Biotinta de matriz extracelular equina para bioengenharia tendínea
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Data
2021-10-27
Autores
Stievani, Fernanda de Castro
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O processo de reparo tendíneo resulta em um tecido de função limitada, sendo assim, as estratégias terapêuticas visando a bioengenharia tendínea com o uso de células tronco, biomateriais funcionais e bioimpressão, vêm se ampliando. Baseado na hipótese de que o hidrogel de matriz extracelular tendínea (MEC), associado a células tronco mesenquimais de origem adiposa (adCTM) é aplicável à bioimpressão e capaz de manter a viabilidade celular in vitro, o presente trabalho objetivou produzir e caracterizar esse hidrogel, avaliar a utilização como biotinta e a biocompatibilidade quando em cultivo com adCTM equinas e quando implantado in vivo. E avaliar a capacidade de induzir a tenogênese nas adCTM quando em cultivo prolongado com e sem força tênsil. Tendões flexores digitais superficiais equinos foram submetidos ao processo de descelularização, liofilização e moagem. O pó de MEC foi solubilizado em solução de ácido clorídrico e pepsina. As soluções de 1% e 2% de MEC foram avaliadas quanto à capacidade de injeção, formação de gel após neutralização, reologia e também as características bioquímicas e microestruturais após a formação de gel. A bioimpressão foi realizada com 1x106 cels/mL por duas bioimpressoras distintas sendo uma pneumática e a outra por extrusão direta. A viabilidade celular foi avaliada após bioimpressão pneumática: 2 horas (M0), 12 horas (M1) e sete dias (M7). O cultivo após bioimpressão por extrusão direta permaneceu por 21 dias, neste foram avaliadas a expressão gênicas dos marcadores de tenogênese e expressão e produção de matriz extracelular tendínea em duas formas distintas de cultivo, com as estruturas recebendo tensão (Gtensil) ou livres (Glivre). A estrutura bioimpressa foi implantada na região subfascial do músculo esplênio bilateral de um equino para avaliar biocompatibilidade. Na avaliação bioquímica observou-se predominância de proteínas colágenas e em sua maioria fibras maiores com poucas fibras intermediárias e pequenas. Na reologia os resultados foram compatíveis com bioimpressão, demonstrando diminuição da viscosidade com o aumento da força aplicada e a adição de transglutaminase antecipou a formação de gel da solução e aumentou a resistência ao hidrogel. A avaliação microestrutural demonstrou maior quantidade de fibrilas no hidrogel com 2% de MEC e rede homogênea quando transglutaminase foi adicionada. A bioimpressão foi satisfatória, com hidrogel de boa qualidade e resistente ao cultivo de 21 dias. A viabilidade celular diminuiu de M0 (87%) para M1 (76%) P = 0,0066 e assim se manteve durante os sete dias de cultivo após bioimpressão pneumática. Após bioimpressão por extrusão direta não houve diferença entre a viabilidade dos momentos avaliados. Houve aumento da expressão tenogênica das adCTM bem como dos marcadores de matriz extracelular, principalmente no Gtênsil. A avaliação in vivo demonstrou infiltrado inflamatório mononuclear de leve a moderado, boa integração com o tecido e poucos sinais clínicos de inflamação. Pode-se concluir que a biotinta de matriz extracelular tendínea equina, quando utilizada com adCTM, foi capaz de promover a expressão de genes de tenogênese e produção de proteínas da matriz extracelular, além de apresentar boa biocompatibilidade, tornando-se potencial estratégia para o tratamento de lesões tendíneas.
