The Dorsal Skinfold Chamber as a New Tympanic Membrane Wound Healing Model: Intravital Insights into the Pathophysiology of Epithelialized Wounds

Abstract

Background: Tympanic membrane perforations (TMPs) are a common complication of trauma and infection. Persisting perforations result from the unique location of the tympanic membrane. The wound is surrounded by air of the middle ear and the external auditory canal. The inadequate wound bed, growth factor, and blood supply lead to circular epithelialization of the perforation's edge and premature interruption of defect closure. Orthotopic animal models use mechanical or chemical tympanic membrane laceration to identify bioactive wound dressings and overcome premature epithelialization. However, all orthotopic models essentially lack repetitive visualization of the biomaterial-wound interface. Therefore, recent progress in 3D printing of customized wound dressings has not yet been transferred to the unique wound setup of the TMP. Here, we present a novel application for the mice dorsal skinfold chamber (DSC) with an epithelialized full-thickness defect as TMP model.

Methods: A circular 2-mm defect was cut into the extended dorsal skinfold using a biopsy punch. The skinfold was either perforated through both skin layers without prior preparation or perforated on 1 side, following resection of the opposing skin layer. In both groups, the wound was sealed with a coverslip or left unclosed (n = 4). All animals were examined for epithelialization of the edge (histology), size of the perforation (planimetry), neovascularization (repetitive intravital fluorescence microscopy), and inflammation (immunohistology).

Results: The edge of the perforation was overgrown by the cornified squamous epithelium in all pre-parations. Reduction in the perforation's size was enhanced by application of a coverslip. Microsurgical preparation before biopsy punch perforation and sealing with a coverslip enabled repetitive high-quality intravital fluorescence microscopy. However, spontaneous reduction of the perforation occurred frequently. Therefore, the direct biopsy punch perforation without microsurgical preparation was favorable: spontaneous reduction did not occur throughout 21 days. Moreover, the visualization of the neovascularization was sufficient in intravital microscopy.

Conclusions: The DSC full-thickness defect is a valuable supplement to orthotopic TMP models. Repetitive intravital microscopy of the epithelialized edge enables investigation of the underlying pathophysiology during the transition from the inflammation to the proliferation phase of wound healing. Using established analysis procedures, the present model provides an effective platform for the screening of bioactive materials and transferring progress in tissue engineering to the special conditions of tympanic membrane wound healing.

Keywords: Biomaterials; Tympanic membrane perforation; Wound dressings; Wound healing models.

Fig. 1

Epithelialization of TMPs. a Ear microscopy of a TMP shows round epithelialized margins in the TMP. b Histology (H&E) of a TMP confirms epithelialization of the defect. The black arrow illustrates the overgrowth of the keratinizing squamous epithelium over the defect margin. EAC, external auditory canal; ME, middle ear; TM, tympanic membrane; TMP, tympanic membrane perforation.

Fig. 2

DSC preparation and experimental groups. SPs were prepared through both layers of skin (1) and panniculus carnosus muscle (2) without prior preparation of the panniculus carnosus muscle. The defect remained open or sealed a coverslip (5). The asymmetrical preparation included microsurgical exposure of the panniculus carnosus muscle (2), before excision of the full-thickness defect (3). Likewise, the chamber remained open or sealed with a coverslip (5). The edges (4) of the full-thickness perforation (3) epithelialized during wound healing (n = 5, transverse plane a, sagittal plane b). SP, symmetric perforation; SP-CS, symmetric perforation with coverslip; AP, asymmetric perforation; AP-CS, asymmetric perforation with coverslip; DSC, dorsal skinfold chamber.

Fig. 3

Study design and experimental procedures. The DSC was prepared on day 0 and a circular full-thickness defect was created using a 2-mm biopsy punch (d 0). After regeneration from the surgical trauma, planimetry, and IVM were performed repetitively (d 4, d 8, d 12, d 16, and d 20). During IVM, the microvascular system and leucocytes were visualized by simultaneous intravenous injection of FITC-dextran and Rhodamine 6G. Following IVM on day 20, the chamber tissue was saved for histology. IVM, intravital fluorescence microscopy; d, day; DSC, dorsal skinfold chamber; FITC, fluorescein isothiocyanate-labeled.

Fig. 4

Premature epithelialization of the wound. HE staining of the DSC tissue was performed on day 20. The remaining defect is illustrated 20 days after preparation of a SP, SP-CS, and AS-CS (×5, ×10, ×20 from left to right). The edge of the defect was overgrown by squamous epithelium in all groups. Premature wound epithelialization without defect closure is characteristic for tympanic membrane defects. DSC, dorsal skinfold chamber; SP, symmetric perforation; SP-CS, symmetric perforation with coverslip; AP-CS, asymmetric perforation with coverslip.

Fig. 5

Repetitive planimetry of the perforation size. All defects were prepared using a 2-mm biopsy punch (Æ 3.14 mm²). Following preparation of a SP (n = 5, dropout 1/5 at day 0, blue) the defect size remains unchanged throughout 20 days. However, preparation of a SP-CS (n = 5, dropout 3/5 at days 12, 16, 20, red) or an AS-CS (n = 5, dropout 1/5 at day 15, green) increases spontaneous wound healing and contraction of the perforation. SE was lowest for the SP (without coverslip). Values are given as means ± SEM of independent experiments. SP, symmetric perforation; SP-CS, symmetric perforation with coverslip; AP-CS, asymmetric perforation with coverslip; SE, standard error.

Fig. 6

Repetitive analysis of functional capillary density. Functional capillary density (cm/cm²) was measured by intravital fluorescence microscopy following iv FITC-dextran injection. Two areas of interest were defined by the distance to the perforation: Central area = close to the perforation (within 1 field of view using ×20 magnification, blue) and peripheral area = distant to the perforation (>2 fields of view using ×20 magnification, red). Computer-guided image analysis was workable in all preparations (SP-CS, AP-CS). The functional capillary density was equal in areas close to the perforation and distant to the perforation. Comparison of central areas (underneath a wound dressing) and peripheral areas (beyond a wound dressing) enables for blood flow assessment depending on the wound therapy. Values are given as means ± SEM of independent experiments (n = 5, dropouts: symmetric perforation 1/5 at day 0; SP-CS 3/5 at days 12, 16, 20; AS-CS 1/5 at day 15). FITC, fluorescein isothiocyanate-labeled; SP, symmetric perforation; SP-CS, symmetric perforation with coverslip; AP-CS, asymmetric perforation with coverslip.

Fig. 7

Representative images of the symmetric DSC perforation. a Macroscopic image of the perforation on day 0. b Intravital fluorescence microscopy (FITC-dextran) of the perforation and the surrounding vessels at day 8 (×50). c Intravital fluorescence microscopy (FITC-dextran) of a central capillary field at day 8 (×200). Visualization of wound healing by means of immunohistochemistry: Ki-67 − proliferation (d); CAE − leukocyte activation (e); F4/80 − macrophages (f). DSC, dorsal skinfold chamber; FITC, fluorescein isothiocyanate-labeled; CAE, chloroacetate esterase.