Bioactivation of 3D Cell-Imprinted Polydimethylsiloxane Surfaces by Bone Protein Nanocoating for Bone Tissue Engineering

Physical and chemical parameters that mimic the physiological niche of the human body have an influence on stem cell fate by creating directional signals to cells. Micro/nano cell-patterned polydimethylsiloxane (PDMS) substrates, due to their ability to mimic the physiological niche, have been widely used in surface modification. Integration of other factors such as the biochemical coating on the surface can achieve more similar microenvironmental conditions and promote stem cell differentiation to the target cell line. Herein, we investigated the effect of physical topography, chemical functionalization by acid bone lysate (ABL) nanocoating, and the combined functionalization of the bone proteins' nanocoated surface and the topographically modified surface. We prepared four distinguishing surfaces: plain PDMS, physically modified PDMS by 3D cell topography patterning, chemically modified PDMS with bone protein nanocoating, and chemically modified nano 3D cell-imprinted PDMS by bone proteins (ABL). Characterization of extracted ABL was carried out by Bradford staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, followed by the MTT assay for evaluation of cell viability on ABL-coated PDMS. Moreover, field emission scanning electron microscopy and profilometry were used for the determination of optimal coating thickness, and the appropriate coating concentration was identified and used in the study. The binding and retention of ABL to PDMS were confirmed by Fourier transform infrared spectroscopy and bicinchoninic acid assay. Sessile drop static water contact angle measurements on substrates showed that the combined chemical functionalization and nano 3D cell-imprinting on the PDMS surface improved surface wettability by 66% compared to plain PDMS. The results of ALP measurement, alizarin red S staining, immunofluorescence staining, and real-time PCR showed that the nano 3D cell-imprinted PDMS surface functionalized by extracted bone proteins, ABL, is able to guide the fate of adipose derived stem cellss toward osteogenic differentiation. Eventually, chemical modification of the cell-imprinted PDMS substrate by bone protein extraction not only improved the cell adhesion and proliferation but also contributed to the topographical effect itself and caused a significant synergistic influence on the process of osteogenic differentiation

In vitro effect of nanosilver toxicity on fibroblast and mesenchymal stem cell lines

Nanotechnology presents countless opportunities to develop new and improved consumer products for the benefit of the society . A most prominent nanoproduct is nanosilver. Nanosilver particles are generally smaller than 100 nm and contain 20–15,000 silver atoms. Despite the wide application of nanomaterials, there is a serious lack of information concerning their impact on human health. In the previous study we reported the cytotoxic of nanosilver on osteoblast G292 cancer cell line and the amount of IC50 determined as 3.42 µg/ml (Moaddab et al., Iran. Nano Lett., Vol. 1, No. 1, January 2011, pp. 11-16). The purpose of the present study is to assess the biological assay of nanosilver on two normal cell lines of fibroblast (HF2), and mesenchymal stem cells . The effect of nanosilver on these cells is evaluated by light microscopy, and by cell proliferation and standard cytotoxicity assays. The results demonstrate a concentration-dependent toxicity for the cells tested, and IC50 was determined as 6.33, and 6.68 µg/ml in mesenchymal stem cell, and fibroblast HF2, respectively. There is no significant difference between the 24 h and 48 h of cells exposure to nanosilver. The results show that Nano-Ag possesses low toxicity to normal cells and can display potential application in cancer chemoprevention and chemotherapy

Biological Evaluation of a Novel Tissue Engineering Scaffold of Layered Double Hydroxides (LDHs)

