Introducing CO2 flux as the carbonate source had an effect on the carbonate content of carbonate apatite (CAp) synthesized by solid state reaction. The reactants were CaCO3 and beta-tricalcium phosphate (β-TCP) and the heat treatment in air was performed at 1250ºC followed by instant cooling in CO2 flux for temperatures ranging from 800ºC room temperature (RT) . The influence of CO2 flux at various temperature drop differences in the cooling process (1250ºC RT, 1250ºC–500ºC, 1250ºC–600ºC, 1250ºC–700ºC, and 1250ºC–800ºC) was tested to optimize the carbonation degree and subsequent effects on the physical and mechanical properties of CAp. Thermally treated samples revealed an increasing degree of carbonation, achieving a maximum of 5.2 wt% at the highest (1250ºC RT) and a minimum of 2.7 wt% at the lowest (1250ºC–800ºC) temperature drop differences, respectively. This showed that the carbonate content was correlated with the increase in exposure to CO2 flux. However, consistent compressive strength, tensile strength, density and porosity were observed against increasing temperature drop differences which indicated that the degree of carbonation exerted no influence on the physical and mechanical properties of CAp. This method enabled the synthesis of solid state CAp simply by exposing calcium phosphate mixtures to CO2 flux. It also allowed the control of carbonate content for desired medical applications.
Immobilization of chlorhexidine (CHX) on stainless steel 316L (SS316L), assisted by a polydopamine film as an intermediate layer is projected as an approach in combating infection while aiding bone regeneration for coating development on orthopedic and dental implants. This study aimed to investigate the ability of CHX coating to promote apatite layer, osteoblast cells viability, adhesion, osteogenic differentiation and mineralization. Stainless steel 316L disks were pre-treated, grafted with a polydopamine film and immobilized with different concentrations of CHX (10-30mM). The apatite layer formation was determined through an in vitro simulated body fluid (SBF) test by ATR-FTIR and SEM-EDX analyses. The osteoblastic evaluations including cells viability, cells adhesion, osteogenic differentiation and mineralization were assessed with human fetal osteoblast cells through MTT assay, morphology evaluation under FESEM, ALP enzyme activity and Alizarin Red S assay. The apatite layer was successfully formed on the CHX coated disks, demonstrating potential excellent bioactivity property. The CHX coatings were biocompatible with the osteoblast cells at low CHX concentration (<20mM) with good adhesion on the metal surfaces. The increment of ALP activity and calcium deposition testified that the CHX coated disks able to support osteoblastic maturation and mineralization. These capabilities give a promising value to the CHX coating to be implied in bone regeneration area.
In this study, randomly oriented hydroxyethyl cellulose/polyvinyl alcohol (HEC/PVA) nanofibers were fabricated by electrospinning. The blend solutions of HEC/PVA with different weight ratio of HEC to PVA were prepared using water as solvent to fabricate nanofibers. These nanofibrous scaffolds were coated with bone-like apatite by immersing into 10x simulated body fluid (SBF) for different time periods. The morphology and structure of the nanofibers were characterized by SEM, FTIR and DSC. FESEM-EDS and FTIR analysis were used to confirm the deposition of apatite on the surface of nanofibers. The results of this study suggest that this apatite coated nanofibrous scaffolds could be a suitable biomaterial for bone tissue engineering.
In present study, a new composition of glass-ceramic was synthesized based on the Na2O-CaO-SiO2-P2O5 glass system. Heat treatment of glass powder was carried out in 2 stages: 600 °C as the nucleation temperature and different temperature on crystallization at 850, 950 and 1000 °C. The glass-ceramic heat-treated at 950 °C was selected as bioactive filler in commercial PMMA bone cement; (PALACOS® LV) due to its ability to form 2 high crystallization phases in comparison with 850 and 1000 °C. The results of this newly glass-ceramic filled PMMA bone cement at 0-16 wt% of filler loading were compared with those of hydroxyapatite (HA). The effect of different filler loading on the setting properties was evaluated. The peak temperature during the polymerization of bone cement decreased when the liquid to powder (L/P) ratio was reduced. The setting time, however, did not show any trend when filler loading was increased. In contrast, dough time was observed to decrease with increased filler loading. Apatite morphology was observed on the surface of the glass-ceramic and selected cement after bioactivity test.
