Modification of Gelatin-Methacrylate, Hyaluronic-Methacrylate and Poly(ethylene) glycol Diacrylate hydrogel bioinks towards the additive manufacturing of articular cartilage
- Authors: Barwick, Matthew William
- Date: 2021-10
- Subjects: Cartilage Diseases , Cartilage Regeneration , Articular cartilage Diseases , Chondrogenesis , Stem cells , Scanning electron microscopy , Fourier transform infrared spectroscopy , Three-dimensional printing , Gelatin-Methacrylate , Hyaluronic-Methacrylate , Poly(ethylene) glycolDiacrylate , Hydrogel bioinks , Real-Time Quantitative Cell Analysis (RTCA) , Bioprinting
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/191181 , vital:45068
- Description: 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, 2021
- Full Text:
- Authors: Barwick, Matthew William
- Date: 2021-10
- Subjects: Cartilage Diseases , Cartilage Regeneration , Articular cartilage Diseases , Chondrogenesis , Stem cells , Scanning electron microscopy , Fourier transform infrared spectroscopy , Three-dimensional printing , Gelatin-Methacrylate , Hyaluronic-Methacrylate , Poly(ethylene) glycolDiacrylate , Hydrogel bioinks , Real-Time Quantitative Cell Analysis (RTCA) , Bioprinting
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/191181 , vital:45068
- Description: 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, 2021
- Full Text:
The creation and validation of aptamers binding to murine 3T3-L1 Preadipocytes: preliminary implications for controlled cellular attachment, differentiation and cell fate
- Authors: Rubidge, Mark Lourens
- Date: 2017
- Subjects: Oligonucleotides , Fat cells , Stem cells , Ligand binding (Biochemistry) , Fluorimetry
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65247 , vital:28714
- Description: The controlled seeding of a variety of stem cells in vitro has been reported to alter the patterns of their subsequent differentiation. This has been attributed to the control of the surface microenvironment onto which adherent stem cells are cultured, especially control of the proximal density of neighbouring cells. Simultaneously, advances in the generation of aptamers - synthetic ligand molecules developed using in vitro selection techniques targeting complex molecules - have aided in the production of molecules capable of selectively binding to a variety of commercial stem cell lines. Combining the aforementioned research fields, the project reported in this thesis aimed to generate DNA-based aptamers capable of assisting with the selective binding of murine 3T3- L1 preadipocytes to a solid surface. This was performed with a view to, eventually, control the seeding densities of the adherent preadipocytes on the surface of the tissue culture dish in subsequent researchers. In the process of meeting this goal, several optimisations of the in vitro process by which aptamers binding to cells are generated (Cell-SELEX) were performed: an analysis into a variety of methods used for the removal of the single stranded aptamer candidate sequences attached to the surface of 3T3-L1 preadipocytes, a comparison of methods for the generation of single-stranded aptamer sequences from double-stranded DNA template molecules and a method for quantifying the removed ssDNA from the cell surface. Their use is further reported in this work. Initially, it was determined that a fluorimetric evaluation of the unbound single stranded DNA was the optimum technique to use to evaluate the relative amounts of aptamer DNA binding to target cells during cell-SELEX; this arose from the release of DNA, and other cell lysate contaminates, which interfered UV/ Vis quantification. The evaluation into different methods of ssDNA removal from the cell surface showed that although trypsinisation of the cells demonstrated the highest level of aptamer detachment (quantified by fluorimetry), there is a decrease the number of potential targets that aptamers could attach to. The most common method for detaching bound DNA aptamer molecules from cellular targets reported in literature, the use of high temperatures, was selected for cell-SELEX to increase the variability in potential target sites on the cell surface. Using techniques optimised in this work, fluorescently-tagged single-stranded oligonucleotide aptamers were later generated with a positive selection pressure to bind to the surface of the 3T3-L1 preadipocytes, but not to their differentiated adipocyte counterparts. After eight cycles of cell-SELEX, fluorescent spectroscopic analysis depicted a 74 % binding retention of the selection pool in the positive preadipocyte selection pool, as opposed to a 0.69 % binding of sequences to the negative differentiated preadipocytes. Following the isolation and identification of candidate sequences, seven separate sequences were identified as being successfully generated from the selection process. Bioinformatic characterisation of these placed sequenced aptamer candidates into two separate families, that were then analysed in opposition to each for their binding affinity toward each other. Using fluorescently-tagged sequences, the binding selectivity of the generated aptamers was validated using both epifluorescent microscopy and confocal microscopy. At this stage, an aptamer sequence selected from prior in-house research to serve as a negative control also demonstrated significant binding to the extracellular matrix of both preadipocytes and mature adipocytes. 5’-thiolated aptamer sequences were used to form self-assembled monolayers on the electrode surfaces of the impedimetric Roche xCELLigence Real-Time Cell Analysis. The use of aptamer sequences to capture the seeded preadipocytes, demonstrated a slight increase in the extent of binding of the preadipocytes to the gold electrode surface and produced some preliminary indications of alterations to the pattern and rate of subsequent differentiation in the preadipocytes. This provides preliminary evidence that aptamers developed to bind specifically to a stem cell line in vitro show potential to be used as to capture said cell when cast in a self- assembled monolayer assembly. This provides a future opportunity to control the seeding densities of the cells in vitro. The effects of cellular differentiation at a set of predefined cellular densities can be demonstrated on a desired stem cell line. , Thesis (MSc) -- Faculty of Faculty of Science, Biotechnology Innovation Centre, 2017
