Engineering Hydrogels with Affinity-Bound Laminin as 3D Neural Stem Cell Culture Systems

Autor: Andrés J. García, Woojin M. Han, Eduardo Conde-Sousa, Isabel F. Amaral, Andreia M Gonçalves, Daniela Barros, Ana Paula Pêgo
Přispěvatelé: Instituto de Investigação e Inovação em Saúde
Jazyk: angličtina
Rok vydání: 2019
Předmět:
Zdroj: Biomater Sci
Repositório Científico de Acesso Aberto de Portugal
Repositório Científico de Acesso Aberto de Portugal (RCAAP)
instacron:RCAAP
ISSN: 1068-2015
Popis: Laminin incorporation into biological or synthetic hydrogels has been explored to recapitulate the dynamic nature and biological complexity of neural stem cell (NSC) niches. However, the strategies currently explored for laminin immobilization within three-dimensional (3D) matrices do not address a critical aspect influencing cell-matrix interactions, which is the control over laminin conformation and orientation upon immobilization. This is a key feature for the preservation of the protein bioactivity. In this work, we explored an affinity-based approach to mediate the site-selective immobilization of laminin into a degradable synthetic hydrogel. Specifically, a four-arm maleimide terminated poly(ethylene glycol) (PEG-4MAL) macromer was functionalized with a mono-PEGylated recombinant human N-terminal agrin (NtA) domain, to promote high affinity binding of laminin. Different NtA concentrations (10, 50 and 100 M) were used to investigate the impact of NtA density on laminin incorporation, hydrogel biophysical properties, and biological outcome. Laminin was efficiently incorporated for all the conditions tested (laminin incorporation > 95%), and the developed hydrogels revealed mechanical properties (average storage modulus (G’) ranging from 187 to 256 Pa) within the values preferred for NSC proliferation and neurite branching and extension. Affinity-bound laminin PEG-4MAL hydrogels better preserve laminin bioactivity, compared to unmodified hydrogels and hydrogels containing physically entrapped laminin, being this effect dependent on NtA concentration. This was evidenced by the 10 M NtA-functionalized PEG-4MAL gels incorporating laminin that support enhanced human NSC proliferation and neurite extension, compared to the latter. Overall, this work highlights the potential of the proposed engineered matrices to be used as defined 3D platforms for the establishment of artificial NSC niches and as extracellular matrix-mimetic microenvironments to support human NSC transplantation. Confocal microscopy was conducted at the Bioimaging i3S Scientific Platform, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122), with the assistance of Maria Lázaro. This work was funded by projects NORTE-01-0145-FEDER-000008 and NORTE-01-0145-FEDER000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020; by Portuguese funds through FCT/MCTES in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER007274), and by Santa Casa da Misericordia de Lisboa through project COMBINE (Prémio Neurociências Melo e Castro 1068-2015). DB was supported by FCT PhD Programs (PD/BD/105953/2014) and Programa Operacional Potencial Humano (POCH), in the scope of the BiotechHealth Program (Doctoral Program on Cellular and Molecular Biotechnology Applied to Health Sciences), Programa FLAD Healthcare 2020 and the project PARES (Prémio Albino Aroso). Eduardo Conde-Sousa was supported by a post-doctoral grant of the project PPBI-POCI-01-0145-FEDER-022122, in the scope of FCT National Roadmap of Research Infrastructures. AJG acknowledges support from the National Institutes of Health (R01 AR062368).
Databáze: OpenAIRE
Externí odkaz: