Dynamic Histological Events and Molecular Changes in Excisional Wound Healing of Diabetic DB/DB Mice Xiao Tian Wang, MD,* Claire C. McKeever, BA, Peter Vonu, BA, Charles Patterson, MD, and Paul Y. Liu, MD Department of Plastic Surgery, Rhode Island Hospital, Warren Alpert School of Medicine, Brown University, Providence, Rhode Island article info Article history: Received 27 March 2018 Received in revised form 18 December 2018 Accepted 17 January 2019 Available online xxx Keywords: Diabetic DB/DB wound closure Wound contraction Re-epithelialization Fibroblast-to-myofibroblast differentiation TGFb signaling pathway abstract Background: Wound contraction and re-epithelialization over the entire healing process had never been histologically examined daily in diabetic mouse wounds. Correlating morphological characters with molecular changes may be essential to understand the potential mechanism of impeded diabetic wound healing. Materials and methods: In 99 db/db and 63 db/m mice, dorsal-paired 8 mm-diameter wounds were created. Wound contraction and re-epithelialization were histologically analyzed dailydsix wounds per group each day. A novel three-dimensional collagen gel model was used to study diabetic dermal fibroblast contractility. Fibroblast-to-myofibroblasts differ- entiation and TGFb-SMAD signaling pathway through the diabetic db/db wound healing process were studied by immunohistochemistry. Results: Db/db wounds presented delayed closure with impaired wound contraction. Re- epithelialization was not slow but showed thinner epithelial formation and irregular ker- atinocyte arrangement. Diabetic dermal fibroblasts had significantly lower contractile ability than nondiabetic fibroblasts. In db/db wounds, a-SMA, the marker of myofibroblasts, showed constantly low through the healing, which represented reduced fibroblast-to- myofibroblasts differentiation. Remarkably weak staining of TGFbRI and low accumula- tion of Smad3 in nuclei were observed. Conclusions: We demonstrated and precisely located downregulated TGFb signaling pathway in db/db wounds by showing low expression of TGFbRI and failure of Smad3 translocation from cytoplasm to nuclei, which was not reported previously. The down- regulated TGFb signaling pathway may contribute to the attenuated fibroblast-to- myofibroblast differentiation. Deficient re-epithelialization and defective wound contrac- tion contribute principally to delayed healing of diabetic db/db wounds. ª 2019 Elsevier Inc. All rights reserved. * Corresponding author. Department of Plastic Surgery, Rhode Island Hospital, Warren Alpert School of Medicine, Brown University, 593 Eddy Street, 239 Multiphasic Building Providence, RI 02903. Tel.: þ1 401 444-9949; fax: þ1 401 444-5351. E-mail address: xwang3@lifespan.org (X.T. Wang). Available online at www.sciencedirect.com ScienceDirect journal homepage: www.JournalofSurgicalResearch.com journal of surgical research  june 2019 (238) 186 e197 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.01.048