Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity.

Histochemical detection of tartrate-resistant acid phosphatase (TRAP) activity is a fundamental technique for visualizing osteoclastic bone resorption and assessing osteoclast activity status in tissues. This approach has mostly employed colorimetric detection, which has limited quantification of activity in situ and co-labelling with other skeletal markers. Here we report simple colorimetric and fluorescent TRAP assays in zebrafish and medaka, two important model organisms for investigating the pathogenesis of bone disorders. We show fluorescent TRAP staining, utilising the ELF97 substrate, is a rapid, robust and stable system to visualise and quantify osteoclast activity in zebrafish, and is compatible with other fluorescence stains, transgenic lines and antibody approaches. Using this approach, we show that TRAP activity is predominantly found around the base of the zebrafish pharyngeal teeth, where osteoclast activity state appears to be heterogeneous.

The effect of naringin on early growth and development of the spheno-occipital synchondrosis as measured by the expression of PTHrP and Sox9--an in vitro model.

The aim of this study was to assess the effect of the flavonoid naringin on the growth of the spheno-occipital synchondrosis by quantifying the levels of expression of Sox9 and PTHrP in an in vitro mouse model. Fifty 1-day-old BALB/c mice were randomly assigned to experimental or control groups, and each group equally divided into five time frames (6, 24, 48, 72 and 168 hours). The mice were sacrificed with phenobarbitone sodium, and the spheno-occipital synchondroses dissected and cultured in control or experimental medium, with the experimental medium supplemented with 0.1 µm naringin. Sections of the specimens underwent immunohistochemical staining for Sox9 and PTHrP, and the amount of expression was quantified using true-colour RGB (red-green-blue) computer-assisted image-analysing system with digital imaging. Data analysis showed there was a significant increase of expression of Sox9 at 6 and 24 hours (P < 0.001) between experimental and control groups, however, there was no significant difference between the levels of expression of PTHrP between experimental and control groups at any of the time frames. There was a very weak correlation found in this study between the expression of PTHrP and Sox9. In conclusion, naringin enhances the growth of the spheno-occipital synchondrosis through over expression of Sox9. This is a successful in vitro model to study factors regulating the growth of the spheno-occipital synchondrosis.

The effect of quercetin on expression of SOX9 and subsequent release of type II collagen in spheno-occipital synchondroses of organ-cultured mice.

OBJECTIVE: To identify the expressions of SOX9 and type II collagen in spheno-occipital synchondrosis in response to quercetin, using a mouse in vitro model. MATERIALS AND METHODS: A total of 50 one-day-old male BALB/c mice were randomly assigned to the control and experimental groups. Each group was subdivided into five different time points, which were 6, 24, 48, 72, and 168 hours, and each subgroup contained 5 mice (n  =  5). In the experimental group, the spheno-occipital synchondrosis was immersed in the BGJb medium + quercetin dihydrate 1 µM. In the control group, the spheno-occipital synchondrosis was immersed in the BGJb medium. Tissue sections were subjected to immunohistochemical staining for SOX9 and type II collagen expressions. RESULTS: Quantitative analysis revealed there was a statistically significant increase of 32.31% (P < .001) in the expression of SOX9 between experimental groups and control groups at 24 hours. Furthermore, there was a statistically significant increase of 22.99% (P < .001) in the expression of type II collagen between experimental groups and control groups at 72 hours. CONCLUSION: The expressions of SOX9 and type II collagen in the spheno-occipital synchondrosis can be increased by quercetin.