Biofuncionalización de superficies a base de fibroína de seda para su potencial uso en aplicaciones cardiovasculares

En el presente trabajo, se biofuncionalizaron con heparina películas fabricadas a base de fibroína (SF) y polivinil alcohol (PVA) utilizando dos técnicas diferentes, la primera por acople de carbodiimida y la segunda por aprovechamiento de interacciones electrostáticas, buscando conseguir un comportamiento antitrombogénico en la superficie de las películas fabricas para su potencial uso como biomateriales para la fabricación de implantes cardiovasculares. Las muestras biofuncionalizadas fueron sometidas a una prueba de coagulación de sangre para verificar el éxito de dicha biofuncionalización. Los resultados mostraron que las muestras biofuncionalizadas por acople de carbodiimida, además de presentar una actividad antitrombogénica superior a las biofuncionalizadas por aprovechamiento de interacciones electrostáticas, presentaban valores de ángulos de contacto más cercanos a los de los materiales para la fabricación de implantes cardiovasculares, y que también, la biofuncionalización no afecta significativamente las propiedades mecánicas y superficiales de las películas fabricadas.

In the present work, fibroin, and polyvinyl alcohol (PVA) - based films were biofunctionalized using two different techniques, the first by carbodiimide coupling and second by exploiting electrostatic interactions, seeking to achieve antithrombogenic behavior on the surface of the manufactured films for their potential use as biomaterials for the manufacture of cardiovascular implants. The biofunctionalized samples were submitted to the blood clotting test to verify the success of said biofunctionalization. The results showed that the samples biofunctionalized by carbodiimide coupling, in addition to presenting a higher antithrombogenic activity than those biofunctionalized by taking advantage of electrostatic interactions, presented contact angles values closer to those of the materials for the manufacture of cardiovascular implants, and that also, the biofunctionalization does not significantly affect the mechanical and surface properties of the fabricated films

Novo sistema de liberação de drogas ­ filme de fibroína de seda carregado ou não com insulina para reparo de feridas em mucosa palatina: estudo in vitro e clínico randomizado / A new drug delivery system ­ silk fibroin film loaded or not with insulin on palatal mucosa wound healing: in vitro study and a randomized clinical trial

Several types of periodontal and peri-implant soft tissue defects require surgical treatment to reestablish function and aesthetics. However, local, and systemic factors can jeopardize tissue repair leading to unexpected outcomes and postoperative discomfort. In order to overcome this problem, new devices have been developed to improve surgical procedures outcomes and patient experience. The aim of the present study was to develop a new silk fibroin (SF)/chitosan (CH) film loaded with insulin as a drug delivery system to improve palatal donor area healing after free gingival graft harvesting for ridge preservation. For this, biomaterial development, characterization and in vitro assessment were performed to evaluate the new delivery system. In addition, 3- months outcomes from palatal wound healing following the use of the proposed delivery system were assessed through clinical, patient centered parameters, immunological, microbiological, and histological evaluations. Sixty-nine patients with indication of tooth extraction were enrolled into 3 groups: Control Group (C) (n=23): open wound on palatal mucosa followed by spontaneous healing; SF/CH Film (F) (n=23): open wound on palatal mucosa and silk fibroin film as dressing; Insulin-loaded SF/CH film (IF) (n=23): open wound on palatal mucosa and an insulin- loaded silk fibroin film as a delivery system. : It was verified some characteristics that are favorable to the oral environment, such as mechanical properties, swelling and permeability to water vapor. The biomaterial presented a standard of a controlled release system through diffusion with delivery stability in human saliva, along with an excellent biocompatibility with the absence of cytotoxicity and genotoxicity increasing cell viability in lineage cells (HaCat). F and IF promoted accelerated palatal wound closure on day 7 and 14 after surgery, as well as an early epithelialization, compared to the C group. Both films were capable to reduce pro-inflammatory cytokines (IL-6, TNF-α, IL-1ß) and modulate biomarkers correlated to tissue degradation/remodeling. Spontaneous healing microbiome reported higher genus/species with pathogenic role in the oral mucosa with reduction in health species following this profile until de end of the follow-up. A tendency of eubiosis was observed in F and IF groups throughout healing process. It seems that this new device has a promising application in oral cavity and positively influence wound healing. (AU)

Diversos tipos de defeitos mucogengivais requerem abordagem cirúrgica para o reestabelecimento funcional e estético. Porém, alterações locais e sistêmicas podem prejudicar o processo de reparo gerando resultados inesperados e desconforto ao paciente. Biomateriais vem sendo desenvolvidos para melhorar os resultados dos procedimentos cirúrgicos e a experiência clínica do paciente. O objetivo do presente estudo foi desenvolver um filme de fibroína de seda (FS) e quitosana (QT) carregado com insulina (INS), atuando como um sistema de liberação, para acelerar a cicatrização de feridas na área doadora palatina após procedimento de preservação de rebordo com uso de enxerto gengival livre. Para isso, foi executado o desenvolvimento, caracterização e avaliação in vitro do biomaterial. Ademais, o resultado de 3 meses do reparo das feridas palatinas foi verificado por meio de avaliações clínicas, imunológica, microbiológica, histológica, bem como parâmetros centrados no paciente. Sessenta e nove pacientes foram alocados aleatoriamente nos grupos Controle (C) (n=23): ferida aberta em palato seguido de cicatrização espontânea; Filme de FS/QT (F) (n=23): ferida aberta em palato associada ao filme na área doadora; Filme de FS/QT carregado com INS (IF) (n=23): ferida aberta em palato associada ao filme carregado com INS na área doadora. Verificou-se propriedades mecânicas, bem como de entumecimento e permeabilidade ao vapor de água, favoráveis ao meio bucal sem nenhuma alteração com a inclusão da INS. O dispositivo apresentou liberação controlada por meio de difusão com estabilidade em saliva humana. Excelente biocompatibilidade com ausência de cito e genotoxicidade foi observada em diversos tipos celulares aumentando a viabilidade celular em células de linhagem (HaCat). F e IF favoreceram um fechamento acelerado da ferida palatina aos 7 e 14 dias pós-injuria, assim como uma epitelização precoce destes comparado ao grupo C. F e IF reduziram citocinas pró-inflamatórias (IL6, TNF-α, IL-1ß) além de apresentarem função modulatória na quantificação de biomarcadores relacionados a degradação tecidual. O Grupo C apresentou gênero/espécies com potencial patogênico e redução de microrganismos relacionados a saúde mantendo este perfil aos 14 e 30 dias. Enquanto isso, uma tendência a eubiose foi observado em F e IF ao longo do processo de cicatrização. Deste modo, verifica-se a aplicação promissora do novo dispositivo na cavidade oral bem como capacidade de influenciar positivamente o reparo da mucosa oral. (AU)

Evaluación del crecimiento de fibroblastos humanos en andamios de fibroma de Bombyx mori L / Evaluation of the growth of human fibroblasts on fibroin scaffolds from Bombyx mori L

RESUMEN La fibroína de Bombyx mori L., es un biomaterial que se ha utilizado por sus características físico/químicas que la hacen útil para la curación de múltiples tejidos. En el contexto de la medicina regenerativa caracterizar a nivel físico y biológico nuevos soportes preparados a partir de fibroína de seda y evaluar su capacidad para la proliferación de fibroblastos humanos, brinda una gran oportunidad para encontrar nuevos biomateriales con aplicaciones favorables en la curación de heridas. Se utilizó fibroína regenerada al 17% para la fabricación de matrices. Estas fueron caracterizadas teniendo en cuenta: estabilidad en condiciones de cultivo, ultraestructura, porosidad, ángulo de contacto y propiedades mecánicas. El grosor promedio de las matrices de fibroína fue 30,1 µm, con una estabilidad superior a 4 semanas en condiciones de cultivo, porosidad del 51% y una capacidad de retención de líquidos del 95%, un ángulo de contacto de 44,5° y un módulo de elasticidad de aproximadamente 200 MPa. Finalmente se evaluó la capacidad del andamio para soportar el crecimiento de fibroblastos humanos. Identificando que los andamios permiten la multiplicación celular, mostrando bajos índices de citotoxicidad (<5%); las células establecieron interacciones fuertes con el andamio, mediante la producción de filopodios y la producción de matriz extracelular propia. Concluyendo esto, que es un andamio compatible de fibroblastos humanos en los procesos para el crecimiento y multiplicación celular en procesos de medicina regenerativa.

ABSTRACT The fibroin of Bombyx mori L, has been used for its physical / chemical characteristics as a biomaterial with applications in the healing of multiple tissues. In the context of regenerative medicine, characterizing at a physical and biological level new supports prepared from silk fibroin and evaluating their capacity for the proliferation of human fibroblasts, offers a very attractive opportunity to find new biomaterials with favorable applications in the healing of wounds. 17% regenerated fibroin was used for the manufacture of matrices. The matrices constructed were characterized in aspects such as: stability under growing conditions, ultra-structure, porosity, contact angle and mechanical properties. Finally, the ability of the scaffold to support the growth of human fibroblasts was evaluated. The average thickness of the fibroin matrices was 30.1 µm, with a stability greater than 4 weeks under culture conditions, porosity of 51% and a liquid retention capacity of 95%, a contact angle of 44.5 ° and a modulus of elasticity of ± 200 MPa. It was identified that the scaffolds allow cell multiplication, showing low cytotoxicity indexes (<5%); the cells established strong interactions with the scaffold, through the production of filopodia and the production of their own extracellular matrix. Constructed scaffolds allow the multiplication of human cells with low cytotoxicity and these establish a close relationship at the cellular level with the biomaterial. This can be used as a compatible scaffold for the growth and multiplication of human cells in a regenerative medicine process of epithelial tissue.

