Phenotype and synthesis activity of joint chondrocytes extracted from newborn rats with prenatal ethanol exposure.

Thirteen female Wistar rats were divided into two groups: one treated with ethanol and the other of untreated. Four newborns from each mother were selected and weighed, measured, and evaluated for physical characteristics. From these neonates, chondrocytes were extracted from the articular cartilages of the femur and tibia, and cultivated in a chondrogenic medium at 37oC and 5% CO2. At 7, 14, and 21 days of cultivation, alkaline phosphatase activity tests, MTT conversion to formazan, and percentage area covered by cells per field were performed. At 21 days, the percentage of PAS+ areas in 3D cultures was performed, as well as the evaluation of gene transcript expression for aggrecan, SOX-9, collagen type II, collagen X, Runx-2, and VEGF by real-time RT-PCR. The means were compared by Student's t-test. The weight of the ethanol group neonates was significantly lower than that of the controls. Chondrocyte cultures from the ethanol group showed significantly higher AP activity, MTT conversion, and cell percentage. There was higher expression of collagen type II and lower expression of SOX-9 in the ethanol group. There was no difference in the percentage of PAS+ areas in pellets and in expression of aggrecan, collagen X, Runx-2, or VEGF between groups. In conclusion, prenatal exposure to ethanol alters the phenotype and activity of offspring chondrocytes, which may be mechanisms by which endochondral bone formation is compromised by maternal ethanol consumption.

Effect of ethanol consumption during pregnancy and lactation on bone histomorphometry and in vitro osteogenic differentiation of bone marrow mesenchymal stem cells in maternal rats.

This study aimed to evaluate the effect of maternal ethanol consumption during gestation and lactation on bone mass and osteogenic differentiation of mesenchymal stem cells of the bone marrow (BMMSCs) in rats. Thirteen adult Wistar rats were used. The rats were mated, and after confirmation of gestation, (day 0) they were distributed in two groups: the control group and the ethanol-treated group. From the ninth day of gestation, the rats of the ethanol and control groups were administered 40% alcoholic solution (4 g ethanol/kg) and distilled water, respectively, daily via gavage until the thirtieth day of lactation. The BMMSCs were extracted from the right femurs and tibiae and cultured using an osteogenic medium for 7, 14, and 21 days. The conversion of MTT to formazan crystals, alkaline phosphatase activity, and percentages of cells per field were analyzed. The number of mineralized nodules per field was examined, and quantification of the gene transcripts for osteopontin, osteocalcin, and BMP-2 was evaluated on day 21 by real-time RT-PCR. Morphometric evaluations of the percentage of trabecular bone and cortical thickness in the left femur and tibia were performed. The means were compared by the Student's t-test, and the differences were considered significant if p < 0.05. The BMMSCs of the rats that consumed ethanol during gestation and lactation, when subjected to osteogenic differentiation in vitro, demonstrated higher conversion of MTT to formazan, higher alkaline phosphatase activity, a higher percentage of cells per field, higher expression of BMP-2, and higher synthesis of mineralized nodules when compared to those of control rat cells. However, there was no significant difference in the percentage of trabecular bone or cortical thickness between both groups. Hence, the consumption of ethanol during pregnancy and lactation did not alter the trabecular and cortical bone tissues of the femur and tibia compared with that of pregnant and lactating control rats that did not consume alcohol, despite BMMSCs showing higher osteogenic differentiation under in vitro conditions.

Estrutura e celularidade de meniscos frescos de coelhos (Oryctolagus cuniculus) preservados em glicerina / Structure and cellularity of the fresh menisci (Oryctolagus cuniculus) of rabbits and the menisci preserved in glycerin

No presente estudo foi avaliada a arquitetura tecidual, a população celular, assim como a integridade e a distribuição dos tipos celulares em meniscos frescos de coelhos e preservados em glicerina 98 por cento. Foram analisados meniscos mediais de coelhos recém abatidos, que foram distribuídos em três grupos: o grupo MF (n=7), composto por meniscos frescos, correspondeu ao grupo controle; o grupo MG (n=7), composto por meniscos preservados em glicerina 98 por cento, por 30 dias, e o grupo MR (n=7), por meniscos preservados em glicerina 98 por cento e reidratados em NaCl 0,9 por cento, por 12 horas. Em todos os meniscos foram identificados e quantificados os diferentes tipos celulares: fibroblastos/fibrócitos e condrócitos. A população celular foi estatisticamente semelhante nos três grupos de meniscos, sendo que os meniscos preservados, grupos MG e MR, apresentaram menor intensidade de coloração e retração das fibras colágenas, diminuição de volume e maior intensidade de coloração dos núcleos (condensação da cromatina), em relação aos meniscos frescos (MF), caracterizando o fenômeno de lise celular. A matriz fibrocartilaginosa dos meniscos preservados revelou- se bem preservada mantendo a arquitetura tecidual dos meniscos. Conclui-se que a glicerina 98 por cento é uma opção de meio de preservação para meniscos objetivando aloenxerto, com matriz colágena desvitalizada.

