Background

The normal biological process of wound healing is a complex dynamic composition of four different phases: hemostasis, inflammation, proliferation, and remodeling. Proper interactions between these different phases are necessary for the wound to heal sufficiently and successfully. Impaired wound healing occurs when these phases do not integrate correctly with one another. Dysfunctional wound healing is currently a major burden in our healthcare system, and 6.5 million people per year suffer from this condition (Sen, 2021). Often, chronic diseases, such as diabetes mellitus, chronic venous insufficiency, or age-associated diseases, are underlying issues behind dysfunctional healing. The healthcare costs of non-healing wounds are high since there are no highly effective therapies currently available (Alexiadou and Doupis, 2012). For example, over the past fifteen years, the success rate for therapeutic trials in wound healing has been zero percent, resulting in no approved therapeutics for wound healing in that time frame (Thomas et al., 2016).

Studying the molecular mechanisms involved in these processes is essential to discover new therapeutic applications to promote healing. Many different species are currently being studied in the field of wound healing research, such as pigs, rats, rabbits, and mice. Pigs have the most similar physiological, anatomical, and functional skin conditions to humans (Ibrahim et al., 2017), but are exceedingly expensive to house and hard to maintain. Rat and rabbit models have been used extensively in the past but with decreasing frequency in recent times. The evolution of various knock-out lineages has made the use of murine models more attractive for studying the effects of various gene targets on healing. The most significant difference between murine and human wound healing is the presence of the panniculus carnosus, an underlying muscle layer that causes wound healing to occur not only through re-epithelization but also through contraction. In rodents, contraction can account for up to 40%–60% of wound closure (Chen et al., 2015), so rodent models are often critiqued for not being translationally applicable to human wound healing kinetics. To overcome this issue, our group invented the splinted excisional wound model in 2004 (Galiano et al., 2004). In this model, silicone splints are sutured around excisional wounds to prevent contraction and instead promote healing through re-epithelialization. This model has become a commonly used wound healing model due to its simplicity and feasibility (Michaels et al., 2007; Nauta et al., 2013; Cho et al., 2016; Chong et al., 2017; Cogan et al., 2018; Hu et al., 2018; Kurt et al., 2018; Dallas et al., 2019; Rhea and Dunnwald, 2020; Liu et al., 2020; Kosaric et al., 2020; Hu et al., 2021; Lintel et al., 2022; Maschalidi et al., 2022; K. Chen et al., 2022b).

Specifically, this wound healing model has been established to test multiple different therapies for wound healing. Several studies have used different wound dressings incorporating various hydrogel compositions. Our group has used the excisional wound model to compare the ability to promote wound healing of four different types of wound dressings made from biocompatible hydrogels (K. Chen et al., 2022b). Casado-Diaz et al. (2022) have used the excisional wound model to evaluate the efficacy of a novel Olea europaea–based hydrogel matrix as a wound dressing. Cell-based therapies can also be studied with the excisional wound model. Our group has shown that treating excisional wounds with Trem2+ macrophages in a hydrogel-based wound dressing enhances angiogenesis and accelerates wound healing (Henn et al., 2021). Our group has also applied an adipose-derived stromal cell-seeded hydrogel wound dressing to excisional burn wounds to evaluate its effect on wound closure (Barrera, 2021). Another application of this model is to evaluate the effect of drug-based therapies on wound healing. For example, adding a recombinant Agrin fragment on excisional wounds has been shown to promote healing (Chakraborty et al., 2021). By utilizing the excisional wound model as well as a burn wound model, our group has shown that wounds treated with the small molecule focal adhesion kinase inhibitor (FAKI) imbued with pullulan hydrogel heal significantly faster than untreated wounds (Ma et al., 2018), establishing the relevance of FAKI therapy to reduce inflammation and fibrosis in rodent models (K. Chen et al., 2021,2022a).

Other wound healing models have been described in mice. One is the dorsal skin fold chamber (Michael et al., 2013), which has been previously used to study burn wounds in rodents. This model also prevents wound healing by contracture by having a titanium frame fixated around the wound. In comparison to that model, our excisional wound model utilizes commercially available silicone sheets as splints to prevent contraction, and the splints necessary to prevent wound contraction are easily manufactured. These important steps allow this model to be easily incorporated at low cost for any research group across the world.

In this protocol, we describe the exact methods of the excisional wound model and how they can be applied to different mouse strains. The wound healing in normal wildtype mice is compared to wound healing in a diabetic mouse strain with a mutation on the leptin gene (db/db). While previous papers have used this model, their methods are usually described vaguely and do not comment on the exact methods necessary to perform this surgery, hindering the reproducibility of the model. Furthermore, methods have become outdated due to improvements in materials and documentation techniques, and our lab has significantly improved our methods compared to our first publication on the model in Galiano et al. (2004). Here, we provide a detailed protocol of this important humanized model to promote reproducibility and present several common troubleshooting situations while performing the surgeries and during post-operative wound care. Additionally, we provide a precise guide on how to perform the main analysis of wound healing curve analysis.