Publicação
Silk fibroin-based scaffolds, hydrogels and calcium-phosphate filled materials aimed for regenerative medicine applications
| Resumo: | Bone and cartilage defects derived from trauma or disease are major problems in orthopedics. Tissue engineering and regenerative medicine provides promising strategies for the regeneration of damaged tissues. Biomaterials, processed into porous scaffolds and hydrogels, have been playing a crucial role in the tissue regeneration. Controlling the physicochemical properties of biomaterials is important for inducing proper cellular response towards tissue formation, thus facilitating the regeneration procedure. While the ideal tissue regeneration outcome has not yet been achieved, great progress had been made in the last decades, in terms of the application of biomaterials for tissue regeneration. The aim of this thesis is to develop novel silk fibroin (SF) based porous scaffolds and hydrogels with adequate properties and controlled conformations for different tissues regeneration. Several strategies were used in this thesis, including the improvement of scaffolds’ strength, biomimetic of the tissue composition and stratified structure, and development of stimuli-responsive hydrogels with injectable or spatial tunable properties. SF derived from Bombyx mori cocoons was chosen as the matrix material because it has many advantages. It is a biodegradable protein based biomaterial with superior in vitro and in vivo biocompatibility. Moreover, its mechanical properties and degradation profile can be tuned by the processing approach. SF can be processed into different shapes and architectures, and it is a readily available supply. Salt-leached SF scaffolds with superior mechanical properties were produced by using highly concentrated aqueous SF solutions. The compressive and storage moduli of the scaffolds were significantly enhanced with increasing the concentration of SF solution. The developed scaffolds were of macro/microporous structure, high porosity and interconnectivity, and presented a homogeneous porosity distribution. The obtained scaffolds present adequate properties for cartilage and meniscus regeneration. Mimicking the composition of natural bone, composite scaffolds composed of SF and calcium phosphate were developed for bone regeneration. Nano calcium phosphate particles were incorporated in the concentrated SF solution using an in-situ synthesis method following saltleaching to develop the silk-nano calcium phosphate (Silk-NanoCaP). These scaffolds maintained the superior mechanical properties of SF scaffolds but demonstrated in vitro bioactivity. The NanoCaP particles were homogeneously distributed in the silk matrix, at both macroscopic and microscopic levels. The leachables of the scaffolds were non-cytotoxic as determined by in vitro cytotoxicity assays. The in vitro and in vivo biological performance of both SF and Silk-NanoCaP scaffolds was further evaluated. These scaffolds supported the viability and proliferation of human adipose tissue derived stromal cells. The formed extracellular matrix improved the mechanical properties of the cell-laden scaffolds or constructs. In vivo, both scaffolds have supported de novo bone formation and ingrowth’s and induced no acute inflammatory response. The Silk NanoCaP scaffold was osteoconductive as new bone grew directly on its surface. This group induced higher amount of new bone formation than the SF group. The Silk-NanoCaP scaffolds can be used in bone regeneration. Considering the stratified/composition characteristics of osteochondral tissue, bilayered scaffolds composed of a SF layer and a Silk-NanoCaP layer were produced for osteochondral defects (OCD) regeneration. The in vitro bioactivity was only observed in the Silk-NanoCaP layer i.e., the bone-like layer. When seeded with marrow mesenchymal stromal cells, the bilayered scaffolds promoted cell viability and proliferation, and the Silk- NanoCaP layer induced a higher alkaline phosphatase level as compared to the SF layer (cartilage-like layer). In vivo subcutaneous implantation showed that the scaffolds supported tissue infiltration and no granulation tissue or acute inflammation were observed. When implanted in the rabbit OCD, the bilayered scaffolds supported cartilage regeneration in the SF layer and promoted bone ingrowths in the Silk-NanoCaP layer. Therefore, they demonstrated to be promising candidates for OCD regeneration. Besides the development of SF based scaffolds, another approach explored in this thesis was to develop injectable and enzymatically cross-linked SF hydrogels that could be suitable for cartilage regeneration. The SF hydrogels were prepared by peroxidase mediated crosslinking of the tyrosine groups in the backbone of SF. These hydrogels could be formed in a few minutes under physiological conditions. Dominant amorphous conformation was presented in these hydrogels. These hydrogels were ionic strength and pH stimuli responsive. Cells were successfully encapsulated into these hydrogels. Subcutaneous implantation showed that these hydrogels did not induce any acute inflammatory reaction. After in vitro cell culture or in vivo implantation, β-sheet conformation was observed in these hydrogels. The developed SF hydrogels can be used as an injectable material for filling tissue defects (such as bone or cartilage) or as a drug delivery system. Finally, SF hydrogels with spatially controllable properties were generated. Core-shell SF hydrogels consisted in a β-sheet conformation in the shell layer and mainly an amorphous conformation in the core layer. These were prepared by the controlled immersion of the peroxidase mediated SF hydrogels in methanol. The thickness of the shell layer and the mechanical properties of the core-shell SF hydrogels increased with increasing the immersion time. When incorporating albumin as a model drug, the core-shell SF hydrogels presented slower and more controllable release profile as compared to the SF hydrogel. The core-shell SF hydrogels can be used as a controlled release system, tissue substitute or both. In this thesis, different strategies for developing novel SF based scaffolds and enzymatically cross-linked hydrogels were explored. In both cases remarkable properties and functions for tissue engineering and regenerative medicine applications were achieved, as well as a high reproducibility of the systems. The SF based scaffolds and enzymatically cross-linked SF hydrogels provided herein can be promising candidates for cartilage, meniscus, bone, and osteochondral regeneration, as well as drug delivery systems or tissue substitutes. |
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| Autores principais: | Yan, Leping |
| Assunto: | Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
| 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: | Bone and cartilage defects derived from trauma or disease are major problems in orthopedics. Tissue engineering and regenerative medicine provides promising strategies for the regeneration of damaged tissues. Biomaterials, processed into porous scaffolds and hydrogels, have been playing a crucial role in the tissue regeneration. Controlling the physicochemical properties of biomaterials is important for inducing proper cellular response towards tissue formation, thus facilitating the regeneration procedure. While the ideal tissue regeneration outcome has not yet been achieved, great progress had been made in the last decades, in terms of the application of biomaterials for tissue regeneration. The aim of this thesis is to develop novel silk fibroin (SF) based porous scaffolds and hydrogels with adequate properties and controlled conformations for different tissues regeneration. Several strategies were used in this thesis, including the improvement of scaffolds’ strength, biomimetic of the tissue composition and stratified structure, and development of stimuli-responsive hydrogels with injectable or spatial tunable properties. SF derived from Bombyx mori cocoons was chosen as the matrix material because it has many advantages. It is a biodegradable protein based biomaterial with superior in vitro and in vivo biocompatibility. Moreover, its mechanical properties and degradation profile can be tuned by the processing approach. SF can be processed into different shapes and architectures, and it is a readily available supply. Salt-leached SF scaffolds with superior mechanical properties were produced by using highly concentrated aqueous SF solutions. The compressive and storage moduli of the scaffolds were significantly enhanced with increasing the concentration of SF solution. The developed scaffolds were of macro/microporous structure, high porosity and interconnectivity, and presented a homogeneous porosity distribution. The obtained scaffolds present adequate properties for cartilage and meniscus regeneration. Mimicking the composition of natural bone, composite scaffolds composed of SF and calcium phosphate were developed for bone regeneration. Nano calcium phosphate particles were incorporated in the concentrated SF solution using an in-situ synthesis method following saltleaching to develop the silk-nano calcium phosphate (Silk-NanoCaP). These scaffolds maintained the superior mechanical properties of SF scaffolds but demonstrated in vitro bioactivity. The NanoCaP particles were homogeneously distributed in the silk matrix, at both macroscopic and microscopic levels. The leachables of the scaffolds were non-cytotoxic as determined by in vitro cytotoxicity assays. The in vitro and in vivo biological performance of both SF and Silk-NanoCaP scaffolds was further evaluated. These scaffolds supported the viability and proliferation of human adipose tissue derived stromal cells. The formed extracellular matrix improved the mechanical properties of the cell-laden scaffolds or constructs. In vivo, both scaffolds have supported de novo bone formation and ingrowth’s and induced no acute inflammatory response. The Silk NanoCaP scaffold was osteoconductive as new bone grew directly on its surface. This group induced higher amount of new bone formation than the SF group. The Silk-NanoCaP scaffolds can be used in bone regeneration. Considering the stratified/composition characteristics of osteochondral tissue, bilayered scaffolds composed of a SF layer and a Silk-NanoCaP layer were produced for osteochondral defects (OCD) regeneration. The in vitro bioactivity was only observed in the Silk-NanoCaP layer i.e., the bone-like layer. When seeded with marrow mesenchymal stromal cells, the bilayered scaffolds promoted cell viability and proliferation, and the Silk- NanoCaP layer induced a higher alkaline phosphatase level as compared to the SF layer (cartilage-like layer). In vivo subcutaneous implantation showed that the scaffolds supported tissue infiltration and no granulation tissue or acute inflammation were observed. When implanted in the rabbit OCD, the bilayered scaffolds supported cartilage regeneration in the SF layer and promoted bone ingrowths in the Silk-NanoCaP layer. Therefore, they demonstrated to be promising candidates for OCD regeneration. Besides the development of SF based scaffolds, another approach explored in this thesis was to develop injectable and enzymatically cross-linked SF hydrogels that could be suitable for cartilage regeneration. The SF hydrogels were prepared by peroxidase mediated crosslinking of the tyrosine groups in the backbone of SF. These hydrogels could be formed in a few minutes under physiological conditions. Dominant amorphous conformation was presented in these hydrogels. These hydrogels were ionic strength and pH stimuli responsive. Cells were successfully encapsulated into these hydrogels. Subcutaneous implantation showed that these hydrogels did not induce any acute inflammatory reaction. After in vitro cell culture or in vivo implantation, β-sheet conformation was observed in these hydrogels. The developed SF hydrogels can be used as an injectable material for filling tissue defects (such as bone or cartilage) or as a drug delivery system. Finally, SF hydrogels with spatially controllable properties were generated. Core-shell SF hydrogels consisted in a β-sheet conformation in the shell layer and mainly an amorphous conformation in the core layer. These were prepared by the controlled immersion of the peroxidase mediated SF hydrogels in methanol. The thickness of the shell layer and the mechanical properties of the core-shell SF hydrogels increased with increasing the immersion time. When incorporating albumin as a model drug, the core-shell SF hydrogels presented slower and more controllable release profile as compared to the SF hydrogel. The core-shell SF hydrogels can be used as a controlled release system, tissue substitute or both. In this thesis, different strategies for developing novel SF based scaffolds and enzymatically cross-linked hydrogels were explored. In both cases remarkable properties and functions for tissue engineering and regenerative medicine applications were achieved, as well as a high reproducibility of the systems. The SF based scaffolds and enzymatically cross-linked SF hydrogels provided herein can be promising candidates for cartilage, meniscus, bone, and osteochondral regeneration, as well as drug delivery systems or tissue substitutes. |
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