Development of an Efficient Murine Tissue Expansion Model for Skin Regeneration Research

Summary

This research presents a practical and efficient protocol for establishing a continuous tissue expansion model in rats, thereby facilitating the mechanism investigation and technique advancement in skin regeneration.

Abstract

Skin regeneration is essential for the healing of various superficial wounds and can be promoted by mechanical stimulation. Tissue expansion is a widely used strategy to accelerate skin regeneration by controlled mechanical stretch; however, its clinical efficacy is often restricted by a mismatch between mechanical loading and the skin's regenerative capacity. To address this limitation, the development of a standardized animal model of tissue expansion is urgently required. In this study, we established a tissue expansion model in Sprague-Dawley rats. Following the dorsal implantation of a 10 mL tissue expander, 5 mL of saline was injected immediately after the surgery. After a two-week recovery period, animals underwent a regimen of twice-weekly saline injections (10 mL per session). The expansion kinetics exhibited a characteristic trend of rapid skin growth followed by a gradual deceleration. In the later stages, severe complications such as skin necrosis, tissue breakdown, and expander exposure were observed. This work establishes a reproducible preclinical framework that closely mimics the physiological and pathological processes of human tissue expansion, providing a foundational model to investigate regenerative mechanisms and optimize clinical protocols.

Introduction

Skin serves as the body's first line of defense, providing mechanical protection and preventing water loss, while also playing crucial roles in temperature regulation, external stimuli response, and immune regulation¹. Skin wounds are common in clinical practice, resulting from trauma, microbial infections, chemical damage, and surgical resection². While small wounds could heal with a normal appearance, large injuries generally leave behind a severe fibrotic scarring that impairs tissue function and may even become life-threatening³. Autologous full-thickness skin grafting is widely considered the gold standard for managing large wounds and preventing scarring⁴. However, acquiring sufficient graftable skin remains a critical clinical challenge. Consequently, the development of innovative skin regeneration technologies has emerged as a central focus in the fields of regenerative medicine and biomaterials⁵.

Since Neumann first introduced the principle of tissue expansion in 1957 by implanting a polyethylene balloon in the postauricular mastoid region to reconstruct an ear deformity⁶, this technique has evolved significantly. Today, it is one of the most widely adopted methods in reconstructive surgery and remains a vital approach for addressing extensive skin defects. Specifically, tissue expansion generates additional autologous tissue by recruiting adjacent skin and stimulating regeneration⁷. Driven by mechanical stimulation, the newly formed skin closely resembles native skin in both appearance and structure, rendering it ideal for clinical application⁸. However, due to a mismatch between the magnitude of the loading force and the growth rate of skin in the late stage, the quantity of harvested skin proves insufficient for large-scale wound coverage⁹. Furthermore, the advancement of tissue expansion technology is hindered by suboptimal expansion efficacy, severe complications, incomplete quantitative evaluation metrics, and poorly characterized molecular mechanisms¹⁰. Given the complexity of regulatory networks in vivo, suitable animal models of tissue expansion are essential to faithfully recapitulate the physiological and pathological responses of skin tissues to mechanical stimulation.

At present, murine (mouse and rat) and porcine models are the predominant choice for in vivo studies¹¹. Mouse dorsal or scalp expansion models are widely used due to their ease of operation; however, the skin is too thin to tolerate prolonged or continuous expansion¹². The rat dorsal expansion model is suitable for continuous expansion due to its extensive subcutaneous space, but the lack of a definitive protocol to terminate expansion limits its clinical applicability^13,14. Although porcine skin shares the highest degree of homology with human skin, its lower elasticity results in delayed responses to mechanical stretch, and the requirement for deep sedation during the process renders experimental manipulation considerably more challenging¹⁵. Therefore, developing an appropriate model of continuous expansion that mimics the clinical process remains a scientific challenge.

The purpose of this study was to develop a rat model of continuous skin expansion. Briefly, a 10 mL silicone expander was inserted into the dorsal region of Sprague-Dawley (SD) rats, and 5 mL of saline was injected immediately after the surgery. Following a two-week recovery period, 10 mL of saline was administered twice per week until a cumulative volume of 155 mL was attained, at which point a significant decline in skin generation was observed, suggesting the exhaustion of regenerative capacity. Regenerative exhaustion signifies a critical decline in skin regenerative capacity, characterized at the cellular level by diminished proliferative potential and breakdown of the extracellular matrix¹⁶. Macroscopically, this manifests as a marked deterioration in skin texture, presenting as skin thinning, ischemia, and striae distensae, potentially progressing to necrosis and tissue breakdown¹⁷. In addition to its technical simplicity, this preclinical model successfully replicates continuous skin expansion, allowing researchers to investigate different expansion stages tailored to specific research objectives. This modeling method can also be applied to genetically modified rats, facilitating the elucidation of key gene functions and thereby uncovering the underlying mechanisms. Furthermore, the ample surface area of the dorsal skin facilitates a self-controlled study design, allowing both experimental and control treatments to be applied within the same animal¹⁸. Collectively, this model provides a useful tool for investigating specific mechanisms involved in tissue expansion and contributes to overcoming the current technological limitations.

