RESUMO
A new tissue sample embedding and processing method is presented that provides downstream compatibility with numerous different histological, molecular biology, and analytical techniques. The methodology is based on the low temperature embedding of fresh frozen specimens into a hydrogel matrix composed of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) and sectioning using a cryomicrotome. The hydrogel was expected not to interfere with standard tissue characterization methods, histologically or analytically. We assessed the compatibility of this protocol with various mass spectrometric imaging methods including matrix-assisted laser desorption ionization (MALDI), desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS). We also demonstrated the suitability of the universal protocol for extraction based molecular biology techniques such as rt-PCR. The integration of multiple analytical modalities through this universal sample preparation protocol offers the ability to study tissues at a systems biology level and directly linking results to tissue morphology and cellular phenotype.
Assuntos
Hidrogéis/química , Derivados da Hipromelose/química , Povidona/química , Manejo de Espécimes/métodos , Inclusão do Tecido/métodos , Animais , Masculino , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
With their low toxicity, high mechanical strength and chemical stability, boron nitride nanotubes (BNNTs) are good candidates to enhance the properties of polymers, composites and scaffolds. Chitosan-based scaffolds are exhaustively investigated in tissue engineering because of their biocompatibility and antimicrobial activity. However, their spontaneous degradation prevents their use in a range of tissue engineering applications. In this study, hydroxylated BNNTs (BNNT-OH) were included into a chitosan scaffold and tested for their mechanical strength, swelling behavior and biodegradability. The results show that inclusion of BNNTs-OH into the chitosan scaffold increases the mechanical strength and pore size at values optimal for high cellular proliferation and adhesion. The chitosan/BNNT-OH scaffold was also found to be non-toxic to Human Dermal Fibroblast (HDF) cells due to its slow degradation rate. HDF cell proliferation and adhesion were increased as compared to the chitosan-only scaffold as observed by scanning electron microscopy (SEM) and fluorescent microscopy images.