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Published in Chad A. Mirkin, Spherical Nucleic Acids, 2020
Aleksandar F. Radovic-Moreno, Natalia Chernyak, Christopher C. Mader, Subbarao Nallagatla, Richard S. Kang, Liangliang Hao, David A. Walker, Tiffany L. Halo, Timothy J. Merkel, Clayton H. Rische, Sagar Anantatmula, Merideth Burkhart, Chad A. Mirkin, Sergei M. Gryaznov
The activity of IR-SNAs was tested in the STAM mouse model (Stelic Institute & Co., Inc.) of NASH. In brief, pathogen-free, 15-d pregnant C57BL/6 mice were obtained from Japan SLC. NASH was induced by s.c. injection of streptozotocin (Sigma-Aldrich) after birth followed by feeding with a high fat diet (CLEA Japan) ad libitum after 4 wk of age. At 6 wk old, mice were randomized into groups of eight and treated every other day from 6 to 9 wk old (3 wk). Compounds or control (PBS) were administered (40 µM oligonucleotide equivalent concentration) in 100 µL by i.p. administration. The impact of treatment administration on NASH and liver fibrosis was determined by collecting livers at 9 wk of age and assessing NASH disease and fibrosis histologically in blinded fashion. In brief, H&E-stained liver sections were scored for steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2), after which a composite nonalcoholic fatty liver disease score was assigned (scale: 0–8). Sections were also stained with Sirius Red for fibrosis and scored as percent positive area according to established protocols.
Methods for Evaluating Articular Cartilage Quality
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
Histologically, general collagen staining can take advantage of the protein’s fibril organization, while collagen typing occurs through immunohistochemical staining. For instance, sirius red, used in a saturated solution of picric acid (also known as picrosirius red), stains collagen fibers pink to red as the dye binds to collagen helices (Figure 5.14). Due to the aspect ratio of the dye molecule, trapping accentuates alignment, especially under polarized light (Junqueira et al. 1979), where binding degree can be assessed as lighter-to-heavier staining transitions from yellow to green. Other stains, such as aniline blue, can be used to stain collagen, and, while polarized light microscopy for collagen alignment can also be performed without staining, the effect for observing alignment is not as dramatic as when sirius red is used. SEM and TEM can also be used to visualize collagen fiber thickness and orientation, although these, like histology, are destructive methods.
Boosting osteogenic potential and bone regeneration by co-cultured cell derived extracellular matrix incorporated porous electrospun scaffold
Published in Journal of Biomaterials Science, Polymer Edition, 2021
Andrew Padalhin, Reiza Ventura, Boram Kim, Tamanna Sultana, Chan Mi Park, Byong-Taek Lee
Biomolecules such as collagen and sulfated glycosaminoglycans were quantified from decellularized membranes and compared to cell seeded samples with either single cell type or co-culture system. Collagen was measured using Sirius Red Total Detection kit (Chondrex) which quantifies total collagen content. Samples were solubilized at room temperature in a solution containing 3.0% v/v acetic acid and 0.01% pepsin overnight. Sirius red (500 µl) was added to the extract solution and was incubated for 20 min. The resulting solution was purified by centrifugation at 10,000 rpm followed by washing. Washed pellets were dissolved in 250 µl of extraction buffer and transferred into a 96 well plate. Absorbance of the solution was read at 540 nm using a microplate reader (F50 Techan Austria). Amount of collagen from each sample was computed using a standard curve.
Effects of latex membrane on guided regeneration of long bones
Published in Journal of Biomaterials Science, Polymer Edition, 2019
Bruna Leonel Carlos, Jéssica Suzuki Yamanaka, Gabriela Rezende Yanagihara, Ana Paula Macedo, Plauto Christopher Aranha Watanabe, João Paulo Mardegan Issa, Rondinelli Donizetti Herculano, Antônio Carlos Shimano
Tibiae from each group were fixed in 4% formaldehyde for 24 h, decalcified in 10% ethylenediamine tetraacetic acid (EDTA), dehydrated in ascending series of alcohol, and diaphanized in xylene. Samples were then embedded in paraffin with the medial aspect of the tibia exposed to the cut. For microscopic analysis, 48 5 µm thick semi-serial sections were obtained from the entire defect region. Two sections were placed on each glass slide using polylysine. Of the 24 slides obtained for each sample, six were stained by Picro sirius red for quantification of type I and III collagen fibers (slides 3, 7, 11, 15, 19, and 23), twelve by the Masson trichrome technique for quantification of bone formation (paired slides), and one for immunohistochemistry (slides 13). The images were assessed at 50× magnification for analyses of bone formation and quantification of collagen and 200× for immunohistochemistry analyses by optical microscope Axio Imager Z2® (Zeiss, Göttingen, Germany).
Mimicking osteochondral interface using pre-differentiated BMSCs/fibrous mesh complexes to promote tissue regeneration
Published in Journal of Biomaterials Science, Polymer Edition, 2022
Lei Fu, Wenwen Zhao, Liwen Zhang, Chenyuan Gao, Xin Zhang, Xiaoping Yang, Qing Cai
Finally, the collagen orientation in the newly formed cartilage tissue at different depths were evaluated by Sirius red staining with the aid of the directionality plugin in ImageJ. As shown in Figure 6A, ROI 1 and ROI 2 represented the superficial and deep cartilage, respectively, the collagen orientations in these two regions were plotted separately in Figure 6B and C for both the groups at the two time points post-surgery. In native articular cartilage, the collagen fibers in the superficial zone were aligned horizontally, while those collagen fibers in the deeper zone grew perpendicularly into the subchondral bone. After 4 W repair with the implanted gradient construct, as the fibrous mesh in the defect being not completely degraded, the Sirius red staining color was only observed on the defect surface. The collagen in ROI 1 tended to be distributed horizontally, while that in ROI 2 was more evenly distributed. The collagen orientation distribution in the two regions had a significant difference, and this difference was more remarkably at 12 W post-implantation. The organization of collagen in the superficial and deep layers of the regenerated cartilage became even hierarchical, that the former (ROI 1) demonstrated more horizontally distributed collagen, while the latter (ROI 2) showed more vertically distributed collagen. In the blank group, whether at 4 W and 12 W, no obvious orientation in collagen arrangement was able to be detected for both the regions, and the uniformly distributed collagen in the area was in accordance with the fact that the defect was mainly filled with fibrous tissue instead of neo-cartilage.