A subscription to JoVE is required to view this content. Sign in or start your free trial.
* These authors contributed equally
This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.
Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction—the conversion of mechanical stimuli into cellular responses—drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.
Wound healing, the process by which the body attempts to repair damaged tissue and rebuild the skin barrier, can result in atypical healing if its processes of hemostasis, inflammation, proliferation, and remodeling are irregular1. Hypertrophic scarring (HTS) is an example of irregular wound healing, characterized by excessive deposition of extracellular matrix and connective tissue at the site of injury resulting in the formation of an enlarged scar tissue area1,2,3. Areas on the body that undergo repeated mechanical stretch stimulations, such as around joints or on the face, are more prone to developing HTS and fibrosis4,5,6,7,8,9,10. We and others have shown that mechanical stretch across a wound bed promotes HTS formation through the activation of mechanotransduction pathways—the conversion of mechanical stimuli into cellular responses9,11.
HTS not only involves complex biological processes but also carries significant social, medical, and economic challenges for the people affected. Affected individuals can struggle with self-esteem and depression, especially when the scars are in visible areas like the face and hands1,9,10,12. Scientific review articles indicate that the prevalence of HTS varies between 32% and 72% in the United States10,13. The severity of these aesthetic concerns, especially in cases of serious burn injuries in the facial region, is underscored by the increasing number of full facial transplantation cases to improve appearance10. These scars can also cause functional impairments by restricting movement6,14, and surgical intervention is often required to excise scars and restore mobility10. The cost of HTS treatment can be substantial, including expenses for surgery, treatments, physical therapy, or even long-term care1,10. In the United States alone, the annual cost of treating HTS exceeds $4 billion10.
Considering the pervasiveness of HTS and the extreme measures taken to address its complications, conventional therapies (e.g., surgical excision, corticosteroid injections, and laser therapy) remain highly variable1,2,15,16,17. While these treatments can offer relief in some cases, they can be insufficient due to the complex nature of scar pathology. Factors such as genetic differences among individuals and an incomplete understanding of the mechanisms driving HTS cause therapeutic strategies to remain clinically unsatisfactory18,19,20. The future of HTS therapy seems to lie in new innovative approaches that target cell mechanistic drivers of HTS, such as mechanotransduction11,21, which we have extensively demonstrated to drive excessive fibrotic scar healing5,6,7,8,11,21,22,23,24,25. Specifically, we had previously developed a murine model that increases wound mechanical strain to promote human-like HTS9. However, after nearly two decades of use, the model has been significantly advanced to improve efficacy and reproducibility. This protocol will allow researchers to best utilize an updated and optimized HTS mouse model to explore the cell populations and drivers behind excessive scarring. The overall goal of this method is to provide researchers with a protocol designed to produce human-like hypertrophic scarring in mice.
Approval from the University of Arizona Institutional Animal Care and Use Committee (IACUC) was obtained for all experiments (control number: 2021-0828). This protocol uses 15-week-old C57BL/6J male mice although it could be applied to other ages and strains9,26.
1. Creating the HTS biomechanical loading device
NOTE: Modifying the palatal expanders into the HTS device can occur at any point before the experiment.
2. Hair removal and initial incision on postoperative day 0 (POD 0)
NOTE: Clean and autoclave several sets of surgical instruments before surgery (e.g., dissection scissors, scalpel, Adson forceps, Needle driver). Prepare sterilized 5-0 sutures for use and have a surgical marker on hand.
3. Placement of HTS biomechanical loading device (POD 4)
NOTE: Clean and autoclave the HTS devices and several sets of surgical instruments before surgery (e.g., dissection scissors, scalpel, Adson forceps, Needle driver, skin stapler, skin staples). Prepare sterilized 5-0 sutures for use. [Optional] If a benchtop fume extractor is available, place the cone near workspace and turn on the suction.
4. Initial stretch of HTS biomechanical loading device (POD 5)
NOTE: Clean and autoclave several sets of surgical instruments (e.g., dissection scissors, scalpel, Adson forceps, Needle driver) prior to surgery. [Optional] If a benchtop fume extractor is available, place the cone near the workspace and turn on the suction.
5. Subsequent stretch of HTS biomechanical loading device (POD 7, 9, 11, 13, 15, 17)
NOTE: Clean and autoclave several sets of surgical instruments (e.g., dissection scissors, scalpel, Adson forceps, Needle driver) prior to surgery. [Optional] If benchtop fume extractor is available, place the cone near the workspace and turn on the suction.
6. Harvesting the HTS tissue (POD 19)
NOTE: Harvesting tissue can take place at any point in the process. We have harvested tissue after only 4 days of stretch to examine early time points; however, tissue is most consistently harvested at POD 19 (2 weeks after strain was initiated). Clean and autoclave several sets of surgical instruments (e.g., dissection scissors, scalpel, Adson forceps) prior to surgery. To get photos of the scar over time, the device can be removed before each stretching step to take a photo of the scar before re-applying the device and re-initiating mechanical strain. [Optional] If a benchtop fume extractor is available, place the cone near the workspace and turn on the suction. The benchtop fume extractor may be turned off when the isoflurane gas is no longer being used.
7. Measuring average scar width
NOTE: This was accomplished with image analysis software ImageJ, and the information was recorded on a spreadsheet.
To clearly demonstrate the effective use of the HTS protocol and identify successful "positive" results, the model was established as shown in Figure 3A. In the representative study, there were two groups: No Stretch Control (n = 6) and Mechanical Stretch HTS group (n = 6) where human-like levels of mechanical strain were induced across the incision to generate an HTS, seen in Figure 3B,C. Within the experimental plan given in
The HTS mouse model is a cost-effective and highly reproducible method for inducing HTS via mechanotransduction and developing potential therapies. While there is an initial learning curve to effectively use the model, the protocol can, with practice, be performed by any researcher without surgical training. Using this model allows researchers to better understand HTS formation and the role of mechanotransduction in wound healing, which may lead to tangible improvements in patient wound care. The video demonstration acco...
The authors have no competing interests or other conflicts associated with the contents of this article.
This work was supported by the Center for Dental, Oral, and Craniofacial Tissue and Organ Regeneration Interdisciplinary Translational Project Awards supported by the National Institute of Dental and Craniofacial Research (U24 DE026914) (G.C.G) and the Plastic Surgery Foundation Translational Research Grant (837107) (K.C.).
Name | Company | Catalog Number | Comments |
100 mL PYREX Griffin beaker | Milipore Signma | CLS1000100 | |
Aesculap Exacta mini trimmer | Aesculap | ||
AutoClip System | Fine Surgical Instruments | 12020-00 | |
BD brand isopropyl alcohol swabs | Fisher Scientific | 13-680-63 | |
Buprenorphine SR (0.5 mg/mL) | Buprenex, Indivior Inc. | 12496-0757-1 | |
C57/BL6 females (6–8 weeks old) | The Jackson Laboratory | 000664 | |
Covidien sterile gauze | Fisher Scientific | 2187 | |
Covidien TelfaTM non-adherent pads | Fisher Scientific, Covidien | 1961 | |
Dental surgical ruler | DoWell Dental Products | S1070 | |
Depilatory cream (Nair Hair Remover Lotion) | Church&Dwight, CVS | 339823 | |
Ethanol 70% solution | Fisher Scientific | 64-17-5 | |
Excel | Microsoft Cooperation | Microsoft.com | software program |
ImageJ | ImageJ, Wayne Rasband | imagej.net | software program |
Inhalation anesthesia system | VetEquip | 922130 | |
Iris scissors 4½ in. stainless | McKesson | 43-2-104 | |
Isoflurane, USP | Dechra Veterinary Products | 17033-094-25 | |
Kaka industrial MUB-1 | Kaka Industrial | 173207 | Only necessary if there is no maker space or fabrication shop available |
Leone Rapid Palatal Expander- 13 mm | Great Lakes Dental Technologies | 125-004 | The key necessary to expand and cotnract the device will come with this product in the box |
Liquid repellent drape 75 x 90 cm with adhesive hole 6 x 9 cm | Omnia S.p.A. | 12.T4362 | |
Medequip Depot Silk Black Braided Sutr 6-0 Rx | Medequip Depot D707N, Fisher Scientific | NCO835822 | |
Needle holder 5 in. with serrated jaws | McKesson | 43-2-842 | |
Prism 9 | GraphPad Holdings, LLC | graphpad.com | software program |
Puralube ophthalmic ointment | Dechra, NDC | 17033-211-38 | |
R studio Desktop | RStudio PBC | rstudio.com | software program |
Surgical skin marker | McKesson | 19-1451_BX | |
Tegaderm, 3 M | VWR | 56222-191 | foam adhesive dressing |
Thermo-peep heating pad | K&H, Amazon | ||
Tissue forceps 4¾ in. stainless 1 x 2 teeth | Mckesson | 43-2-775 | |
Vetbond (3 M) | Saint Paul, MN | 1469SB |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved