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. 2020 Apr 10;2020:2045969. doi: 10.1155/2020/2045969

Figure 5.

Figure 5

Human recombinant Asporin (rhAsp) treatment decreases matrix mineralization in osteogenic (OS) media-induced avian adult AVICs in vitro. (a, b) Alizarin Red S staining data is showing (a, nI–nIII) bright-field microscopic images (nI (×10) and nII and nIII (×16)) of OS-induced AVICs (arrowheads) and (nI–nIII) bright-field microscopic images (nI (×10) and nII and nIII (×16)) of rhAsp-treated OS-induced AVICs (arrowheads). (c) Quantitative assay of Alizarin Red S shows that absorbance of rhAsp-treated OS-induced AVICs decreased (0.064) compared to only OS-induced AVICs (0.089) (p ≤ 0.05, n = 3). (d, e) Here, immunostaining data show that a number of Asporin-positive (Asp+) cells increased in rhAsp-treated OS-induced AVICs compared to OS-induced AVICs (arrowheads). (f) Quantitative data show 1.75% increase in Asp+ cells in rhAsp-treated OS-induced AVICs compared to OS-induced AVICs only. (g) (I and VI): qRT-PCR data show that Asporin is upregulated by 0.9 fold (I), osteogenic markers Osteopontin and Runx2 are downregulated by 0.29 fold (II) and 0.79 fold (III), and Wnt signaling-specific markers, β-catenin and Wnt3a, are downregulated by 0.55 fold (IV) and 0.71 fold (V), whereas Dkk1 is upregulated by 0.3 fold (VI), respectively, in rhAsp-treated OS-induced AVICs compared to OS-induced AVICs only. Statistical significance was determined by Student's t-test, where ∗ denotes p ≤ 0.05, ∗∗ denotes p ≤ 0.01, and ∗∗∗ denotes p ≤ 0.001 and n = 3.