Mineralization parameter analysis of subcutaneously implanted control and galanin (GAL)–coated collagen scaffolds. For this study, collagen sponges were coated with phosphate buffered saline (control), GAL only, GAL antagonist galantide only (GT), or both (GAL + GT) and subcutaneously implanted for 4 wk. (A–D) Hematoxylin and eosin revealed trabecular structures in GAL- and GAL + GT–coated scaffolds. (E–H) Newly formed connective tissues as revealed by Masson’s trichrome staining. (F) Note the dense connective tissue blocks and red coloration indicative of osteoid. (I–L) Alizarin red staining and (M–P) von Kossa staining illustrate key mineralized tissue components (J, L) calcium and (N) phosphorus indicative of mineralized tissue formation in GAL- and GAL + GT–coated scaffolds. (Q) Quantitative real-time polymerase chain reaction analysis of bone-related protein expression in controls, GAL, GT, and GAL + GT treatment groups at the mRNA level. (R, S) Western blotting comparison of bone-related proteins subjected to GAL, GT, and GAL + GT treatment and corresponding densitometry analysis. #P < 0.05 downregulation. *P < 0.01. ***P < 0.001. Con, control.