A Novel Animal Model for Simulating Scarring After Cleft Lip Repair

Introduction

Excessive scarring after cleft lip repair may occur in as many as 47% of cases.¹ Management of postsurgical scarring starts with lip massage, frequently progressing to therapeutic injections (e.g., steroids, botulinum toxin) or even revision surgery.² Historically, animal models of scarring have been utilized for investigating potential therapies, however, most models do not incorporate factors such as wound tension, frequent movement, or exposure to oral flora, all of which may affect lip scarring.^3,4 Herein we describe a simple standardized animal model to simulate scarring after lip repair.

Materials and Methods

All experiments were approved by the Tufts University Institutional Animal Care and Use Committee. Twelve, 6-week-old male New Zealand white rabbits weighing 2–3 kg were utilized. The naturally occurring, median cleft lip of each rabbit was surgically widened and repaired to simulate closure of a left unilateral cleft lip, similar to a technique described by Bardach and Eisbach but with distinct modifications.^5,6

Description of procedure

Anesthesia was induced using intramuscular ketamine (30 mg/kg) and xylazine (5 mg/kg), followed by inhaled isoflurane (1–5%). The rabbit was orotracheally intubated and secured in a Trendelenburg position to avoid aspiration. The upper lip was shaved and a cream containing calcium hydroxide (Nair^®, Church & Dwight) was applied. After sterile preparation, 100 mm² of soft tissue was resected from the left lateral lip element (Fig. 1). The incisions were beveled to preserve some mucosa, assuring an adequate gingivolabial sulcus for eating. A 10 mm subnasal horizontal back cut with a 5 mm equilateral Burow's triangle was created (Fig. 1), followed by a rotation-advancement lip closure performed in layers, using 4-0 plain gut suture for mucosa and 4-0 silk for the muscle layer. The skin edges were cauterized and the dermis left open (Supplementary Fig. S1). Antibiotics (Baytril 5 mg/kg SQ) were provided intraoperatively and once daily through postoperative day 3. A dose of long-acting analgesic was given preoperatively (buprenorphine SR 0.1 mg/kg SQ). Animals were kept in individual cages, received water ad libitum, and consumed a moistened soft diet for 3–4 days before transitioning to dry rabbit chow (Envigo, Global 2031 Rabbit Diet).

Fig. 1.

Intraoperative photographs. The naturally occurring median cleft lip in a rabbit (a). Markings were made to create a standardized defect within the left lateral lip element, simulating asymmetric tension following left unilateral cleft lip repair. A 10 mm horizontal lateral back-cut with a 5 mm Burow's triangle was planned to prevent a standing cone and distortion of the nares after closure (b). The full-thickness defect following excision of 100 cm² of tissue and additional incisions for closure (c). Rotation-advancement repair with the dermal edges left open to encourage scarring (d).

Photodocumentation of healing was performed at predetermined intervals. Subjects were followed for 42 days to determine the latency between surgery and scarring. Animals were then euthanized with intravenous sodium pentobarbital (100 mg/kg), allowing for histologic examination of the surgical scars (Supplementary Data).

Results

All animals underwent successful surgery and recovery from anesthesia; lip closure did not affect feeding or respiration. The first two surgeries, performed before the others as a pilot experiment, incorporated careful approximation of skin edges with plain gut suture resulting in minimal scarring (Supplementary Fig. S2). After observing this finding, the method of skin closure was altered as described above. In the remaining 10 animals, one rabbit developed partial wound separation on postoperative day 7 and was sacrificed given the concern for outlier status on final scar review. The other nine animals demonstrated a consistent time line of healing (Supplementary Fig. S3), with scarring detectable as early as 21 days postoperatively. Mature and distinct scar formation was apparent visually and histologically by postoperative day 35 (Fig. 2 and Supplementary Fig. S4).

Fig. 2.

Clinical and histologic documentation of scarring. Scarring was detectable as early as 21 days postoperatively (a), and became more mature and distinct by day 42 (b) (outlined with blue dots). Microscopic examination of representative tissue specimens captured at the midline of the incision confirmed dense inflammatory cell infiltration (H&E; 100 × magnification) (c) and demonstrates high collagen density within scar tissue (Masson's trichrome; 200 × magnification) (d).

Discussion

We report a reliable animal model of scarring after lip repair, which imparts consistent scar formation as early as 3 weeks postoperatively. While several animal models of cutaneous scarring already exist, the model described herein incorporates additional factors that affect healing of orofacial defects, including frequent movement, minor trauma, exposure to oral flora, and healing of multiple tissue types under tension. This novel model may simulate a variety of lip defects, including orofacial clefts, soft tissue trauma, and cancer resections. Further studies aiming at quantifying histological measures of scar formation in this animal model are ongoing. Such analyses will quantitatively examine potential variance in scarring between animals and establish normative values for control subjects before examining potential therapeutic treatments.

Authors' Contributions

E.P. contributed to the conception, design, animal experiments and interpretation of the data, drafted and critically revised the article. C.B. and A.R.S. contributed to the conception, design, animal experiments, interpretation of the data, and critically revised the article. C.A.T. and T.E.V.D. contributed to the conception, design, interpretation of the data, and critically revised the article. All authors gave the final approval and agree to be accountable for all aspects of the work.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

Supported by USPHS grants K08DE027119 to E.P., R01DE025020 to T.E.V.D. and U01DE024503 to C.A.T. from the National Institute of Dental and Craniofacial Research (NIDCR) and National Institute of Health (NIH).

Supplementary Material

Supplementary Data

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4