Animal Models of Osteoarthritis
Yuehuei H. An, Richard J. Friedman in Animal Models in Orthopaedic Research, 2020
Most investigators use histopathology as an end point in their studies. However, one major limitation is an adequate validated grading scheme. Many grading schemes have been developed and used to describe the changes that occur in the articular cartilage observed in osteoarthritis,38,,108,109 and some have even been semi-automated.110 The most widely adopted grading method for evaluating microscopically hyaline cartilage changes was first described by Mankin et al. in 1971.109 However, the Mankin scheme has been described by one group to be reproducible,111 and by others to be inadequate.38,112 Furthermore, the use of the histochemical stain Safranin O has also been questioned by some researchers for its lack of reproducibility.113 Thus the need for a reproducible, standardized and validated grading scheme still exists and may involve scoring of area and severity of the surface fibrillation. For small animals, if no other analyses are planned, it may be possible to do this by scanning electron microscopy.14,19
Methods for Evaluating Articular Cartilage Quality
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi in Articular Cartilage, 2017
Dyes commonly used for staining cartilage glycosaminoglycans take advantage of the negative charge of molecules, and these include Safranin O, toluidine blue, and Alcian blue (Figure 5.17). Safranin O is a red to orange stain, often used with Fast Green (a neutral dye) as a counterstain. Articular cartilage should first be protonated in an acidic solution before dye binding occurs. Toluidine blue is a metachromatic dye that changes from blue to pink-purple when the sulfated groups of a glycosaminoglycan molecule bring several dye molecules close together. This color change is only visible under aqueous conditions, and dried toluidine blue complexes will appear blue. Toluidine blue has been used in cartilage histology with aqueous mounts to distinguish sulfated glycosaminoglycans from other anionic species.
Bone, Muscle, and Tooth
Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard in Toxicologic Pathology, 2018
Similar to bone, a variety of specialized techniques are available to detect and characterize changes in joint structure. While not commonly used in routine toxicity studies, they are widely used in a variety of animal models of arthritis (Bendele 2011). In many of the models, the use of a frontal trimming plane is preferred to allow for examination of both the medial and lateral components of the joint. The most commonly used stains are H&E for a routine evaluation of overall joint histology and the cationic stain toluidine blue to evaluate the hyaline cartilage, including detection of changes in proteoglycan content. Safranin O and other special stains, as well as immunohistochemical techniques to evaluate various collagen types, can be employed.
Sabinene suppresses growth, biofilm formation, and adhesion of Streptococcus mutans by inhibiting cariogenic virulence factors
Published in Journal of Oral Microbiology, 2019
Bog-Im Park, Beom-Su Kim, Kang-Ju Kim, Yong-Ouk You
Biofilm formation was evaluated using a petri dish model and a resin teeth model according to a previously described method [13]. Briefly, various concentrations (0.05–0.4 mg/mL) of sabinene were added to BHI broth supplemented with 0.1% sucrose in petri dishes, 24-well plates (Nunc, Copenhagen, Denmark), or 24-well plates containing resin teeth (Endura, Shofu Inc., Japan). The setups were inoculated with 5 × 105 CFU/mL S. mutans culture and incubated for 24 h at 37°C. The supernatants were removed, and the dishes and plates were washed with distilled water. Biofilm formation was analyzed by staining with 0.1% safranin and subsequent photographic documentation. The stained safranin was dissolved using 30% acetic acid and the optical density was measured at 530 nm wavelength for quantity analysis.
Mitochondrial dysfunction: maternal protein restriction as a trigger of reactive species overproduction and brainstem energy failure in male offspring brainstem
Published in Nutritional Neuroscience, 2019
D. J. S. Ferreira, A. A. Pedroza, G. R. F. Braz, M. P. Fernandes, C. J. Lagranha
Several methods can be used to evaluate the membrane potential, herein we used the safranin-o uptake to estimate the electron difference between the mitochondrial spaces. In the assay, we evaluated the fluorescence decay, which has been correlated with the safranin attachment to internal mitochondria membrane in response to the increase in membrane potential. The animals that suffered protein restriction presented lower safranin-o uptake when compared to NP animals (NP= 35.61 ± 4.8 vs. LP= 19.32 ± 2.43% safranin uptake, P = 0.02) (Fig. 2a). Because ΔΨm can be regulated by UCP, which consumes proton motive force (pmf) without ATP formation, we evaluated the mRNA of UCP2. Our results demonstrated that UCP2 expression was expressed at higher levels in the brainstems of LP animals than in NP (NP = 0.063 ± 0.003 vs. LP = 0.204 ± 0.043, P = 0.03) (Fig. 2b).
The red-light emitting diode irradiation increases proliferation of human bone marrow mesenchymal stem cells preserving their immunophenotype
Published in International Journal of Radiation Biology, 2021
Rafał B. Lewandowski, Małgorzata Stępińska, Andrzej Gietka, Monika Dobrzyńska, Mariusz P. Łapiński, Elżbieta A. Trafny
For chondrogenesis, the hBM-MSC cells were cultured in MSCGM, until they reached 80–90% confluence in T75 culture flask (Nunc, Roskilide, Denmark). Cells were detached with trypsin/EDTA solution (Lonza, CC-3232) and Dulbecco-PBS (Gibco, Life Technologies) with 10% FBS (Standard FBS, EuroClone S.p.A., Lonza ECS0180L) were added to culture flask to inhibit trypsinization. The cell suspension was centrifuged at 150 × g for 5 min in room temperature to pellet the cells. The total number of cells in the pellet was 2.5 × 105, and they were subsequently cultured in 0.5 ml hMSC Differentiation BulletKit™, Chondrogenic (Lonza, PT-3003) supplemented with 10 ng/ml, freshly prepared growth factor-beta 3 (TGFβ3) for three weeks. The culture medium was refreshed every 3–4 days. The control cell pellets were cultured in MSCGM for the same period. After that period to confirm the in vitro chondrogenesis process, the pellets were embedded in paraffin and sectioned into 5–7 mm sections. The sections were deparaffinized with xylene (three times for 3 min) and ethanol in decreasing percentage concentrations as follows: 100% (two times for 3 min), 95% (two times for 3 min), and 70% (3 min). The sections were slide-mounted and subjected to the staining with 0.1% aqueous solution of safranin-O dye for 5 min. Safranin-O dye stains glycosaminoglycans on red color (Rosenberg 1971). Finally, the sections after dehydration process in 95% (two times for 5 min.), 100% ethanol (three times for 5 min), and xylene (three times for 5 min) were secured with a mounting medium (Dako Agilent Pathology Solutions, Santa Clara, CA).
Related Knowledge Centers
- Cartilage
- Cell Biology
- Counterstain
- Histology
- Mucin
- Cell Nucleus
- Mast Cell
- Staining
- Gram Stain
- Endospore Staining