Nanosized Hydroxyapatite Crystals Research Articles

Effects of physical properties of nano-sized hydroxyapatite crystals on cellular toxicity in renal epithelial cells

Hydroxyapatite (HAP) is not only a common component of most idiopathic CaOx stones, but also the core of Randall's plaque. HAP is a nest that can induce the formation of Randall's plaques and even kidney stones. We studied the toxic effects and mechanisms of four different types of nano-HAP crystals (H-Sphere, 72.5 nm × 72.5 nm; H-Needle, 37.2 nm × 162.7 nm; H-Rod, 42.3 nm × 115.3 nm; and H-Plate, 145.5 nm × 272.9 nm) on human renal proximal tubular epithelial cells (HK-2). HAP crystals could cause oxidative stress that triggered a series of cell dysfunction problems, resulting in decreased cell viability, loss of cell membrane integrity, cell swelling, and cell necrosis. The toxic effect of HAP was mainly attributed to its entry into cell by endocytosis and its accumulation in the lysosomes, causing the level of intracellular reactive oxygen species (ROS) to rise, the mitochondrial membrane potential (Δψm) to decrease, the lysosomal integrity to be destroyed, and the cell cycle blocked during the G0/G1 phase. The cytotoxicity of the four kinds of HAP crystals was ranked as follows: H-Sphere > H-Needle > H-Rod > H-Plate. The cytotoxicity of each crystal was positively correlated with low absolute zeta potential, conduciveness to internalized morphology, large specific surface area and aspect ratio, and small particle size. These results indicated that nano-HAP could damage HK-2 cells, and the physical properties of HAP crystals play a vital effect in their cytotoxicity.

Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells.

ObjectiveThis study aimed to investigate the damage mechanism of nanosized hydroxyapatite (nano-HAp) on mouse aortic smooth muscle cells (MOVASs) and the injury-inhibiting effects of diethyl citrate (Et2Cit) and sodium citrate (Na3Cit) to develop new drugs that can simultaneously induce anticoagulation and inhibit vascular calcification.MethodsThe change in cell viability was evaluated using a cell proliferation assay kit, and the amount of lactate dehydrogenase (LDH) released was measured using an LDH kit. Intracellular reactive oxygen species (ROS) and mitochondrial damage were detected by DCFH-DA staining and JC-1 staining. Cell apoptosis and necrosis were detected by Annexin V staining. Intracellular calcium concentration and lysosomal integrity were measured using Fluo-4/AM and acridine orange, respectively.ResultsNano-HAp decreased cell viability and damaged the cell membrane, resulting in the release of a large amount of LDH. Nano-HAp entered the cells and damaged the mitochondria, and then induced cell apoptosis by producing a large amount of ROS. In addition, nano-HAp increased the intracellular Ca2+ concentration, leading to lysosomal rupture and cell necrosis. On addition of the anticoagulant Et2Cit or Na3Cit, cell viability and mitochondrial membrane potential increased, whereas the amount of LDH released, ROS, and apoptosis rate decreased. Et2 Cit and Na3Cit could also chelate with Ca+ to inhibit the intracellular Ca2+ elevations induced by nano-HAp, prevent lysosomal rupture, and reduce cell necrosis. High concentrations of Et2Cit and Na3Cit exhibited strong inhibitory effects. The inhibitory capacity of Na3Cit was stronger than that of Et2Cit at similar concentrations.ConclusionBoth Et2Cit and Na3Cit significantly reduced the cytotoxicity of nano-HAp on MOVASs and inhibited the apoptosis and necrosis induced by nano-HAp crystals. The chelating function of citrate resulted in both anticoagulation and binding to HAp. Et2Cit and Na3Cit may play a role as anticoagulants in reducing injury to the vascular wall caused by nano-HAp.

On the electrocrystallization of pure hydroxyapatite nanowalls on Nitinol alloy using a bipolar pulsed current

In this study a bipolar pulsed current was used for the electrochemical deposition of calcium phosphate coatings on the Nitinol superelastic alloy. The coatings were characterized using FE-SEM, XRD, FTIR, HRTEM, and gas adsorption techniques. Results show that the electrodeposited coating is a crystalline film which is composed of pure hydroxyapatite (HA) nanowalls. According to the FTIR data, the coating is free from other metastable calcium phosphate phases like octacalcium phosphate. It was shown that both monoclinic and hexagonal phases can be detected in HRTEM images and related SAED patterns. The coating, which is deposited at direct and reverse current densities of −3.0 and 0.1 mA/cm2 respectively, revealed a surface area of 38.5 m2/g and ultra-fine pores were detected in the surface of the coating plates. Study on the effect of the reverse pulse current revealed that the volume of porosity is increased by increasing in the current density of the reverse pulse. Also in this condition the resulted film was composed of nanosized HA crystals. It seems that the reverse current step can influence the porosity and the structure of the coatings by dissolution of the unstable phases which are formed during direct current step of the bipolar pulse deposition.

Bioinspired Synthesis of Large-Pore, Mesoporous Hydroxyapatite Nanocrystals for the Controlled Release of Large Pharmaceutics

Mesoporous hydroxyapatite (HA) rod-like nanocrystals (MHANs) with large and tunable mesopores and small particle sizes were synthesized using a novel bioinspired route where mesoporous silica nanoparticles (MSNs) acted as effective nucleating agents, and fetuin (an abundant serum protein) inhibited the growth of HA. Under the cooperative influences of the MSNs and fetuin, the nucleation and growth processes of MHANs were effectively controlled such that nanosized HA crystals with dumbbell morphologies, mesoporous structures, and large surface areas were produced. This synthetic strategy was also controllable such that MHANs with particle sizes of 235–515 nm, pore sizes of 21.1–30.5 nm, and surface areas of 130–225 m2 g–1 could be systematically produced by tuning the fetuin concentration. The MHANs were then investigated as drug delivery vehicles and exhibited a high loading capacity and sustained release of large pharmaceutics, as shown through a model large-molecule protein. Demonstration of effective i...

Controlled growth of hydroxyapatite on the surface of natural stilbite from Ethiopia: application in mitigation of fluorosis

This contribution reports the preparation of a composite material based on a natural zeolite rich in calcium, stilbite, on which surface nanosized hydroxyapatite is grown under controlled conditions. Low crystallisation temperatures enable the crystallisation of nano-sized hydroxyapatite crystals, which display a very high intrinsic capacity (based on weight of hydroxyapatite) of fluoride removal. The low cost and easy accessibility of the materials employed for its preparation, as well as the simplicity of the synthesis procedure, make these materials suitable candidates for their use in fluoride removal from drinking water.

Micro- and nanostructured hydroxyapatite-collagen microcarriers for bone tissue-engineering applications

Novel hydroxyapatite (HA)-collagen microcarriers (MCs) with different micro/nanostructures were developed for bone tissue-engineering applications. The MCs were fabricated via calcium phosphate cement (CPC) emulsion in oil. Collagen incorporation in the liquid phase of the CPC resulted in higher MC sphericity. The MCs consisted of a porous network of entangled hydroxyapatite crystals, formed as a result of the CPC setting reaction. The addition of collagen to the MCs, even in an amount as small as 0.8 wt%, resulted in an improved interaction with osteoblast-like Saos-2 cells. The micro/nanostructure and the surface texture of the MCs were further tailored by modifying the initial particle size of the CPC. A synergistic effect between the presence of collagen and the nanosized HA crystals was found, resulting in significantly enhanced alkaline phosphatase activity on the collagen-containing nanosized HA MCs.

Characterization and structural analysis of zinc-substituted hydroxyapatites

The substitution of Zn in hydroxyapatite (HA) crystals was examined via comprehensive characterization techniques. Nanosized HA crystals were synthesized by the wet chemical method in aqueous solutions including various amounts of Zn ions. X-ray fluorescent spectroscopy was used to examine the amount of Zn in the HA precipitates. Scanning electron microscopy and high-resolution transmission electron microscopy were employed to examine the effects of Zn on the morphology and crystal size of the precipitates. Conventional powder X-ray diffraction and the Rietveld refinement method revealed the apatite lattice parameters and phase changes with the inclusion of Zn. The results indicated that Zn ions partially substituted for Ca ions in the apatite structure. They were not simply adsorbed on the apatite surface or in the amorphous phase. The precipitates maintained the apatite phase up to a Zn:(Zn + Ca) ratio of 15–20 mol.% in the solution. Pure HA was well crystallized and the crystals had regular shapes, whereas the Zn-substituted apatite crystals became irregular and formed agglomerates. The lattice parameters, a and c, decreased at a Zn:(Zn + Ca) ratio of 10 mol.%.

Nonwoven silk fibroin net/nano‐hydroxyapatite scaffold: Preparation and characterization

The purpose of this study was to prepare and characterize a three-dimensional scaffold consisting of porous nonwoven silk fibroin net/nano-apatite composite. The silk fibroin net was mineralized with apatite by alternative soaking in calcium and phosphate solutions. The scaffold exhibited a porous microstructure with open porosity (70-78%), with an average pore size of about 163 +/- 40.0 microm. The swelling ratio and water uptake were 4.55% and 81.93%, respectively, indicating excellent hydrophilicity. The nano-sized hydroxyapatite crystals were needle like, with a length of approximately 100-300 nm and a diameter of approximately 20-60 nm. The results of in vitro cell culture study using rat osteoblast cells demonstrated that the nonwoven silk fibroin net/nano-apatite composite showed excellent cytocompatibility for the growth of osteoblasts and had the capability to improve the viability of osteoblasts.

Effect of silk sericin on assembly of hydroxyapatite nanocrystals into enamel prism-like structure

Taking the inspiration from the biomineral, the silk sericin was selected to modulate the assembly of nanosized hydroxyapatite crystals via a modified co-precipitation method. The effect of silk sericin concentration and mineralization time on the formation of hydroxyapatite nanocrystals, including their nucleation, growth, aggregation, especially assembly dynamics, were investigated using the transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. The results revealed that both the concentration of silk sericin and the mineralization time could affect the size, morphology and assembly of hydroxyapatite crystals. Higher concentration of silk sericin in the reaction system was apt to form the larger hydroxyapatite crystals and longer mineralization time helped to improve the crystallinity of hydroxyapatite crystals together with an increase of crystal size. More interested thing was that an arrangement of nanocrystals in order was observed when the concentration of silk sericin was kept at 1(w/v)% in the reaction system. The larger rod-like hydroxyapatite crystals of 300–500 nm in length and 50–80 nm in diameter were assembled along the c-axis by the smaller crystals of ca. 20 nm under the control of silk sericin, whose arrangement method was similar to the natural enamel. A proposed formation mechanism of the special structure was elucidated. In addition, the biocompatibility of obtained crystals was evaluated in vitro and their strong ability to promote the cell differentiation and proliferation was proved in the paper, which may be important for the biomedical applications and also for the fundamental studies of cell–matrix interactions.

Strong, macroporous, and in situ-setting calcium phosphate cement-layered structures

Calcium phosphate cement (CPC) is highly promising for clinical uses due to its in situ-setting ability, excellent osteoconductivity and bone-replacement capability. However, the low strength limits its use to non-load-bearing applications. The objectives of this study were to develop a layered CPC structure by combining a macroporous CPC layer with a strong CPC layer, and to investigate the effects of porosity and layer thickness ratios. The rationale was for the macroporous layer to accept tissue ingrowth, while the fiber-reinforced strong layer would provide the needed early-strength. A biopolymer chitosan was incorporated to strengthen both layers. Flexural strength, S (mean±sd; n=6) of CPC-scaffold decreased from (9.7±1.2) to (1.8±0.3) MPa ( p<0.05), when the porosity increased from 44.6% to 66.2%. However, with a strong-layer reinforcement, S increased to (25.2±6.7) and (10.0±1.4) MPa, respectively, at these two porosities. These strengths matched/exceeded the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. Relationships were established between S and the ratio of strong layer thickness/specimen thickness, a/h: S=(17.6 a/h+3.2) MPa. The scaffold contained macropores with a macropore length (mean±sd; n=147) of (183±73) μm, suitable for cell infiltration and tissue ingrowth. Nano-sized hydroxyapatite crystals were observed to form the scaffold matrix of CPC with chitosan. In summary, a layered CPC implant, combining a macroporous CPC with a strong CPC, was developed. Mechanical strength and macroporosity are conflicting requirements. However, the novel functionally graded CPC enabled a relatively high strength and macroporosity to be simultaneously achieved. Such an in situ-hardening nano-apatite may be useful in moderate stress-bearing applications, with macroporosity to enhance tissue ingrowth and implant resorption.

Development of a nonrigid, durable calcium phosphate cement for use in periodontal bone repair

Calcium phosphate cement (CPC) hardens in situ to form hydroxyapatite and has been used in dental and craniofacial restorative applications. However, when CPC was used in periodontal osseous repair, tooth mobility resulted in the fracture and exfoliation of the brittle CPC implant. The objective of the authors' study was to develop a strong and nonrigid CPC to provide compliance for tooth mobility without fracturing the implant. The authors used tetracalcium phosphate, dicalcium phosphate anhydrous and biopolymer chitosan to develop a strong and nonrigid CPC. They used a powder:liquid ratio of 2:1, compared with the 1:1 ratio of a previously developed nonrigid CPC control. Specimens were characterized using a flexural test, scanning electron microscopy and powder X-ray diffraction. After 28 days of immersion, the new cement had a flexural strength (mean +/- standard deviation; n = 6) of 5.2 +/- 1.0 megapascals, higher than 1.8 +/- 1.5 MPa for the control (P < .05) and overlapping the reported strengths of sintered hydroxyapatite implants and cancellous bone. This cement showed a high ductility with a strain at peak load of 6.5 +/- 1.3 percent, compared with 4.4 +/- 1.9 percent for the control; both were 20-fold higher than the 0.2 percent of the conventional CPC. Nanosized hydroxyapatite crystals, similar to those in teeth and bones, were formed in the cements. The new nonrigid cement, containing nanohydroxyapatite crystals, possessed a high ductility and superior fracture resistance. This strong, tough and nonrigid CPC may be useful in periodontal repair to provide compliance for tooth mobility without fracture. The results of this study may yield the first self-hardening and nonrigid hydroxyapatite composite with high strength and durability and large deformation capability to be useful in the regeneration of periodontal osseous defects.