In the present study was evaluated the tissue architecture, the percentage of cellular population, as well as viability and distribution of cells in fresh menisci of rabbits and preserved in 98 percent glycerin. Were analyzed medial menisci of rabbits freshly slaughtered, which were distributed into three groups: the MF group (n=7), composed of fresh menisci, corresponded to the control group; the MG group (n=7), composed by menisci preserved in 98 percent glycerin, for 30 days, and the MR group (n=7) by menisci preserved in 98 percent glycerin and rehydrated in NaCl 0.9 percent for 12 hours. In all menisci were identified and quantified the different cell types: fibroblasts/fibrocytes and condrocytes. The cell population percentage was statistically similar in all groups. All menisci preserved in the MG and MR groups showed a lower intensity of color and shrinkage of collagen fibers, reduced volume and higher intensity of staining of nucleus (chromatin condensation), as compared to fresh menisci (MF), featuring the phenomenon of cell lysis. The cartilaginous matrix of preserved menisci proved to be well preserved because the tissue architecture was maintained. It was concluded that 98 percent glycerin is an optional preservation mean for meniscal allografts with a devitalized collagenous matrix.

Avaliação das fibras colágenas de meniscos frescos e preservados em glicerina: estudo experimental em coelhos (Oryctolagus cuniculus) / Evaluation of the collagen fibers of the fresh meniscus and the meniscuses preserved in glycerine: experimental study in rabbits (Oryctolagus cuniculus)

No presente estudo foi avaliado o efeito da glicerina 98 por cento sobre as fibras colágenas, arquitetura tecidual e o tamanho de meniscos mediais de coelhos da raça Nova Zelândia. Os meniscos foram separados em três grupos: (1) Grupo MF com meniscos frescos (grupo controle), (2) Grupo MG com meniscos preservados em glicerina 98 por cento por 30 dias, e (3) Grupo MR com meniscos preservados em glicerina 98 por cento por 30 dias e reidratados em NaCl 0,9 por cento, por 12 horas. Os cortes histológicos foram corados com sirius red para identificação dos tipos de colágenos e observados em microscópio de luz polarizada, avaliando-se a concentração total de colágeno Tipo I e III e a disposição das fibras. Os meniscos frescos apresentaram significativamente maior concentração de fibras colágenas Tipo I e menor concentração de fibras colágenas Tipo III que os meniscos preservados (MG e MR); isto ocorreu devido à perda de água e conseqüente redução do tamanho dos meniscos e retração das fibras colágenas dos meniscos dos Grupos MG e MR; isto pode ter feito com que as fibras Tipo I, mais espessas e em maior quantidade, se tornassem mais evidentes do que as fibras colágenas Tipo III, que são mais delgadas e frágeis (fibrilas). Nos três grupos estudados, as fibras colágenas apresentaram-se de forma circunferencial, interpostas por fibras orientadas radialmente. Entretanto, nos grupos tratados (MG e MR) foi observado, em pequenas áreas, leve desorganização das fibras colágenas, o que correspondeu a 42,8 por cento e 14,3 por cento dos meniscos, respectivamente. O grupo de meniscos em glicerina apresentou redução significativa (p<0,05) no tamanho em relação ao Grupo MF. No Grupo MR, 85,7 por cento dos meniscos retornaram ao seu tamanho inicial após a reidratação. A glicerina 98 por cento é eficaz na preservação de meniscos, mantendo o tamanho, a arquitetura estrutural, a integridade e percentagem do colágeno dos meniscos preservados semelhante à de...

In the present study was evaluated the effect of 98 percent glycerin on the collagen fibers, tissue architecture and size of medial meniscuses of New Zealand rabbits. The animals were separated into three groups: (1) Group MF of fresh meniscus, (2) Group MG of meniscus preserved in glycerin for 30 days, and (3) Group MR of meniscus preserved in glycerin for 30 days and rehydrated in NaCl 0.9 percent for 12 hours. Histological sections were stained with sirius red for identification of collagen types, which were examined with a polarized light microscope. The total collagen concentration and the fiber arrangement were evaluated. Group MF presented higher Type I collagen concentration and lower Type III collagen concentration when compared with Group MG and MR. This fact is due to the water loss and consequent reduction in size and subsequent retraction of the collagen fibers of the meniscus from these groups, caused by dehydration. This may have occurred because those Type I fibers, thicker and in larger quantities, become more evident than Type III collagen fibers, which are more slender and fragile (fibrils). In the three groups studied, the collagen fibers presented themselves in a circumference form, interposed by radially oriented fibers. All of the meniscuses from Group MF presented fibers arranged obliquely, while in the treated groups, a slight disorganization of collagen fibers could be observed in some areas, what corresponds to 42.8 percent and 14.3 percent of the meniscuses, respectively. The MG group presented a significant decrease (p<0.05) in size if compared with the MF group. In the MR group, 85.7 percent of the meniscuses went back to the original size after rehydratation. The 98 percent glycerin is effective in preserving the meniscus, following rehydratation, maintaining size, structural architecture, integrity and percentage of collagen of the meniscus preserved similar to the fresh one.