Microfluidic fabrication of alendronate-loaded chitosan nanoparticles for enhanced osteogenic differentiation of stem cells

Aims: In this study, we used a cross-junction microfluidic device for preparation of alendronate-loaded chitosan nanoparticles with desired characteristics to introduce a suitable element for bone tissue engineering scaffolds. Main methods: By controlling the reaction condition in microfluidic device, six types of alendronate-loaded chitosan nanoparticles were fabricated which had different physical properties. Hydrodynamic diameter of synthetized particles was evaluated by dynamic light scattering (102 to 215 nm). Nanoparticle morphology was determined by SEM and AFM images. The osteogenic effects of prepared selected nanoparticles on human adipose stem cells (hA-MSCs) were evaluated by assessment of alkaline phosphatase (ALP) activity, calcium deposition, ALP and osteopontin gene expression. Key findings: The highest loading efficiency percentage (LE) was 32.42 ± 2.02. Based on MTT assessment, two samples which had no significant cytotoxicity were chosen for further studies (particle sizes and LE were 142 ± 6.1 nm, 198 ± 16.56 nm, 16.76 ± 3.91 and 32.42 ± 2.02, respectively). In vitro release behavior of nanoparticles displayed pH responsive characteristics. Significant faster release was seen in acidic pH = 5.8 than neutral pH = 7.4. The selected nanoparticles demonstrated higher ALP activity at 14 days in comparison to selected blank sample and osteogenic differentiation media (ODM) and a downregulation at 21 days in comparison to 14 days. Calcium content assay at 21 days displayed significant differences between alendronate-loaded nanoparticles and ODM. ALP and osteopontin mRNA expression was significantly higher than the cells cultured in ODM at 14 and 21 days. Significance: We concluded that our prepared nanoparticles significantly enhanced osteogenic differentiation of hA-MSCs and can be a suitable compartment of bone tissue engineering scaffolds. © 202

Real-Time Assay as A Tool for Detecting lytA Gene in Streptococcus pneumoniae Isolates Citation

Abstract Objective: In-time diagnosis of Streptococcus pneumoniae (S. pneumonia) can play a significant role in decreasing morbidity and mortality rate. Applying molecular methods has gained popularity due to the existing limits of routine diagnostic methods. Examining the expression of different genes of this bacterium through different molecular methods suggests that lytA gene has a higher sensitivity and specificity in diagnosis of Streptococcus pneumoniae. The aim of this study was to evalutate lytA gene expression in diagnosis of invasive S. pneumonia in culture positive specimens by real-time polymerase chain reaction (PCR). Materials and Methods: IIn this a descriptive study, All received specimens were isolated to identify S. pneumoniae. DNA was then extracted and after optimizing the test and determining the detection limit, samples were tested by real-time PCR using lytA gene primers. Results: Twenty seven isolates were diagnosed as S. pneumoniae. In all, the extracted DNA was positive in real-time method. The electrophoresis of the products also confirmed the presence of single product b along with the 53 base pair fragment. The detection limit of the test was less 6 colony forming unit (CFU). Conclusion: Real-Time PCR seems to provide reliable and rapid results. We suggest that this test should be conducted on the preliminary isolated specimens, since applying various biochemical tests need one extra working day