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

Khan Sharun; S. Amitha Banu; Merlin Mamachan; Athira Subash; Karikalan Mathesh; Rohit Kumar; Obli Rajendran Vinodhkumar; Kuldeep Dhama; Laith Abualigah; Abhijit M. Pawde; Amarpal

doi:10.18006/2023.11(5).822.833

Authors

Khan Sharun Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0003-1040-3746
S. Amitha Banu Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0002-7182-8169
Merlin Mamachan Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0009-0002-5861-1878
Athira Subash Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0009-0007-0947-704X
Karikalan Mathesh Centre for Wildlife Conservation, Management and Disease Surveillance, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India. https://orcid.org/0000-0002-2013-9179
Rohit Kumar Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0003-3134-0772
Obli Rajendran Vinodhkumar Division of Epidemiology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0002-7232-4122
Kuldeep Dhama Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0001-7469-4752
Laith Abualigah Computer Science Department, Prince Hussein Bin Abdullah Faculty for Information Technology, Al al-Bayt University, Mafraq 25113, Jordan https://orcid.org/0000-0002-2203-4549
Abhijit M. Pawde Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0001-7399-5596
Amarpal Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0002-4827-3433

DOI:

https://doi.org/10.18006/2023.11(5).822.833

Keywords:

Collagen content, Digital image analysis, Differential staining, ImageJ, Histopathology, Wound healing

Abstract

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.

Author Biography

Laith Abualigah, Computer Science Department, Prince Hussein Bin Abdullah Faculty for Information Technology, Al al-Bayt University, Mafraq 25113, Jordan

Hourani Center for Applied Scientific Research, Al-Ahliyya Amman University, Amman 19328, Jordan

MEU Research Unit, Middle East University, Amman 11831, Jordan

Department of Electrical and Computer Engineering, Lebanese American University, Byblos 13-5053, Lebanon

School of Computer Sciences, Universiti Sains Malaysia, Pulau Pinang 11800, Malaysia

School of Engineering and Technology, Sunway University Malaysia, Petaling Jaya 27500, Malaysia

Applied Science Research Center, Applied Science Private University, Amman 11931, Jordan

References

Banu, S. A., Pawde, A. M., Sharun, K., Kalaiselvan, E., Shivaramu, S., et al. (2023). Evaluation of bone marrow-derived mesenchymal stem cells with eggshell membrane for full-thickness wound healing in a rabbit model. Cell and tissue banking, 10.1007/s10561-023-10105-0. DOI: https://doi.org/10.1007/s10561-023-10105-0

Barington, K., Dich-Jørgensen, K., & Jensen, H. E. (2018). A porcine model for pathomorphological age assessment of surgically excised skin wounds. Acta veterinaria Scandinavica, 60(1), 33. DOI: https://doi.org/10.1186/s13028-018-0387-3

Bist, D., Pawde, A. M., Amarpal, Kinjavdekar, P., Mukherjee, R., et al. (2021). Evaluation of canine bone marrow-derived mesenchymal stem cells for experimental full-thickness cutaneous wounds in a diabetic rat model. Expert opinion on biological therapy, 21(12), 1655–1664. DOI: https://doi.org/10.1080/14712598.2022.1990260

Chang, J. Y., & Kessler, H. P. (2008). Masson trichrome stain helps differentiate myofibroma from smooth muscle lesions in the head and neck region. Journal of the Formosan Medical Association, 107(10), 767–773. DOI: https://doi.org/10.1016/S0929-6646(08)60189-8

Chen, Y., Yu, Q., & Xu, C. B. (2017). A convenient method for quantifying collagen fibers in atherosclerotic lesions by ImageJ software. International Journal of Clinical and Experimental Medicine, 10(10): 14904-14910.

Constantine, V. S., & Mowry, W. (1968). Selective staining of human dermal collagen. The Journal of Investigative Dermatology, 50(5), 414-18. DOI: https://doi.org/10.1038/jid.1968.67

Costa, G. M., Araujo, S. L., Xavier Júnior, F. A. F., Morais, G. B. D., Silveira, J. A. D. M., Viana, D. D. A., & Evangelista, J. S. A. M. (2019). Picrosirius red and Massons Trichrome staining techniques as tools for detection of collagen fibers in the skin of dogs with endocrine dermatopathologies. Ciência Animal Brasileira. e-55398. DOI: https://doi.org/10.1590/1089-6891v20e-55398

