Carbonate-substituted Hydroxyapatite Research Articles

Carbonate substituted hydroxyapatite: Resorption by osteoclasts modifies the osteoblastic response

Carbonate substitution within the hydroxyapatite (HA) lattice improves osteoconduction, although the mechanism by which this occurs is unclear. Discs of dense, sintered, phase-pure HA and AB-type carbonate substituted hydroxyapatite (CHA) were cultured for 21 days with human CD14+ cells in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of nuclear factor (NF)-kappaB (sRANKL), during which time osteoclasts developed and resorbed the ceramic surface. Discs were then seeded with human osteoblasts (HOBs), and proliferation and collagen synthesis were measured. On some discs, the conditioned proteinaceous layer left behind by the osteoclasts was preserved. Proliferation of HOBs was increased on resorbed compared to control (unresorbed) surfaces on both materials, provided this osteoclast-conditioned layer was left intact. Collagen synthesis by HOBs was increased on previously resorbed surfaces compared to unresorbed surfaces. This effect was seen on both materials but was seen at an earlier time point on CHA. The results suggest that osteoclasts can condition synthetic bioceramic surfaces and alter the responses of osteoblasts that subsequently populate them. Carbonate substitution may enhance osteoconduction indirectly via effects on enhanced bioresorption.

In vitro study of matrix surface properties of porous granulated calcium phosphate ceramic materials made in Russia

We performed in vitro screening of monophasic (hydroxyapatite, beta-tricalcium phosphate, carbonate-substituted hydroxyapatite with 0.59 and 5.9 wt% substitution with CO(3)(2-)) and biphasic (hydroxyapatite-tricalcium phosphate with various percentage of the components 80/20, 60/40, 20/80, silicon-substituted hydroxyapatite with 0.79 wt% SiO(2)) porous granulated ceramics composed of calcium phosphate powders synthesized by methods of heterophasic interaction of reagents and precipitation from aqueous solutions using MTT test and cultured human fibroblasts. Acute toxicity of materials (24-h incubation with cell culture) and matrix properties (3, 5, 7, 14, 18, 21, 28 days in culture) were evaluated. We selected a batch of materials obtained by precipitation from aqueous solutions, which were non-toxic and were characterized by good matrix properties (for cells). Biphasic ceramics with hydroxyapatite-tricalcium phosphate ratio of 80/20 exhibited best characteristics, and ceramics on the basis of silicon-substituted hydroxyapatite showed moderate characteristics.

Preparation and Characterization of Macroporous Carbonate-Substituted Hydroxyapatite Scaffold

Human bone mineral is different in composition from stoichiometric hydroxyapatite (Ca10(PO4)6(OH)2) in that it contains additional ions, of which CO32- is the most abundant species. Two macroporous carbonate-substituted hydroxyapatite (CHA) scaffolds were prepared by treating a macroporous CaCO3 column (porosity, 88%; pore and hole size, 160 and 71 μm), prepared in this study, hydrothermally at 120 °C in 1 M phosphate solutions of (NH4)2HPO4 and K2HPO4. Especially, it was found that the CaCO3 was transformed into CHA after hydrothermal treatment (HT) for 24 h irrespective of the type of phosphate solution. Under HT for 24 h, the crystallite size and crystal shape of CHA prepared in K2HPO4 solution (KCHA) and those of CHA prepared in (NH4)2HPO4 (NH4CHA) were quite different from each other. Moreover, NH4CHA contained 2.09 wt % A-type CO32- out of 6.05 wt % CO32-, but KCHA contained a small amount of A-type CO32- (0.08 wt % out of 8.24 wt % CO32-). The chemical formulas were Ca9.23(NH4)0.12(PO4)5.36(CO3)0.64(OH)0.54(CO3)0.34 for NH4CHA and Ca8.18K0.5(PO4)4.72(CO3)1.28(OH)0.1(CO3)0.02 for KCHA. Therefore, the properties, such as carbonate type and content, and crystal shape, of CHA are significantly affected by the type of phosphate solution.

In Vitro Study on Different Cell Response to Spherical Hydroxyapatite Nanoparticles

Hydroxyapatite (HA) is widely used in filling of bone defects and coating on metal parts of prosthetic implants due to its excellent biocompatibility, bioactivity, and bone-bonding properties. It has been demonstrated that micro-sized HA particles cause inflammatory reaction, especially for the needle shaped particles. However, little effort has been concentrated on the cell responses of the spherical HA nanoparticles. The aim of the present work is to chemically and physically characterize the synthesized HA nanoparticles and to investigate the in vitro cell responses. X-ray diffraction, electron microscopy, nitrogen adsorption, and Fourier transform infrared spectroscopy revealed that the particles consisted of nearly spherical crystallites of carbonate-substituted HA with size of 20-40 nm and specific surface area of 75 m(2)/g. L929 cell proliferation experiments demonstrate that the spherical HA nanoparticles is more biocompatible than commercially available HA. On the other hand, U2-OS cell test results show that the inhibition rate of the spherical HA nanoparticles increases with time and concentration. The half effective inhibitory concentration (IC50) of the nanoparticles was determined to be 50.8 mug/mL at 72 h. All these data indicated that the synthesized spherical nanocrystalline HA particles can function as an effective biomaterial for bone tumorectomy repair, while having little adverse effect.

In vitro evaluation of nanosized carbonate‐substituted hydroxyapatite and its polyhydroxyethylmethacrylate nanocomposite

Nanometer scale carbonate-substituted hydroxyapatite (nanoCHA) particles were prepared and examined using transmission electron microscopy, which revealed their polycrystalline nature with a rod-like morphology (20-30 nm in width and 50-80 nm in length). In vitro cytotoxicity study showed that there was some evidence of lactate dehydrogenase (LDH) release when macrophages were in contact with high concentrations of nanoCHA particles. The levels of LDH release decreased significantly with a reduction in nanoCHA concentration. A similar trend was observed for the inflammatory cytokine TNF-alpha. nanoCHA particles with high carbonate content induced a high level of TNF-alpha release. Biological testing using a human osteoblast (HOB) cell model found that HOB cells were able to grow and proliferate on a nanoCHA deposited surface. Well organized actin fibers were observed for HOB cells in contact with nanoCHA particles with low carbonate content and the cell proliferation rate was higher on these particles in comparison with those of high carbonate nanoCHA particles. Therefore, low carbonate nanoCHA particles were incorporated into poly-(2-hydroxyethylmethacrylate) matrix to make a nanocomposite. It was found that the nanoCHA composite was hydrophilic and became rubber-like after hydration. Both 20 wt % and 40 wt % composites were able to induce the formation of bone-like apatite after immersion in simulated body fluid. A high bioactivity of the composite was obtained with high loading of the nanoCHA filler. These results demonstrate the potential of formulating nanocomposites for biomedical applications.

The fallacy of the calcium-phosphorus product

Scattered through the practice of medicine are dogmas with little or no scientific basis. One of these is the product of the serum calcium and phosphorus concentrations, the so-called calcium-phosphorus product or Ca x P. The assumption that ectopic calcification will occur when the product of the serum calcium and phosphorus concentrations exceeds a particular threshold has become standard practice in nephrology even though there is little scientific basis. Experimental support is lacking, the chemistry underlying the use of the product is oversimplified and the concept that ectopic calcification is simply the result of supersaturation is biologically flawed. The evidence that the Ca x P is an independent risk factor for mortality and morbidity is also questionable. Although ectopic calcification can occur in many sites, this review will focus on vascular calcification, as it is the most common site and the site most likely to affect patient outcomes.

