Retraction Note: In Vitro Transfection of Plasmid DNA by Amine Derivatives of Gelatin Accompanied with Ultrasound Irradiation.

The Editor-in-Chief has retracted this article [1] at the request of the corresponding author. Figure 1B appears to be identical to Figure 1D, despite being under different experimental conditions.

Bone defect healing is induced by collagen sponge/polyglycolic acid.

We have evaluated the capability of a collagen/poly glycolic acid (PGA) scaffold in regeneration of a calvarial bone defects in rabbits. 4 bone critical size defects (CSD) were created in the calvarial bone of each rabbit. The following 4 treatment modalities were tested (1) a collagen/PGA scaffold (0.52% w/w); (2) the collagen/PGA scaffold (0.52% w/w) seeded with adipose-derived mesenchymal stem cells (AD-MSCs, 1 × 106 cells per each defect); (3) AD-MSCs (1 × 106 cells) no scaffold material, and (4) blank control. The rabbits were then divided into 3 random groups (of 5) and the treatment outcomes were evaluated at 4, 8 and 12 weeks. New bone formation was histologically assessed. Experimental groups were analyzed by CT scan and real-time PCR. Histological analysis of bone defects treated with collagen/PGA alone exhibited significant fibrous connective tissue formation at the 12 weeks of treatments (P ≤ 0.05). There was no significant difference between collagen/PGA alone and collagen/PGA + AD-MSCs groups. The results were confirmed by CT scan data showing healing percentages of 34.20% for the collage/PGA group alone as compared to the control group and no difference with collagen/PGA containing AD-MSCs (1 × 106 cells). RT-PCR analysis also indicated no significant differences between collagen/PGA and collagen/PGA + AD-MSC groups, although both scaffold containing groups significantly express ALP and SIO rather than groups without scaffolds. Although there was no significant difference between the scaffolds containing cells with non-cellular scaffolds, our results indicated that the Collagen/PGA scaffold itself had a significant effect on wound healing as compared to the control group. Therefore, the collagen/PGA scaffold seems to be a promising candidate for research in bone regeneration.

Long bone mesenchymal stem cells (Lb-MSCs): clinically reliable cells for osteo-diseases.

Mesenchymal stem cells (MSCs) have been designated as the most reliable cells in clinics to treat osteo-diseases because of their versatile nature. MSCs, isolated from long bone (Lb-MSCs) are rarely reported and named as RIA-MSCs because of the reamer-irrigator-aspirator (RIA) device. The potential of these cells in the treatment of non-union bone fractures made them the ideal candidates to be studied for clinical practices. In this work, effect of cryopreservation on the proliferation and differentiation capabilities of long bone MSCs (Lb-MSCs) has been studied. For this purpose, Lb-MSCs were isolated via RIA device and characterized using flow cytometry and differentiation assays. Cells were cryopreserved for 3, 6 and 12 months and thereafter were characterized using differentiation assays and genetic markers specific for osteogenic, chondrogenic, and adipogenic potential quantitatively by qRT-PCR. Lb-MSCs were found expressing MSC characteristic markers defining their identity. The population doubling time (PDT) was about 2.5 ± 0.5 days and colonies appeared after 7-10 days. Differentiation potential and gene expression of 3, 6 and 12 months cryopreserved Lb-MSCs were unaltered. The results show that cryopreservation did not have an effect on the differentiation potential of human Lb-MSCs. Therefore, our work offers Lb-MSCs as clinically cells for treating osteo-diseases.

Application of a Cu-chitosan/multiwalled carbon nanotube film-modified electrode for the sensitive determination of rutin.

A new sensitive electrochemical sensor, a glassy carbon electrode modified with chemically cross-linked copper-complexed chitosan/multiwalled carbon nanotubes (Cu-CS/MWCNT/GCE), for rutin analysis was constructed. Experimental investigations of the influence of several parameters showed that the rutin can effectively accumulate on the surface of the Cu-CS/MWCNT/GCE, which accumulation caused a pair of well-defined redox peaks in the electrochemical signal when measurements were carried out in Britton-Robinson buffer solution (pH 3, 0.04 M). The surface of the Cu-CS/MWCNT/GCE was characterized by field-emission scanning electron microscopy, transmission electron microscopy, and X-ray diffractometry analysis. In a rutin concentration range of 0.05-100 µM and under optimized conditions, a linear relationship between the oxidation peak current of rutin and its concentration was obtained with a detection limit of 0.01 µM. The Cu-CS/MWCNT/GCE showed good selectivity, stability, and reproducibility. Moreover, the sensor was used to determine the presence of rutin in fruits with satisfactory results.