Bone Tissue Engineering (BTE) Composed of Three Main Parts: Scaffold, Cells and Signaling Factors. Several Materials and Composites Are Suggested as a Scaffold for BTE. Biocompatibility is One of the Most Important Property of a BTE Scaffold. in This Work Synthesis of a Novel Nanocomposite Including Layered Double Hydroxides (LDH) and Gelatin is Carried Out and its Biological Properties Were Studied. the Co-Precipitation (PH=11) Method Was Used to Prepare the LDH Powder, using Calcium Nitrate, Magesium Nitrate and Aluminum Nitrate Salts as Starting Materials. the Resulted Precipitates Were Dried. X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM) Analyses Were Used to Characterize the Synthesized Powders. the Results Demonstrated the Presence of Nanocrystals of Ca-LDH and Mg-LDH as Hexagonal and Layered Morphology. the Obtained Powders Were Composed to Gelatin Via Solvent Casting Method Then Freez Dried. the Scaffold Was Prepared Via Membrane Lamination Method from the Resulted Layers that Linked Together with Gelatin as Binder. in Order to Investigate the Scaffold Cytotoxicity MTT Assay Was Done with a Osteosarcoma Cell Line. No Toxic Response Was Observed in Specimens. as a Major Result, It Was Demonstrated that the Specimen Showed a Significant Cellular Response. Then Osteosarcoma Cells Were Cultured for 7-Day and 14-Day Extract of Powders. the Composites Osteoconductivity Was Investigate with Cells Alkaline Phosphatase Extraction. the Results Demonstrated that the Ca-LDH/gelatin Composite Scaffold Has a Good Potential for Bone Tissue Engineering Applications and Mg-LDH Specimen Has a Better Osteconductivity. © (2012) Trans Tech Publications

Targeting delivery of lipocalin 2-engineered mesenchymal stem cells to colon cancer in order to inhibit liver metastasis in nude mice

One of the major obstacles in cancer therapy is the lack of anticancer agent specificity to tumor tissues. The strategy of cell-based therapy is a promising therapeutic option for cancer treatment. The specific tumor-oriented migration of mesenchymal stem cells (MSCs) makes them a useful vehicle to deliver anticancer agents. In this study, we genetically manipulated bone marrow-derived mesenchymal stem cells with their lipocalin 2 (Lcn2) in order to inhibit liver metastasis of colon cancer in nude mice. Lcn2 was successfully overexpressed in transfected MSCs. The PCR results of SRY gene confirmed the presence of MSCs in cancer liver tissue. This study showed that Lcn2-engineered MSCs (MSC-Lcn2) not only inhibited liver metastasis of colon cancer but also downregulated the expression of vascular endothelial growth factor (VEGF) in the liver. Overall, MSCs by innate tropism toward cancer cells can deliver the therapeutic agent, Lcn2, and inhibit cancer metastasis. Hence, it could be a new modality for efficient targeted delivery of anticancer agent to liver metastasis. © 2015, International Society of Oncology and BioMarkers (ISOBM)

Transplacentally Acquired Maternal Antibody against Hepatitis B Surface Antigen in Infants and its Influence on the Response to Hepatitis B Vaccine

BACKGROUND: Passively acquired maternal antibodies in infants may inhibit active immune responses to vaccines. Whether maternal antibody against hepatitis B surface antigen (anti-HBs) in infants may influence the long-term immunogenicity of hepatitis B vaccine remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Totally 338 pairs of mothers and children were enrolled. All infants were routinely vaccinated against hepatitis B based on 0-, 1- and 6-month schedule. We characterized the transplacental transfer of maternal anti-HBs, and compared anti-HBs response in children of mothers with or without anti-HBs. In a prospective observation, all 63 anti-HBs positive mothers transferred anti-HBs to their infants; 84.1% of the infants had higher anti-HBs concentrations than their mothers. One and half years after vaccination with three doses of hepatitis B vaccine, the positive rate and geometric mean concentration (GMC) of anti-HBs in 32 infants with maternal anti-HBs were comparable with those in 32 infants without maternal antibody (90.6% vs 87.5%, P = 0.688, and 74.5 vs 73.5 mIU/ml, P = 0.742, respectively). In a retrospective analysis, five and half years after vaccination with three doses vaccine, the positive rates of anti-HBs in 88 children of mothers with anti-HBs ≥1000 mIU/ml, 94 children of mothers with anti-HBs 10-999 mIU/ml, and 61 children of mothers with anti-HBs <10 mIU/ml were 72.7%, 69.2%, and 63.9% (P = 0.521), respectively; anti-HBs GMC in these three groups were 38.9, 43.9, and 31.7 mIU/ml (P = 0.726), respectively. CONCLUSIONS/SIGNIFICANCE: The data demonstrate that maternal anti-HBs in infants, even at high concentrations, does not inhibit the long-term immunogenicity of hepatitis B vaccine. Thus, current hepatitis B vaccination schedule for infants will be still effective in the future when most infants are positive for maternal anti-HBs due to the massive vaccination against hepatitis B