Due to a tissue with limited function results from tendon repair, therapeutic strategies targeting tendon engineering with stem cells and bioprinting of functional biomaterials are rising. Based on the hypothesis that extracellular matrix (ECM) hydrogel loaded with equine adipose tissue mesenchymal stem cells (adMSC) is suitable for bioprinting and preserve cell viability in vitro. This study aimed to produce and characterize this hydrogel and evaluate as bioink and its cytocompatibility when in culture with adMSC and when implanted in vivo. And to evaluate tenogenesis when in long-term free culture or with tensile strength with adCTM. Equine digital flexor tendons were decellularized, lyophilized and milled, The ECM powder was solubilized in hydrochloride acid and pepsin solution. One and two percent ECM solutions (1% e 2% ECM) were evaluated as injectability, jellified after neutralization, rheology and biochemical and microstructural properties after gel formation. The hydrogel was loaded with 1x106 cells/mL for bioprinting and two different prints were used, pneumatic and direct extrusion. The viability was evaluated two hours after printing (M0), 12 hours after printing (M1) and seven days after printing (M7) using pneumatic printing. Direct extrusion bioprinting lasted 21 days and tenogeneis and matrix genes expression and production were also evaluated using free constructs (Glivre) or with tensile strength (Gtensil). For in vivo biocompatibility evaluation the construct was implanted sub-fascial bilateral in splenic muscle of a horse. The biochemistry evaluation revealed majority collagen proteins with predominance of larger fibrils and with less thin and intermediate. The Rheology showed bioprinting suited characteristics with viscosity decreasing while applied force increased. The transglutaminase supplementation anticipated gel formation and increased hydrogel deformation resistance. 2% ECM showed higher fiber content and a homogeneous distribution after transglutaminase adding. The bioprinting was effective with good quality hydrogel and durable to 21 days culture. Cellular viability decreased from 87% in M0 to 76% in M1, P = 0,0066, and remained for seven days with pneumatic printing. And after direct extrusion viability remained unchanged during evaluated moments. Enhancement of tenogenesis expression and matrix production were significant higher in Gtensil. In vivo showed mild to moderate mononuclear infiltrate, good tissue integration and minor clinical inflammation sign. In conclusion this tendon ECM bioink loaded with adCTM induces tenogenesis, matrix protein production and presents good biocompatibility being suitable for future studies in tendon lesion treatments.
Due to a tissue with limited function results from tendon repair, therapeutic strategies targeting tendon engineering with stem cells and bioprinting of functional biomaterials are rising. Based on the hypothesis that extracellular matrix (ECM) hydrogel loaded with equine adipose tissue mesenchymal stem cells (adMSC) is suitable for bioprinting and preserve cell viability in vitro. This study aimed to produce and characterize this hydrogel and evaluate as bioink and its cytocompatibility when in culture with adMSC and when implanted in vivo. And to evaluate tenogenesis when in long-term free culture or with tensile strength with adCTM. Equine digital flexor tendons were decellularized, lyophilized and milled, The ECM powder was solubilized in hydrochloride acid and pepsin solution. One and two percent ECM solutions (1% e 2% ECM) were evaluated as injectability, jellified after neutralization, rheology and biochemical and microstructural properties after gel formation. The hydrogel was loaded with 1x106 cells/mL for bioprinting and two different prints were used, pneumatic and direct extrusion. The viability was evaluated two hours after printing (M0), 12 hours after printing (M1) and seven days after printing (M7) using pneumatic printing. Direct extrusion bioprinting lasted 21 days and tenogeneis and matrix genes expression and production were also evaluated using free constructs (Glivre) or with tensile strength (Gtensil). For in vivo biocompatibility evaluation the construct was implanted sub-fascial bilateral in splenic muscle of a horse. The biochemistry evaluation revealed majority collagen proteins with predominance of larger fibrils and with less thin and intermediate. The Rheology showed bioprinting suited characteristics with viscosity decreasing while applied force increased. The transglutaminase supplementation anticipated gel formation and increased hydrogel deformation resistance. 2% ECM showed higher fiber content and a homogeneous distribution after transglutaminase adding. The bioprinting was effective with good quality hydrogel and durable to 21 days culture. Cellular viability decreased from 87% in M0 to 76% in M1, P = 0,0066, and remained for seven days with pneumatic printing. And after direct extrusion viability remained unchanged during evaluated moments. Enhancement of tenogenesis expression and matrix production were significant higher in Gtensil. In vivo showed mild to moderate mononuclear infiltrate, good tissue integration and minor clinical inflammation sign. In conclusion this tendon ECM bioink loaded with adCTM induces tenogenesis, matrix protein production and presents good biocompatibility being suitable for future studies in tendon lesion treatments.
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Palavras-chave
Tenogênese, Bioimpressão, Células tronco, Biomateriais