Publicação
Development of vascularizable hydrogels to promote spinal cord injury repair: from RGD to mimetic ligands
| Resumo: | A major contributor for the severe deficits spinal cord injury (SCI) patients face lies on the disruption of the blood-spinal cord barrier (BSCB), and consequent deficient angiogenic response. Therefore, during this PhD thesis we proposed to develop vascularizable cell transplantation vehicles based on modified gellan gum (GG) hydrogels aiming to promote SCI repair. We started by understanding whether human umbilical vein endothelial cells (HUVECs) were able to assemble into vascular-like constructs when cultured on in-house GRGDS-modified GG (GG-GRGDS). HUVECs only developed stable vascular-like constructs in the presence of adipose-derived stem cells (ASCs). Moreover, the co-culture secretome had an enrichment on defined neurotrophic factors in comparison to the individual culture of both types of cells. We also observed a significant promotion on axonal growth in comparison to the hydrogels without cells. The next part of the work involved the modification of GG with two integrin-specific ligands (against αvβ3 and α5β1) to improve the bioactivity of the polymer. Cell adhesion studies showed that the αvβ3 mimetic promoted significant adhesion of ASCs to the matrix in comparison to RGD and the other mimetic ligand. This was even more evident for the formulation modified using a GG:ligand ratio of 10:1 (GGp10αv). Despite integrin specificity, it were only observed vascular-like structures only when HUVECs were co-cultured with ASCs. The axonal growth experiments did not reveal a statistical increase when comparing the co-culture with the hydrogel alone. Finally, the in vivo evaluation of both in-house GG-GRGDS and GGp10αv on a T8 transection model of SCI showed statistically significant motor improvements for GGp10αv encapsulating ASCs+HUVECs in comparison to lesioned animals. Furthermore, both GG-GRGDS and GGp10αv (with and without cells) lead to significant gains of sensation 8 weeks after SCI. It was also observed that during the acute phase of SCI GGp10αv+cells promoted a marked decrease on pro-inflammatory cytokines such as IL-1 α, IL-1 β, TNF-α and MCP-1 that are associated with the exacerbation of damage following SCI. On the other hand, IL-10 and IL-4, which are more connected to a pro-regenerative inflammation, were augmented relative to injured animals. Even though the histological and proteomic characterization of the tissue is still lacking, these indications point out towards a neuroprotective effect of GGp10αv+cells on injured tissue. |
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| Autores principais: | Rocha, Luís António Ferreira |
| Assunto: | Integrin-specific biomaterials Mesenchymal stem cells Neurovascular Spinal cord injury Vascular engineering Biomateriais com especificidade para integrinas Células estaminais mesenquimatosas Engenharia vascular Lesões medulares Neurovascular Ciências Médicas::Ciências da Saúde |
| Ano: | 2021 |
| 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: | A major contributor for the severe deficits spinal cord injury (SCI) patients face lies on the disruption of the blood-spinal cord barrier (BSCB), and consequent deficient angiogenic response. Therefore, during this PhD thesis we proposed to develop vascularizable cell transplantation vehicles based on modified gellan gum (GG) hydrogels aiming to promote SCI repair. We started by understanding whether human umbilical vein endothelial cells (HUVECs) were able to assemble into vascular-like constructs when cultured on in-house GRGDS-modified GG (GG-GRGDS). HUVECs only developed stable vascular-like constructs in the presence of adipose-derived stem cells (ASCs). Moreover, the co-culture secretome had an enrichment on defined neurotrophic factors in comparison to the individual culture of both types of cells. We also observed a significant promotion on axonal growth in comparison to the hydrogels without cells. The next part of the work involved the modification of GG with two integrin-specific ligands (against αvβ3 and α5β1) to improve the bioactivity of the polymer. Cell adhesion studies showed that the αvβ3 mimetic promoted significant adhesion of ASCs to the matrix in comparison to RGD and the other mimetic ligand. This was even more evident for the formulation modified using a GG:ligand ratio of 10:1 (GGp10αv). Despite integrin specificity, it were only observed vascular-like structures only when HUVECs were co-cultured with ASCs. The axonal growth experiments did not reveal a statistical increase when comparing the co-culture with the hydrogel alone. Finally, the in vivo evaluation of both in-house GG-GRGDS and GGp10αv on a T8 transection model of SCI showed statistically significant motor improvements for GGp10αv encapsulating ASCs+HUVECs in comparison to lesioned animals. Furthermore, both GG-GRGDS and GGp10αv (with and without cells) lead to significant gains of sensation 8 weeks after SCI. It was also observed that during the acute phase of SCI GGp10αv+cells promoted a marked decrease on pro-inflammatory cytokines such as IL-1 α, IL-1 β, TNF-α and MCP-1 that are associated with the exacerbation of damage following SCI. On the other hand, IL-10 and IL-4, which are more connected to a pro-regenerative inflammation, were augmented relative to injured animals. Even though the histological and proteomic characterization of the tissue is still lacking, these indications point out towards a neuroprotective effect of GGp10αv+cells on injured tissue. |
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