Mutations in Thalassemia Carrier Couples: The Importance of Prenatal Diagnostic Tests

Background: Thalassemia carrier couples play an important role in increasing thalassemia patients. The study of thalassemia genotypes in carrier couples is also effective in improving genetic counseling for them. The aim of this study was to investigate the prevalence of thalassemia mutations and genotypes in couples. Methods: This cross-sectional study was performed on 241 couples who were suspected of thalassemia from April 2018 to March 2020 in Lorestan province. Statistical analysis of data was performed using SPSS software 16.0 (SPSS Inc., Chicago, IL, USA). Online tools such as www.ithanet.eu/db/ithagenes and http://globin.bx.psu.edu/hbvar/menu.html were also used to match patients' mutations with known cases. Results: IVSII-1 (G>A), CD36-37 (-T), IVSI-110 (G>A), —Med, and alpha(3.7 )were the most common mutations in the beta and alpha genes, respectively. IVSII-1 (G>A) 130/13 (26.1), CD36-37 (-T) beta 0/beta (21.1), and IVSI-110 (G>A) beta 0/beta (10.3) genotypes were the most common in women. The frequency of these genotypes in men were 24.8, 28.6, and 12.8, respectively. Among alpha thalassemia carriers, the alpha(3.7)alpha/alpha alpha genotype had the highest frequency among women (3.7) and men (5.3). Alpha and beta-thalassemia were 15 and 13 times higher in related women and 18 and 9 times higher in related men than non-related ones, respectively. This difference was statistically significant (p < 0.001). In addition, 12.8 of fetuses were thalassemia major, 31.9 beta thalassemia minor, and 10.3 normal. Conclusions: Thalassemia screening in related couples plays an important role in reducing thalassemia major infants

Chemoselective PEGylation of cysteine analogs of human basic fibroblast growth factor (hbFGF) - design and expression

Purpose: To improve the stability and bioactivity of human basic fibroblast growth factor (hbFGF) by site-specific pegylationMethods: Four new mutants of hbFGF were designed with substituted Asp68, Lys77, Glu78 and Arg81 with cysteine with the aid of bioinformatics technique, and then cloned into pET21a plasmid, transferred into E. coli BL21 (DE3). The expressed proteins were purified using cation exchange and heparin affinity chromatography. Cysteine analogs of hbFGF were PEGylated with 10 KDa PEG and purified using size exclusion chromatography. Mitogenic activity and resistance against denaturation agents were evaluated by MTT assay and fluorescence spectrophotometry, respectively, and the results obtained were compared with the non-PEGylated form.Results: Despite greater resistance against denaturation agent (1.2 M guanidine hydrochloride for denaturation of PEGylated mutants compared with 0.8 M for non-PEGylated forms), the mitogenic activities of the four mutants Asp68, Lys77, Glu78 and Arg81were retained at 79, 78.6, 83.3 and 75.6 %, respectively.Conclusion: PEGylated hbFGF shows decreased mitogenic activity and increased resistance against denaturation agent. Keywords: Bioinformatics, Fibroblast growth factor, Cysteine analog, PEGylation, Denaturation agent, Guanidine hydrochloride, Mitogenic activit