Protocol

All animal procedures were approved by the Ethics Committee of Shanghai Ninth People's Hospital (No. SH9H-2025-A1673-1) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Preparation

1. Acclimatize all SD rats for 7 days prior to the study. House SD rats in an animal facility with a 12 h light/12 h dark cycle. Provide a standard wheat/soy-based diet and water ad libitum throughout the experiment.
2. Design 10 mL semicircle tissue expanders for experimental use (Figure 1A), which comprise an expansile balloon, an injection port, and a connective catheter. Sterilize the expanders using electron-beam irradiation (25 kGy). Prior to surgery, check the integrity of each expander to ensure safety. Inject 10 mL of saline through the injection port, then observe the patency of the connective catheter and expansion to confirm the absence of water leakage.
   NOTE: Drain the pre-injected normal saline.

2. Pre-operative and intra-operative animal care

1. Acquire male SD rats, 6-8 weeks of age, 240-260 g of weight.
2. Anesthetize the SD rats by inhalation of isoflurane. Place the rats into the induction chamber and induce anesthesia using 5% isoflurane at a flow rate of 3 L/min. Maintain anesthesia during the operation using 2% isoflurane at a flow rate of 1.5 L/min.
3. Place the anesthetized SD rats on a cotton pad to maintain warmth during the surgery, and place them on a heated pad (approximately 28 ℃) for 20 min after recovery from anesthesia to prevent hypothermia.
4. Use the toe pinch withdrawal reflex to assess the depth of anesthesia.
   NOTE: Monitor the respiratory rate and pattern, the body temperature, and the toe skin color of anaesthetized SD rats.

3. Tissue expander implantation

1. Shave the dorsal skin of SD rats with a hair clipper. Assess skin color, texture, and presence of any lesions to determine the suitability for expansion.
2. Draw a horizontal dotted line in the center area of the back to mark the incision location. Disinfect the shaved area with 75% alcohol, and cover with a sterile aperture drape.
3. Administer normal saline subcutaneously along the marked line using a 1mL syringe with a 30 G (0.31 mm in diameter) needle, then perform a horizontal incision using a scalpel. Perform an extensive dissection of the subcutaneous tissue above and below the incision along the dorsal muscular fascia. Create a pocket large enough to accommodate the expansion balloon and injection port using mosquito forceps. Position the inferior pole of the expansion balloon 3-5 cm from the incision, with the apex facing the skin layer and the baseplate resting on the muscle. Additionally, suture the fascia on both sides adjacent to the junction of the connective catheter and the injection port to stabilize the port. Adjust the positions of the balloon and the injection port to ensure the connecting tube is free of sharp bends or kinks (Figure 1B).
   NOTE: Normal saline injection facilitates skin incision and anatomical plane identification. Orient the injection port with the soft side facing the skin layer to enable subsequent solution injections.
4. Close the incisions using interrupted 4-0 sutures. Subsequently, inject an initial 5 mL of saline with a 25 G (0.5 mm in diameter) scalp needle.
   NOTE: Suture the incision carefully to avoid puncturing the expander with the suture needle tip. Insert the scalp needle vertically into the injection port and withdraw it vertically.
5. Allow the SD rat to recover from anesthesia, and place it into an individual cage.

4. Continuous expansion process

1. Allow SD rats bearing expanders to recover for 2 weeks, ensuring complete incision healing. Mark a 2.5 × 2.5 cm dashed-line box on the expansion area to facilitate visualization of skin changes (Figure 2A).
2. Anesthetize SD rats using the method described in steps 2.2 to 2.4. Shave the expansion region to facilitate observation. Terminate the expansion procedure immediately if skin expansion is complicated by infection, ecchymosis, or necrosis.
   NOTE: Shave the expanding area at least once weekly to accommodate rapid hair growth.
3. Shave the region around the injection port, and disinfect using 75% alcohol. Fix the port, then inject 10 mL of normal saline slowly using a 25 G scalp needle. Press the injection point with aseptic gauze for 2 min to confirm hemostasis.
4. Allow SD rats to recover from anesthesia, place them on a heated pad for 20 min, and then transfer them to individual cages.
5. Inject normal saline twice weekly. Monitor expansion-related complications during follow-up, including expander leakage, kinking of the connecting tube, expander exposure, skin ecchymosis, necrosis, infection, and breakdown (Figure 2E). Investigate potential catheter kinking if resistance is encountered during saline injection. Suspect an expander leakage if the expansion balloon softens. Identify localized ecchymosis as a sign of compromised perfusion, and skin darkening as necrosis. Suspect infection upon observing marked localized redness, swelling, and elevated local skin temperature. Classify non-healing wounds as skin breakdown and examine for possible expander exposure.
   NOTE: Examine the expanded skin before and after each injection to facilitate the early detection of adverse events. Terminate the expansion procedure immediately if serious complications occur, including expander leakage, skin breakdown or necrosis, and expander exposure, to maximize the salvage of viable skin.

