ABSTRACT
Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5-7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson's trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.
Subject(s)
Tissue Scaffolds , Trachea , Dogs , Animals , Tissue Scaffolds/chemistry , Trachea/metabolism , Detergents/pharmacology , Detergents/analysis , Detergents/metabolism , Vacuum , Tissue Engineering/methods , Extracellular Matrix/metabolism , Proteoglycans/metabolism , Glycosaminoglycans/metabolism , DNA/metabolismABSTRACT
SUMMARY: Dystrophin disfunction results in sarcolemma destabilization, leading muscle cell damage by continuous degeneration cycles and limited regeneration. In muscle dystrophy, caused by dystrophin dysfunction, inflammation, necrosis and fibrosis are pathophysiological muscle function loss characteristics. As a genetic disease, this muscle dystrophy has no cure, however, advances in drug therapy using glucocorticoids can decrease the disease progression. Subsequently, alternative therapies were studied, such as ursolic acid (UA), that inhibits muscle atrophy and increases muscle mass and strength. Herein, we used 10 mg/kg daily supplementation in mdx mice for 4 weeks to evaluate serum creatine phosphokinase (CPK), muscle strength (Kondziela test), muscular organization (histology) and expression of fibrosis related genes (TGF-ß, TNF-α, mstn and ostn). UA supplementation increased muscle morphological organization, motor strength and decreased muscular TGF-ß expression. Altogether, the gene expression profile, histological organization and strength could suggest that UA treatment did not stop the fibrogenesis but decreased its progress.
RESUMEN: La disfunción de la distrofina resulta en la desestabilización del sarcolema, llevando al daño de las células musculares por ciclos continuos de degeneración y regeneración limitada. En la distrofia muscular, debido a la disfunción de la distrofina, la inflamación, la necrosis y la fibrosis, son características fisiopatológicas de la pérdida de la función muscular. Como enfermedad genetica no es possible remediar esta distrofia muscular, sin embargo, los avances en la terapia de medicamentos con glucocorticoides pueden disminuir la progresión de la enfermedad. Se estudiaron terapias alternativas, como el ácido ursólico (UA), que inhibe la atrofia muscular y aumenta la masa y la fuerza muscular. En este estudio, utilizamos una suplementación diaria de 10 mg / kg en ratones mdx durante 4 semanas para evaluar la creatina fosfoquinasa (CPK) sérica, la fuerza muscular (prueba de Kondziela), la organización muscular (histología) y la expresión de genes relacionados con la fibrosis (TGF-ß, TNF- α, mstn y ostn). La suplementación con AU aumentó la organización morfológica muscular, la fuerza motora y la disminución de la expresión muscular de TGF-ß. El perfil de expresión génica, la organización histológica y la fuerza simultáneamente podrían sugerir que el tratamiento con AU no detuvo la fibrogénesis sino que disminuyó su progreso.
Subject(s)
Animals , Male , Mice , Oleanolic Acid/analogs & derivatives , Muscular Dystrophies , Oleanolic Acid/administration & dosage , Fibrosis , Transforming Growth Factor beta , Mice, Inbred mdx , Creatine Kinase/blood , Muscle StrengthABSTRACT
One of the dogmas of mammalian reproduction states is that primordial germ cells in females are restricted to the intrauterine phase, and that only a small portion of oocytes is available for ovulation during the adult life. Among the rare exceptions to this rule is the plains viscacha. This specie polyovulates up to 800 oocytes per cycle, from which 10 to 12 are implanted, but only 1-2 conceptuses survive. To better understand the key mechanisms of this pattern of embryonic to uterine interactions, we analyzed 19 female genital systems by means of gross morphology, histology, stereology and immunohistochemistry. Data showed that a specialized, highly convoluted structure of the ovarian cortex developed during the intrauterine phase as a prerequisite for the massive super-ovulation, likely associated with the inhibition of apoptosis and continued proliferation of germ cells, as well as maintenance of several corpora lutea during the adult life. In addition, specializations of uterine vasculature and musculature were demonstrated. Altogether, these key morphological characteristics evolved in order to contribute as compensatory or controlling mechanism for polyovulation and polyimplantation that led these species into becoming an unique enigma in reproductive biology, and a potential animal model to provide explanations regarding to developmental specializations.