Elshazly, N., Saad, M. M., El Backly, R. M., Hamdy, A., Patruno, M., et al. (2023). Nanoscale borosilicate bioactive glass for regenerative therapy of full-thickness skin defects in rabbit animal model. Frontiers in Bioengineering and Biotechnology, 11, 1036125. DOI: https://doi.org/10.3389/fbioe.2023.1036125

Juengsomjit, R., Meesakul, O., Arayapisit, T., Larbcharoensub, N., & Janebodin, K. (2022). Polarized Microscopic Analysis of Picrosirius Red Stained Salivary Gland Pathologies: An Observational Study. European journal of dentistry, 16(4), 930–937. DOI: https://doi.org/10.1055/s-0042-1743145

Junqueira, L.C., Bignolas, G., & Brentani, R.R. (1979). Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Journal of Histochemistry & Cytochemistry, 11(04):447–455. DOI: https://doi.org/10.1007/BF01002772

Kular, J. K., Basu, S., & Sharma, R. I. (2014). The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering. Journal of tissue engineering, 5, 2041731414557112. DOI: https://doi.org/10.1177/2041731414557112

Landini, G., Martinelli, G., & Piccinini, F. (2021). Colour deconvolution: stain unmixing in histological imaging. Bioinformatics, 37(10), 1485-1487. DOI: https://doi.org/10.1093/bioinformatics/btaa847

Lattouf, R., Younes, R., Lutomski, D., Naaman, N., Godeau, G., Senni, K., & Changotade, S. (2014). Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues. The journal of histochemistry and cytochemistry, 62(10), 751–758. DOI: https://doi.org/10.1369/0022155414545787

Lee, E. S., Kim, J. H., Im, S., Lee, K. B., Sohn, S., & Kang, W. H. (2001). Application of computerized image analysis in pigmentary skin diseases. International Journal of Dermatology, 40(1), 45-49. DOI: https://doi.org/10.1046/j.1365-4362.2001.00084.x

López De Padilla, C. M., Coenen, M. J., Tovar, A., De la Vega, R. E., Evans, C. H., & Müller, S. A. (2021). Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon. The journal of histochemistry and cytochemistry, 69(10), 633–643. DOI: https://doi.org/10.1369/00221554211046777

Marcos-Garcés, V., Harvat, M., Molina Aguilar, P., Ferrández Izquierdo, A., & Ruiz-Saurí, A. (2017). Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome, Picrosirius red and confocal microscopy techniques. Journal of microscopy, 267(2), 130–142. DOI: https://doi.org/10.1111/jmi.12553

Meyer, P. F., de Oliveira, P., Silva, F. K. B. A., da Costa, A. C. S., Pereira, C. R. A., Casenave, S., Valentim Silva, R. M., et al. (2017). Radiofrequency treatment induces fibroblast growth factor 2 expression and subsequently promotes neocollagenesis and neoangiogenesis in the skin tissue. Lasers in Medical Science, 32(8): 1727-1736. DOI: https://doi.org/10.1007/s10103-017-2238-2

Owens, P., Engelking, E., Han, G., Haeger, S. M., & Wang, X. J. (2010). Epidermal Smad4 deletion results in aberrant wound healing. The American journal of pathology, 176(1), 122-133. DOI: https://doi.org/10.2353/ajpath.2010.090081

Ruifrok, A. C., & Johnston, D. A. (2001). Quantification of histochemical staining by color deconvolution. Analytical and quantitative cytology and histology. 23(4):291-9.

Segnani, C., Ippolito, C., Antonioli, L., Pellegrini, C., Blandizzi, C., Dolfi, A., & Bernardini, N. (2015). Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon. PloS one, 10(12), e0144630. DOI: https://doi.org/10.1371/journal.pone.0144630

Sharma, R., Rehani, S., Mehendiratta, M., Kardam, P., Kumra, M., et al. (2015). Architectural Analysis of Picrosirius Red Stained Collagen in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma using Polarization Microscopy. Journal of clinical and diagnostic research, 9(12), EC13–EC16. DOI: https://doi.org/10.7860/JCDR/2015/13476.6872

Street, J. M., Souza, A. C., Alvarez-Prats, A., Horino, T., Hu, X., Yuen, P. S., & Star, R. A. (2014). Automated quantification of renal fibrosis with Sirius Red and polarization contrast microscopy. Physiological reports, 2(7), e12088. DOI: https://doi.org/10.14814/phy2.12088

Van De Vlekkert, D., Machado, E., & d'Azzo, A. (2020). Analysis of Generalized Fibrosis in Mouse Tissue Sections with Masson's Trichrome Staining. Bio-protocol, 10(10), e3629. DOI: https://doi.org/10.21769/BioProtoc.3629