number of histochemical markers of the osteoclast phenotype. Resorption of the ceramic increased with increasing carbonate content up to 2.35 wt %, when resorption trails and pits characteristic of osteoclast activity were seen. Controlling carbonate content may be one way of controlling the rate of resorption of synthetic HA ceramics., ((c) 2009 Wiley Periodicals, Inc.)

The crystallization process in recently developed toothpastes, containing nanoparticles of carbonate substituted hydroxyapatite (nano-CHA), was investigated. For this purpose, the non-conventional Energy Dispersive X-Ray Diffraction technique, that demonstrated to be a powerful tool to follow in situ phase transformations, was applied, for the first time, to products of pharmaceutical-cosmetic interest. Two types of toothpastes, containing 15 and 20 wt% of nano-CHA, respectively, have been studied. It was observed that, after mixing the toothpastes with water and saliva in order to reproduce in vivo conditions, a crystallization of nano-CHA takes place. Such process occurs in a characteristic time of (22 +/- 1) min for the toothpaste containing 15 wt% of nano-CHA and of (3.9 +/- 0.5) min for the one containing 20% of nano-CHA. For both toothpastes, a 10% increase in grain dimensions was observed over an average characteristic time of (55 +/- 5) min.

Carbonated hydroxyapatites were formed via reactions in NaHCO(3)/NaH(2)PO(4) solutions from a mixture of particulate tetracalcium phosphate (TetCP) and anhydrous dicalcium phosphate (DCPA). Reactions were followed by determinations of pH and ion concentrations. The solids formed were analyzed by XRD and FTIR. Rates of heat evolution were established by isothermal calorimetry. Reactions in the absence of NaH(2)PO(4) did not reach completion within 24 h. Constitution of reactants to achieve a DCPA-to-NaHCO(3) ratio of 1, in conjunction with the presence of NaH(2)PO(4) as a buffer, was found to be optimal for formation of apatite with no remaining reactant. The amount of carbonate incorporated in this apatite was 4-5 wt%. Calorimetry indicated the reaction mechanism to depend on the bicarbonate concentration in solution. The presence of NaH(2)PO(4) was found to increase the reaction rate but decrease the extent of carbonate uptake.