Postembedding Decalcification of Mineralized Tissue Sections Preserves the Integrity of Implanted Biomaterials and Minimizes Number of Experimental Animals

Figure 6

Silver stain visualizes the completely decalcified biomaterial and the osteocytes. Undecalcified femur with SrCPC in PMMA T9100 sections shows the clear portions of minerals and organic compounds. (a) Ca⁺ distribution accounts for CPC and mineralized bone matrix. (b) Strict presence of Sr⁺ within the SrCPC area and its vicinity. (c) Organic compound at the bone matrix areas and not the CPC area. (d) Overlay of minerals (Ca⁺: green; Sr⁺: blue; and organic compound: red) serves as a map of portions distribution before decalcification. (e-f) Collagenous portion before (e) decalcification in the same areas as after (f) decalcification. (g) The Ag⁺ staining corresponds to the areas of organic compounds. (h) Overlay of Ca⁺ (red) before decalcification and Ag⁺ after decalcification (green) hints that the Ag⁺ is visible after mineral removal. (i-j) Light microscopic imaging of decalcified bone section shows the comparative view of the implant and the surrounding tissue. Furthermore, osteocytes and cell canaliculi are evident in the bone matrix. Image (i) was acquired by 5x magnification objective; then individual tiles were stitched together by Leica application suite (LASX) software. (j) was acquired by 40x magnification objective whereas inset was acquired by 100x magnification objective.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)