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
Electrode surface modification using metallophthalocyanines and metal nanoparticles : electrocatalytic activity
- Authors: Maringa, Audacity
- Date: 2015
- Subjects: Phthalocyanines , Nanoparticles , Electrocatalysis , Scanning electron microscopy , X-ray photoelectron spectroscopy , Electrochemistry , Scanning electrochemical microscopy
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4541 , http://hdl.handle.net/10962/d1017921
- Description: Metallophthalocyanines and metal nanoparticles were successfully synthesized and applied for the electrooxidation of amitrole, nitrite and hydrazine individually or when employed together. The synthesized materials were characterized using the following techniques: predominantly scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), electrochemistry and scanning electrochemical microscopy (SECM). Different electrode modification methods were used to modify the glassy carbon substrates. The methods include adsorption, electrodeposition, electropolymerization and click chemistry. Modifying the glassy carbon substrate with MPc (electropolymerization) followed by metal nanoparticles (electrodeposition) or vice versa, made a hybrid modified surface that had efficient electron transfer. This was confirmed by electrochemical impedance studies with voltammetry measurements having lower detection potentials for the analytes. This work also describes for the first time the micropatterning of the glassy carbon substrate using the SECM tip. The substrate was electrografted with 4-azidobenzenediazonium salt and then the click reaction was performed using ethynylferrocene facilitated by Cu⁺ produced at the SECM tip. The SECM imaging was then used to show the clicked spot.
- Full Text:
- Date Issued: 2015
- Authors: Maringa, Audacity
- Date: 2015
- Subjects: Phthalocyanines , Nanoparticles , Electrocatalysis , Scanning electron microscopy , X-ray photoelectron spectroscopy , Electrochemistry , Scanning electrochemical microscopy
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4541 , http://hdl.handle.net/10962/d1017921
- Description: Metallophthalocyanines and metal nanoparticles were successfully synthesized and applied for the electrooxidation of amitrole, nitrite and hydrazine individually or when employed together. The synthesized materials were characterized using the following techniques: predominantly scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), electrochemistry and scanning electrochemical microscopy (SECM). Different electrode modification methods were used to modify the glassy carbon substrates. The methods include adsorption, electrodeposition, electropolymerization and click chemistry. Modifying the glassy carbon substrate with MPc (electropolymerization) followed by metal nanoparticles (electrodeposition) or vice versa, made a hybrid modified surface that had efficient electron transfer. This was confirmed by electrochemical impedance studies with voltammetry measurements having lower detection potentials for the analytes. This work also describes for the first time the micropatterning of the glassy carbon substrate using the SECM tip. The substrate was electrografted with 4-azidobenzenediazonium salt and then the click reaction was performed using ethynylferrocene facilitated by Cu⁺ produced at the SECM tip. The SECM imaging was then used to show the clicked spot.
- Full Text:
- Date Issued: 2015
Screening of entomopathogenic fungi against citrus mealybug (Planococcus citri (Risso)) and citrus thrips (Scirtothrips aurantii (Faure))
- FitzGerald, Véronique Chartier
- Authors: FitzGerald, Véronique Chartier
- Date: 2014
- Subjects: Entomopathogenic fungi , Citrus mealybug -- South Africa -- Eastern Cape , Citrus thrips -- South Africa -- Eastern Cape , Citrus -- Diseases and pests , Citrus mealybug -- Biological control , Citrus thrips -- Biological control , Biological pest control agents , Scanning electron microscopy , Mycoses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4166 , http://hdl.handle.net/10962/d1020887
- Description: Mealybugs (Planococcus citri) and thrips (Scirtothrips aurantii) are common and extremely damaging citrus crop pests which have proven difficult to control via conventional methods, such as chemical pesticides and insect growth regulators. The objective of this study was to determine the efficacy of entomopathogenic fungi against these pests in laboratory bioassays. Isolates of Metarhizium anisopliae and Beauveria bassiana from citrus orchards in the Eastern Cape, South Africa were maintained on Sabouraud Dextrose 4% Agar supplemented with Dodine, chloramphenicol and rifampicin at 25°C. Infectivity of the fungal isolates was initially assessed using 5th instar false codling moth, Thaumatotibia leucotreta, larvae. Mealybug bioassays were performed in 24 well plates using 1 x 107 ml-1 conidial suspensions and kept at 26°C for 5 days with a photoperiod of 12 L:12 D. A Beauveria commercial product and an un-inoculated control were also screened for comparison. Isolates GAR 17 B3 (B. bassiana) and FCM AR 23 B3 (M. anisopliae) both resulted in 67.5% mealybug crawler mortality and GB AR 23 13 3 (B. bassiana) resulted in 64% crawler mortality. These 3 isolates were further tested in dose-dependent assays. Probit analyses were conducted on the dose-dependent assays data using PROBAN to determine LC₅₀ values. For both the mealybug adult and crawlers FCM AR 23 B3 required the lowest concentration to achieve LC₅₀ at 4.96 x 10⁶ conidia ml-1 and 5.29 x 10⁵ conidia ml-1, respectively. Bioassays on adult thrips were conducted in munger cells with leaf buds inoculated with the conidial suspensions. Isolate GAR 17 B3 had the highest mortality rate at 70% on thrips while FCM AR 23 B3 resulted in 60% mortality. Identification of the isolates, FCM AR 23 B3, GAR 17 B3 and GB AR 23 13 3, were confirmed to be correct using both microscopic and molecularly techniques. ITS sequences were compared to other sequences from GenBank and confirmed phylogenetically using MEGA6. Mealybug infection was investigated using scanning electron microscopy, mycosis was confirmed but the infection process could not be followed due to the extensive waxy cuticle. These results indicate that there is potential for the isolates FCM AR 23 B3 and GAR 17 B3 to be developed as biological control agents for the control of citrus mealybug and thrips. Further research would be required to determine their ability to perform under field conditions.
- Full Text:
- Date Issued: 2014
- Authors: FitzGerald, Véronique Chartier
- Date: 2014
- Subjects: Entomopathogenic fungi , Citrus mealybug -- South Africa -- Eastern Cape , Citrus thrips -- South Africa -- Eastern Cape , Citrus -- Diseases and pests , Citrus mealybug -- Biological control , Citrus thrips -- Biological control , Biological pest control agents , Scanning electron microscopy , Mycoses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4166 , http://hdl.handle.net/10962/d1020887
- Description: Mealybugs (Planococcus citri) and thrips (Scirtothrips aurantii) are common and extremely damaging citrus crop pests which have proven difficult to control via conventional methods, such as chemical pesticides and insect growth regulators. The objective of this study was to determine the efficacy of entomopathogenic fungi against these pests in laboratory bioassays. Isolates of Metarhizium anisopliae and Beauveria bassiana from citrus orchards in the Eastern Cape, South Africa were maintained on Sabouraud Dextrose 4% Agar supplemented with Dodine, chloramphenicol and rifampicin at 25°C. Infectivity of the fungal isolates was initially assessed using 5th instar false codling moth, Thaumatotibia leucotreta, larvae. Mealybug bioassays were performed in 24 well plates using 1 x 107 ml-1 conidial suspensions and kept at 26°C for 5 days with a photoperiod of 12 L:12 D. A Beauveria commercial product and an un-inoculated control were also screened for comparison. Isolates GAR 17 B3 (B. bassiana) and FCM AR 23 B3 (M. anisopliae) both resulted in 67.5% mealybug crawler mortality and GB AR 23 13 3 (B. bassiana) resulted in 64% crawler mortality. These 3 isolates were further tested in dose-dependent assays. Probit analyses were conducted on the dose-dependent assays data using PROBAN to determine LC₅₀ values. For both the mealybug adult and crawlers FCM AR 23 B3 required the lowest concentration to achieve LC₅₀ at 4.96 x 10⁶ conidia ml-1 and 5.29 x 10⁵ conidia ml-1, respectively. Bioassays on adult thrips were conducted in munger cells with leaf buds inoculated with the conidial suspensions. Isolate GAR 17 B3 had the highest mortality rate at 70% on thrips while FCM AR 23 B3 resulted in 60% mortality. Identification of the isolates, FCM AR 23 B3, GAR 17 B3 and GB AR 23 13 3, were confirmed to be correct using both microscopic and molecularly techniques. ITS sequences were compared to other sequences from GenBank and confirmed phylogenetically using MEGA6. Mealybug infection was investigated using scanning electron microscopy, mycosis was confirmed but the infection process could not be followed due to the extensive waxy cuticle. These results indicate that there is potential for the isolates FCM AR 23 B3 and GAR 17 B3 to be developed as biological control agents for the control of citrus mealybug and thrips. Further research would be required to determine their ability to perform under field conditions.
- Full Text:
- Date Issued: 2014
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