Modification of Gelatin-Methacrylate, Hyaluronic-Methacrylate and Poly(ethylene) glycol Diacrylate hydrogel bioinks towards the additive manufacturing of articular cartilage

Cartilage degradation is most commonly associated with Rheumatoid arthritis and Osteoarthritis, affecting millions of people worldwide. Joint transplants commonly use titanium alloys, which have a shelf life of between 10-15 years. Although the titanium transplant restores partial mobility, side effects such as inflammation, swelling, faulty implants, and metal poisoning in some cases resulting from the transplant. The use of additive manufacturing of articular cartilage sheds new, innovative prospects for joint replacements. This study sets out to formulate and characterize five different hydrogel types towards the additive manufacturing of articular cartilage. Chondrogenic and Adipogenic differentiation was carried out on two separate adipose-mesenchymal stem cell lines A270620-01A, and A311019-02T and validation and efficiency of the differentiation and chondrogenic gene expression was carried out using Alcian Blue stain, Oil Red O stain and Quantitative Reverse Transcription PCR (RT-qPCR). Hydrogel formulation and characterisation of 10 % Gelatin-methacryloyl (GelMA), 10 % Poly (ethylene) glycol diacrylate (PEGDA), 5 % GelMA/5 % PEGDA, 10 % GelMA/0.5 % Hyaluronic Acid Methacrylate (HAMA) and 10 % PEGDA/0.5 % HAMA was carried out through swelling and degradation ratios, surface area and porosity characterisation using Scanning Electron Microscopy (SEM). Hydrogel component and spectroscopic analysis were carried using Real-Time Quantitative Cell Analysis (RTCA) and Fourier-transform Infrared Spectroscopy (FTIR) analysis for each formulated hydrogel's chemical characterisation. Three-dimensional printing (3D) of 10 % PEGDA/0.5 % HAMA and 5 % GelMA/5 % PEGDA was performed using the Zortrax INKSPIRE Resin Ultra-Violet (UV) LCD Desktop 3D Printer. Hydrogel sterility and cell viability were carried out for each hydrogel type using fluorescence microscopy. Both A270620-01A and A311019-02T cell lines showed adipogenic and chondrogenic differentiation ability, with A311019-02T cell line showing greater chondrogenic differentiation of Alcian blue staining. The A270620-01A cell line resulted in a greater collagen gene expression based on the RT-qPCR results. The hydrogel 10 % GelMA showed the greatest swelling ratio of 1260 % in DPBS and 1192 % in DMEM. A significant difference between hydrogel swelling and swelling with Dulbecco's Phosphate Buffered Saline (DPBS) and Dulbecco’s Modified Eagle Medium (DMEM) was observed. The 10 % PEGDA hydrogel had the greatest degradation ratio of 59 % mass remaining, where the 10 % GelMA/0.5 % HAMA showed the least amount of degradation with a mass remaining at 91 %. The 10 % GelMA showed the greatest porosity will the largest pore size of 14 μm in diameter. Hydrogel component and spectroscopic analysis showed no cytotoxic effects for the visible light photoinitiator used to polymerize the hydrogel and no cytotoxic effects for the concentrations used in chondrogenic differentiation. The FTIR analysis showed partial gelatin and hyaluronic acid modification with methacrylic anhydride; however, the distinction between the hybrid hydrogels and single polymer hydrogels could not be made effectively. UV and ethanol washing showed to completely sterilise the hydrogel disks from any contaminants, making them suitable for tissue culture. The cell viability analysis showed the 10 % GelMA/HAMA having the highest cell viability of 77.3 % using 5000 cells/disk and 89.64 % viability using 50 000 cells/disk over a 7-day incubation period. Overall, the combination of two polymers, GelMA and HAMA, has good potential as a 3D hydrogel scaffold towards additive manufacturing of articular cartilage.Thesis (MSc) -- Faculty of Science, Biotechnology Innovation Centre, 202

Generating hair follicle inductive dermal papillae cells from adipose derived mesenchymal stem cells

