This paper discusses the successful fabrication of a novel triple-layered poly(lactic-co-glycolic acid) (PLGA)-based composite membrane using only a single step that combines the techniques of solvent casting and thermally induced phase separation/solvent leaching. The resulting graded membrane consists of a small pore size layer-1 containing 10 wt% non-stoichiometric nanoapatite (NAp)+1-3 wt% lauric acid (LA) for fibroblastic cell and bacterial inhibition, an intermediate layer-2 with 20-50 wt% NAp+1 wt% LA, and a large pore size layer-3 containing 30-100 wt% NAp without LA to allow bone cell growth. The synergic effects of 10-30 wt% NAp and 1 wt% LA in the membrane demonstrated higher tensile strength (0.61 MPa) and a more elastic behavior (16.1% elongation at break) in 3 wt% LA added membrane compared with the pure PLGA (0.49 MPa, 9.1%). The addition of LA resulted in a remarkable plasticizing effect on PLGA at 3 wt% due to weak intermolecular interactions in PLGA. The pure and composite PLGA membranes had good cell viability toward human skin fibroblast, regardless of LA and NAp contents.
A study was conducted at Universiti Putra Malaysia to determine the effect of phosphate-solubilizing bacteria (PSB) and organic acids (oxalic & malic) on phosphate (P) solubilization from phosphate rock (PR) and growth of aerobic rice. Four rates of each organic acid (0, 10, 20, and 30 mM), and PSB strain (Bacillus sp.) were applied to aerobic rice. Total bacterial populations, amount of P solubilization, P uptake, soil pH, and root morphology were determined. The results of the study showed significantly high P solubilization in PSB with organic acid treatments. Among the two organic acids, oxalic acid was found more effective compared to malic acid. Application of oxalic acid at 20 mM along with PSB16 significantly increased soluble soil P (28.39 mg kg(-1)), plant P uptake (0.78 P pot(-1)), and plant biomass (33.26 mg). Addition of organic acids with PSB and PR had no influence on soil pH during the planting period. A higher bacterial population was found in rhizosphere (8.78 log10 cfu g(-1)) compared to the nonrhizosphere and endosphere regions. The application of organic acids along with PSB enhanced soluble P in the soil solution, improved root growth, and increased plant biomass of aerobic rice seedlings without affecting soil pH.
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.
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.
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.
Nanocomposites of magnetite (Fe3O4) and reduced graphene oxide (rGO) generate heat under an alternating magnetic field and therefore have potential applications as thermoseeds for cancer hyperthermia treatment. However, the properties of such nanocomposites as biomaterials have not been sufficiently well characterized. In this study, the osteoconductivity of Fe3O4-rGO nanocomposites of various compositions was evaluated in vitro in terms of their apatite-forming ability in simulated body fluid (SBF). Furthermore, the heat generation of the nanocomposites was measured under an alternating magnetic field. The apatite-forming ability in SBF improved as the Fe3O4 content in the nanocomposite was increased. As the Fe3O4 content was increased, the nanocomposite not only rapidly raised the surrounding temperature to approximately 100 °C, but the specific absorption rate also increased. We assumed that the ionic interaction between the Fe3O4 and rGO was enhanced and that Brown relaxation was suppressed as the proportion of rGO in the nanocomposite was increased. Consequently, a high content of Fe3O4 in the nanocomposite was effective for improving both the osteoconductivity and heat generation characteristics for hyperthermia applications.
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.
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.
Plasmid DNA is one of the indispensable components in molecular biology research and a potential biomaterial for gene therapy and DNA vaccination. Both quality and quantity of extracted plasmid DNA are of the great interests in cloning and subsequent expression of genes in vitro and in vivo for basic research and therapeutic interventions. Bacteria with extremely short generation times are the valuable source of plasmid DNA that can be isolated through a number of existing techniques. However, the current methods have some limitations in isolating high quality plasmid DNA since the multimeric plasmid which is believed to be more efficiently transcribed by RNA polymerase than the monomeric form, is almost lost during the extraction process. Recently, we developed a rapid isolation technique for multimeric plasmid based on generation of a 'protein aggregate' using a zwitterionic detergent and alkali. Here we have investigated the roles of different parameters in the whole extraction process to optimise the production of high quality multimeric plasmid DNA. Moreover, we have showed the advantageous effects of nanoparticles to effectively sediment the 'protein aggregate' for smooth elution of multimeric plasmid DNA from it. Finally, quality assessment study has revealed that the isolated multimeric DNA is at least 10 times more transcriptionally active than the monomeric form isolated by the commercially available Qiaget kit.
Autologous bone grafting remains the gold standard for almost all bone void-filling orthopedic surgery. However, autologous bone grafting has several limitations, thus scientists are trying to identify an ideal synthetic material as an alternative bone graft substitute. Magnesium-doped biphasic calcium phosphate (Mg-BCP) has recently been in the spotlight and is considered to be a potential bone substitute. The Mg-BCP is a mixture of two bioceramics, that is, hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), doped with Mg2+ , and can be synthesized through chemical wet-precipitation, sol-gel, single diffusion gel, and solid state reactions. Regardless of the synthesis routes, it is found that the Mg2+ preferentially accommodates in β-TCP lattice instead of the HA lattice. The addition of Mg2+ to BCP leads to desirable physicochemical properties and is found to enhance the apatite-forming ability as compared to pristine BCP. In vitro results suggest that the Mg-BCP is bioactive and not toxic to cells. Implantation of Mg-BCP in in vivo models further affirmed its biocompatibility and efficacy as a bone substitute. However, like the other bioceramics, the optimum physicochemical properties of the Mg-BCP scaffold have yet to be determined. Further investigations are required regarding Mg-BCP applications in bone tissue engineering.
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.
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.
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.