RESUMO
SUMMARY: The normal sequential development of the hip joint (HJ) was considered for the evaluation of the morphological and ultrastructural aspects of the joint cartilage of the proximal femoral head epiphysis in human fetuses between 16 to 31 weeks of intra uterine life (IUL). Twenty human fetuses were fixed in 10 % formalin solution. Fetuses were divided into 4 groups (n=5): Group 1 (G1): 16-19 weeks IUL; Group 2 (G2): 20-23 weeks IUL; Group 3 (G3): 24-27 weeks IUL and Group 4 (G4): 28-31 weeks IUL. The right moieties of the HJ were subjected to light microscopy to determine the chondrocyte area, volume, and density and the extracellular matrix (ECM) density. The collagen component in ECM was qualitatively evaluated using Safranin-O and picrosirius techniques under polarized light. The left portions were analyzed using scanning electron microscopy (SEM). The advance of age revealed a gradual increase in chondrocyte area and volume and in ECM density, and a decrease in chondrocyte density. The apparent prevalence of type II collagen fibers in G1 and type III collagen fibers in G4, as well as a balance between type I and III collagen fibers in G2 and G3 suggest a process of cartilaginous evolution and repair. The pantographic organization of the collagen fiber meshes from the depth to the cartilage surface of the femoral head suggests that the arcade collagen network architecture starts at the fetal stage, regardless of the compressive forces applied. The morphological data may contribute not only to a better understanding of the maturation and cartilage organization in this area but also to serve as a theoretical basis for aspects related to diseases and joint malformations.
RESUMEN: El desarrollo secuencial normal de la articulación de la cadera (AC) se consideró para la evaluación de los aspectos morfológicos y ultraestructurales del cartílago articular de la epífisis proximal y de la cabeza femoral en fetos humanos entre 16 y 31 semanas de vida intrauterina (SVIU). Veinte fetos humanos fueron fijados en solución de formalina al 10 %. Los fetos se dividieron en 4 grupos (n = 5): Grupo 1 (G1), 1619 semanas de IUL; Grupo 2 (G2), 20-23 semanas SVIU; Grupo 3 (G3), 24-27 semanas SVIU y Grupo 4 (G4), 28-31 semanas SVIU. Las muestras derechas de la AC se sometieron a microscopía óptica para determinar el área, el volumen y la densidad de los condrocitos y la densidad de la matriz extracelular (MEC). El componente de colágeno en la MEC se evaluó cualitativamente utilizando técnicas de safranina-O y picrosirius bajo luz polarizada. Las muestras de la AC izquierda se analizaron utilizando microscopía electrónica de barrido (MEB). El avance de la edad reveló un aumento gradual en el área y el volumen de los condrocitos y en la densidad de la MEB, y una disminución en la densidad de los condrocitos. La aparente prevalencia de las fibras de colágeno tipo II en G1 y tipo III en G4, así como el equilibrio entre las fibras de colágeno tipo I y III en G2 y G3 sugieren un proceso de evolución y reparación cartilaginosa. La organización pantográfica de las mallas de fibra de colágeno desde la profundidad a la superficie del cartílago de la cabeza femoral sugiere que la arquitectura de la red de colágeno comienza en la etapa fetal, independientemente de las fuerzas compresivas aplicadas. Los datos morfológicos pueden contribuir no solo a una mejor comprensión de la organización de la maduración y el cartílago en esta área, sino también servir de base teórica para los aspectos relacionados con enfermedades y malformaciones articulares.
Assuntos
Humanos , Feto , Articulação do Quadril/ultraestrutura , Microscopia Eletrônica de Varredura , Colágeno/ultraestrutura , Condrócitos/ultraestrutura , Matriz Extracelular , Articulação do Quadril/embriologiaRESUMO
Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.