ABSTRACT
Tissue engineering (TE) has become an alternative for auricular reconstruction based on the combination of cells, molecular signals and biomaterials. Scaffolds are biomaterials that provide structural support for cell attachment and subsequent tissue development. Ideally, a scaffold should have characteristics such as biocompatibility and bioactivity to adequate support cell functions. Our purpose was to evaluate biocompatibility of microtic auricular chondrocytes seeded onto a chitosan-polyvinyl alcohol-epichlorohydrin (CS-PVA-ECH) hydrogel to propose this material as a scaffold for tissue engineering application. After being cultured onto CS-PVA-ECH hydrogels, auricular chondrocytes viability was up to 81%. SEM analysis showed cell attachment and extracellular matrix formation that was confirmed by IF detection of type II collagen and elastin, the main constituents of elastic cartilage. Expression of elastic cartilage molecular markers during in vitro expansion and during culture onto hydrogels allowed confirming auricular chondrocyte phenotype. In vivo assay of tissue formation revealed generation of neotissues with similar physical characteristics and protein composition to those found in elastic cartilage. According to our results, biocompatibility of the CS-PVA-ECH hydrogel makes it a suitable scaffold for tissue engineering application aimed to elastic cartilage regeneration.
La ingeniería de tejidos (TE) es una alternativa para la reconstrucción auricular basada en la combinación de células, señales moleculares y biomateriales. Los andamios fabricados con biomateriales brindan un soporte estructural que favorece la adhesión cellular y el desarrollo del tejido. Un andamio debe poseer características como biocompatibilidad y bioactividad para soportar adecuadamente funciones celulares. Nuestro objetivo fue evaluar la biocompatibilidad de condrocitos auriculares de microtia cultivados sobre un hidrogel a base de quitosano-alcohol polivinílico-epiclorhidrina (CS-PVA-ECH) y proponerlo como andamio con aplicaciones en ingeniería de tejidos. La viabilidad de los condrocitos auriculares es superior al 81% después de ser cultivados sobre el hidrogel. El análisis por SEM reveló la unión celular y formación de matriz extracellular sobre el hidrogel; confirmada mediante detección por IF de colágena tipo II y elastina. La expresión de marcadores moleculares durante la expansión in vitro y el cultivo sobre los hidrogeles confirmaron el fenotipo condral. El ensayo de formación de tejido in vivo demostró la generación de neotejidos con características físicas y composición similar al cartílago elástico. Nuestros resultados indican que la biocompatibilidad del hidrogel de CS-PVA-ECH lo hace un andamio adecuado para aplicaciones en ingeniería de tejidos enfocadas a regeneración de cartílago elástico.
Subject(s)
Humans , Chondrocytes/cytology , Tissue Engineering/methods , Chitosan/chemistry , Ear Cartilage/cytology , Polyvinyls/chemistry , Biocompatible Materials , Immunohistochemistry , Cell Culture Techniques , Chondrocytes/metabolism , Hydrogels , Epichlorohydrin/chemistryABSTRACT
The purpose of this study was to evaluate the influence of different types of PLGA scaffolds on the formation of human auricular and septal cartilages. The scaffolds were formed in tubular shape from 110,000 g/mol PLGA (poly lactic glycolic acid) and 220,000 g/mol one. Elastic cartilage was taken from the ear of a patient aged under 20 years old and hyaline cartilage from the nasal septum. The chondrocytes cells were then isolated by Klausburn method. After second passages, the chondrocytes were seeded on the PLGA scaffolds followed by in vitro culture for one week. The cells-PLGA scaffold complex was implanted at the back of nude mouses for 8 weeks. The tissue engineered cartilages were separated from nude mouse and examined histologically after staining with the Hematoxylin Eosin and Verhoeff. The formation of extracellular matrix and the porosity of the scaffolds were examined by scanning electron microscopy. The pores were well formed and uniformly distributed in both 110,000 g/mol and 220,000 g/mol PLGA scaffolds. The extracellular matrix was formed better in 110,000 g/mol PLGA compared to 220,000 g/mol one. And hyaline cartilage was proliferated better in vitro culture than elastic cartilage. After 8 weeks in vivo culture, cartilage was well formed with 110,000 g/mol PLGA, however lumen was collapsed. In contrast with 220,000 g/mol PLGA scaffold, neocartilage was formed in minimal amount while the architecture of scaffold was well preserved. Elastic cartilage seems to be better than hyaline one in terms of neocartilage formation. From the analysis after Verhoeff staining the cartilages, the neocartilage from elastic cartilage was proved to be elastic cartilage. In summary, there was no significant difference between elastic cartilage and hyaline cartilage in their morphologies, proliferation rates and the degree of cartilage formation since they were tissue engineered, however marked difference was found in neocartilage formation and preservation of scaffold architecture between 110,000 g/mol PLGA scaffold and 220,000 one. From the present findings, it is concluded that the influence of scaffold materials is significantly higher than that of different types of cells on the formation of new tissues.