Characterization of macroporous carbonate-substituted hydroxyapatite bodies prepared in different phosphate solutions

Bone mineral of human is different in composition from the stoichiometric hydroxyapatite (Ca10(PO4)6(OH)2) in that it contains additional ions, of which CO 3 2− is the most abundant species. Carbonate-substituted hydroxyapatite (CHA) bodies were prepared by the hydrothermal treatment of highly porous calcium carbonate (CaCO3) body at 120 °C in 1 M M2HPO4 and M3PO4 solutions (M = NH4 or K). It was found that CaCO3 body was almost transformed into CHA body after hydrothermal treatment for 24 h irrespective of type of phosphate solution. However, a small amount of CaCO3 still remained after the treatment in K3PO4 for 48 h. Crystal shape of CHA bodies prepared in those solutions except for K2HPO4 was flake-like, which was different from that (stick-like) of original CaCO3 body used for the preparation of CHA body. CHA prepared in the K2HPO4 showed globule-like crystal. Average pore size and hole size of the CHA bodies were 150, 70 μm and their porosities were about 89% irrespective of the solution. Carbonate content was slightly higher in the CHA bodies obtained from potassium phosphate solutions than in those obtained from ammonium phosphate solutions. Mostly B-type CHA was obtained after the hydrothermal treatment in the potassium phosphate solutions. On the other hand, mixed A- and B-type CHA (ca. 1–2 in molar ratio) was obtained in the ammonium phosphate solutions. The content of CO 3 2− in the CHA body depended on the type of phosphate solution and was slightly larger in the potassium phosphate solutions.

Preparation and Characterization of Antheraea pernyi Silk Fibroin Based Nanohydroxyapatite Composites

In the present study, Antheraea pernyi silk fibroin (Ap-SF) is employed to regulate the mineralization of hydroxyapatite (HAp) nanocrystals. Calcium phosphate crystals precipitate in the aqueous solution of regenerated Ap-SF at pH 7.4 and room temperature. The samples are charaterized with X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Based on the XRD results, the inorganic phase in the mineralized Ap-SF composites is HAp as small particles with low crystallinity. The FTIR analysis reveals that the HAp crystals are carbonate-substituted HAp and compounded with Ap-SF. The TEM images show mineralized nanofibrils in the composites as rod-like shapes 4—5 nm in diameter. Electron diffraction (SAED) patterns of selected areas of the composites exhibit polycrystalline rings that are ascribed to HAp. SEM images show mineralized Ap-SF composites with several mineralized rods 49—74 nm in diameter.

Preparation of bioactive glasses with controllable degradation behavior and their bioactive characterization

Bioactive glasses and ceramics have been widely investigated for bone repair because of their excellent bioactive characteristics. However, these biomaterials undergo incomplete conversion into a bone-like material, which severely limits their biomedical application. In this paper, borosilicate bioactive glasses were prepared by traditional melting process. The results showed that borosilicate glasses possessed high biocompatibility and bioactivity. In addition, when immersed in a 0.02 mol/L K2HPO4 solution, particles of a borate glass were fully converted to HA. The desirable conversion rate to HA may be achieved through the adjustment of the B2O3/SiO2 ratio. The results of XRD and FTIR analysis indicated that the degradation product was carbonate-substituted hydroxyapatite, which was similar to the inorganic component of bone

Preparation and physicochemical properties of a novel hydroxyapatite/chitosan–silk fibroin composite

A novel hydroxyapatite/chitosan–silk fibroin (HA/CTS–SF) composite was prepared for bone repair and replacement by a coprecipitation method. It was revealed that the inorganic phase in the composite was carbonate-substituted HA with low crystallinity. The HA crystallites were found to be needle-like in shape with a typical size of 20–50 nm in length and around 10 nm in width. The composite exhibited a higher compressive strength than the precipitated HA without any organic source involved, which was closely related to the perfect incorporation of chitosan and SF macromolecules into the composite. The chemical interactions occurring between the mineral phase and the organic matrix were thought to improve the interfacial bonding and thus resulted in the enhanced mechanical property of the composite.

Collagen Scaffolds Reinforced with Biomimetic Composite Nano-Sized Carbonate-Substituted Hydroxyapatite Crystals and Shaped by Rapid Prototyping to Contain Internal Microchannels

The next generation of tissue engineering scaffolds will be made to accommodate blood vessels and nutrient channels to support cell survival deep in the interior of the scaffolds. To this end, we have developed a method that incorporates microchannels to permit the flow of nutrient-rich media through collagen-based scaffolds. The scaffold matrix comprises nano-sized carbonate-substituted hydroxyapatite (HA) crystals internally precipitated in collagen fibers. The scaffold therefore mimics many of the features found in bone. A biomimetic precipitation technique is used whereby a collagen membrane separates reservoirs of calcium and phosphate solutions. The collision of calcium and phosphate ions diffusing from opposite directions results in the precipitation of mineral within the collagen membrane. Transmission electron microscopy analysis showed the dimension of the mineral crystals to be approximately 180 x 80 x 20 nm, indicating that the crystals reside in the intermicrofibril gaps. Electron diffraction indicated that the mineral was in the HA phase, and infrared spectroscopy confirmed type A carbonate substitution. The collagen-HA membrane is then used to make 3-dimensional (3D) scaffolds: the membrane is shredded and mixed in an aqueous-based collagen dispersion and processed using the critical point drying method. Adjusting the pH of the dispersion to 5.0 before mixing the composite component preserved the nano-sized carbonate-substituted HA crystals. Branching and interconnecting microchannels in the interior of the scaffolds are made with a sacrificial mold manufactured by using a 3D wax printer. The 3D wax printer has been modified to print the mold from biocompatible materials. Appropriately sized microchannels within collagen-HA scaffolds brings us closer to fulfilling the mass transport requirements for osteogenic cells living deep within the scaffold.

Collagen Scaffolds Reinforced with Biomimetic Composite Nano-Sized Carbonate-Substituted Hydroxyapatite Crystals and Shaped by Rapid Prototyping to Contain Internal Microchannels

Collagen Scaffolds Reinforced with Biomimetic Composite Nano-Sized Carbonate-Substituted Hydroxyapatite Crystals and Shaped by Rapid Prototyping to Contain Internal Microchannels

Kinetics and mechanisms of the conversion of silicate (45S5), borate, and borosilicate glasses to hydroxyapatite in dilute phosphate solutions

Bioactive glasses with controllable conversion rates to hydroxyapatite (HA) may provide a novel class of scaffold materials for bone tissue engineering. The objective of the present work was to comprehensively characterize the conversion of a silicate bioactive glass (45S5), a borate glass, and two intermediate borosilicate glass compositions to HA in a dilute phosphate solution at 37 degrees Celsius. The borate glass and the borosilicate glasses were derived from the 45S5 glass by fully or partially replacing the SiO(2) with B(2)O(3). Higher B(2)O(3) content produced a more rapid conversion of the glass to HA and a lower pH value of the phosphate solution. Whereas the borate glass was fully converted to HA in less than 4 days, the silicate (45S5) and borosilicate compositions were only partially converted even after 70 days, and contained residual SiO(2) in a Na-depleted core. The concentration of Na(+) in the phosphate solution increased with reaction time whereas the PO(4) (3-) concentration decreased, both reaching final limiting values at a rate that increased with the B(2)O(3) content of the glass. However, the Ca(2+) concentration in the solution remained low, below the detection limit of atomic absorption, throughout the reaction. Immersion of the glasses in a mixed solution of K(2)HPO(4) and K(2)CO(3) produced a carbonate-substituted HA but the presence of the K(2)CO(3) had little effect on the kinetics of conversion to HA. The kinetics and mechanisms of the conversion process of the four glasses to HA are compared and used to develop a model for the process.

Carbonate Substituted Hydroxyapatite; Development and Function of Osteoclasts

Human osteoclasts derived from CD14+ precursors were cultured on discs of stoichiometric hydroxyapatite (HA) and carbonate substituted hydroxyapatite (CHA) of varying carbonate contents. Resorption of the ceramic increased with increasing carbonate content up to 2.35 wt. %. Development of osteoclasts is qualitatively different on ceramics compared to dentine, occurring in discrete, confluent subpopulations, which suggests local cell signalling may be important in the process. Resorption appears to drive further development of osteoclasts. Controlling carbonate content may be one way of controlling the rate of resorption of synthetic HA ceramics.

Osteoblastic Response to Resorbed Ceramic Surfaces; The Role of the Osteoclast in Osteoconduction

The mechanism by which carbonate substitution within the hydroxyapatite (HA) lattice improves osteoconduction is unclear. Discs of dense, sintered, phase-pure HA and carbonate substituted hydroxyapatite (CHA) were cultured with human CD14+ cells in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of nuclear factor (NF)-κB (sRANKL), during which time osteoclasts developed and resorbed the ceramic surface. Discs were then seeded with human osteoblasts (HOBs), and proliferation and collagen synthesis measured. Proliferation was increased on resorbed compared to control (unresorbed) surfaces on both materials. Collagen synthesis was increased on CHA compared to HA, an increase accelerated on a previously resorbed surface. The results suggest that osteoclasts can condition synthetic bioceramic surfaces and alter the responses of osteoblasts which subsequently populate them. Carbonate substitution may enhance osteoconduction via effects on enhanced bioresorption.

Effect of carbonate substitution on the ultrastructural characteristics of hydroxyapatite implants

Carbonate ion substitution has been shown to be beneficial for increasing the amount of in vivo osseointegration to hydroxyapatite (HA). Nevertheless, mechanisms by which carbonate ions increase in vivo bioactivity are not fully understood. Sintered granules of HA and carbonate-substituted hydroxyapatite (CHA) were implanted for 6 and 12 weeks in an ovine model. Samples containing the bone-implant interface were prepared for transmission electron microscopy (TEM) and TEM was used to compare the in vivo reactivity of sintered granules of HA and CHA. The current findings demonstrated that CHA (1.2 and 2.05 wt.%) is more soluble than pure HA in vivo. More dissolution was observed from the CHA, at the bone-implant interface and within the implant, when compared to pure HA. A less crystalline phase was formed between the 2.05 wt.% CHA and bone at 12 weeks in vivo. Bone surrounding both the pure HA and 1.2 wt.% CHA was relatively disorganised at 12 weeks. In comparison, bone surrounding the 2.05 wt.% CHA was considerably more organised and in many regions collagen fibrils were present. Despite increased quality of bone surrounding 2.05 wt.% CHA, compared to 1.2 wt.% CHA, the amount of dissolution from both materials was similar.

Porous scaffolds of gelatin–hydroxyapatite nanocomposites obtained by biomimetic approach: Characterization and antibiotic drug release

Gelatin-hydroxyapatite (HA) nanocomposite porous scaffolds were fabricated biomimetically, and their feasibility as a drug-delivery carrier for tissue-regeneration and wound-healing treatments was addressed. The composite sols were prepared by the precipitation of HA up to 30 wt % within a gelatin solution with the use of calcium and phosphate precursors, and the porous scaffold was obtained by casting the sols and further freeze drying. The obtained bodies were crosslinked with carbodiimide derivatives to retain chemical and thermal integrity. The apatite precipitates were observed to be a poorly crystallized carbonate-substituted HA. The nanocomposite scaffolds had porosities of approximately 89-92% and exhibited a bimodal pore distribution, that is, the macropores (approximately 300-500 microm) of the framework structure, and micropores (approximately 0.5-1 microm) formed on the framework surface. Transmission electron microscopy (TEM) observation revealed the precipitation of highly elongated HA nanocrystals on the gelatin network. The well-developed porous structure and organized nanocomposite configurations were in marked contrast to the directly mixed gelatin-HA powder conventional composites. For drug-release tests, tetracycline, an antibiotic drug, was entrapped within the scaffold, and the drug-release profile was examined with processing parameters, such as HA amount in gelatin, crosslinking degree, and initial drug addition. The drug entrapment decreased with increasing HA amount, but increased with increasing crosslinking degree and initial drug addition. The crosslinking of the gelatin was the prerequisite to sustaining and controlling the drug releases. Compared to pure gelatin, the gelatin-HA nanocomposites had lower drug releases, because of their lower water uptake and degradation. All the nanocomposite scaffolds released drugs in proportion to the initial drug addition, suggesting their capacity to deliver drugs in a controlled manner. Based on the findings of the well-developed morphological feature and controlled drug-release profile, the gelatin-HA nanocomposite porous scaffolds are suggested to be potentially useful for hard-tissue regeneration.

