Comparative Evaluation of Masson's Trichrome and Picrosirius Red Staining for Digital Collagen Quantification Using ImageJ in Rabbit Wound Healing Research

The therapeutic potential of Pluronic F127 (PF127) hydrogel loaded with adipose-derived stromal vascular fraction (AdSVF), mesenchymal stem cells (AdMSC), and conditioned media (AdMSC-CM) for repairing full-thickness skin wounds was evaluated using a rabbit model. The rabbits were randomly divided into eight groups with six animals each and treatment was given as per the predetermined protocol (3 doses at one-week interval): Group A (Control), Group B (AdSVF), Group C (AdMSC), Group D (AdMSC-CM), Group E (PF127), Group F (AdSVF + PF127), Group G (AdMSC + PF127), and Group H (AdMSC-CM + PF127). Skin tissue samples were collected from the healing wounds on day 28 for staining and collagen quantification. Collagen density (Area %) was quantified using tissue sections stained with Masson's Trichrome (MT) and Picrosirius Red (PSR) stain using the Colour Deconvolution plugin of ImageJ and RGB stack method, respectively. These techniques function based on separating different colour channels in the stained tissue sections to isolate the collagen fibers and then quantifying them through thresholding and image analysis. Across the treatment groups, both staining methods generally showed a trend of increased collagen density compared to the control group. For most groups, PSR staining consistently indicated slightly lower collagen densities than MT staining. However, the overall trends were similar in both staining. The comparison between PSR and MT staining methods revealed that both techniques effectively assess collagen density in healing wounds. However, there were subtle differences in the absolute values obtained, with PSR staining tending to yield slightly lower collagen density measurements than MT. These differences can be attributed to the distinct mechanisms of these staining methods. Therefore, both staining methods can digitally quantify collagen density in wound healing research.