The bone marrow niche represents a specialized environment that regulates mesenchymal stem cell quiescence and self-renewal, yet fosters stem cell migration and differentiation upon demand. An in vitro model that embodies these features would open up the ability to perform detailed study of stem cell behavior. In this paper we present a simple bone marrow-like niche model, which comprises of nanomagnetically levitated stem cells cultured as multicellular spheroids within a type I collagen gel. The stem cells maintained are nestin positive and remain quiescent until regenerative demand is placed upon them. In response to coculture wounding, they migrate and appropriately differentiate upon engraftment. This tissue engineered regeneration-responsive bone marrow-like niche model will allow for greater understanding of stem cell response to injury and also facilitate as a testing platform for drug candidates in a multiwell plate format.

Mesenchymal stem cells are multipotent adult stem cells capable of generating bone, cartilage and fat, and are thus currently being exploited for regenerative medicine. When considering osteogenesis, developments have been made with regards to chemical induction (e.g. differentiation media) and physical induction (e.g. material stiffness, nanotopography), targeting established early transcription factors or regulators such as runx2 or bone morphogenic proteins and promoting increased numbers of cells committing to osteo-specific differentiation. Recent research highlighted the involvement of microRNAs in lineage commitment and terminal differentiation. Herein, gold nanoparticles that confer stability to short single stranded RNAs were used to deliver MiR-31 antagomiRs to both pre-osteoblastic cells and primary human MSCs in vitro. Results showed that blocking miR-31 led to an increase in osterix protein in both cell types at day 7, with an increase in osteocalcin at day 21, suggesting MSC osteogenesis. In addition, it was noted that antagomiR sequence direction was important, with the 5 prime reading direction proving more effective than the 3 prime. This study highlights the potential that miRNA antagomiR-tagged nanoparticles offer as novel therapeutics in regenerative medicine.

Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrin- medicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake. Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake.

Mesenchymal stem cells (MSCs) reside quiescently within a specialised ‘niche’ environment in the bone marrow. However, following appropriate signalling cues, MSCs mobilise and migrate out from the niche, typically toward either sites of injury (a regenerative response) or toward primary tumours (an intrinsic homing response, which promotes MSCs as cellular vectors for therapeutic delivery). To date, very little is known about MSC mobilisation. By adopting a 3D MSC niche model, whereby MSC spheroids are cultured within a type I collagen gel, recent studies have highlighted interleukin-6 (IL-6) as a key cytokine involved in MSC migration. Herein, the ability of IL-6 to induce MSC migration was further investigated, and the key matrix metalloproteinases used to effect cell mobilisation were identified. Briefly, the impact of IL-6 on the MSC migration in a two-dimensional model systems was characterised—both visually using an Ibidi chemotaxis plate array (assessing for directional migration) and then via a standard 2D monolayer experiment, where cultured cells were challenged with IL-6 and extracted media tested using an Abcam Human MMP membrane antibody array. The 2D assay displayed a strong migratory response toward IL-6 and analysis of the membrane arrays data showed significant increases of several key MMPs. Both data sets indicated that IL-6 is important in MSC mobilisation and migration. We also investigated the impact of IL-6 induction on MSCs in 3D spheroid culture, serving as a simplistic model of the bone marrow niche, characterised by fluorescently tagged magnetic nanoparticles and identical membrane antibody arrays. An increase in MMP levels secreted by cells treated with 1 ng/mL IL-6 versus control conditions was noted in addition to migration of cells away from the central spheroid mass.

Aims: RNAi is a powerful tool for gene silencing that can be used to reduce undesirable overexpression of oncogenes as a novel form of cancer treatment. However, when using RNAi as a therapeutic tool there is potential for associated gene effects. This study aimed to utilize gold nanoparticles to deliver siRNA into HeLa cells. Results: Knockdown of the c-myc oncogene by RNAi, at the RNA, protein and cell proliferation level was achieved, while also identifying associated gene responses. Discussion: The gold nanoparticles used in this study present an excellent delivery platform for siRNA, but do note associated gene changes. Conclusion: The study highlights the need to more widely assess the cell physiological response to RNAi treatment, rather than focus on the immediate RNA levels.

The respiratory epithelium is exposed to assault by toxins and pathogens through the process of inhalation, which has numerous implications on both human and animal health. As such, there is a need to develop and characterise an in vitro model of the airway epithelium to study respiratory pathologies during infection or toxicology experiments. This has been achieved by growing airway epithelial cells at an air-liquid interface (ALI). Characterisation of ALI models are not well-defined for airway epithelial cells derived from non-human species. In this study we have fully characterised a bovine airway epithelial cell models (AECM) grown at an ALI in relation to ex vivo tissue. The morphology of the model was monitored at three day intervals, to identify the time-period at which the culture was optimally differentiated. The model was shown to be fully-differentiated by day 21 post-ALI. The culture formed a stereotypical pseudostratified, columnar epithelium containing the major cell types of the bronchial epithelium (ciliated-, goblet- and basal cells). Once fully differentiated the bovine AECM displayed both barrier function, through the formation of tight-junctions, and active mucociliary clearance, important properties of the mucosal barrier. The bovine bronchial epithelial cells remained stable for three weeks, with no evidence of deterioration or dedifferentiation. The window in which the model displayed full differentiation was determined to be between day 21-42 post-ALI. Through comparison with ex vivo tissue derived from donor animals, our bovine AECM was shown to be highly representative of the in vivo bovine bronchial epithelium and can be utilised in the study of respiratory pathologies.