Blood Coagulation Disorders Among the Iranian Population: a Systematic Review

Background: Blood coagulation disorders are one of the causes of mortality. Therefore, the study of coagulation disorders is also important. This systematic review was conducted to investigate blood coagulation disorders in the Iranian population. Methods: Searches in electronic databases such as Web of Science, PubMed, Scopus, SID, ProQuest, and Magiran from May 10, 1990 to May 10, 2019 were performed according to PRISMA guidelines. Cross-sectional, cohort, experimental, and case-control studies were included according to the inclusion criteria without gender and language restrictions. Results: After screening and selection, 14 studies were selected for data extraction. Accordingly, the most common blood coagulation disorder in the south of Iran was a defect in FXIII (599 of 1,165). C.559T>C (27 of 189) and c.562T>C (20 of 189) mutations had the highest frequency. The most common FXIII polymorphism among the Iranian Azerbaijanis was Val34Leu (203 of 410). The second most common coagulation disorder was FV Leiden (396 of 1,165). Then, c.1691G>A (151 of 396) was the most common mutation. Conclusions: This study shows that the most critical coagulation disorder among the Iranian population is FXIII deficiency and the most common mutation is c.562T>C. © 2023 Verlag Klinisches Labor GmbH. All rights reserved

Evaluation of Immunogenicity of Divalent DNA Vaccine Encoding Brucella melitensis Omp31 and P39 Genes in Balb/c Mice

Introduction & Objective: Brucella is a facultative intracellular pathogen and one of the etiologic agents of brucellosis that can infect humans and domestic animals. Attenuated strains such as B. melitensis Rve1 and B. abortus S19 and Rb51 are being used to control brucellosis in domestic animals. However, no safe and effective vaccine is available for human use. This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the B. melitensis Omp31 protein and P39 protein, designated pCDNA3 recombinant vector. Materials & Methods: This experimental study was performed in Biotechnology Research Center of Islamic Azad University, Shahrekord branch in summer, 1386. Construction of pCDNA3 recombinant vector containing Omp31 and P39 genes of B. melitensis was completed. Then, 12 Balb/c mice were immunized intramuscularly with 100 mg per 50 micro liters of this DNA vaccine. Control mice, 12 Balb/c mice, were simultaneously injected with PBS. During the 1st, 7th, 15th and 30th days the mice received the injections. Afterwards, the ELISA cytokine assay was performed and data were analyzed by SPSS software. Results: Intramuscular injection of the divalent DNA vaccine elicited cellular immune responses in Balb/c mice. The ELISA cytokine assay with serum of vaccinated mice showed high level of IFN-γ and low changes of IL-4 in compare with control mice. Conclusion: Use of divalent genetic vaccine based on the Omp31 and P39 genes can elicit a strong cellular immune response against Brucellosis

Characterization of poly(3-hydroxybutyrate)/nano-hydroxyapatite composite scaffolds fabricated without the use of organic solvents for bone tissue engineering applications

Poly(3-hydroxybutyrate)/nano-hydroxyapatite (PHB/nHA) composite scaffolds were fabricated without the use of organic solvents at different mass fractions of HA nanoparticles. HA nanoparticles were homogeneously dispersed as primary particles in the polymer matrix of the scaffolds at 10 and 15 wt.% nHA content. Agglomeration of HA nanoparticles occurred when the nHA content of the scaffolds reached 20 wt.%. All the scaffolds had high porosities with interconnected porous structure and optimized pore size ranges. Mechanical properties of all the scaffolds were in the range of mechanical properties of cancellous bone. Scaffolds were biocompatible to MG-63 cells inthe indirect method of cytotoxicity evaluation. Also, the morphology of the attached MG-63 cellsin direct contact with the scaffolds indicated the appropriate cell-scaffold interaction. Thus, the PHB/nHA composite scaffolds investigated in this study tend to be favorable for bone tissue engineering applications