Synthesis of AS1411-aptamer-conjugated CdTe quantum dots with high fluorescence strength for probe labeling tumor cells.

In this paper, we report microwave-assisted, one-stage synthesis of high-quality functionalized water-soluble cadmium telluride (CdTe) quantum dots (QDs). By selecting sodium tellurite as the Te source, cadmium chloride as the Cd source, mercaptosuccinic acid (MSA) as the capping agent, and a borate-acetic acid buffer solution with a pH range of 5-8, CdTe nanocrystals with four colors (blue to orange) were conveniently prepared at 100 °C under microwave irradiation in less than one hour (reaction time: 10-60 min). The influence of parameters such as the pH, Cd:Te molar ratio, and reaction time on the emission range and quantum yield percentage (QY%) was investigated. The structures and compositions of the prepared CdTe QDs were characterized by transmission electron microscopy, energy-dispersive X-ray spectroscopy, selective area electron diffraction, and X-ray powder diffraction experiments. The formation mechanism of the QDs is discussed in this paper. Furthermore, AS1141-aptamer-conjugated CdTe QDs in the U87MG glioblastoma cell line were assessed with a fluorescence microscope. The obtained results showed that the best conditions for obtaining a high QY of approximately 87% are a pH of 6, a Cd:Te molar ratio of 5:1, and a 30-min reaction time at 100 °C under microwave irradiation. The results showed that AS1141-aptamer-conjugated CdTe QDs could enter tumor cells efficiently. It could be concluded that a facile high-fluorescence-strength QD conjugated with a DNA aptamer, AS1411, which can recognize the extracellular matrix protein nucleolin, can specifically target U87MG human glioblastoma cells. The qualified AS1411-aptamer-conjugated QDs prepared in this study showed excellent capabilities as nanoprobes for cancer targeting and molecular imaging.

Biomedical nanostructured coating for minimally invasive surgery devices applications: characterization, cell cytotoxicity evaluation and an animal study in rat.

BACKGROUND: Thermal injury and tissue sticking, which influence wound remodeling, are major concerns in electrosurgery. In this study, the effect of lateral thermal injury caused by different electrosurgical electrodes on hepatic remodeling was investigated. METHODS: A monopolar electrosurgical unit equipped with untreated stainless steel (SS) and chromium nitride coated stainless steel (CrN-SS) electrodes was used to create lesions on the liver lobes of adult rats. Animals were sacrificed for evaluations at 0, 3, 7, and 28 days postoperatively. RESULTS: CrN-SS needles generated lower levels of sticking tissue, and the thermographs showed that recorded highest temperature in liver tissue from the CrN-SS needle group was significantly lower than in the SS needle group. The total injury area of livers treated with CrN-SS needles was significantly lower than livers treated with SS needles at each time point. Moreover, the CrN-SS needles caused a relatively smaller area of lateral thermal injury, a smaller area of fibrotic tissue, and a faster process of hepatic remodeling in rat liver than the SS needles. Immunofluorescence staining and Western blot analysis showed that rats treated with CrN-SS needles expressed lower levels of NF-κB and caspase-3 postoperatively. CONCLUSIONS: This study reveals that the plating of electrodes with a CrN film is an efficient method for improving the performance of electrosurgical units and should benefit wound remodeling. However, more tests must be performed to confirm these promising findings in human patients.

Safety profile of dextran-spermine gene delivery vector in mouse lungs.

A nano-sized polymer, dextran-spermine (D-SPM), was shown to have the capacity to deliver gene to the lung of mouse via intranasal route. In this study, assessments on the safety profile of D-SPM were performed to complement the gene expression results. African green monkey kidney fibroblast (COS-7) and human adenocarcinoma breast (MCF-7) cells transfected with D-SPM/pDNA showed massive reduction in the number of viable cells. As for in vivo study, elevated level of neutrophils was observed, despite the minimal level of pro-inflammatory cytokines (TNF-alpha, IL-12, IFN-gamma) detected in the bronchoalveolar lavage fluid (BALF) of mice treated with the D-SPM/pDNA complexes. Histology profile examinations of the lungs showed mild inflammatory responses, with inflamed areas overlap with healthy areas. Although reduction of mice weight was seen at day 1 post administration, the mice did not show any sign of abnormal behavior or physical appearance. Biodistribution study was performed to determine the ability of the D-SPM/pDNA complexes to infiltrate to other non-intended organs. The result showed that the D-SPM/pDNA complexes were only localized at the lung and no gene expression was detected in other organs or blood. In short, these results indicate that the D-SPM/pDNA exhibited mild toxicity in the mouse lungs.

Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

Development of 3D in vitro technology for medical applications.

In the past few years, biomaterials technologies together with significant efforts on developing biology have revolutionized the process of engineered materials. Three dimensional (3D) in vitro technology aims to develop set of tools that are simple, inexpensive, portable and robust that could be commercialized and used in various fields of biomedical sciences such as drug discovery, diagnostic tools, and therapeutic approaches in regenerative medicine. The proliferation of cells in the 3D scaffold needs an oxygen and nutrition supply. 3D scaffold materials should provide such an environment for cells living in close proximity. 3D scaffolds that are able to regenerate or restore tissue and/or organs have begun to revolutionize medicine and biomedical science. Scaffolds have been used to support and promote the regeneration of tissues. Different processing techniques have been developed to design and fabricate three dimensional scaffolds for tissue engineering implants. Throughout the chapters we discuss in this review, we inform the reader about the potential applications of different 3D in vitro systems that can be applied for fabricating a wider range of novel biomaterials for use in tissue engineering.

Gene Therapy for Regenerative Medicine.

The development of biological methods over the past decade has stimulated great interest in the possibility to regenerate human tissues. Advances in stem cell research, gene therapy, and tissue engineering have accelerated the technology in tissue and organ regeneration. However, despite significant progress in this area, there are still several technical issues that must be addressed, especially in the clinical use of gene therapy. The aims of gene therapy include utilising cells to produce a suitable protein, silencing over-producing proteins, and genetically modifying and repairing cell functions that may affect disease conditions. While most current gene therapy clinical trials are based on cell- and viral-mediated approaches, non-viral gene transfection agents are emerging as potentially safe and effective in the treatment of a wide variety of genetic and acquired diseases. Gene therapy based on viral vectors may induce pathogenicity and immunogenicity. Therefore, significant efforts are being invested in non-viral vectors to enhance their efficiency to a level comparable to the viral vector. Non-viral technologies consist of plasmid-based expression systems containing a gene encoding, a therapeutic protein, and synthetic gene delivery systems. One possible approach to enhance non-viral vector ability or to be an alternative to viral vectors would be to use tissue engineering technology for regenerative medicine therapy. This review provides a critical view of gene therapy with a major focus on the development of regenerative medicine technologies to control the in vivo location and function of administered genes.

Developing 3D Technology for Drug Discovery.

Developing 3D living systems will open many doors and lead to significant improvements in biological tools, drug discovery process, lead identification as well as therapeutic approaches. The miniaturization of this approach allows one to perform many more experiments than previously possible more simply. 3D in vitro technology aims to develop a set of tools that are simple, inexpensive, portable, and robust that could be commercialized and used in various fields of biomedical sciences, such as drug discovery, diagnostic tools, therapeutic approaches, and regenerative medicine.

Self-assembly of tissue transglutaminase into amyloid-like fibrils using physiological concentration of Ca2+.

Tissue transglutaminase (tTG or TG2) is a member of the transglutaminase family that catalyzes calcium dependent formation of isopeptide bonds. It has been shown that the expression of TG2 is elevated in neurodegenerative diseases such as Parkinson's, Huntington's, and Alzheimer's. We have investigated the self-assembly of TG2 in vitro. First, using software, hot spots, which are prone for aggregation, were identified in domain 2 of the enzyme. Next we expressed and purified recombinant TG2 and its truncated version that contains only the catalytic domain, and examined their amyloidogenic behavior in various conditions including different temperatures and pHs, in the presence of metal ions and Guanosine triphosphate (GTP). To analyze various stages leading to TG2 fibrillation, we employed various techniques including Thioflavin T (ThT) binding assay, Congo-Red, birefringence, Circular Dichroism (CD), 8-anilino-1-naphthalene sulfonic acid (ANS) binding, Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). Our results indicated that using low concentrations of Ca(2+), TG2 self-assembled into amyloid-like fibrils; this self-assembly occurred at the physiological temperature (37 °C) and at a higher temperature (57 °C). The truncated version of TG2 (domain 2) also forms amyloid-like fibrils only in the presence of Ca(2+). Because amyloid formation has occurred with domain 2 alone where no enzymatic activity was shown, self-cross-linking by the enzyme was ruled out as a mechanism of amyloid induction. The self-assembly of TG2 was not significant with magnesium and zinc ions, indicating specificity of the self-assembly for calcium ions. The calcium role in self-assembly of TG2 into amyloid may be extended to other proteins with similar biophysical properties to produce novel biomaterials.

Safety Evaluation of Nanotechnology Products.

Nanomaterials are now being used in a wide variety of biomedical applications. Medical and health-related issues, however, have raised major concerns, in view of the potential risks of these materials against tissue, cells, and/or organs and these are still poorly understood. These particles are able to interact with the body in countless ways, and they can cause unexpected and hazardous toxicities, especially at cellular levels. Therefore, undertaking in vitro and in vivo experiments is vital to establish their toxicity with natural tissues. In this review, we discuss the underlying mechanisms of nanotoxicity and provide an overview on in vitro characterizations and cytotoxicity assays, as well as in vivo studies that emphasize blood circulation and the in vivo fate of nanomaterials. Our focus is on understanding the role that the physicochemical properties of nanomaterials play in determining their toxicity.

Gene transfer into the lung by nanoparticle dextran-spermine/plasmid DNA complexes.

A novel cationic polymer, dextran-spermine (D-SPM), has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.

Cyclopropenium Nanoparticles and Gene Transfection in Cells.

Non-viral vectors for the transfection of genetic material are at the frontier of medical science. In this article, we introduce for the first time, cyclopropenium-containing nanoparticles as a cationic carrier for gene transfection, as an alternative to the common quaternary ammonium transfection agents. Cyclopropenium-based cationic nanoparticles were prepared by crosslinking poly(ethylene imine) (PEI) with tetrachlorocyclopropene. These nanoparticles were electrostatically complexed with plasmid DNA into nanoparticles (~50 nm). Their cellular uptake into F929 mouse fibroblast cells, and their eventual expression in vitro have been described. Transfection is enhanced relative to PEI with minimal toxicity. These cyclopropenium nanoparticles possess efficient gene transfection capabilities with minimal cytotoxicity, which makes them novel and promising candidates for gene therapy.

Quantitative analysis of cell adhesion on aligned micro- and nanofibers.

In this study, we quantitatively analyzed the affinity of cell adhesion to aligned nanofibers composed of composites of poly(glycolic acid) (PGA) and collagen. Electrospun composite fibers were fabricated at various PGA/collagen weight mixing ratio (7, 18, 40, 67, and 86%) to generate fibers that ranged in diameter from 10 mum to 500 nm. Scanning electron microscopy (SEM) observation revealed that the PGA/collagen fibers were long and uniformly aligned, irrespective of the PGA/collagen weight mixing ratio. In addition, it was observed that a significantly higher number of NIH3T3 fibroblasts adhered to nanofibers with smaller diameters in comparison to fibers with larger diameters. The highest affinity of cell adhesion was observed in the PGA/collagen fibers with diameter of 500 nm and PGA/collagen weight mixing ratio of 40%. Furthermore, the adherent cells were more elongated on fibers with smaller diameters. Thus, based on the results here, PGA/collagen composite fibers are suitable for tissue culture studies and provide an attractive material for tissue engineering applications.

Technology of RNA Interference in Advanced Medicine.

BACKGROUND: RNA interference (RNAi) and related pathways involving small interfering RNAs (siRNAs), microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs) regulate processes such as antiviral defense, genome surveillance, heterochromatin formation, and gene expression in animals, plants, and fungi. Studies on RNAi have revealed a two-step mechanism: (i) Degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. (ii) The siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the mRNA and degrades it. OBJECTIVE: Molecular structures of Dicer, Argonaute proteins, and RNA-bound complexes have offered insights into the underlying mechanisms of RNA-silencing pathways. METHODS: Sequence specific gene silencing using small interfering RNA (siRNA) is now being evaluated as a novel therapeutic strategy. RESULTS: Recently, promising data have been obtained from clinical trials for the treatment of respiratory syncytial virus and age-related macular degeneration. The exact mechanism of the RNAi pathways is still unclear. CONCLUSION: Our review summarizes the RNAi pathways and the known functions of siRNAs, miRNAs, and piRNAs in lower and higher organisms (mostly focusing on mammals) and discusses the potential applications of RNAi.

Tissue engineering strategies for the induction of angiogenesis using biomaterials.

Angiogenesis is touted as a fundamental procedure in the regeneration and restoration of different tissues. The induction of de novo blood vessels seems to be vital to yield a successful cell transplantation rate loaded on various scaffolds. Scaffolds are natural or artificial substances that are considered as one of the means for delivering, aligning, maintaining cell connection in a favor of angiogenesis. In addition to the potential role of distinct scaffold type on vascularization, the application of some strategies such as genetic manipulation, and conjugation of pro-angiogenic factors could intensify angiogenesis potential. In the current review, we focused on the status of numerous scaffolds applicable in the field of vascular biology. Also, different strategies and priming approaches useful for the induction of pro-angiogenic signaling pathways were highlighted.