5. Expansion area measurement and regenerated skin collection

1. Measure the expansion area using a High-Precision 3D Scanner. Through scanning the skin above the expander, the surface area of the scanned region is automatically calculated (Figure 2B).
   NOTE: Perform scans on anesthetized SD rats prior to each injection.
2. Photograph the expanded skin using a digital camera; measure and weigh the area demarcated by the dashed-line box, and record the changes in surface area (Figure 2C) and mass (Figure 2D).
3. Euthanize SD rats by exposure to excessive carbon dioxide at the endpoint of the experiment.
   NOTE: In this research, define the endpoint as a total injection volume of 155 mL to replicate the entire expansion process in clinical practice.
4. At post-mortem, mark sampling areas on both the expanded skin and the adjacent normal skin. After aspirating half of the total volume from the expander, incise the skin overlying the injection port horizontally using scissors. Dissect the plane between the expander and the expanded skin extensively using mosquito forceps to allow complete and intact removal of the expander. Excise skin samples using scissors and bisect the tissue. Fix them in 4% paraformaldehyde solution and process for histological staining.
   NOTE: Demarcate the areas of interest for sampling prior to expander removal to prevent misidentification due to skin retraction, ensuring accurate identification of localized treatment regions (Figure 3A).
5. Place collected animal carcasses separately in yellow medical waste bags. Dispose of used expanders, syringes, and gauze in yellow medical waste bags. Discard blades, needles, and other sharps into designated sharps containers.
   NOTE: All waste disposal in this study complies with the management regulations of the Experimental Animal Center of Shanghai Ninth People's Hospital.

6. Skin histological staining

1. Embed skin samples in paraffin, cut into 5 µm sections, and place onto adhesive slides.
2. Use hematoxylin and eosin staining (HE staining) to evaluate the skin thickness (Figure 3B,C), and Masson trichrome staining to assess the collagen deposition (Figure 3D,E). Scan the sections using a whole slide scanner and analyze with ImageJ version 1.54 software.
3. Use CD31 immunofluorescence staining to evaluate vascularization. Apply primary antibodies and incubate overnight at 4 °C, use FITC-conjugated secondary antibodies, and then examine under a fluorescence microscope (Figure 3F,G).
4. Analyze quantitative data using GraphPad Prism version 10.0 software, and present results as mean ± standard deviation (SD). Perform statistical comparisons using Student's t-test, defining statistical significance as *p < 0.05.

Results

In this study, we established a standard protocol for a continuous tissue expansion model. After confirming the safety of the expander (Figure 1A), it was surgically implanted subcutaneously into the dorsal region of SD rats. Its inflating surface was positioned toward the skin. To prevent expander displacement, the connection point between the catheter and injection port was secured to the muscular fascia (Figure 1B). Following the twice-weekly saline injection...

Discussion

Tissue expansion has emerged as a widely used technique to generate additional skin that closely resembles the neighboring healthy skin in terms of texture and color¹⁹. It has been applied in diverse clinical conditions, including scars repair, breast reconstruction, and reconstruction following the resection of body surface tumors²⁰. However, the long-term tissue expansion still remains a critical challenge, primarily due to exhausted skin regenerative capacity

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CD31 antibody	Abcam Shanghai Trading Co.,Ltd	ab182981	IHC staining
Electron-beam irradiation instrument	Tongwei Xinda Technology (Jiangsu) Co,. Ltd	High-energy electron accelerator (10 MeV, 24 kW)	Sterilization
FITC-conjugated secondary antibody	Proteintech Group, Inc	SA0003-2	IHC staining
Graghpad Prism software	GraphPad Software	Version 10.0	Statistical analysis
Hematoxylin and Eosin Staining Kit	Beyotime Biotech Inc	C0105S	Histological staining
High-Precision 3D Scanner	SHINING 3D	EinScan Pro 2X V2	Area measurement
Image J software	National Institutes of Health	Version 1.54	Statistical analysis
Masson's Trichrome Staining Kit	Beyotime Biotech Inc	C0189S	Histological staining
Tissue expanders	Shanghai Winner Plastic surgery Products Co.,Ltd.	N/A	Special customization