Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in “tagging DNA synthesis” is the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using ‘Huisgen’s reaction’ (1,3-dipolar cycloaddition or ‘click chemistry’). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other “unnatural bases”. These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.
Diabetic foot ulcers (DFUs) are chronic wounds, with 20% of cases resulting in amputation, despite intervention. A recently approved tissue engineering product—a cell‐free collagen‐glycosaminoglycan (GAG) scaffold—demonstrates 50% success, motivating its functionalization with extracellular matrix (ECM). Induced pluripotent stem cell (iPSC) technology reprograms somatic cells into an embryonic‐like state. Recent findings describe how iPSCs‐derived fibroblasts (“post‐iPSF”) are proangiogenic, produce more ECM than their somatic precursors (“pre‐iPSF”), and their ECM has characteristics of foetal ECM (a wound regeneration advantage, as fetuses heal scar‐free). ECM production is 45% higher from post‐iPSF and has favorable components (e.g., Collagen I and III, and fibronectin). Herein, a freeze‐dried scaffold using ECM grown by post‐iPSF cells (Post‐iPSF Coll) is developed and tested vs precursors ECM‐activated scaffolds (Pre‐iPSF Coll). When seeded with healthy or DFU fibroblasts, both ECM‐derived scaffolds have more diverse ECM and more robust immune responses to cues. Post‐iPSF‐Coll had higher GAG, higher cell content, higher Vascular Endothelial Growth Factor (VEGF) in DFUs, and higher Interleukin‐1‐receptor antagonist (IL‐1ra) vs. pre‐iPSF Coll. This work constitutes the first step in exploiting ECM from iPSF for tissue engineering scaffolds.
Gene-activated scaffolds have been shown to induce controlled, sustained release of functional transgene both in vitro and in vivo. Bone morphogenetic proteins (BMPs) are potent mediators of osteogenesis however we found that the delivery of plasmid BMP-2 (pBMP-2) alone was not sufficient to enhance bone formation. Therefore, the aim of this study was to assess if the use of a series of modified BMP-2 plasmids could enhance the functionality of a pBMP-2 gene-activated scaffold and ultimately improve bone regeneration when implanted into a critical sized bone defect in vivo. A multi-cistronic plasmid encoding both BMP-2 and BMP-7 (BMP-2/7) was employed as was a BMP-2-Advanced plasmid containing a highly truncated intron sequence. With both plasmids, the highly efficient cytomegalovirus (CMV) promoter sequence was used. However, as there have been reports that the elongated factor 1-α promoter is more efficient, particularly in stem cells, a BMP-2-Advanced plasmid containing the EF1α promoter was also tested. Chitosan nanoparticles (CS) were used to deliver each plasmid to MSCs and induced transient up-regulation of BMP-2 protein expression, in turn significantly enhancing MSC-mediated osteogenesis when compared to untreated controls (p < 0.001). When incorporated into a bone mimicking collagen-hydroxyapatite scaffold, the BMP-2-Advanced plasmid, under the control of the CMV promotor, induced MSCs to produce approximately 2500 μg of calcium per scaffold, significantly higher (p < 0.001) than all other groups. Just 4 weeks post-implantation in vivo, this cell-free gene-activated scaffold induced significantly more bone tissue formation compared to a pBMP-2 gene-activated scaffold (p < 0.001) as indicated by microCT and histomorphometry. Immunohistochemistry revealed that the BMP-2-Advanced plasmid accelerated differentiation of osteoprogenitor cells to mature osteoblasts, thus causing rapid healing of the bone defects. This study confirms that optimising the plasmid construct can enhance the functionality of gene-activated scaffolds and translate to accelerated bone formation in a critical sized defect.
The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.
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