Publicação
New nanotechnology approaches using dendrimers modified with natural polymers for controlling stem cells behaviour in tissue engineering strategies
| Resumo: | In the recent years, great progress has been done in the emerging field of tissue engineering. Despite the important advances the performance of cells-scaffold constructs, one of the several tissue engineering approaches, remains limited in part due to the need for optimize cell culture techniques and culture media. Nanocarrier systems have generated a significant amount of interest in the ex vivo cell maintenance, and control of the cellular fate in vivo mainly due to their internalization efficiency, drug loading capacity, and to favorably modulate the solubility and pharmacokinetics of drugs. Dendrimers are synthetic, monodispersive, spherical and highly branched macromolecules that present unique advantages and fulfills most requirements as carriers for drug delivery; however, it has been found that high generation dendrimers are often cytotoxic. Thus, in this thesis we focused our attention in this fundamental problem and explore the development of novel nanobiomaterials based on the grafting of carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers, the socalled CMCht/PAMAM dendrimer nanoparticles. These macromolecular vehicles were developed to explore a new concept consisting on the intracellular and controlled delivery of bioactive molecules aimed at control stem cells functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. Thus, by combining nanotechnology-based systems and traditional tissue engineering strategies, we expect to develop a novel therapeutic solution for the efficient treatment of damage/diseased cells and tissues. To validate this new concept, there is the need to evaluate the performance of the developed nanocarriers, in vitro and in vivo. Firstly, the uptake efficiency and internalization mechanism of fluorescent-labeled CMCht/PAMAM dendrimer nanoparticles was investigated using different cell types. Fluorescence microscopy studies revealed that the developed nanocarriers could be efficiently internalized, either by cell lines and primary cultures, after a few hours. Flow cytometry studies revealed that rat bone marrow stromal cells (RBMSCs) cultured in the presence of colchicine, an alkaloid that inhibits endocytosis, decreased the internalization of the nanoparticles. These data showed that uptake by cells was primarily via an active endocytotic mechanism, but not exclusively. Preliminary studies were also carried out to evaluate the possible applicability of the CMCht/PAMAM dendrimer nanoparticles in the central nervous system. Internalization rate, cell viability and metabolic activity studies were performed using rat post-natal hippocampal neurons and cortical glial cells that revealed their ability for being taken up by these cells and its non cytotoxicity. Complementarily, dexamethasone (Dex), a glucocorticoid known to have important role on the proliferation and expression of osteoblastic differentiation markers, was used as a model drug and incorporated into the bulk of the nanoparticles. Physicochemical characterization and in vitro biological studies have demonstrated that the Dex-loaded CMCht/PAMAM dendrimer nanoparticles were successfully synthesized, were not cytotoxic in the range of concentration below 1 mg.mL-1 and promote osteogenesis (2-D system). To assess the true value of the Dex-loaded CMCht/PAMAM dendrimer nanoparticles systems for application in tissue engineering strategies, we use different biomaterials to develop a set of novel scaffolds both ceramic and polymeric or formulations. These scaffolds were found to be suitable for applications in bone, cartilage and osteochondral tissue engineering. In vitro studies have shown that combination of scaffolds, bone marrow stromal cells and Dex-loaded CMCht/PAMAM dendrimer nanoparticles (3-D system) enhanced osteogenesis. Finally, in vivo studies have shown that the novel Dex-loaded CMCht/PAMAM dendrimer nanoparticles may be beneficial as intracellular nanocarrier, supplying Dex in a regimented manner, while avoiding the need of culturing stem cells for long periods of time in vitro, towards promoting the osteogenic differentiation. Remarkably, the proposed strategy allow modulate and direct stem cells differentiation towards osteogenic phenotype, enhance in vivo proteoglycan extracellular matrix synthesis and promote superior de novo bone formation. This thesis mark the transition from the ‘proof-of-concept’ to useful intracellular nanocarrier tool, as the Dex-loaded CMCht/PAMAM dendrimer nanoparticles show promise for application in direct stem cell to a particular cell fate, in vitro and in vivo. |
