Publicação
Biochemical and physical surface functionalization of polycaprolactone as a key mediator of osteogenic differentiation and vascularized tissue morphogenesis
| Resumo: | The orthopedic implants represent an ever-growing biomedical market, as the population is continuously expanding and the average life expectancy is increasing. Tissue engineering and regenerative medicine aim at opening a pathway of therapy and treatment of bone tissue loss or end stage damage. Envisioning this goal, biodegradable solutions, such as polycaprolactone (PCL), offer a number of physiological and clinical advantages over permanent implants. This work aims to determine the aptitude of PCL surface roughness and the density-specific role of fibronectin (FN) adsorbed on PCL to induce differentiation of adult stem cells towards the osteoblastic lineage. Furthermore, as the establishment of a microvascular blood supply, necessary to assure cell survival, is still identified as a major challenge for the clinical application of (bone) engineered biomaterials, the combination of physical regular surface pattern motifs of PCL and density-specific fibronectin coating was investigated to determine the biomaterial effectiveness on the morphogenesis of microvascular tissue, both in vitro and in vivo. Our findings indicate that the optimal FN density regime of ~48 ng/cm2 could consistently and significantly support higher expression of osteocommitment biomarkers, such as cuboidal cytoskeleton morphology, alkaline phosphatase (ALP) activity and collagen type 1deposition. Furthermore, it was verified that such a density can be used as relevant alternative to the potent synthetic osteogenic supplement dexamethasone (Dex), in the osteogenic commitment of stem cells in vitro. Our analysis also demonstrated the differential regulation of the osteogenic differentiation of adult stem cells (from early ALP activity to end-process mineralization) by different roughness average (Ra) along an engineered surface roughness gradient. Faster osteogenic commitment and strongest osteogenic expression was obtained at Ra ~ 2.1 – 3.1 μm. Importantly, the removal of Dex, and even the removal of all osteogenesis-inducing supplements from the cell culture medium, did not prevent the differentiation process from occurring. Indeed, the PCL Ra ~ 0.9 – 2.1 μm showed the ability to alone direct the osteogenic differentiation of the stem cells, in vitro. Finally, we showed that geometrically defined micropatterns of PCL, in association with human density-specific FN adsorption (determined from a gradient study), can induce/instruct the endothelial cells (ECs) to mature into a luminized capillary network, both in vitro and in vivo. These results cumulatively enrich our knowledge on the biochemical and physical cues which evoke osteogenic stem cell modulation and successful recapitulation of supportive microvascularization, highlighting the potential for creating effective solutions for orthopedic applications featuring a clinically relevant biodegradable material. |
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| Autores principais: | Torres, Ana B. Faia |
| Assunto: | Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
| Ano: | 2015 |
| 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: | The orthopedic implants represent an ever-growing biomedical market, as the population is continuously expanding and the average life expectancy is increasing. Tissue engineering and regenerative medicine aim at opening a pathway of therapy and treatment of bone tissue loss or end stage damage. Envisioning this goal, biodegradable solutions, such as polycaprolactone (PCL), offer a number of physiological and clinical advantages over permanent implants. This work aims to determine the aptitude of PCL surface roughness and the density-specific role of fibronectin (FN) adsorbed on PCL to induce differentiation of adult stem cells towards the osteoblastic lineage. Furthermore, as the establishment of a microvascular blood supply, necessary to assure cell survival, is still identified as a major challenge for the clinical application of (bone) engineered biomaterials, the combination of physical regular surface pattern motifs of PCL and density-specific fibronectin coating was investigated to determine the biomaterial effectiveness on the morphogenesis of microvascular tissue, both in vitro and in vivo. Our findings indicate that the optimal FN density regime of ~48 ng/cm2 could consistently and significantly support higher expression of osteocommitment biomarkers, such as cuboidal cytoskeleton morphology, alkaline phosphatase (ALP) activity and collagen type 1deposition. Furthermore, it was verified that such a density can be used as relevant alternative to the potent synthetic osteogenic supplement dexamethasone (Dex), in the osteogenic commitment of stem cells in vitro. Our analysis also demonstrated the differential regulation of the osteogenic differentiation of adult stem cells (from early ALP activity to end-process mineralization) by different roughness average (Ra) along an engineered surface roughness gradient. Faster osteogenic commitment and strongest osteogenic expression was obtained at Ra ~ 2.1 – 3.1 μm. Importantly, the removal of Dex, and even the removal of all osteogenesis-inducing supplements from the cell culture medium, did not prevent the differentiation process from occurring. Indeed, the PCL Ra ~ 0.9 – 2.1 μm showed the ability to alone direct the osteogenic differentiation of the stem cells, in vitro. Finally, we showed that geometrically defined micropatterns of PCL, in association with human density-specific FN adsorption (determined from a gradient study), can induce/instruct the endothelial cells (ECs) to mature into a luminized capillary network, both in vitro and in vivo. These results cumulatively enrich our knowledge on the biochemical and physical cues which evoke osteogenic stem cell modulation and successful recapitulation of supportive microvascularization, highlighting the potential for creating effective solutions for orthopedic applications featuring a clinically relevant biodegradable material. |
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