- Full Text:
- Authors: Rubidge, Mark Lourens
- Date: 2017
- Subjects: Oligonucleotides , Fat cells , Stem cells , Ligand binding (Biochemistry) , Fluorimetry
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65247 , vital:28714
- Description: The controlled seeding of a variety of stem cells in vitro has been reported to alter the patterns of their subsequent differentiation. This has been attributed to the control of the surface microenvironment onto which adherent stem cells are cultured, especially control of the proximal density of neighbouring cells. Simultaneously, advances in the generation of aptamers - synthetic ligand molecules developed using in vitro selection techniques targeting complex molecules - have aided in the production of molecules capable of selectively binding to a variety of commercial stem cell lines. Combining the aforementioned research fields, the project reported in this thesis aimed to generate DNA-based aptamers capable of assisting with the selective binding of murine 3T3- L1 preadipocytes to a solid surface. This was performed with a view to, eventually, control the seeding densities of the adherent preadipocytes on the surface of the tissue culture dish in subsequent researchers. In the process of meeting this goal, several optimisations of the in vitro process by which aptamers binding to cells are generated (Cell-SELEX) were performed: an analysis into a variety of methods used for the removal of the single stranded aptamer candidate sequences attached to the surface of 3T3-L1 preadipocytes, a comparison of methods for the generation of single-stranded aptamer sequences from double-stranded DNA template molecules and a method for quantifying the removed ssDNA from the cell surface. Their use is further reported in this work. Initially, it was determined that a fluorimetric evaluation of the unbound single stranded DNA was the optimum technique to use to evaluate the relative amounts of aptamer DNA binding to target cells during cell-SELEX; this arose from the release of DNA, and other cell lysate contaminates, which interfered UV/ Vis quantification. The evaluation into different methods of ssDNA removal from the cell surface showed that although trypsinisation of the cells demonstrated the highest level of aptamer detachment (quantified by fluorimetry), there is a decrease the number of potential targets that aptamers could attach to. The most common method for detaching bound DNA aptamer molecules from cellular targets reported in literature, the use of high temperatures, was selected for cell-SELEX to increase the variability in potential target sites on the cell surface. Using techniques optimised in this work, fluorescently-tagged single-stranded oligonucleotide aptamers were later generated with a positive selection pressure to bind to the surface of the 3T3-L1 preadipocytes, but not to their differentiated adipocyte counterparts. After eight cycles of cell-SELEX, fluorescent spectroscopic analysis depicted a 74 % binding retention of the selection pool in the positive preadipocyte selection pool, as opposed to a 0.69 % binding of sequences to the negative differentiated preadipocytes. Following the isolation and identification of candidate sequences, seven separate sequences were identified as being successfully generated from the selection process. Bioinformatic characterisation of these placed sequenced aptamer candidates into two separate families, that were then analysed in opposition to each for their binding affinity toward each other. Using fluorescently-tagged sequences, the binding selectivity of the generated aptamers was validated using both epifluorescent microscopy and confocal microscopy. At this stage, an aptamer sequence selected from prior in-house research to serve as a negative control also demonstrated significant binding to the extracellular matrix of both preadipocytes and mature adipocytes. 5’-thiolated aptamer sequences were used to form self-assembled monolayers on the electrode surfaces of the impedimetric Roche xCELLigence Real-Time Cell Analysis. The use of aptamer sequences to capture the seeded preadipocytes, demonstrated a slight increase in the extent of binding of the preadipocytes to the gold electrode surface and produced some preliminary indications of alterations to the pattern and rate of subsequent differentiation in the preadipocytes. This provides preliminary evidence that aptamers developed to bind specifically to a stem cell line in vitro show potential to be used as to capture said cell when cast in a self- assembled monolayer assembly. This provides a future opportunity to control the seeding densities of the cells in vitro. The effects of cellular differentiation at a set of predefined cellular densities can be demonstrated on a desired stem cell line. , Thesis (MSc) -- Faculty of Faculty of Science, Biotechnology Innovation Centre, 2017
- Full Text:
Establishment of human OCT4 as a putative HSP90 client protein: a case for HSP90 chaperoning pluripotency
- Authors: Sterrenberg, Jason Neville
- Date: 2015
- Subjects: Induced pluripotent stem cells , Heat shock proteins , Stem cells , Transcription factors , Molecular chaperones
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/194010 , vital:45415 , 10.21504/10962/194010
- Description: The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Authors: Sterrenberg, Jason Neville
- Date: 2015
- Subjects: Induced pluripotent stem cells , Heat shock proteins , Stem cells , Transcription factors , Molecular chaperones
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/194010 , vital:45415 , 10.21504/10962/194010
- Description: The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
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