Evaluación de la fibroína de seda como biomaterial de soporte para el crecimiento de células mesenquimales estromales de pulpa dental / Evaluation of silk fibroin as a support biomaterial for the growth of stromal mesenchymal cells of dental pulp

La regeneración de tejidos usando células, factores de crecimiento y soportes constituyen una alternativa en la Medicina Regenerativa. La fibroína de seda es un excelente biosoporte, sus propiedades mecánicas únicas le permiten soportar procesos de adhesión y crecimiento celular. Objetivo. Evaluar la fibroína de la seda obtenida del gusano de seda Bombyx mori L como material de soporte para el crecimiento de células mesenquimales estromales de pulpa dental (CMPD). Métodos. La fibroína obtenida a partir de capullos de gusanos de seda Bombyx mori L criados en la Granja El Pílamo, propiedad de la Universidad Tecnológica de Pereira, se empleó para la fabricación de películas de fibroína íntegras y resistentes a condiciones de cultivo. Las CMPD fueron obtenidas a partir de un donante de diente premolar, la pieza dental se cortó con disco de diamante para la obtención de la pulpa que fue sometida a disgregación enzimática. Las células obtenidas se subcultivaron hasta el segundo pase, para posteriormente transferirse a cajas de cultivo que contenían películas de fibroína, se sometieron a condiciones inherentes al proceso de incubación siguiendo su crecimiento y viabilidad celular durante 27 días. Resultados. Al final del periodo de incubación, se observaron películas integras, estables y resistentes que permitieron el crecimiento celular. Conclusión. Se plantea el uso de fibroína como un biopolímero natural que brinda un soporte mecánico, un microambiente óptimo y un mimetismo de la estructura organizacional de los tejidos, postulándose como un potencial biomaterial para procesos de crecimiento celular en Medicina Regenerativa e Ingeniería de Tejidos.(AU)

Tissue regeneration using cells, growth factors and supports are an alternative in Regenerative Medicine. Silk fibroin is an excellent biosupport, its unique mechanical properties allow it to support processes of cell adhesion and growth. Objective: Evaluating the obtained silk´s fibroin from silkworm (Bombyx mori L) as a scaffold material for growth of dental pulp mesenchymal stromal cells (CMPD). Methods: The fibroin was obtained from silkworm (Bombyx mori L) cocoons reared at "The Pilamo" Farm, owned by the Universidad Tecnológica de Pereira. Procedures for obtaining full and resistant fibroin films to culture conditions were performed. The CMPD were obtained from a premolar tooth, the tooth was cut with a diamond blade to obtain the pulp which was subjected to enzymatic digestion. The cells obtained were subcultured until the second pass, and then, transferred to culture dishes containing fibroin films. This cells were cultured in standard conditions and tracking incubation with cell viability and growth for 27 days. Results: At the end of the incubation period, we realize that the fibroin films were intact and also this fibroin films allows cell growth.Conclusion: The use of fibroin as a natural biopolymer provides mechanical support, also an optimal microenvironment and mimic the organizational structure of tissues, so we postulated fibroin as a potential biomaterial for cell growth to be used in regenerative medicine and tissue engineering.(AU)

Uso de biomateriales a partir de la fibroína de la seda de gusano de seda (bombyx mori L. ) para procesos de medicina regenerativa basada en ingeniería de tejidos / Fibroin from silkworm (bombyx mori L ) as biomaterial used in regenrative medicine process based on tissue engineering

La regeneración de tejidos usando células, andamios y factores de crecimiento apropiados es un enfoque clave en las terapias de regeneración de tejido o de órganos. La fibroína de la seda, ha demostrado que puede ser utilizada eficazmente como un material de andamiaje en estos tratamientos. Las fibras de seda se obtienen de diversas fuentes animales, tales como arañas, gusanos de seda, escorpiones, ácaros y las moscas. La seda extraída a partir de capullos del gusano de seda (Bombyx mori L), se caracteriza por sus excelentes propiedades mecánicas, biocompatibilidad y biodegradabilidad que le permiten ser una fuente adecuada para el desarrollo de dispositivos biomédicos. La combinación única de elasticidad, resistencia y compatibilidad con células de mamíferos hace de la fibroína de la seda un material atractivo para la ingeniería de tejidos. Esta revisión aborda el procesamiento de fibroína de la seda en diferentes formas de biomateriales, sus aplicaciones, ventajas y limitaciones como biomaterial de andamiaje en la ingeniería ósea, vascular, de piel, cartílagos, ligamentos, tendones y de tejidos cardíaco, nervioso, ocular y vesical.

Tissue regeneration using cells, scaffolds and appropriate growth factors is a key approach in therapy for tissue or organs regeneration. The fibroin from silk has been shown to be effectively used as a scaffold material in these treatments. Silk fibers are obtained from various animal sources, such as spiders, silkworms, scorpions, mites and flies. The silk extracted from silkworm's (Bombyx mori L.) cocoons, is characterized by its excellent mechanical properties, biocompatibility and biodegradability this characteristics makes silk be a suitable source for the development of biomedical devices. The unique combination of elasticity, strength and compatibility with mammalian cells made of silk fibroin attractive for tissue engineering material. This review addresses the processing of silk fibroin in different forms of biomaterials, applications, advantages and limitations as a biomaterial scaffold in tissue engineering for bone, vascular tissues, skin, cartilage, ligaments, tendons, heart tissue, nervous , eye and bladder.