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:
- Date Issued: 2021-10
- 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:
- Date Issued: 2021-10
Development of a low-cost bioprinting system for the fabrication of cell-laden sodium alginate hydrogels
- Authors: Honiball, John Robert
- Date: 2018
- Subjects: Regenerative medicine , Tissue engineering , Alginates , Colloids , Three-dimensional printing
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59204 , vital:27470
- Description: Bioprinting is a rapidly expanding technology with the ability to fabricate in vitro 3D tissues in a layer-by-layer manner to ultimately produce a living tissue which physiologically resembles native in vivo tissue functionality. Unfortunately, large costs associated with commercially available bioprinters severely limit the amount of people/research groups with access to the technology. Here, we investigated the potential for modifying a commercially available RepRap Prusa iteration 3 (i3) three-dimensional (3D) printer, by replacing the traditional plastic-based print-head with various open-source syringe-housed microextrusion print-head units, such that deposition of composite bioinks consisting of cells, biopolymer scaffolds and/or biomolecules may be achieved at a relatively low cost. Using adipose-derived human mesenchymal stromal stem cells (ad-HMSC) induced for adipogenic differentiation, as well as human umbilical vein endothelial cells (HUVEC), the potential for fabricating vascularised adipose tissue was investigated. The non-toxic, inexpensive algal polysaccharide, sodium alginate, was used to test the printability of the system, as well as for investigating the functionality unmodified sodium alginate has for use as a potential bioink in adipose tissue engineering. Cell viability assays, namely WST-1 and fluorescein diacetate (FDA)/propidium iodide (PI) live/dead cell staining, revealed that ad-HMSC were viable after 7 days of culture. However, viability of HUVEC encapsulated hydrogels revealed significantly lower cell viability. Live/dead cell staining revealed that the modified printing system was able to print ad-HMSC/HUVEC co-cocultures with a large degree of cell viability after 1 day of culture. However, after 7 days of culture, the majority of cells were revealed to be dead. Furthermore, due to the lack of mechanical integrity possessed by alginate in a liquid-like state, printing sodium alginate hydrogels in air consistently resulted in deformation of printed constructs. The newly developed 3D printing technique termed freeform reversible embedding of suspended hydrogels (FRESH) was therefore investigated as a means for achieving 3D spatial control of printed hydrogels using the modified system. Printing cell-free sodium alginate hydrogels within gelatin sacrificial support baths allowed for fabricating constructs in a spatially defined manner. However, overprinting and swelling of alginate hydrogels negatively affected the overall printing accuracy. The present study aimed to pave the way for further system modifications and refinements, such that the ultimate goal of low-cost bioprinting may be achieved. Further optimisation of printing parameters, hydrogel characteristics and sterilisation techniques may allow for fabricating viable, physiologically relevant tissues using the modified system developed.
- Full Text:
- Date Issued: 2018
- Authors: Honiball, John Robert
- Date: 2018
- Subjects: Regenerative medicine , Tissue engineering , Alginates , Colloids , Three-dimensional printing
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59204 , vital:27470
- Description: Bioprinting is a rapidly expanding technology with the ability to fabricate in vitro 3D tissues in a layer-by-layer manner to ultimately produce a living tissue which physiologically resembles native in vivo tissue functionality. Unfortunately, large costs associated with commercially available bioprinters severely limit the amount of people/research groups with access to the technology. Here, we investigated the potential for modifying a commercially available RepRap Prusa iteration 3 (i3) three-dimensional (3D) printer, by replacing the traditional plastic-based print-head with various open-source syringe-housed microextrusion print-head units, such that deposition of composite bioinks consisting of cells, biopolymer scaffolds and/or biomolecules may be achieved at a relatively low cost. Using adipose-derived human mesenchymal stromal stem cells (ad-HMSC) induced for adipogenic differentiation, as well as human umbilical vein endothelial cells (HUVEC), the potential for fabricating vascularised adipose tissue was investigated. The non-toxic, inexpensive algal polysaccharide, sodium alginate, was used to test the printability of the system, as well as for investigating the functionality unmodified sodium alginate has for use as a potential bioink in adipose tissue engineering. Cell viability assays, namely WST-1 and fluorescein diacetate (FDA)/propidium iodide (PI) live/dead cell staining, revealed that ad-HMSC were viable after 7 days of culture. However, viability of HUVEC encapsulated hydrogels revealed significantly lower cell viability. Live/dead cell staining revealed that the modified printing system was able to print ad-HMSC/HUVEC co-cocultures with a large degree of cell viability after 1 day of culture. However, after 7 days of culture, the majority of cells were revealed to be dead. Furthermore, due to the lack of mechanical integrity possessed by alginate in a liquid-like state, printing sodium alginate hydrogels in air consistently resulted in deformation of printed constructs. The newly developed 3D printing technique termed freeform reversible embedding of suspended hydrogels (FRESH) was therefore investigated as a means for achieving 3D spatial control of printed hydrogels using the modified system. Printing cell-free sodium alginate hydrogels within gelatin sacrificial support baths allowed for fabricating constructs in a spatially defined manner. However, overprinting and swelling of alginate hydrogels negatively affected the overall printing accuracy. The present study aimed to pave the way for further system modifications and refinements, such that the ultimate goal of low-cost bioprinting may be achieved. Further optimisation of printing parameters, hydrogel characteristics and sterilisation techniques may allow for fabricating viable, physiologically relevant tissues using the modified system developed.
- Full Text:
- Date Issued: 2018
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