Caracterização morfofuncional de veias safenas magnas descelularizadas como arcabouço para engenharia de tecidos
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Data
2021-09-01
Autores
Rezende, Karise Naves de
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Universidade Estadual Paulista (Unesp)
Resumo
Introdução: O tratamento para a doença arterial obstrutiva periférica em situação de isquemia crítica geralmente requer um procedimento cirúrgico de revascularização, que pode ser realizado por técnicas endovasculares ou cirurgia convencional com bypass. Em cirurgias convencionais, o substituto vascular deve apresentar as mesmas características do vaso sanguíneo que se deseja substituir. No entanto, nem sempre isso é possível. A engenharia de tecidos em vasos sanguíneos surge como alternativa de fornecimento de vasos sanguíneos bioengenheirados, baseados em uma medicina personalizada. Objetivo: Realizar a descelularização de veias safenas magnas humanas (VSM), para produção de um scaffold tridimensional, para futuro uso em engenharia de tecidos em vasos sanguíneos. Comparar VSM em condição de suficiência e insuficiência, e congeladas ou não, para identificação dos melhores processos de descelularização, mantendo suas características biomecânicas. Metodologia: Foi realizada a caracterização morfofuncional de VSM descelularizadas por dois agentes descelularizantes, dodecil sulfato de sódio (SDS) e deoxicolato de sódio (DS), em duas concentrações (2% e 3%) mantidas por 1 e 2 horas em agitação contínua e analisadas pela identificação de células íntegras (contagem nuclear) em lâminas coradas em hematoxilina e eosina (HE). Foi analisado também a integridade da matriz extracelular (ME) através dos mesmos cortes histológicos. Os scaffolds foram avaliados comparativamente quanto as suas propriedades biomecânicas, estabelecendo-se os grupos: B1 - VSM insuficiente (in natura); B2 -VSM suficiente (in natura); B3 - VSM insuficiente (congelada); B4 - VSM suficiente (congelada); B5 - VSM insuficiente (SDS – 2% 2h); B6 - VSM suficiente (SDS – 2% 2h); B7 - VSM insuficiente (DS – 3% 2h); B8 - VSM suficiente (DS – 3% 2h), pela análise de limite de proporcionalidade (LP), coeficiente de rigidez (CR) e carga máxima (CM). Resultados: Foram utilizados VSM de 22 participantes para todas as análises. Os protocolos de descelularização de SDS 2% e DS 3% ambos com 2 horas de VSM suficientes e insuficientes se mostraram efetivos, sem diferença estatística entre eles, tanto para VSM suficientes como insuficientes (p>0,05). A ME se manteve integra em todos os protocolos de descelularização realizados. Para os testes biomecânicos, não se observou diferença estatística entre os grupos para LP (p>0,051). Quanto ao CR, observou-se que há diferença estatística entre os grupos (p=0,003), sendo observado que o grupo padrão B2 foi superior aos grupos, B6 (p=0,007) e B8(p=0,046); e B4 é superior a B6 (p=0,016). Para CM, houve diferença estatística significativa entre os grupos (p<0,040), sendo queo grupo B8 foi superior aos grupos B1 (p=0,0276), B5 (p=0,0092), B6 (p=0,0024) e B7 (p=0,0111); e o B2 foi superior ao grupo B6 (p=0,0339). Conclusão: A descelularização de veias safenas humanas pode ser realizada de forma simples e eficiente, mantendo relativa integridade da ME, utilizando SDS a 2% e DS 3%, ambos com 2h de exposição. Veias safenas magnas suficientes e insuficientes, congeladas ou não, demonstram-se semelhantes durante a realização dos testes biomecânicos, porém, após o processo de descelularização VSM suficientes tiveram aumento de fragilidade após a descelularização. O mesmo não aconteceu para as VSM insuficientes que se mantiveram estáveis após a descelularização. Novos estudos devem ser realizados para se estabelecer o possível uso desses arcabouços na engenharia de tecidos de vasos sanguíneos.
Background: Treatment for peripheral arterial disease in critically ischemic situations usually requires a surgical revascularization procedure, which can be performed using endovascular techniques or conventional bypass surgery. In conventional surgeries, the vascular substitute must present the same characteristics as the blood vessel to be replaced. However, this is not always possible. Tissue engineering in blood vessels emerges as an alternative to supplying bioengineered blood vessels, based on personalized medicine. Objective: To perform the decellularization of human great saphenous veins (GSV) to produce a three-dimensional scaffold for future use in tissue engineering in blood vessels. Compare GSV in sufficiency and insufficiency conditions, and frozen or not, to identify the best decellularization processes, maintaining its biomechanical characteristics. Methodology: The morphofunctional characterization of GSV decellularized by two decellularizing agents, sodium dodecyl sulfate (SDS) and sodium deoxycholate (DS) was performed at two concentrations (2% and 3%) maintained for 1 and 2 hours under continuous agitation and analyzed by identifying intact cells (nuclear count) on slides stained with hematoxylin and eosin (HE). The integrity of the extracellular matrix (ME) was also analyzed through the same histological sections. The scaffolds were comparatively evaluated for their biomechanical properties, establishing the groups: B1 - Insufficient GSV (in natura); B2 - Sufficient GSV (in natura); B3 - Insufficient GSV (in frozen); B4 - Sufficient GSV (in frozen); B5 - Insufficient GSV (SDS – 2% 2h); B6 - Sufficient GSV (SDS – 2% 2h); B7 - Insufficient GSV (DS – 3% 2h); B8 - Sufficient GSV (DS – 3% 2h), by proportionality limit (PL), stiffness coefficient (SC) and maximum load (ML) analysis. Results: GSV from 22 participants were used for all analyses. The decellularization protocols of SDS 2% and DS 3% both with 2 hours of sufficient and insufficient GSV proved to be effective, with no statistical difference between them, both for sufficient and insufficient GSV (p>0.05). The extracellular membrane remained integrated into all decellularization protocols performed. For the biomechanical tests, there was no statistical difference between the groups for PL (p>0.051). As for the SC, it was observed that there is a statistical difference between the groups (p=0.003), being observed that the standard group B2 was superior to the groups, B6 (p=0.007) and B8 (p=0.046); and B4 is greater than B6 (p=0.016). For ML, there was a statistically significant difference between groups (p<0.040), with group B8 being superior to groups B1 (p=0.0276), B5 (p=0.0092), B6 (p=0.0024), and B7 (p=0.0111); and B2 was superior to group B6 (p=0.0339). Conclusion: The decellularization of human saphenous veins can be performed simply and efficiently, maintaining relative integrity of the ME, using 2% SDS and 3% DS, both with 2h of exposure. Sufficient and insufficient great saphenous veins, frozen or not, are similar during the performance of biomechanical tests, however, after the decellularization process, sufficient VSM had an increase in fragility after decellularization. The same did not happen for the insufficient VSM that remained stable after decellularization. Further studies should be carried out to establish the possible use of these frameworks in blood vessel tissue engineering.
Background: Treatment for peripheral arterial disease in critically ischemic situations usually requires a surgical revascularization procedure, which can be performed using endovascular techniques or conventional bypass surgery. In conventional surgeries, the vascular substitute must present the same characteristics as the blood vessel to be replaced. However, this is not always possible. Tissue engineering in blood vessels emerges as an alternative to supplying bioengineered blood vessels, based on personalized medicine. Objective: To perform the decellularization of human great saphenous veins (GSV) to produce a three-dimensional scaffold for future use in tissue engineering in blood vessels. Compare GSV in sufficiency and insufficiency conditions, and frozen or not, to identify the best decellularization processes, maintaining its biomechanical characteristics. Methodology: The morphofunctional characterization of GSV decellularized by two decellularizing agents, sodium dodecyl sulfate (SDS) and sodium deoxycholate (DS) was performed at two concentrations (2% and 3%) maintained for 1 and 2 hours under continuous agitation and analyzed by identifying intact cells (nuclear count) on slides stained with hematoxylin and eosin (HE). The integrity of the extracellular matrix (ME) was also analyzed through the same histological sections. The scaffolds were comparatively evaluated for their biomechanical properties, establishing the groups: B1 - Insufficient GSV (in natura); B2 - Sufficient GSV (in natura); B3 - Insufficient GSV (in frozen); B4 - Sufficient GSV (in frozen); B5 - Insufficient GSV (SDS – 2% 2h); B6 - Sufficient GSV (SDS – 2% 2h); B7 - Insufficient GSV (DS – 3% 2h); B8 - Sufficient GSV (DS – 3% 2h), by proportionality limit (PL), stiffness coefficient (SC) and maximum load (ML) analysis. Results: GSV from 22 participants were used for all analyses. The decellularization protocols of SDS 2% and DS 3% both with 2 hours of sufficient and insufficient GSV proved to be effective, with no statistical difference between them, both for sufficient and insufficient GSV (p>0.05). The extracellular membrane remained integrated into all decellularization protocols performed. For the biomechanical tests, there was no statistical difference between the groups for PL (p>0.051). As for the SC, it was observed that there is a statistical difference between the groups (p=0.003), being observed that the standard group B2 was superior to the groups, B6 (p=0.007) and B8 (p=0.046); and B4 is greater than B6 (p=0.016). For ML, there was a statistically significant difference between groups (p<0.040), with group B8 being superior to groups B1 (p=0.0276), B5 (p=0.0092), B6 (p=0.0024), and B7 (p=0.0111); and B2 was superior to group B6 (p=0.0339). Conclusion: The decellularization of human saphenous veins can be performed simply and efficiently, maintaining relative integrity of the ME, using 2% SDS and 3% DS, both with 2h of exposure. Sufficient and insufficient great saphenous veins, frozen or not, are similar during the performance of biomechanical tests, however, after the decellularization process, sufficient VSM had an increase in fragility after decellularization. The same did not happen for the insufficient VSM that remained stable after decellularization. Further studies should be carried out to establish the possible use of these frameworks in blood vessel tissue engineering.
Descrição
Palavras-chave
Veias, Vasos sanguíneos, Engenharia de tecidos, Descelularização, Veins, Blood vessels, Tissue engineering, Decellularization