Microsc. Microanal. 21, 1482–1490, 2015 doi:10.1017/S1431927615015366 © MICROSCOPY SOCIETY OF AMERICA 2015 Time-Dependent Resolution of Collagen Deposition During Skin Repair in Rats: A Correlative Morphological and Biochemical Study Rômulo D. Novaes, 1, * Marli C. Cupertino, 2 Mariaurea M. Sarandy, 2 André Souza, 3 Evelise A. Soares, 4 and Reggiani V. Gonçalves 3 1 Department of Structural Biology, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 700, Campus Universitário, Centro, Alfenas, MG 37130-000, Brazil 2 Department of General Biology, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil 3 Department of Animal Biology, Federal University of Viçosa, Viçosa, MG 36570-000, Brazil 4 Department of Anatomy, Federal University of Alfenas, Alfenas, MG 37130-000, Brazil Abstract: Skin samples were used to compare microscopy methods used to quantify collagen with potential applicability to resolve time-dependent collagen deposition during skin wound healing in rats. Skin wounds by secondary intention were made in rats and tissue fragments were collected every 7 days for 21 days. Collagen content determined by biochemical analysis was compared with collagen measured by point counting (PC) on histological skin sections stained by Gomori’s trichrome method (Trichrome/PC), Sirius red under polarized light (PL) microscopy (Sirius red/PL-PC), and computational color segmentation (CS) applied to sections stained with Sirius red (Sirius red/PL-CS). All microscopy methods investigated resolved the time-dependent dynamics of collagen deposition in scar tissue during skin wound healing in rats. Collagen content measured by Sirius red/PL-PC and Sirius red/PL-CS was significantly lower when compared with Trichrome/PC. The Trichrome/PC method provided overestimated values of collagen compared with biochemical analysis. In the early stages of wound healing, which shows high production of noncollagenous molecules, Sirius red/PL-CS and Sirius red/PL-PC methods were more suitable for quantification of collagen fibers. Trichrome staining did not allow clear separation between collagenous and noncollagenous elements in skin samples, introducing a marked bias in collagen quantification. Key words: dermis, extracellular matrix, morphology, pathology, wound healing I NTRODUCTION Skin wound healing is a complex chemically mediated pro- cess that restores the structural and/or functional integrity of damaged tissue (Seifert & Maden, 2014). This process is divided into three continuous and complementary phases represented by inflammation, proliferation, and maturation (Gonçalves et al., 2010; Seifert & Maden, 2014). In the early stages, in response to regulatory molecules (i.e., cytokines and fibrinopeptides), inflammatory cells migrate to the injured tissue to remove clotted blood and cellular and extracellular matrix (EM) debris (Gonçalves et al., 2010; Seifert & Maden, 2014). In this phase, viable cells at the wound edges and leucocytes secrete modulatory molecules (i.e., cytokines and growth factors) that drive the subsequent phase (Pierce et al., 1991; Werner & Grose, 2003). Stimulated by these molecules, the proliferative phase ensues with intense proliferation of fibroblasts, endothelial cells, and keratinocytes, accompanied by an increased deposition of EM molecules, especially collagen (Werner & Grose, 2003; Seifert & Maden, 2014). In the final phase (maturation), the mitotic rate slows and excessive cellularity in scar tissue is reduced by apoptotic mechanisms. Maturation processes then determine EM reorganization, especially by replacing thin (type III) with thick (type I) collagen fibers, reinforcing the mechanical strength of the newly formed tissue (Hodde, 2002; Gonçalves et al., 2010). The synthesis of collagen fibers is an important factor directly related to the quality of the wound healing process (Hodde, 2002; Novaes et al., 2014; Seifert & Maden, 2014). These fibers provide a tridimensional scaffold by which resident cells and leukocytes migrate and spread over the entire wound area in repair (Hodde, 2002; Seifert & Maden, 2014). Furthermore, collagen fibers are involved in mechanotransduction processes, offering stimuli to activate signaling pathways that directly regulates cell phenotype, survival, division, migration, and synthesis of biomolecules (Grinnell, 2003; Wong et al., 2012). Owing to its pivotal structural and functional role, several analytical methods have been developed to quantify collagen content in skin samples (Tasanen et al., 1996; El-Domyati et al., 2002; Prabhu et al., 2012, 2014). Biochemical procedures are considered gold-standard quantitative methods applied in collagen analysis (Kiernan, 2002; Prabhu et al., 2012). However, owing to its simplicity, low cost, and runtime, microscopy evaluation of collagen content in scar tissue has been systematically applied in *Corresponding author. romuonovaes@yahoo.com.br Received July 8, 2015; accepted September 24, 2015