Current management options for cutaneous burn wounds, including split thickness skin grafts and cultured epithelial autografts, generate an epithelial barrier which lacks a dermal layer and skin adnexae including hair follicles and sebaceous glands. This results in a loss of pliability and contractures that cause functional and cosmetic impairment. Embryological hair follicle morphogenesis results from a complex series of mesenchymal-epithelial interactions and to date a method of generating de novo folliculogenesis from human cells has yet to be accomplished. Existing models rely on combining 'inductive’ dermal and 'receptive’ epithelial components and placing them within a suitable model. Epithelial cells are easily obtainable from skin biopsies therefore obtaining sufficient quantities of 'trichogenic’ dermal cells remains the most significant challenge of this approach. The main aim of this project is to contribute to the achievement of de novo folliculogenesis by generating dermal papillae (DP) like-spheroids using adipose derived mesenchymal stem cells (ASCs) that, when combined with responsive epithelial cells, would be capable of inducing hair follicle formation. ASCs were directed towards a hair follicle DP-like fate by culture using the hanging drop method and exposure to Wnt, mimicking signalling and mesenchymal condensation in embryological hair follicle induction. Gene expression analysis using RT-PCR showed that the DP-cell marker Versican is expressed at high levels in ASCs under routine culture conditions and the exposure of ASCs to Wnt results in a more than threefold increase in this expression. These results suggest that Wnt/β-catenin signalling may regulate DP cell aggregative growth through modifying versican expression possibly through binding of β-catenin to the TCF transcription factor complex. Culture of ASCs using the hanging drop method produces spheroids similar in size to human hair follicle DP. Histology of these spheroids demonstrates viable cells that flatten around the outside. The spheroids grow out when replated onto Matrigel in a 3D culture model and exhibit a morphology similar to that of primary hair follicle DP cells. Analysis of mRNA expression demonstrates that Versican expression is significantly upregulated in DP-like spheroids in the absence or presence of Wnt demonstrating that Versican may be responsible for both induction and maintenance of mesenchymal cell condensates. Alpha smooth muscle actin is expressed in low levels in ASC spheroids compared to ASCs in a monolayer and this may reflect a 'migratory’ myofibroblast like phenotype of ASCs in a monolayer similar to cells with the hair follicle dermal sheath. The addition of Wnt to ASC spheroids has no additional effect on Versican expression possibly reflecting a negative feedback loop resulting from high local concentrations of endogenous Wnt expression from ASCs. The results of this study show that spheroid cell culture and exposure to Wnt of ASCs results in cell clusters with similar morphology and gene expression to hair follicle DP cells. The novel method of DP-like cell generation described in this study makes use of cells that are readily obtainable from patients and require minimal time and manipulation in culture and therefore could potentially be rapidly translatable to clinical trials

Tissue-specific adaptations of cell types

Cells are the building blocks of life, forming the vast diversity of tissues and organisms in Nature. Across these, common cellular morphologies and functions have been identified. High-throughput, multifactorial profiling of cells has grown exponentially in recent years with the advent of single-cell RNA-sequencing (scRNA-seq), increasingly unravelling cell diversity. Nonetheless, it is not yet known how different environments affect cellular phenotypes. The work presented on this Thesis reports on the transcriptional variation of cell types across tissues, by use of single-cell RNA-sequencing. This technology, developed in the last 10 years, has greatly impacted our ability to distinguish cellular heterogeneity by their gene expression in various tissues or conditions. Chapter 1 outlines the impact of single-cell RNA-sequencing in cell biology, presenting the technology as the natural progression of lower throughput or low-resolution methods. The chapter then shows how cellular heterogeneity can be deconstructed by analysing this type of genomics data. It then expands on how individual datasets can be used to build models of cell type identity for automatic annotation, ultimately outlining the need to create a global cell type census of a whole organism. A cell compendium like this should be useful for automatic annotation, as well as to obtain a cross-tissue integrative overview of cell identity. The same chapter also delves into the topic of heterogeneity in immune cells. Due to the evolutionary pressure they are subject to and ubiquitous nature across the organism, these are some of the most diverse cell types in multicellular organisms. Chapter 2 presents a deconstruction of T-regulatory cells’ phenotypes in different mouse and human tissues using single-cell RNA-sequencing. The analysis in this chapter will show how these cells are structured in subpopulations, and how they adapt when migrating between lymphoid and non-lymphoid tissues. It will also assess the conservation of gene expression programmes for the same populations between mouse and human. The creation of a global cell type reference is an endeavour that can facilitate analysis of new data, and reveal novel insights about cell and tissue biology. Several datasets have now been produced, and a method that can efficiently integrate them and prepare them for use as a reference is necessary. Chapter 3 details the development of such method, exploring its strengths and how it can be improved, in a mouse dataset. Chapter 4 then applies this pipeline to a collection of human data, and shows how cell types relate across tissues, as well as how the human reference can be used in a practical case. Lastly, Chapter 5 summarises all chapters, providing an overview on how single cell sequencing has changed what we know about tissue biology, and how listing cell types and compiling them as a functional reference can help future developments in life sciences.Funded by the European Union’s H2020 research and innovation programme ‘ENLIGHT-TEN’ under Marie Skłodowska-Curie grant agreement no. 675395

Membrane vesicles: Examination of biophysical properties with atomic force microscopy

Extracellular vesicles (EVs) are not only intensively studied to increase our fundamental knowledge on their functioning, but also for diagnosis, therapeutics and drug delivery purposes. To improve the current and potential applications of EVs, a fundamental understanding of their stability, structure, and function is crucial. Such studies can be conducted at the single particle level to gain biological and physical information about the vesicles and the particle to particle variability. A suitable technique to investigate EVs under near- to physiological conditions is atomic force microscopy (AFM). Operated in liquid, it provides images of the EVs while mechanical properties of the particles can be obtained as well. Here we present our approach and the latest results in studying the structure and mechanics of these particles

Biointerface Coatings for Biomaterials and Biomedical Applications

This succinct reprint provides students and researchers the latest studies to the world of surface coatings in biomedical applications. This eBook contains one editorial, one review paper, and 10 research papers. The technology covers vapor phase coating, wet chemistry coating, and plasma spray. The research areas focus on antifouling, anti-corrosion, and tissue engineering. This specific and accessible reprint is the ideal example of surface coatings for students in bioengineering and materials science