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Abstract(s)
Substâncias poliméricas extracelulares de cianobactérias (EPS) são heteropolissacáridos complexos e anfifílicos de alto peso molecular, que possuem a vantagem de serem facilmente produzidos e isolados. A produção de polissacáridos bacterianos pode ocorrer sob condições controladas, o que permite o desenvolvimento de biopolímeros com características reprodutíveis. Cyanothece sp. é uma cianobactéria unicelular fixadora de N2 que cresce em ambientes marinhos (isolado da zona costeira de Zanzibar). Esta cianobactéria é uma das mais eficientes produtoras de EPS, libertando cerca de 74% dos carboidratos para o meio sobre a forma de RPS (“released polysaccharides”). A adequabilidade dos EPS da Cyanothece na área biomédica foi estudada e reportada, nomeadamente em entrega de fármacos e na sua aplicação em revestimentos. O nosso projeto representa os primeiros estudos efetuados com o objetivo de produzir microambientes tridimensionais baseados nos EPS de cianobactérias, adequados para cultura e entrega celular in vitro tendo em conta que, até ao presente momento, nenhum estudo do uso de EPS de cianobactérias para estratégias de engenharia de tecidos foi reportado. Neste trabalho, o desenvolvimento de hidrogéis poliméricos foto-polimerizáveis e funcionalizados com anidrido metacrílico, foi realizado com sucesso. As propriedades mecânicas e químicas do material obtido foram investigadas, bem como a adequabilidade do material como veículo de transporte e entrega de células. O polímero foi funcionalizado exibindo 48% de conjugação com grupos metacrilato. Após polimerização, foi possível obter hidrogéis com um módulo elástico até 10 kPa, estáveis em água e meio de cultura celular. Fibroblastos Neonatais Dérmicos humanos (hDNFs) e Células Estaminais Mesenquimais humanas (hMSCs) foram incorporadas nos hidrogéis e as suas viabilidades celulares, atividades metabólicas e deposição de matriz extracelular foram estudadas. Os resultados obtidos demonstraram que, em ambos os casos, as células foram capazes de aderir, proliferar e estabelecer contatos entre as mesmas durante todo o período de cultura, sem haver necessidade de realizar funcionalizações adicionais com o objetivo de incorporar componentes promotores de adesão celular, o que levou à confirmação da cito compatibilidade deste material. Além disso, com estimulação osteogénica adequada, as hMSCs foram capazes de iniciar diferenciação osteogénica, que foi mais tarde confirmada por ensaios colorimétricos (von Kossa e Fosfatase Alcalina). Apesar de estudos adicionais serem necessários para validar e suportar as presentes descobertas, os resultados obtidos sugerem que os RPS são um biomaterial promissor para o uso em estratégias de engenharia de tecidos e como veículo de transporte e entrega de células.
Cyanobacterial extracellular polymeric substances (EPS), mainly composed of heteropolysaccharides with high molecular weight, can be easily produced and isolated. The production of bacterial polysaccharides can occur under controlled conditions, allowing the development of biopolymers with reproducible characteristics. Cyanothece sp. is a unicellular N2-fixing cyanobacterium that grows in marine environments (isolated from the coastal waters of Zanzibar). This cyanobacterial strain is one of the most efficient EPS producers, releasing approximately 74 ± 4% of its carbohydrates to the external medium as RPS (released polysaccharides). The suitability of Cyanothece’s EPS in the biomedical field has been studied and reported in drug delivery and coating applications. Our work represents the first studies aiming to produce a 3D microenvironment based on cyanobacterial EPS suitable for in vitro cell culture and cell delivery, since no previous reports of the use of cyanobacterial EPS in tissue engineering strategies exist on the literature, to the best of our knowledge. In this work, we successfully developed a photocrosslinkable polymeric hydrogel based on Cyanothece’s RPS by functionalization with methacrylic anhydride. The structural and mechanical properties of the obtained material were investigated, as well as, its suitability as a cell delivery vehicle. The polymer was functionalized, exhibiting 48% of methacrylated moieties grafted to the polymer backbone. After polymerization, it was possible to obtain hydrogels with an elastic modulus reaching 10 kPa, which were stable both in water and cell culture medium. The RPS-based hydrogels were embedded with human Dermal Neonatal Fibroblasts (hDNFs) and human Mesenchymal Stem Cells (hMSCs) and their cellular viability, metabolic activity and extracellular matrix deposition were studied. The results showed that, in both cases, the cells were able to adhere, proliferate and establish cell-to cell contact throughout the culture, without the need of further functionalization with cell-binding motifs, confirming the cytocompatibility of this novel material. Moreover, with proper osteogenic stimulation, the hMSCs were able to undergo osteogenic differentiation, which was further confirmed by colorimetric stains (von Kossa and Alkaline Phosphatase). Although further studies are needed to validate and support the present findings, overall, the obtained results suggest that RPS are a promising biomaterial to be used in cell delivery and Tissue Engineering strategies.
Cyanobacterial extracellular polymeric substances (EPS), mainly composed of heteropolysaccharides with high molecular weight, can be easily produced and isolated. The production of bacterial polysaccharides can occur under controlled conditions, allowing the development of biopolymers with reproducible characteristics. Cyanothece sp. is a unicellular N2-fixing cyanobacterium that grows in marine environments (isolated from the coastal waters of Zanzibar). This cyanobacterial strain is one of the most efficient EPS producers, releasing approximately 74 ± 4% of its carbohydrates to the external medium as RPS (released polysaccharides). The suitability of Cyanothece’s EPS in the biomedical field has been studied and reported in drug delivery and coating applications. Our work represents the first studies aiming to produce a 3D microenvironment based on cyanobacterial EPS suitable for in vitro cell culture and cell delivery, since no previous reports of the use of cyanobacterial EPS in tissue engineering strategies exist on the literature, to the best of our knowledge. In this work, we successfully developed a photocrosslinkable polymeric hydrogel based on Cyanothece’s RPS by functionalization with methacrylic anhydride. The structural and mechanical properties of the obtained material were investigated, as well as, its suitability as a cell delivery vehicle. The polymer was functionalized, exhibiting 48% of methacrylated moieties grafted to the polymer backbone. After polymerization, it was possible to obtain hydrogels with an elastic modulus reaching 10 kPa, which were stable both in water and cell culture medium. The RPS-based hydrogels were embedded with human Dermal Neonatal Fibroblasts (hDNFs) and human Mesenchymal Stem Cells (hMSCs) and their cellular viability, metabolic activity and extracellular matrix deposition were studied. The results showed that, in both cases, the cells were able to adhere, proliferate and establish cell-to cell contact throughout the culture, without the need of further functionalization with cell-binding motifs, confirming the cytocompatibility of this novel material. Moreover, with proper osteogenic stimulation, the hMSCs were able to undergo osteogenic differentiation, which was further confirmed by colorimetric stains (von Kossa and Alkaline Phosphatase). Although further studies are needed to validate and support the present findings, overall, the obtained results suggest that RPS are a promising biomaterial to be used in cell delivery and Tissue Engineering strategies.
Description
Keywords
Substâncias poliméricas extracelulares (eps) Cyanothece Polissacáridos livres (rps) Engenharia de tecidos Hidrogéis Extracellular polymeric substances (eps) Released polysaccharides (rps) Tissue engineering Hydrogels