Mechanochemical synthesis of nanocrystalline carbonate-substituted hydroxyapatite

Abstract Carbonate-substituted hydroxyapatite has been produced by solid-state reactions using a mechanochemical synthesis method. Ammonium phosphate monobasic and calcium carbonate were used as raw materials. Mixtures of the initial powders were prepared using a Ca/P molar ratio similar to that of non-substituted hydroxyapatite, Ca10(PO4)6(OH)2. Dry milling of the powders was performed by using a ball to powder weight ratio equals 20:1. The obtained product was characterized by means of infrared spectroscopy, transmission electron microscopy and X-ray diffraction. Under the employed synthesis conditions hydroxyapatite is obtained after 2 h of milling. The crystallinity and the amount of the hydroxyapatite increase as milling time does. After milling for 6 h, nanosized apatite which has a possible composition Ca9+y(CO3)(PO4)5(OH)1+2y is obtained.

Basic calcium phosphate crystals as a unique therapeutic target in osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis that occurs in humans. Despite its prevalence, the pathogenesis of OA is not fully understood. Intraarticular basic calcium phosphate (BCP) (an inclusive term for partially carbonate-substituted hydroxyapatite, octacalcium phosphate and tricalcium phosphate) crystals are implicated in OA and are associated with severe degenerative arthritis characterized by marked synovial hyperplasia, aggravated joint degeneration and large joint effusions. Their pathogenicity relates, at least in part, to their ability to stimulate cellular mitogenesis in a number of cell types including macrophages, porcine articular chondrocytes (PAC) and human fibroblasts (HF) and induce prostaglandin, cytokine and matrix metalloproteinase synthesis and secretion in HF and PAC. Identification of BCP crystals in OA joints remains problematic because of the lack of a simple and reliable analytic procedure. There is currently no drug available that prevents the formation or modifies the biological effects of BCP crystals. This review highlights the recent advances in our knowledge of BCP crystal deposition diseases and discusses the potential therapeutic strategies for BCP crystal-associated OA.

Silk fibroin regulated mineralization of hydroxyapatite nanocrystals

In the present study, silk fibroin was employed to regulate the mineralization of hydroxyapatite (HA) nanocrystals. The calcium phosphate crystals precipitated in the aqueous solution of silk fibroin at pH 8 and room temperature. The depositions collected at different reaction time were detected by X-ray diffraction analysis to investigate the mineralization process of calcium phosphate. The results indicated that fibroin protein could significantly promote the crystal growth of HA. The formed HA crystals were also studied by Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The FTIR results revealed that the HA crystals are carbonate-substituted HA and compounded with fibroin. There are strong chemical interactions between HA and fibroin protein, which can be derived from the blue shift of amide II peak (from the position of 1517–1539cm−1). TEM images showed that the mineralized nanofibrils in the composites are rod like in shape with the diameter of about 2–3nm. Selected area electron diffraction patterns from the composites exhibit polycrystalline rings, which were well indexed as the HA phase with 002 preferential orientations.