Tissue engineering; strategies, tissues, and biomaterials.

Current tissue regenerative strategies rely mainly on tissue repair by transplantation of the synthetic/natural implants. However, limitations of the existing strategies have increased the demand for tissue engineering approaches. Appropriate cell source, effective cell modification, and proper supportive matrices are three bases of tissue engineering. Selection of appropriate methods for cell stimulation, scaffold synthesis, and tissue transplantation play a definitive role in successful tissue engineering. Although the variety of the players are available, but proper combination and functional synergism determine the practical efficacy. Hence, in this review, a comprehensive view of tissue engineering and its different aspects are investigated.

Evaluation of cationic dendrimer and lipid as transfection reagents of short RNAs for stem cell modification.

Nowadays a large number of clinical trials suffer from lacking an efficient method for drug delivery into target cells with minimal side effects. Due to the great significance of this issue in novel and effective therapies, more attempts are required in order to distinguish better conditions for biomedical drug delivery. Since embryonic stem cells (ESCs) are under scrutiny of many new studies, development of novel methods for their genetical and functional modifications is of great value. On the other hand, the application of short nucleic acids in new therapeutic approaches is increasing. In this study the efficiency of small interfering RNA (siRNA) uptake with two transfection reagents generation five of polyamidoamine dendrimer (PAMAM G5) as a cationic dendrimer and N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methyl-sulfate (DOTAP) as a cationic lipid and one commercially available reagent were evaluated in mouse ESCs using flow cytometry. Prior to the cellular investigations; atomic force microscopy; gel electrophoresis; siRNA binding and release assays; and size and zeta potential measurements were utilized to characterize the physicochemical properties of reagent-siRNA nano-complexes. The safety of the nano-complexes was subsequently assessed by MTT assay. Functional effects of siRNA (complementary strand for OCT4 transcript) transfection in ESCs with the mentioned reagents were analyzed using a quantitative real-time polymerase chain reaction (qPCR). Surprisingly DOTAP at higher molar ratios and PAMAM at lower molar ratios could successfully knock down the OCT4 transcription relatively better than commercial reagent. Our findings supported the appropriate efficiency of the mentioned transfection reagents for short nucleic acid transfection. From a clinical point of view, this research helps allocation of short nucleic acids into stem cells therapies.

Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pDNA transfection.

BACKGROUND: Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. METHODS: The physicochemical properties of the PAMAM-pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. RESULTS: Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. CONCLUSION: Presented data shows that the rate-limiting step of PAMAM-pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression.