Preparação de scaffolds por combinação das técnicas de fotopolimerização e electrospinning para engenharia de tecidos

Abstract

Stents vasculares são dispositivos biomédicos de valor incontestável no tratamento e regeneração de artérias ou veias que ficaram obstruídas ou enfraquecidas. Ao longo dos últimos anos, muito se tem vindo a desenvolver procurando um equilíbrio entre a estabilidade mecânica, biocompatibilidade e o recurso à ação farmacológica de modo a que se combatam as tromboses/embolias vasculares e a ocorrência de complicações como a reestenose após implantes. Atualmente existe no mercado uma elevada variedade de stents vasculares de diversos materiais, desde metálicos a polímeros biodegradáveis com incorporação, ou não, de um fármaco. Os stents metálicos foram os primeiros a serem comercializados, contudo têm problemas inerentes de inflamação ou mesmo incompatibilidade com o organismo dada a durabilidade de aplicação. Por outro lado, os stents vasculares biodegradáveis estão cada vez mais em ascensão, apesar das propriedades mecânicas e sua durabilidade serem ainda um desafio. Polímeros biodegradáveis, sintéticos ou naturais, com grupos terminais fotoreticuláveis têm-se demostrado promissores e úteis em aplicações in situ. Dois dos polímeros mais usados na área biomédica, concretamente na preparação de hidrogéis e scaffolds, são a gelatina e a policaprolactona, respetivamente. A sua mistura pode ser explorada na construção de stents versáteis, com melhores propriedades biológicas para tecidos vasculares ao mesmo tempo que exibem boas propriedades mecânicas. Com esta dissertação pretendeu-se produzir scaffolds por combinação de estratégias de fotoreticulação e electrospinning usando duas abordagens de síntese diferentes, com vista à utilização dos materiais finais como stents vasculares. Em ambas as abordagens de síntese usou-se a gelatina (polímero natural) e a PCL (polímero sintético). A gelatina foi inicialmente funcionalizada com anidrido metacrílico para a formação do hidrogel Gel- MA. Na primeira abordagem, as fibras de PCL foram produzidas por electrospinning sendo de seguida revestidas por hidrogéis de diferentes composições (tendo por base a Gel-MA). Na segunda abordagem, fibras foram preparadas a partir de soluções distintas de PCL e Gel-MA, que foram misturadas e induzidas no electrospinning. Desta forma obteve-se um compósito fibroso. Em ambos os processos, sujeitaram-se os materiais a irradiação UV para garantir a adequada reticulação da gelatina, usando o Irgacure® 2959 como fotoiniciador. Para os scaffolds produzidos foram avaliadas propriedades químicas, físicas, térmicas e biológicas. A análise de ATR-FTIR permitiu observar as ligações características de cada polímero em ambos os scaffolds. Na técnica SEM verificou-se que a disposição das fibras nos scaffolds era aleatória e no caso dos scaffolds revestidos com hidrogel era notório o efeito desse revestimento. A análise TGA mostrou que os scaffolds eram termicamente estáveis. Quando incubados em uma solução tampão fosfato (PBS) durante 28 dias, os scaffolds revestidos com Gel-MA 10% foram os que apresentaram maior diferença de perda de hidrogel. Nos ensaios de hemólise, por contacto direto, só os scaffolds compósitos mostraram valores bastante satisfatórios, revelando-se não hemolíticos. Relativamente aos scaffolds revestidos estes só exibiram esse carácter após uma lavagem prévia do material com PBS. Todavia, os scaffolds apresentaram um carácter trombogénico, o que não é favorável para a aplicação pretendida. Os valores de viabilidade celular, após contacto dos materiais com células endoteliais circulantes foram bastante satisfatórios para os scaffolds compósitos, porém, os scaffolds revestidos de hidrogel com macrómeros apresentaram baixa viabilidade celular. Recomenda-se, neste caso, novos estudos de biocompatibilidade mas também um esforço de síntese para perceber o que pode ser modificado nas formulações. No estudo preliminar de libertação de fármaco para os scaffolds revestidos, obtiveram-se perfis de libertação interessantes tendo em conta a aplicação. Face aos resultados alcançados, pode-se concluir que os scaffolds das duas abordagens apresentam características promissoras para utilização como stents vasculares, podendo ser importantes ferramentas em Engenharia de Tecidos.

Vascular stents are biomedical devices of an unquestionable value in the treatment and regeneration of injured and weakened arteries or veins. Over the years, the development of these materials with promising performances requires a thorough understanding and a stringent balance between mechanical stability, biocompatibility and pharmacological activity, fighting against thrombosis, vascular embolism episodes or the possibility of several complications after implant, such as restenosis. Nowadays in the market there are several options of vascular stents, produced from different metals or biodegradable polymers, with or without entrapped drugs. The metallic stents were the first commercially available, nevertheless with inherent issues like inflammations during application or even incompatibility in the body after long usage. On the other hand, biodegradable vascular stents are increasingly in the field, but their mechanical properties and durability remains a challenge. Biodegradable polymers, either synthetic or natural, with photocurable end-groups have proved to be promising and useful for in situ applications. Two of the most commonly used polymers in the biomedical field, particularly in hydrogels and scaffolds development, are gelatine and polycaprolactone (PCL), respectively. Their mixtures may be used and tuned to produce flexible and robust stents with improved biological properties for vascular tissues while exhibiting good mechanical properties. This work aimed at exploring the production of several scaffolds by combination of electrospinning and photocrosslinking strategies. Two different approaches in material synthesis were tested to obtain new materials to be used and characterized as vascular stents. In both synthesis approaches, gelatine (natural polymer) and PCL (synthetic polymer) were used. The gelatine was firstly functionalized with carbon-carbon double bonds by methacrylic anhydride, leading to a Gel-MA hydrogel. In the first approach, PCL fibers were produced by electrospinning and then coated with several hydrogels of different compositions based on Gel-MA. In the second approach, fibers were prepared from diverse PCL and Gel-MA solutions and finally mixed and induced in electrospinning apparatus. In this case, fibrous composites were obtained. In both strategies, the final materials were subjected to UV irradiation, using Irgacure® 2959 as Vascular stents are biomedical devices of an unquestionable value in the treatment and regeneration of injured and weakened arteries or veins. Over the years, the development of these materials with promising performances requires a thorough understanding and a stringent balance between mechanical stability, biocompatibility and pharmacological activity, fighting against thrombosis, vascular embolism episodes or the possibility of several complications after implant, such as restenosis. Nowadays in the market there are several options of vascular stents, produced from different metals or biodegradable polymers, with or without entrapped drugs. The metallic stents were the first commercially available, nevertheless with inherent issues like inflammations during application or even incompatibility in the body after long usage. On the other hand, biodegradable vascular stents are increasingly in the field, but their mechanical properties and durability remains a challenge. Biodegradable polymers, either synthetic or natural, with photocurable end-groups have proved to be promising and useful for in situ applications. Two of the most commonly used polymers in the biomedical field, particularly in hydrogels and scaffolds development, are gelatine and polycaprolactone (PCL), respectively. Their mixtures may be used and tuned to produce flexible and robust stents with improved biological properties for vascular tissues while exhibiting good mechanical properties. This work aimed at exploring the production of several scaffolds by combination of electrospinning and photocrosslinking strategies. Two different approaches in material synthesis were tested to obtain new materials to be used and characterized as vascular stents. In both synthesis approaches, gelatine (natural polymer) and PCL (synthetic polymer) were used. The gelatine was firstly functionalized with carbon-carbon double bonds by methacrylic anhydride, leading to a Gel-MA hydrogel. In the first approach, PCL fibers were produced by electrospinning and then coated with several hydrogels of different compositions based on Gel-MA. In the second approach, fibers were prepared from diverse PCL and Gel-MA solutions and finally mixed and induced in electrospinning apparatus. In this case, fibrous composites were obtained. In both strategies, the final materials were subjected to UV irradiation, using Irgacure® 2959 as Vascular stents are biomedical devices of an unquestionable value in the treatment and regeneration of injured and weakened arteries or veins. Over the years, the development of these materials with promising performances requires a thorough understanding and a stringent balance between mechanical stability, biocompatibility and pharmacological activity, fighting against thrombosis, vascular embolism episodes or the possibility of several complications after implant, such as restenosis. Nowadays in the market there are several options of vascular stents, produced from different metals or biodegradable polymers, with or without entrapped drugs. The metallic stents were the first commercially available, nevertheless with inherent issues like inflammations during application or even incompatibility in the body after long usage. On the other hand, biodegradable vascular stents are increasingly in the field, but their mechanical properties and durability remains a challenge. Biodegradable polymers, either synthetic or natural, with photocurable end-groups have proved to be promising and useful for in situ applications. Two of the most commonly used polymers in the biomedical field, particularly in hydrogels and scaffolds development, are gelatine and polycaprolactone (PCL), respectively. Their mixtures may be used and tuned to produce flexible and robust stents with improved biological properties for vascular tissues while exhibiting good mechanical properties. This work aimed at exploring the production of several scaffolds by combination of electrospinning and photocrosslinking strategies. Two different approaches in material synthesis were tested to obtain new materials to be used and characterized as vascular stents. In both synthesis approaches, gelatine (natural polymer) and PCL (synthetic polymer) were used. The gelatine was firstly functionalized with carbon-carbon double bonds by methacrylic anhydride, leading to a Gel-MA hydrogel. In the first approach, PCL fibers were produced by electrospinning and then coated with several hydrogels of different compositions based on Gel-MA. In the second approach, fibers were prepared from diverse PCL and Gel-MA solutions and finally mixed and induced in electrospinning apparatus. In this case, fibrous composites were obtained. In both strategies, the final materials were subjected to UV irradiation, using Irgacure® 2959 as the radical photoinitiator. In order to evaluate materials feasibility for the intended application, chemical, physical, thermal and biological properties were assessed. The ATR-FTIR analysis allowed a follow up of each polymer bonds in both scaffolds. The SEM technique confirmed a random arrangement of the fibers in the scaffolds, and in the case of the coated materials, the effect of hydrogels coating was noticeably detected. TGA analysis showed that all materials were thermally stable. When the samples were incubated in phosphate buffer solution for 28 days, the scaffolds coated with Gel-MA 10% presented the highest mass loss. In the haemolysis assays, by direct contact, just composite scaffolds exhibited good values of haemolysis percentage, revealing a non-haemolytic nature. The coated scaffolds only presented a non-haemolytic character after washing their by-products with phosphate buffer solution. However, all scaffolds presented a thrombogenic nature, which is not favourable for the purposed application. The results of cell viability studies, after direct contact of the materials with circulating endothelial cells, were quite acceptable for the composite samples. Nonetheless, the coated scaffolds with hydrogels showed poor cell viability. In this case, further biocompatibility studies are recommended in the future but also a research effort must be accomplished to obtain improved formulations. In the preliminary assessment of drug release profiles, the overall performance of the coated scaffolds showed potential in the field, according to therapeutic needs. Altogether, characterization results showed that the produced materials presented a set of properties suitable for Biomedical and Tissue Engineering applications, particularly as vascular stents.