Existing nonviral gene delivery systems to lungs are inefficient and associated with dose limiting toxicity in mammalian cells. Therefore, carbonate apatite (CO3Ap) nanoparticles were examined as an alternative strategy for effective gene delivery to the lungs. This study aimed to (1) assess the gene delivery efficiency of CO3Ap in vitro and in mouse lungs, (2) evaluate the cytotoxicity effect of CO3Ap/pDNA in vitro, and (3) characterize the CO3Ap/pDNA complex formulations. A significantly high level of reporter gene expression was detected from the lung cell line transfected with CO3Ap/pDNA complex prepared in both serum and serum-free medium. Cytotoxicity analysis revealed that the percentage of the viable cells treated with CO3Ap to be almost similar to the untreated cells. Characterization analyses showed that the CO3Ap/pDNA complexes are in a nanometer range with aggregated spherical structures and tended to be more negatively charged. In the lung of mice, highest level of transgene expression was observed when CO3Ap (8 μL) was complexed with 40 μg of pDNA at day 1 after administration. Although massive reduction of gene expression was seen beyond day 1 post administration, the level of expression remained significant throughout the study period.
Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.
The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial.
Interconnected porous tricalcium phosphate ceramics are considered to be potential bone substitutes. However, insufficient mechanical properties when using tricalcium phosphate powders remain a challenge. To mitigate these issues, we have developed a new approach to produce an interconnected alpha-tricalcium phosphate (α-TCP) scaffold and to perform surface modification on the scaffold with a composite layer, which consists of hybrid carbonate apatite / poly-epsilon-caprolactone (CO3Ap/PCL) with enhanced mechanical properties and biological performance. Different CO3Ap combinations were tested to evaluate the optimal mechanical strength and in vitro cell response of the scaffold. The α-TCP scaffold coated with CO3Ap/PCL maintained a fully interconnected structure with a porosity of 80% to 86% and achieved an improved compressive strength mimicking that of cancellous bone. The addition of CO3Ap coupled with the fully interconnected microstructure of the α-TCP scaffolds coated with CO3Ap/PCL increased cell attachment, accelerated proliferation and resulted in greater alkaline phosphatase (ALP) activity. Hence, our bone substitute exhibited promising potential for applications in cancellous bone-type replacement.
Substantial amount of research has been done in recent decades for the development of nanoparticle systems to selectively deliver drugs to cancer cells for concurrently enhancing and reducing anti-cancer and off-target effects, respectively. pH-sensitive carbonate apatite (CA) was originally developed for efficient and targeted delivery of DNA, siRNA and proteins to various cancer cell lines. Recently, the CA particles were employed to deliver anti-cancer drugs, cyclophosphamide, doxorubicin and methotrexate to cancer cells. Here, we report on the fabrication and characterization of gemcitabine- loaded CA particles, followed by the evaluation of their roles in enhancement of cytotoxicity in two human and one murine breast cancer cell lines. HPLC was performed to measure binding efficiency of the drug to the apatite particles whereas particle size and zeta potential were evaluated to characterize drug/apatite complex. Depending on the initial doses of the drug, its bind binding affinity towards the particles varied from 3.85% to 4.45%. The particle size was found to surprisingly decrease with an increase of the initial drug concentration. In vitro chemosensitivity assay revealed that apatite/drug nanoparticle complexes presented significantly higher cytotoxicity to breast cancer cells compared to free drugs, which could be correlated with the enhanced cellular uptake of the small size drug-loaded particles through endocytosis compared to the passive diffusion of the free drug.
Most of the classical drugs used today to destroy cancer cells lead to the development of acquired resistance in those cells by limiting cellular entry of the drugs or exporting them out by efflux pumps. As a result, higher doses of drugs are usually required to kill the cancer cells affecting normal cells and causing numerous side effects. Accumulation of the therapeutic level of drugs inside the cancer cells is thus required for an adequate period of time to get drugs' complete therapeutic efficacy minimizing the side effects on normal cells. In order to improve the efficacy of chemotherapeutic drugs, nanoparticles of carbonate apatite and its strontium (Sr(2+))-substituted derivative were used in this study to make complexes with three classical anticancer drugs, methotrexate, cyclophosphamide and 5-flurouracil. The binding affinities of these drugs to apatite were evaluated by absorbance and HPLC analysis and the therapeutic efficacy of drug-apatite complexes was determined by cell viability assay. Carbonate apatite demonstrated significant binding affinity towards methotrexate and cyclophosphamide leading to more cellular toxicity than free drugs in MCF-7 and 4T1 breast cancer cells. Moreover, Sr(2+) substitution in carbonate apatite with resulting tiny particles less than 100 nm in diameter further promoted binding of methotrexate to the nanocarriers indicating that Sr(2+)-substituted apatite nanoparticles have the high potential for loading substantial amount of anti-cancer drugs with eventual more therapeutic effectiveness.
Diabetes mellitus is a chronic disease accompanied by a multitude of problems worldwide with subcutaneously administered insulin being the most common therapy currently. Controlledrelease insulin is assumed to be of high importance for long-term glycaemic control by reducing the number of daily injections. Long-acting insulin also mimics the basal insulin levels in normal individuals that may be lacking in diabetic patients. Nanoparticles of carbonate apatite as established for efficient intracellular transport of DNA and siRNA have the potential to be used for sustained release of insulin as responsive nano-carriers. The flexibility in the synthesis of the particles over a wide range of pHs with eventual adjustment of pH-dependent particle dissolution and the manageable variability of particle-integrity by incorporating selective ions into the apatite structure are the promising features that could help in the development of sustained release formulations for insulin. In particular strontium-incorporated carbonate apatite particles were formulated and compared with those of unsubstituted apatite in the context of insulin binding and subsequent release kinetics in DMEM, simulated buffer and finally human blood over a period of 20 hours. Clearly, the former demonstated to have a stronger electrostatic affinity towards the acidic insulin molecules and facilitate to some extent sustained release of insulin by preventing the initial burst effect at physiological pH in comparison with the latter. Thus, our findings suggest that optimization of the carbonate apatite particle composition and structure would serve to design an ideal insulin nano-carrier with a controlled release profile.
Despite being widely used for treating cancer, chemotherapy is accompanied by numerous adverse effects as a result of systemic distribution and nonspecific interactions of the drugs with healthy tissues, eventually leading to therapeutic inefficacy and chemoresistance. Cyclophosphamide (Cyp) as one of the chemotherapeutic pro-drugs is activated in liver and used to treat breast cancer in high dose and in combination with other drugs. In an attempt to reduce the off-target effects and enhance the therapeutic efficacy, pH-sensitive carbonate apatite nanoparticles that had predominantly and size-dependently been localized in liver following intravenous administration, were employed to electrostatically immobilize Cyp and purposely deliver it to the liver for activation. Cyp-loaded particles formed by simple 30 min incubation at 37ºC of the DMEM (pH 7.4) medium containing CaCl2 and Cyp, enhanced in vitro cytotoxicity at different degrees depending on the cell types. The size of the particles could be tightly controlled by the amount of CaCl2 required to prepare the particles and thus the bio-distribution pattern inside different organs of the body. Unlike the small particles (~ 200 nm), the large size particles (~ 600 nm) which were more efficiently accumulated in liver, significantly reduced the tumor volume following intravenous injection in 4T1-induced murine breast cancer model at a very low dose (0.17 mg/Kg) of the drug initially added for complex formation, thus shedding light on the potential applications of the Cyp-loaded nano-formulations in the treatment of breast cancer.
Fission tracks are linear trails of intense radiation damage in the crystal structure of a mineral, produced by spontaneous fissioning of uranium-238 (238U) atoms. Detail information on the low-temperature thermal histories of rocks, below∼120 °C for tracks in apatite and below∼350 °C for zircon, can be provided by Fission-track (FT) analysis. The purpose of this article is to present apatite and zircon fission-track data, and U-Pb granite ages that provide information about the cooling histories of a rock which can be crucial in comprehending the exhumation episodes of the study area, in particular, and the region, in general. Granite samples were collected along the same vertical profile at different elevation, 178-944 m.a.s.l. These samples were used to determine Fission-Track and crystallization ages. HeFTy software was employed to interpret the cooling histories of the samples using forward and inverse models. The inverse model was an approach of reproducing the observed data, and it was carried out only for fission-track data from the apatite grains. And it was constructed after generating a number of forward models, where in each of these models the predicted apatite fission-track parameters were compared to the measured values. The apatite fission track (AFT) and zircon fission track (ZFT) data indicated expected age trends, i.e. the older ages at higher elevations and the younger ages at lower elevations. Similarly, the data shows that the apatite and zircon FT ages appear younger than the age of the rock crystallization. The U-Pb age in zircon consistently suggest the age of the granite is Late Triassic.
The purpose of this study was to characterize commercial glass polyalkenoate cement (GPC) or glass ionomer cement (GIC), Glass Carbomer(®), which is designed to promote remineralization to fluorapatite (FAp) in the mouth. The setting reaction of the cement was followed using magic angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy.
A guided bone regeneration (GBR) membrane has been extensively used in the repair and regeneration of damaged periodontal tissues. One of the main challenges of GBR restoration is bacterial colonization on the membrane, constitutes to premature membrane degradation. Therefore, the purpose of this study was to investigate the antibacterial efficacy of triple-layered GBR membrane composed of poly(lactic-co-glycolic acid) (PLGA), nanoapatite (NAp) and lauric acid (LA) with two types of Gram-negative periodontal bacteria, Fusobacterium nucleatum and Porphyromonas gingivalis through a disc diffusion and bacterial count tests. The membranes exhibited a pattern of growth inhibition and killing effect against both bacteria. The increase in LA concentration tended to increase the bactericidal activities which indicated by higher diameter of inhibition zone and higher antibacterial percentage. It is shown that the incorporation of LA into the GBR membrane has retarded the growth and proliferation of Gram-negative periodontal bacteria for the treatment of periodontal disease.
Cancer cells lose their control on cell cycle by numerous genetic and epigenetic alterations. In a tumor, these cells highly express growth factor receptors (GFRs), eliciting growth, and cell division. Among the GFRs, epidermal growth factor receptor-1 (EGFR1) (Her1/ERBB1) and epidermal growth factor receptor-2 (EGFR2) (Her2/ERBB2) from epidermal growth factor (EGF) family and insulin-like growth factor-1 receptor (IGF1R) are highly expressed on breast cancer cells, thus contributing to the aggressive growth and invasiveness, have been focused in this study. Moreover, overexpression of these receptors is related to suppression of cell death and conferring resistance against the classical drugs used to treat cancer nowadays. Therefore, silencing of these GFRs-encoding genes by using selective small interfering RNAs (siRNAs) could be a powerful approach to treat breast cancer. The inorganic pH sensitive carbonate apatite nanoparticles (NPs) were used as a nano-carrier to deliver siRNA(s) against single or multiple GFR genes in breast cancer cells as well as in a mouse model of breast carcinoma. Silencing of egfr1 and erbb2 simultaneously led to a reduction in cell viability with an increase in cell death signal in the cancer cells and regression of tumor growth in vivo.
Multidrug resistance, a major impediment to successful cancer chemotherapy, is the result of overexpression of ATP-binding cassette (ABC) transporters extruding internalized drugs. Silencing of ABC transporter gene expression with small interfering RNA (siRNA) could be an attractive approach to overcome multidrug resistance of cancer, although delivery of siRNA remains a major hurdle to fully exploit the potential of siRNA-based therapeutics. Recently, we have developed pH-sensitive carbonate apatite nanoparticles to efficiently carry and transport siRNA across the cell membrane, enabling knockdown of the cyclin B1 gene and consequential induction of apoptosis in synergy with anti-cancer drugs.
Treatment of breast cancer, the second leading cause of female deaths worldwide, with classical drugs is often accompanied by treatment failure and relapse of disease condition. Development of chemoresistance and drug toxicity compels compromising the drug concentration below the threshold level with the consequence of therapeutic inefficacy. Moreover, amplification and over-activation of proto-oncogenes in tumor cells make the treatment more challenging. The oncogene, ROS1 which is highly expressed in diverse types of cancers including breast carcinoma, functions as a survival protein aiding cancer progression. Thus we speculated that selective silencing of ROS1 gene by carrier-mediated delivery of siRNA might sensitize the cancer cells to the classical drugs at a relatively low concentration. In this investigation we showed that intracellular delivery of c-ROS1-targeting siRNA using pH-sensitive inorganic nanoparticles of carbonate apatite sensitizes mouse breast cancer cells (4T1) to doxorubicin, but not to cisplatin or paclitaxel, with the highest enhancement in chemosensitivity obtained at 40 nM of the drug concentration. Although intravenous administrations of ROS1-loaded nanoparticles reduced growth of the tumor, a further substantial effect on growth retardation was noted when the mice were treated with the siRNA- and Dox-bound particles, thus suggesting that silencing of ROS1 gene could sensitize the mouse breast cancer cells both in vitro and in vivo to doxorubicin as a result of synergistic effect of the gene knockdown and the drug action, eventually preventing activation of the survival pathway protein, AKT1. Our findings therefore provide valuable insight into the potential cross-talk between the pathways of ROS1 and doxorubicin for future development of effective therapeutics for breast cancer.
In this study, the unaffected, affected and heavily affected teeth were studied by using X-Ray diffraction method. The D-values, which are related to the crystallite size of the apatite crystals, were calculated according to the Sherrer equation. The crystallite size of unaffected enamel was found to be 1530.95Å, while the affected enamel was 1490.22Å and the heavily affected enamel was 1484.16Å respectively. This study showed that the unaffected enamel has greater crystallite size as compared to the affected and heavily affected enamel.