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| Autores principais: | Oliveira, Joaquim M. |
| Ano: | 2009 |
| 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 the recent years, great progress has been done in the emerging field of tissue engineering. Despite the important advances the performance of cells-scaffold constructs, one of the several tissue engineering approaches, remains limited in part due to the need for optimize cell culture techniques and culture media. Nanocarrier systems have generated a significant amount of interest in the ex vivo cell maintenance, and control of the cellular fate in vivo mainly due to their internalization efficiency, drug loading capacity, and to favorably modulate the solubility and pharmacokinetics of drugs. Dendrimers are synthetic, monodispersive, spherical and highly branched macromolecules that present unique advantages and fulfills most requirements as carriers for drug delivery; however, it has been found that high generation dendrimers are often cytotoxic. Thus, in this thesis we focused our attention in this fundamental problem and explore the development of novel nanobiomaterials based on the grafting of carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers, the socalled CMCht/PAMAM dendrimer nanoparticles. These macromolecular vehicles were developed to explore a new concept consisting on the intracellular and controlled delivery of bioactive molecules aimed at control stem cells functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. Thus, by combining nanotechnology-based systems and traditional tissue engineering strategies, we expect to develop a novel therapeutic solution for the efficient treatment of damage/diseased cells and tissues. To validate this new concept, there is the need to evaluate the performance of the developed nanocarriers, in vitro and in vivo. Firstly, the uptake efficiency and internalization mechanism of fluorescent-labeled CMCht/PAMAM dendrimer nanoparticles was investigated using different cell types. Fluorescence microscopy studies revealed that the developed nanocarriers could be efficiently internalized, either by cell lines and primary cultures, after a few hours. Flow cytometry studies revealed that rat bone marrow stromal cells (RBMSCs) cultured in the presence of colchicine, an alkaloid that inhibits endocytosis, decreased the internalization of the nanoparticles. These data showed that uptake by cells was primarily via an active endocytotic mechanism, but not exclusively. Preliminary studies were also carried out to evaluate the possible applicability of the CMCht/PAMAM dendrimer nanoparticles in the central nervous system. Internalization rate, cell viability and metabolic activity studies were performed using rat post-natal hippocampal neurons and cortical glial cells that revealed their ability for being taken up by these cells and its non cytotoxicity. Complementarily, dexamethasone (Dex), a glucocorticoid known to have important role on the proliferation and expression of osteoblastic differentiation markers, was used as a model drug and incorporated into the bulk of the nanoparticles. Physicochemical characterization and in vitro biological studies have demonstrated that the Dex-loaded CMCht/PAMAM dendrimer nanoparticles were successfully synthesized, were not cytotoxic in the range of concentration below 1 mg.mL-1 and promote osteogenesis (2-D system). To assess the true value of the Dex-loaded CMCht/PAMAM dendrimer nanoparticles systems for application in tissue engineering strategies, we use different biomaterials to develop a set of novel scaffolds both ceramic and polymeric or formulations. These scaffolds were found to be suitable for applications in bone, cartilage and osteochondral tissue engineering. In vitro studies have shown that combination of scaffolds, bone marrow stromal cells and Dex-loaded CMCht/PAMAM dendrimer nanoparticles (3-D system) enhanced osteogenesis. Finally, in vivo studies have shown that the novel Dex-loaded CMCht/PAMAM dendrimer nanoparticles may be beneficial as intracellular nanocarrier, supplying Dex in a regimented manner, while avoiding the need of culturing stem cells for long periods of time in vitro, towards promoting the osteogenic differentiation. Remarkably, the proposed strategy allow modulate and direct stem cells differentiation towards osteogenic phenotype, enhance in vivo proteoglycan extracellular matrix synthesis and promote superior de novo bone formation. This thesis mark the transition from the ‘proof-of-concept’ to useful intracellular nanocarrier tool, as the Dex-loaded CMCht/PAMAM dendrimer nanoparticles show promise for application in direct stem cell to a particular cell fate, in vitro and in vivo. |
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