Hydroxyapatite Crystal Growth Research Articles

The effect of interfacial bonding of calcium phosphate cements containing bio-mineralized multi-walled carbon nanotube and bovine serum albumin on the mechanical properties of calcium phosphate cements

The aim of this study was to investigate the effect of adding bio-mineralized hydroxyl functionalized multi-walled carbon nanotubes (MWCNTs–OH) on the compressive strength of calcium phosphate cements (CPCs). Bovine serum albumin (BSA) was also incorporated as a protein which acts as promoter of hydroxyapatite (HA) crystal growth when bounded to CPC granules. The results show that the strong interfacial bonding of CPC/MWCNTs–OH is essential to improve the mechanical properties of CPC/bio-mineralized MWCNTs–OH/BSA composite.

Preparation and In Vitro Bioactivity of Novel Mesoporous Borosilicate Bioactive Glass Nanofibers

Mesoporous borosilicate bioactive glass nanofibers consisting of a network of interconnected macropores and mesopores were developed using electrospinning technology combined with a polymer/surfactant cotemplate. The morphology and structure of the nanofibrous scaffolds were studied by scanning electron microscopy, energy‐dispersive spectroscopy, Fourier transform infrared spectroscopy, and X‐ray diffraction. The morphology and mesoporous structure of the borosilicate glass nanofibers can be adjusted by altering the concentrations of the glass precursor solution and surfactant. The in vitro bioactivity of the bioglass nanofibers was investigated by immersion in simulated body fluid. Results from these experiments suggested that the mesoporous borosilicate glass nanofibrous scaffold had great potential for bone regeneration because it promoted the rapid growth of hydroxyapatite crystals.

Coating of hydroxyapatite films on metal substrates by seeded hydrothermal deposition

Hydroxyapatite (HAP) was successfully coated on various metal substrates by a novel seeded hydrothermal deposition method. A nanoscale HAP seed layer was first formed by a short electrochemical synthesis. The seed layer promotes HAP crystal growth onto the surface during a subsequent hydrothermal crystallization step. The surface morphology and microstructure of the HAP coatings can be regulated by varying the reaction temperature, solution pH, calcium-to-phosphorus molar ratio in the starting solution, and hydrothermal deposition time. The new method has advantages over many other reported HAP deposition techniques in that it produces highly crystalline, crack-free, adherent films of uniform thickness. In all the films, the HAP crystals are preferentially oriented with the c-axis normal to the substrate. The as-developed HAP coatings are attractive for applications in the area of bioactive surface modification of metallic implants where the microstructure of the film is advantageous for promoting bone growth.

Biomimetic growth of hydroxyapatite on super water-soluble carbon nanotube-protein hybrid nanofibers

We report here on a facile strategy for the creation of super water-soluble carbon nanotube (CNT)-protein hybrid nanofibers. Toluene-soluble CNTs were first synthesized by noncovalent functionalizing CNTs with a bifunctional linker, Z-glycine N-succinimidyl ester (Z-Gly-OSu), and then water-soluble CNT-protein hybrid nanofibers were created by phase-transferring the Z-Gly-OSu-functionalized CNTs from the toluene phase to protein aqueous solution. CNT-fibronectin (CNT-FN) and CNT-hemoglobin (CNT-HGB) hybrid nanofibers were successfully prepared in this work. Raman and XPS spectra confirmed the successful functionalization of CNTs with Z-Gly-OSu. TEM results indicated the formation of CNT-protein hybrid nanofibers. Biomimetic mineralization was conducted on the CNT-FN hybrid nanofibers and nanofiber film to explore the effects of protein and CNTs on the formation of hydroxyapatite (HA) crystals. SEM, Raman spectra, and TEM results indicated that the FN molecules promote the nucleation of crystalline nuclei and the CNT templates control the orientation of nuclei and the crystal growth of HA to form flake-like HA crystals.

Hexagonal hydroxyapatite formation on TiO2 nanotubes under urea modulation

Hexagonal prism-like hydroxyapatite (HA) crystals were synthesized under hydrothermal conditions utilizing the combination modulation of TiO2 nanotubes and urea. TiO2 nanotubes were used as templates to induce HA crystal nucleation under hydrothermal conditions. Comparison of HA formation on nanotubular surfaces and flat surfaces demonstrated that nanotubular surfaces favored prism-like hexagonal HA crystallization. Urea was selected as the precipitation agent to control the HA growth and mediate its morphology. The hydrothermal temperature should be high enough to lead the decomposition of urea in order to ensure HA formation. Density functional theory (DFT) calculation showed that the urea had higher binding energy with (100) surfaces than with (001) surfaces, and therefore could inhibit the growth of (100) surfaces of HA, which possibly influenced the morphological development of HA crystals. This study demonstrated that HA crystal growth could be modulated in its nucleation and growth stage. It also implied that computer simulation might play an important role to select the suitable precipitation agent in order to obtain crystals with specially designed morphology.

Influence of magnesium ions and amino acids on the nucleation and growth of hydroxyapatite

It has long been known that magnesium (Mg) ions are effective inhibitors of hydroxyapatite (HAP) crystal nucleation and growth. During the HAP nucleation, it is now generally recognized that the involvement of assembled amorphous calcium phosphate (ACP) nuclei and prenuclei may be of greater importance in the overall crystallization mechanism. In the calcium-phosphate (Ca–P) biomineralization process significant amounts of Mg ions are present in animal skeletons where they are thought to stabilize ACP mineral phases. However, there is still some uncertainty concerning the precise roles of Mg in the nucleation and growth of apatite minerals. Herein, a slow, well-controlled, Ca–P crystallization study under a precisely defined thermodynamic driving force shows that Mg ions are able to prevent the nucleation of HAP in supersaturated Ca–P solutions by stabilizing gel-like ACP phases, prolonging the induction and subsequent transformation times, under near-physiological conditions (pH 7.400, ionic strength (I) = 0.150 mol L−1, 37.0 °C). Relatively large HAP crystals growing from the ACP surfaces were characterized by HRTEM and SEM. Surprisingly, EDS spectra revealed that no Mg ions were present in either the gel-like ACP or final crystalline phase. Following the addition of amino acids to the supersaturated solutions both in the presence and absence of Mg ions, the nucleation of HAP was promoted by shortening the induction and transformation times, and at the same time, the sizes of ACP and HAP crystals decreased as compared with the controls. Our results suggest that the formation of magnesium-phosphate (Mg–P) ion pairs may reduce the thermodynamic driving force for nucleation and phase transformation to HAP.

Modifications of a calcium phosphate cement with biomolecules—Influence on nanostructure, material, and biological properties

Calcium phosphate cements (CPC), forming hydroxyapatite during the setting reaction, are characterized by good biocompatibility and osteoconductivity, however, their remodeling into native bone tissue is slow. One strategy to improve remodeling and bone regeneration is the directed modification of their nanostructure. In this study, a CPC was set in the presence of cocarboxylase, glucuronic acid, tartaric acid, α-glucose-1-phosphate, L-arginine, L-aspartic acid, and L-lysine, respectively, with the aim to influence formation and growth of hydroxyapatite crystals through the functional groups of these biomolecules. Except for glucuronic acid, all these modifications resulted in the formation of smaller and more agglomerated hydroxyapatite particles which had a positive impact on the biological performance indicated by first experiments with the human osteoblast cell line hFOB 1.19. Moreover, adhesion, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) as well as binding of the growth factors BMP-2 and VEGF was investigated on CPC modified with cocarboxylase, arginine, and aspartic acid. Initial adhesion of hBMSC was improved on these three modifications and proliferation was enhanced on CPC modified with cocarboxylase and arginine whereas osteogenic differentiation remained unaffected. Modification of the CPC with arginine and aspartic acid, but not with cocarboxylase, led to a higher BMP-2 binding.

Mineralization of breast capsular tissue: changes in factors affecting apatite formation and growth

Experiments were designed to investigate isolatable factors that could either promote or inhibit formation of hydroxyapatite (HA) crystals associated with mineralization of the fibrous capsule surrounding silicone breast implants. Explanted capsular tissue was minced, filtered (0.2μm), inoculated into culture plates containing DMEM/10% FBS and kept at 37°C, 95%O2. Within 14 days, biofilm‐like and suspending (planktonic) aggregates containing HA formed to a greater extent in plates inoculated with sample from calcified (CC) than non‐calcified (NC) capsules (158 vs 4 nephelometric turbidity units, respectively). Confocal microscopy of formalin‐fixed sections of NC showed the major cell types to be macrophages and fibroblasts. These cells were immunopositive for fetuin, a blood protein. In contrast, CC were largely fibrous; fetuin labeling was sparse except for the calcific plaque itself on the surface of the capsule adjacent to the implant. Thus, a factor which affects HA formation, possibly a HA nucleator, or a modifier of nucleator activity, can be isolated from mineralized fibrous breast capsules. Localization of fetuin within NC but not CC cells is consistent with its known role in inhibiting HA crystal growth. Supported by grants from the Fetzer Foundation, NIH HL88988 and Division of Experimental Surgery, Mayo Clinic.

Physiological role of alkaline phosphatase explored in hypophosphatasia

Hypophosphatasia (HPP) is the instructive rickets or osteomalacia caused by loss-of-function mutation(s) within TNSALP, the gene that encodes the "tissue nonspecific" isoenzyme of alkaline phosphatase (TNSALP). HPP reveals a critical role for this enzyme in skeletal mineralization. Increased extracellular levels of pyridoxal 5'-phosphate and inorganic pyrophosphate (PP(i)) demonstrate that TNSALP is a phosphomonoester phosphohydrolase and a pyrophosphatase that hydrolyzes much lower concentrations of natural substrates than the artificial substrates of laboratory assays. Clearly, TNSALP acts at physiological pH and "alkaline phosphatase" is a misnomer. Aberrations of vitamin B(6) metabolism in HPP revealed that TNSALP is an ectoenzyme. PP(i) excesses cause chondrocalcinosis and sometimes arthropathy. The skeletal disease is due to PP(i) inhibition of hydroxyapatite crystal growth extracellularly so that crystals form within matrix vesicles but fail to enlarge after these structures rupture. Trials of alkaline phosphatase replacement therapy for HPP suggest that TNSALP functions at the level of skeletal tissues.

Induction of Hydroxyapatite Particles Formation on PEGDA-Based Hydrogels by Nanobacteria

Nanobacteria is a tiny structure with size varying 80 to 500nm, commonly occurring in clusters and producing a biofilm which contains carbonate or hydroxyl apatite. In this study, the bioactive synthetic hydrogel materials were prepared with polyethylene glycol diacrylate (PEGDA) and 2-hydroxyethyl mathacrylate (HEMA) by UV photo-polymerization. Bone marrow mesenchymal stem cells (BMSCs) were seeded onto hydrogel surface for five days. The BMSCs cell adhesion on hydrogels was confirmed by SEM to evaluate the biocompatibility of the materials. It was found groups of nanoparticles on the hydrogel surface and the particles were analyzed by SEM. The particles were analyzed for its inorganic chemical constituents using energy dispersive X-ray microanalysis (EDS). The predominant components were found to be calcium (24.40%) and phosphorus (13.98%). The most likely source of cell culture contamination by such organisms is bovine serum albumin (BSA) used as supplement in culture media. Nanobacteria in BSA may be the important factor which accelerated hydroxyapatite crystal growth on hydrogels. It is important to study the biomineralization in biological system and has potential application in biomaterials science and biotechnology.

Hydroxyapatite nucleation and growth mechanism on electrospun fibers functionalized with different chemical groups and their combinations

Controlled nucleation and growth of hydroxyapatite (HA) crystals on electrospun fibers should play important roles in fabrication of composite scaffolds for bone tissue engineering, but no attempt has been made to clarify the effects of chemical group densities and the cooperation of two and more groups on the biomineralization process. The aim of the current study was to investigate into HA nucleation and growth on electrospun poly( dl-lactide) fibers functionalized with carboxyl, hydroxyl and amino groups and their combinations. Electrospun fibers with higher densities of carboxyl groups, combination of hydroxyl and carboxyl groups with the ratio of 3/7, and combination of amino, hydroxyl and carboxyl groups with the ratio of 2/3/5 were favorable for HA nucleation and growth, resulting in higher content and lower crystal size of formed HA. Carboxyl groups were initially combined with calcium ions through electrostatic attraction, and the introduction of hydroxyl groups could modulate the distance between carboxyl groups. The introduction of amino groups may lead to the inner ionic bonding with carboxyl groups, but can accelerate phosphate ions to form HA through a chelate ring with the calcium ion and carbonyl oxygen. The biological evaluation indicated that the mineralized scaffolds acted as an excellent cell support to maintain desirable cell–substrate interactions, to provide favorable conditions for cell proliferation and to stimulate the osteogenic differentiation.

Dynamic Effect of Citrate on Electrolytic Deposition of Hydroxyaptite on Ti Surface

During electrolytic deposition process of hydroxyapatite(HA) on Ti surface,2.4mmol/L citrate is added into the electrolyte containing 0.6mmol/L Ca2+ ion and 0.36mmol/L H2PO-4 ion.The results show that the incubation period of the HA is prolonged significantly,and the HA crystals take the morphology of needlelike cone rather than the typical hexagonal prism.Through analyzing the deposition current,coating weight increment and crystal morphology changes,the whole deposition process is divided into 5 procedures: adsorption period,incubation period,eruptive growth period,extra-layer growth period and balanced growth period.Based on the further discussion of micro-mechanism of each period,the HA crystal growth model during electrolytic deposition process is proposed.

Rapid growth of hydroxyapatite nanoparticles using ultrasonic irradiation

A rapid, environmental friendly and low-cost method to prepare hydroxyapatite nanoparticles is proposed. In this method, hydroxyapatite is produced in a sonicated pseudo-body solution. The sonication time was found effective in the formation of the crystalline phase of nanoparticles. In our experimental condition, 15 min sonication resulted in the most pure hydroxyapatite phase. Also it was shown that growth temperature is a crucial factor and hydroxyapatite crystallizes only at 37 °C. The particles formed by sonication were generally smaller and more spherical than those obtained without sonication. Sonication increased the hydroxyapatite crystal growth rate up to 5.5 times compared to non-sonication condition. The comparison between the specific surface area of hydroxyapatite nanoparticles obtained by sonication and without sonication demonstrated that sonication increased the specific surface area from 63 m 2/g to 107 m 2/g and decreased the size of nanoparticles from 30 nm to 18 nm. Analysis on the pore structure demonstrated that the fractal structures obtained with and without sonication were considerably different.

Loss-of-Function ENPP1 Mutations Cause Both Generalized Arterial Calcification of Infancy and Autosomal-Recessive Hypophosphatemic Rickets

The analysis of rare genetic disorders affecting phosphate homeostasis led to the identification of several proteins that are essential for the renal regulation of phosphate homeostasis; for example, fibroblast growth factor 23 (FGF23), which inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D synthesis. Here, we report presumable loss-of-function mutations in the ENPP1 gene (ectonucleotide pyrophosphatase/phosphodiesterase) in members of four families affected with hypophosphatemic rickets. We provide evidence for the conclusion that ENPP1 is the fourth gene-in addition to PHEX, FGF23, and DMP1-that, if mutated, causes hypophosphatemic rickets resulting from elevated FGF23 levels. Surprisingly, ENPP1 loss-of-function mutations have previously been described in generalized arterial calcification of infancy, suggesting an as yet elusive mechanism that balances arterial calcification with bone mineralization.

New application of poly(butylene succinate) (PBS) based ionomer as biopolymer: a role of ion group for hydroxyapatite (HAp) crystal formation

Sodium sulfonate ionic group bearing PBS ionomer (PBSi) and hydroxyapatite (HAp) composites were prepared by soaking in the simulated body fluid (SBF) solution, which is the biomimetic method, as well as the effects of the ionic group on the HAp crystal formation and growth were investigated. The introduced sodium sulfonate ionic groups operated as the functional groups with negative charge densities, which can bind plentiful Ca2+ ions efficiently, consequently serving as active sites allowing HAp crystals to grow on the surfaces of the PBSi matrix. By SEM analysis, it was observed that HAp became growing as the shape with the porous holes. These holes are thought to be very suitable for the ingrowths of the surrounding tissue and the assistance to the bone formation. Based on this finding, it can be clearly concluded that the ionic groups in the PBSi may be decisive factors in growing HAp, and it is anticipated that this novel materials can contribute to excellent biopolymer.

Cobalt, chromium and nickel affect hydroxyapatite crystal growth in vitro

Metals are widely used in orthopaedics and recent studies have reported that patients with metal implants have a significant increase of metal levels in serum and synovial fluid. Femoral neck fracture occurred in some patients with metal-on-metal implants for unknown reasons. Recently, bone quality has emerged as an important factor of bone strength and few studies have investigated the effects of metal ions on hydroxyapatite properties. In the present study, we investigated the effects of Co 2+, Cr 3+ and Ni 2+ on hydroxyapatite (HA) growth in vitro, using carboxymethylated poly(2-hydroxyethyl methacrylate) (pHEMA) as a biomaterial for calcification. We have demonstrated that metal ions reduced the quantity of mineral formed at the surface of the polymer and decreased the ratio Ca/P by 1.12-, 1.05- and 1.08-fold for Cr 2+, Cr 3+ and Ni 2+ respectively. Furthermore, the size of calcospherites was significantly increased in the metal-doped HA compared to the controls, indicating a possible effect of metal ions on the crystal lattice. Indeed, the presence of metal ions increased the crystal size as well as the crystallinity of HA and reduce the lattice parameter c of the HA framework. The information obtained from this work suggests that the quality of the mineral around metallic implants could be altered. However, further investigation should be conducted to further elucidate the effects of metal incorporation on bone mineral and the functional consequences.

Long‐term effects of magnesium carbonate on coronary artery calcification and bone mineral density in hemodialysis patients: A pilot study

Observational data suggest that elevated magnesium levels in dialysis patients may prevent vascular calcification and in vitro magnesium can prevent hydroxyapatite crystal growth. However, the effects of magnesium on vascular calcification and bone mineral density have not been studied prospectively. Seven chronic hemodialysis patients participated in this open label, prospective pilot study to evaluate the effects of a magnesium-based phosphate binder on coronary artery calcification (CAC) scores and vertebral bone mineral density (V-BMD) in patients with baseline CAC scores >30. Magnesium carbonate/calcium carbonate (elemental Mg: 86 mg/elemental Ca 100 mg) was administered as the principal phosphate binder for a period of 18 months and changes in CAC and V-BMD were measured at baseline, 6, 12, and 18 months. Serum magnesium levels averaged 2.2+/-0.4 mEq/L (range: 1.3-3.9 mEq/L). Phosphorus levels (4.5+/-0.6 mg/dL) were well controlled throughout the 18 months study. Electron beam computed tomography results demonstrated a small not statically significant increase in absolute CAC scores, no significant change in median percent change, and a small none significant change in V-BMD. Magnesium may have a favorable effect on CAC. The long-term effect on bone mineral density remains unclear. Larger studies are needed to confirm these findings.

Kinetic and morphological effects of biopolymers on the growth of hydroxyapatite crystals

Kinetic and morphological effects of biopolymers on the growth of hydroxyapatite crystals

A (13)C{(31)P} REDOR NMR investigation of the role of glutamic acid residues in statherin- hydroxyapatite recognition.

The side chain carboxyl groups of acidic proteins found in the extra-cellular matrix (ECM) of mineralized tissues play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), the principal mineral component of bone and teeth. Among the acidic proteins found in the saliva is statherin, a 43-residue tyrosine-rich peptide that is a potent lubricant in the salivary pellicle and an inhibitor of both HAP crystal nucleation and growth. Three acidic amino acids-D1, E4, and E5-are located in the N-terminal 15 amino acid segment, with a fourth amino acid, E26, located outside the N-terminus. We have utilized (13)C{(31)P} REDOR NMR to analyze the role played by acidic amino acids in the binding mechanism of statherin to the HAP surface by measuring the distance between the delta-carboxyl (13)C spins of the three glutamic acid side chains of statherin (residues E4, E5, E26) and (31)P spins of the phosphate groups at the HAP surface. (13)C{(31)P} REDOR studies of glutamic-5-(13)C acid incorporated at positions E4 and E26 indicate a (13)C-(31)P distance of more than 6.5 A between the side chain carboxyl (13)C spin of E4 and the closest (31)P in the HAP surface. In contrast, the carboxyl (13)C spin at E5 has a much shorter (13)C-(31)P internuclear distance of 4.25 +/- 0.09 A, indicating that the carboxyl group of this side chain interacts directly with the surface. (13)C T(1rho) and slow-spinning MAS studies indicate that the motions of the side chains of E4 and E5 are more restricted than that of E26. Together, these results provide further insight into the molecular interactions of statherin with HAP surfaces.

Chronic Recurrent Multifocal Osteomyelitis Mimicked in Childhood Hypophosphatasia

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by low serum alkaline phosphatase (ALP) activity caused by inactivating mutations within TNSALP, the gene that encodes the "tissue-nonspecific" isoenzyme of ALP (TNSALP). In HPP, extracellular accumulation of inorganic pyrophosphate, a TNSALP substrate, inhibits hydroxyapatite crystal growth leading to rickets or osteomalacia. Chronic recurrent multifocal osteomyelitis (CRMO) is the pediatric syndrome of periarticular pain and radiographic changes resembling infectious osteomyelitis but without lesional pathogens. Some consider CRMO to be an autoinflammatory disease. An unrelated boy and girl with the childhood form of HPP suffered chronic, multifocal, periarticular pain, and soft tissue swelling. To investigate this unusual complication, we evaluated their cumulative clinical, biochemical, radiological, and histopathological findings and performed mutation analysis of their TNSALP alleles. The earliest radiographic disturbances were typical of childhood HPP. Subsequently, changes consistent with CRMO developed at sites where there was pain, including lucencies, osteosclerosis, and marked expansion of the underlying metaphyses. Bone marrow edema was shown by MRI. Biopsies of affected bone showed nonspecific histopathological findings and no pathogens. The boy was heterozygous (c.1133A>T, p.D378V) and the girl compound heterozygous (c.350A>G, p.Y117C, c.400_401AC>CA, p.T134H) for different TNSALP missense mutations. Nonsteroidal anti-inflammatory drugs diminished their pain, which improved or resolved at maturity. HPP should be considered when CRMO is a diagnostic possibility. Metaphyseal radiographic changes and marrow edema associated with periarticular bone pain and soft tissue swelling suggestive of osteomyelitis can complicate childhood HPP.