Publicação
BoneIntegra: engineering bone tissue constructs through Integrated hydrostatic pressure
| Resumo: | The continuously rising life expectancy has led to an increasing incidence of bone-related disorders, driving the demand for innovative strategies in bone tissue engineering (BTE). Among the most promising approaches are scaffolds, particularly hydrogel-based ones, often combined with cells to support tissue regeneration. Scaffold design plays a crucial role in promoting cell adhesion and growth, as well as in ensuring mechanical stability in biological environments. To explore complex and functional designs, there is a parallel need for advanced manufacturing techniques. In the first phase of this thesis, volumetric bioprinting, a light-based technique capable of producing entire 3D structures in a single step within seconds, was used to fabricate GelMA hydrogels (without cells and cell-laden) in cubic geometries with unit cells defined by: i) cylindrical (three intersecting cylinders) and ii) spherical pores (a single sphere at the center of the cubic unit cell). GelMA is here used for its tunable mechanical properties and its ability to mimic the native extracellular matrix. Firstly, functionalization of gelatine into GelMA was performed, achieving a modification degree similar to commercial GelMA. By optimizing light intensity, cylindrical and spherical-based architectures were successfully fabricated via volumetric printing. Under an optimized light power (of 270 mJ/cm2) and a defined voxel unit of 40 μm, both architectures were obtained with high shape fidelity. With respect to dimensional accuracy, experimental pore size showed a variation of 11.46% and 32.52% and total porosity about 55.63 ± 10.59 % and 69.30 ± 4.69 % for cylindrical and spherical-based constructs, respectively. Young’s modulus analysis revealed that both architectures responded to mechanical compression at a macroscale in a similar range. The Y201 immortalized human bone marrow mesenchymal stem cell (hBM-MSCs) was explored to fabricate the cell-laden constructs, which show viability and proliferation up to 14 days. Cylindrical unit cells exhibited higher proliferation at day 14 which was attributed to the higher surface area that characterizes this architecture. Conducted at Newcastle University, this work confirmed the ability to create intricate, cell-supportive constructs, with cells showing gene expression that may indicate osteogenic activity. When conceptualizing bone tissue engineering scaffolds, besides scaffold design, one must consider the mechanical microenvironment to which the cells are exposed. Thus, the second phase focused on how hydrostatic pressure (HP), a poorly explored but physiologically relevant mechanical cue, affects cells in 3D environments. Optimization of an HP chamber was performed considering the suitable materials to be used in terms of sterilization and performance in working conditions. Troubleshooting included materials interface to seal the chamber, protocol to adjust the HP via controlling the pressure and controlling the actuator displacement. Afterwards, HP was applied into 3D scaffolds. Due to material shipping issues, it was not possible to use the hydrogel constructs fabricated via volumetric printing. As an alternative, composite scaffolds were used as 3D porous platforms to culture hBM-MSCs. In contrast to the very few reports existent on HP application of 2D monocultures, 2 hours of cyclic stimulation in 3D porous scaffolds induced cell senescence, and the assay was interrupted mid-culture. Therefore, a control experiment was designed in order to establish the suitable time for stimulation without risking causing cell death likely due to oxygen deprivation. A period of 30 minutes was found to allow cells to be stimulated with a cyclic application of HP up to 300 kPa maintaining viability. Therefore, for future studies with human primary cells from the human bone marrow, stimulation should be based on periods of 30 minutes. Despite challenges, this work contributed towards more physiologically relevant scaffolds for bone tissue engineering, including novel architectures fabricated by volumetric printing and establishment of suitable HP stimulation time for human bone marrow-derived mesenchymal stem cells. |
|---|---|
| Autores principais: | Martins, Inês Coutinho |
| Assunto: | GelMA Hydrogel Porous architectures 3D volumetric bioprinting Bone tissue engineering Hydrostatic pressure Bioreactor |
| Ano: | 2025 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Aveiro |
| Idioma: | inglês |
| Origem: | RIA - Repositório Institucional da Universidade de Aveiro |
| Resumo: | The continuously rising life expectancy has led to an increasing incidence of bone-related disorders, driving the demand for innovative strategies in bone tissue engineering (BTE). Among the most promising approaches are scaffolds, particularly hydrogel-based ones, often combined with cells to support tissue regeneration. Scaffold design plays a crucial role in promoting cell adhesion and growth, as well as in ensuring mechanical stability in biological environments. To explore complex and functional designs, there is a parallel need for advanced manufacturing techniques. In the first phase of this thesis, volumetric bioprinting, a light-based technique capable of producing entire 3D structures in a single step within seconds, was used to fabricate GelMA hydrogels (without cells and cell-laden) in cubic geometries with unit cells defined by: i) cylindrical (three intersecting cylinders) and ii) spherical pores (a single sphere at the center of the cubic unit cell). GelMA is here used for its tunable mechanical properties and its ability to mimic the native extracellular matrix. Firstly, functionalization of gelatine into GelMA was performed, achieving a modification degree similar to commercial GelMA. By optimizing light intensity, cylindrical and spherical-based architectures were successfully fabricated via volumetric printing. Under an optimized light power (of 270 mJ/cm2) and a defined voxel unit of 40 μm, both architectures were obtained with high shape fidelity. With respect to dimensional accuracy, experimental pore size showed a variation of 11.46% and 32.52% and total porosity about 55.63 ± 10.59 % and 69.30 ± 4.69 % for cylindrical and spherical-based constructs, respectively. Young’s modulus analysis revealed that both architectures responded to mechanical compression at a macroscale in a similar range. The Y201 immortalized human bone marrow mesenchymal stem cell (hBM-MSCs) was explored to fabricate the cell-laden constructs, which show viability and proliferation up to 14 days. Cylindrical unit cells exhibited higher proliferation at day 14 which was attributed to the higher surface area that characterizes this architecture. Conducted at Newcastle University, this work confirmed the ability to create intricate, cell-supportive constructs, with cells showing gene expression that may indicate osteogenic activity. When conceptualizing bone tissue engineering scaffolds, besides scaffold design, one must consider the mechanical microenvironment to which the cells are exposed. Thus, the second phase focused on how hydrostatic pressure (HP), a poorly explored but physiologically relevant mechanical cue, affects cells in 3D environments. Optimization of an HP chamber was performed considering the suitable materials to be used in terms of sterilization and performance in working conditions. Troubleshooting included materials interface to seal the chamber, protocol to adjust the HP via controlling the pressure and controlling the actuator displacement. Afterwards, HP was applied into 3D scaffolds. Due to material shipping issues, it was not possible to use the hydrogel constructs fabricated via volumetric printing. As an alternative, composite scaffolds were used as 3D porous platforms to culture hBM-MSCs. In contrast to the very few reports existent on HP application of 2D monocultures, 2 hours of cyclic stimulation in 3D porous scaffolds induced cell senescence, and the assay was interrupted mid-culture. Therefore, a control experiment was designed in order to establish the suitable time for stimulation without risking causing cell death likely due to oxygen deprivation. A period of 30 minutes was found to allow cells to be stimulated with a cyclic application of HP up to 300 kPa maintaining viability. Therefore, for future studies with human primary cells from the human bone marrow, stimulation should be based on periods of 30 minutes. Despite challenges, this work contributed towards more physiologically relevant scaffolds for bone tissue engineering, including novel architectures fabricated by volumetric printing and establishment of suitable HP stimulation time for human bone marrow-derived mesenchymal stem cells. |
|---|
Atividades financiadas
Não foram encontrados projetos financiados.