Publicação
Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications
| Resumo: | In a typical tissue engineering approach, cells are collected from the patient and then seeded into a threedimensional scaffold where they proliferate to generate a tissue-like substitute to be re-implanted back into the defect site. However, human tissues possess various degrees of complexity which often makes them impossible to be reproduced in such a simplified way. In fact, many tissues such as bone, for example, exhibit specific architectures and shapes, mechanical properties and cellular content that are very challenging to reproduce, in particular when combined together into a single construct. In order to overcome the limitations found in the generation of complex tissues such as bone and bone interfaces with other tissues, various automated technologies have been adopted by tissue engineering approaches to help generate viable tissue substitutes in a time- and cost-effective way. Bioreactors are automated systems where cell-seeded scaffolds can be cultured under highly controlled conditions to generate replacement tissues. They improve the mobility of nutrients and avoid cell death in internal regions of constructs and influence cellular development through biomechanical stimuli. Bioreactors show the potential to generate constructs in a more standardized, traceable, cost-effective, safe and regulatory-compliant way. Additive manufacturing is another highly automated technology which has more recently been introduced into tissue engineering. Its main use has been in producing 3D scaffolds with highly defined architectures by layer-by-layer deposition of materials. Herein we proposed the utilization of additive manufacturing to build simultaneously and concomitantly a 3D scaffold and bioreactor chamber, from different materials, as a single object. This would allow to produce scaffolds readily contained into bioreactor chambers, reducing the necessity for assembling, hence reducing production time and cost as well as the contamination risk due to the significantly decreased manipulation of both the scaffold and bioreactor. In a first approach we aimed at applying this concept to the generation of tailor-made constructs for defectand patient-specific applications. By resorting to medical imaging, a 3D model of a tibia bone tissue section was obtained and utilized as a template for generating a porous tissue replica scaffold as well as an enclosing culture chamber tightly fitting the outer shape of the porous scaffolds. The device showed to be able to homogenously distribute cells throughout the scaffold and to keep them viable along a 6 weeks culture period. In a second approach, the same concept was used to simultaneouslly seed and culture multiple scaffolds contained into one single upscalable perfusion culture device. Additionally, the device was used for coating the scaffolds contained in its interior with a calcium phosphate layer in order to enhance their osteoinductivity upon implantation. Cultured scaffolds showed homogeneous cell distribution and high cell viability throughout a 4 weeks culture period and calcium phosphate-coated scaffolds resulted in a significant increase in cell number. Such device may also find applications in the high throughput screening of combinations of multiple variable factors such as the selected biomaterials, scaffold architectures, cell types and culture regimes. Furthermore, this device might be applicable in the simultaneous generation of large amounts of tissue substitutes in a scenario of widespread adoption of tissue engineering-based therapies. Additive manufacturing was also applied to a specific tissue engineering application that requires the development of a biphasic construct, targeting the generation of bone-periodontal ligament-teeth interfaces in a guided tissue regeneration strategy. In this case additive manufacturing was combined with the electrospinning technology to fabricate the biphasic construct, that is potentially able to accommodate a bone and a periodontal ligament tissue construct in separate cavities. The additively manufactured 3D scaffold was treated by means of a calcium phosphate coating in order to increase its osteoinductivity and then attached to a fine electrospun fibrilar mesh. First the additively manufactured part of the scaffold was seeded with osteoblasts and later the electrospun part was used for depositing cell sheets of periodontal ligament acting as a biomechanical support. After further culture, the complex constructs were finally attached to dentin blocks simulating the surface of teeth and subcutaneously implanted into rats. After 8 weeks of implantation, increased bone formation was observed when comparing to non-coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted the vascularization of the periodontal cell sheets and the formation of a tissue similar to native periodontal ligament tissue at the interface with dentin. Given the promising results achieved, this new and complex biphasic scaffold represents therefore a new hope in the regeneration of complex tissue defects resulting from serious forms of periodontitis. Finally, a study was performed to evaluate the feasibility of cryopreservation-based storage and later off-theshelf utilization of cell/scaffold constructs showing that cell and scaffold properties can be maintained upon cryopreservation and that the architecture of porous scaffolds may favor the retention and viability of construct’s cellular content. In summary, the work performed in this thesis resulted in significant advances towards automation and mass production of tailor-made and off-the-shelf tissue engineered products that might facilitate the widespread clinical adoption of tissue engineering strategies holding the promise to revolutionize the treatment of damaged tissues. |
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| Autores principais: | Costa, Pedro Ferreira da |
| Assunto: | Engenharia e Tecnologia::Biotecnologia Industrial |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | In a typical tissue engineering approach, cells are collected from the patient and then seeded into a threedimensional scaffold where they proliferate to generate a tissue-like substitute to be re-implanted back into the defect site. However, human tissues possess various degrees of complexity which often makes them impossible to be reproduced in such a simplified way. In fact, many tissues such as bone, for example, exhibit specific architectures and shapes, mechanical properties and cellular content that are very challenging to reproduce, in particular when combined together into a single construct. In order to overcome the limitations found in the generation of complex tissues such as bone and bone interfaces with other tissues, various automated technologies have been adopted by tissue engineering approaches to help generate viable tissue substitutes in a time- and cost-effective way. Bioreactors are automated systems where cell-seeded scaffolds can be cultured under highly controlled conditions to generate replacement tissues. They improve the mobility of nutrients and avoid cell death in internal regions of constructs and influence cellular development through biomechanical stimuli. Bioreactors show the potential to generate constructs in a more standardized, traceable, cost-effective, safe and regulatory-compliant way. Additive manufacturing is another highly automated technology which has more recently been introduced into tissue engineering. Its main use has been in producing 3D scaffolds with highly defined architectures by layer-by-layer deposition of materials. Herein we proposed the utilization of additive manufacturing to build simultaneously and concomitantly a 3D scaffold and bioreactor chamber, from different materials, as a single object. This would allow to produce scaffolds readily contained into bioreactor chambers, reducing the necessity for assembling, hence reducing production time and cost as well as the contamination risk due to the significantly decreased manipulation of both the scaffold and bioreactor. In a first approach we aimed at applying this concept to the generation of tailor-made constructs for defectand patient-specific applications. By resorting to medical imaging, a 3D model of a tibia bone tissue section was obtained and utilized as a template for generating a porous tissue replica scaffold as well as an enclosing culture chamber tightly fitting the outer shape of the porous scaffolds. The device showed to be able to homogenously distribute cells throughout the scaffold and to keep them viable along a 6 weeks culture period. In a second approach, the same concept was used to simultaneouslly seed and culture multiple scaffolds contained into one single upscalable perfusion culture device. Additionally, the device was used for coating the scaffolds contained in its interior with a calcium phosphate layer in order to enhance their osteoinductivity upon implantation. Cultured scaffolds showed homogeneous cell distribution and high cell viability throughout a 4 weeks culture period and calcium phosphate-coated scaffolds resulted in a significant increase in cell number. Such device may also find applications in the high throughput screening of combinations of multiple variable factors such as the selected biomaterials, scaffold architectures, cell types and culture regimes. Furthermore, this device might be applicable in the simultaneous generation of large amounts of tissue substitutes in a scenario of widespread adoption of tissue engineering-based therapies. Additive manufacturing was also applied to a specific tissue engineering application that requires the development of a biphasic construct, targeting the generation of bone-periodontal ligament-teeth interfaces in a guided tissue regeneration strategy. In this case additive manufacturing was combined with the electrospinning technology to fabricate the biphasic construct, that is potentially able to accommodate a bone and a periodontal ligament tissue construct in separate cavities. The additively manufactured 3D scaffold was treated by means of a calcium phosphate coating in order to increase its osteoinductivity and then attached to a fine electrospun fibrilar mesh. First the additively manufactured part of the scaffold was seeded with osteoblasts and later the electrospun part was used for depositing cell sheets of periodontal ligament acting as a biomechanical support. After further culture, the complex constructs were finally attached to dentin blocks simulating the surface of teeth and subcutaneously implanted into rats. After 8 weeks of implantation, increased bone formation was observed when comparing to non-coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted the vascularization of the periodontal cell sheets and the formation of a tissue similar to native periodontal ligament tissue at the interface with dentin. Given the promising results achieved, this new and complex biphasic scaffold represents therefore a new hope in the regeneration of complex tissue defects resulting from serious forms of periodontitis. Finally, a study was performed to evaluate the feasibility of cryopreservation-based storage and later off-theshelf utilization of cell/scaffold constructs showing that cell and scaffold properties can be maintained upon cryopreservation and that the architecture of porous scaffolds may favor the retention and viability of construct’s cellular content. In summary, the work performed in this thesis resulted in significant advances towards automation and mass production of tailor-made and off-the-shelf tissue engineered products that might facilitate the widespread clinical adoption of tissue engineering strategies holding the promise to revolutionize the treatment of damaged tissues. |
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