Sarah A. Plautz

MR elastography monitoring of tissue-engineered constructs

The objective of tissue engineering (TE) is to create functional replacements for various tissues; the mechanical properties of these engineered constructs are critical to their function. Several techniques have been developed for the measurement of the mechanical properties of tissues and organs; however, current methods are destructive. The field of TE will benefit immensely if biomechanical models developed by these techniques could be combined with existing imaging modalities to enable noninvasive, dynamic assessment of mechanical properties during tissue growth. Specifically, MR elastography (MRE), which is based on the synchronization of a mechanical actuator with a phase contrast imaging pulse sequence, has the capacity to measure tissue strain generated by sonic cyclic displacement. The captured displacement is presented in shear wave images from which the complex shear moduli can be extracted or simplified by a direct measure, termed the shear stiffness. MRE has been extended to the microscopic scale, combining clinical MRE with high‐field magnets, stronger magnetic field gradients and smaller, more sensitive, radiofrequency coils, enabling the interrogation of smaller samples, such as tissue‐engineered constructs. The following topics are presented in this article: (i) current mechanical measurement techniques and their limitations in TE; (ii) a description of the MRE system, MRE theory and how it can be applied for the measurement of mechanical properties of tissue‐engineered constructs; (iii) a summary of in vitro MRE work for the monitoring of osteogenic and adipogenic tissues originating from human adult mesenchymal stem cells (MSCs); (iv) preliminary in vivo studies of MRE of tissues originating from mouse MSCs implanted subcutaneously in immunodeficient mice with an emphasis on in vivo MRE challenges; (v) future directions to resolve current issues with in vivo MRE in the context of how to improve the future role of MRE in TE. Copyright

Network analysis of endogenous gene expression profiles after polyethyleneimine-mediated DNA delivery

DNA delivery systems, which transport exogenous DNA to cells, have applications that include gene therapy, tissue engineering and medical devices. Although the cationic nonviral DNA carrier polyethyleneimine (PEI) has been widely studied, the molecular factors and pathways underlying PEI‐mediated DNA transfer remain largely unknown, preventing the design of more efficient delivery systems.

In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix

Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96h. Every 24h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17β-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P<0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P<0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC-), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P<0.001) in 17β-oestradiol was observed in culture media from ENC+ compared with ENC- and CONT embryos. These results illustrate that preimplantation pig embryos encapsulated in alginate hydrogels can undergo morphological changes with increased expression of steroidogenic transcripts and oestrogen production, consistent with in vivo-developed embryos. This alginate culture system can serve as a tool for evaluating specific mechanisms of embryo elongation that could be targeted to improve pregnancy outcomes.

Glucocorticoid Cell Priming Enhances Transfection Outcomes in Adult Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) are one of the most widely researched stem cell types with broad applications from basic research to therapeutics, the majority of which require introduction of exogenous DNA. However, safety and scalability issues hinder viral delivery, while poor efficiency hinders nonviral gene delivery, particularly to hMSCs. Here, we present the use of a pharmacologic agent (glucocorticoid) to overcome barriers to hMSC DNA transfer to enhance transfection using three common nonviral vectors. Glucocorticoid priming significantly enhances transfection in hMSCs, demonstrated by a 3-fold increase in efficiency, 4-15-fold increase in transgene expression, and prolonged transgene expression when compared to transfection without glucocorticoids. These effects are dependent on glucocorticoid receptor binding and caused in part by maintenance of normal metabolic function and increased cellular (5-fold) and nuclear (6-10-fold) DNA uptake over hMSCs transfected without glucocorticoids. Results were consistent across five human donors and in cells up to passage five. Glucocorticoid cell priming is a simple and effective technique to significantly enhance nonviral transfection of hMSCs that should enhance their clinical use, accelerate new research, and decrease reliance on early passage cells.

Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection

Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection.

Temporal endogenous gene expression profiles in response to lipid‐mediated transfection

Design of efficient nonviral gene delivery systems is limited as a result of the rudimentary understanding of the specific molecules and processes that facilitate DNA transfer.