Ultrafast Laser Microlaryngeal Surgery for In Vivo Subepithelial Void Creation in Canine Vocal Folds

Abstract

Background/objectives: Tightly-focused ultrafast laser pulses (pulse widths of 100 fs-10 ps) provide high peak intensities to produce a spatially confined tissue ablation effect. The creation of sub-epithelial voids within scarred vocal folds (VFs) via ultrafast laser ablation may help to localize injectable biomaterials to treat VF scarring. Here, we demonstrate the feasibility of this technique in an animal model using a custom-designed endolaryngeal laser surgery probe.

Methods: Unilateral VF mucosal injuries were created in two canines. Four months later, ultrashort laser pulses (5 ps pulses at 500 kHz) were delivered via the custom laser probe to create sub-epithelial voids of ~3 × 3-mm² in both healthy and scarred VFs. PEG-rhodamine was injected into these voids. Ex vivo optical imaging and histology were used to assess void morphology and biomaterial localization.

Results: Large sub-epithelial voids were observed in both healthy and scarred VFs immediately following in vivo laser treatment. Two-photon imaging and histology confirmed ~3-mm wide subsurface voids in healthy and scarred VFs of canine #2. Biomaterial localization within a void created in the scarred VF of canine #2 was confirmed with fluorescence imaging but was not visualized during follow-up two-photon imaging. As an alternative, the biomaterial was injected into the excised VF and could be observed to localize within the void.

Conclusions: We demonstrated sub-epithelial void formation and the ability to inject biomaterials into voids in a chronic VF scarring model. This proof-of-concept study provides preliminary evidence towards the clinical feasibility of such an approach to treating VF scarring using injectable biomaterials.

Level of evidences: N/A Laryngoscope, 133:3042-3048, 2023.

Keywords: biomaterials; endoscopy; microlaryngeal surgery; phonosurgery.

Fig. 1.

Experimental setup. (a) Schematic of the mobile laser surgery system. The system consists of (1) the probe, (2) three-axis motorized stage with stepper motors, (3) two linear stages, (4) articulating arm, (5) fiber laser laptop, (6) fiber laser, (7) Kagome fiber coupling optics, (8) 2’×3’ optical table, (9) mobile cart, (10) fiber laser chiller, and (11) power supplies and controllers for the laser, shutter, stepper motors, and piezo-fiber scanning element. (b) Picture of the mobile laser surgery system built for use in operating room at UT Southwestern. (c) Laser surgery probe inserted through rigid laryngoscope into whole excised porcine larynx as performed in the lab. (d) Same setup as (c), except laryngoscope is removed and the probe tip orientation on the inferior porcine VF is shown in a hemilarynx. (c) and (d) illustrate the representative geometry of the laser surgery system that was used for the in vivo canine experiments.

Fig. 2.

In vivo laser ablation of healthy canine VF using miniaturized ultrafast laser surgery system. (a) 0° telescopic image of healthy VF (left) and scarred VF (right) in canine #2. (b) 70° telescopic image of sub-epithelial void immediately after laser ablation in healthy VF of canine #2. Red arrow points to the top left corner of the void. (c) H&E of void shown in (b). The left-right direction of the figure is lined up with the anterior-posterior direction of the vocal fold mucosa. Scale bar is 500 μm.

Fig. 3.

In vivo laser ablation of scarred canine VF using miniaturized ultrafast laser surgery system. (a) 70° telescopic image of sub-epithelial void immediately after ablation in scarred VF of canine #2 (blue arrow). Image of the void created in healthy VF (Fig. 2) of canine #2 is still visible ~30 minutes after ablation (red arrow). (b) TPF/SHG images of void in scarred VF shown in (a). Center image shows the void 123 μm deep into the VF. A clearly defined square-shaped void is observed at this depth and collagen fibers near the void margin indicate ablation within the scarred SLP. Cross sections through the centerlines of the center image are shown below and to the right. Black arrows in cross section images indicate the x-y image plane shown in center image. (c) Montage of selected frames from the z-stack. Images in epithelium are taken with TPF (first column), deeper imaging planes acquired with SHG (second column). Scale bars are 500 μm. (d) Trichrome stain of void in scarred VF shown in (a), (b), and (c). The left-right direction of the figure is lined up with the anterior-posterior direction of the vocal fold mucosa. Scale bar is 100 μm.

Fig. 4.

Injection and localization of Rhodamine tagged PEG30 biomaterial within sub-epithelial voids created in vivo with miniaturized ultrafast laser surgery system. (a) White light image of biomaterial injected into void. The void can be seen as a raised “blister”, similar to previous results. (b) Void + biomaterial imaged with fluorescence stereo-microscope. The white dotted ovals in (a) and (b) circumscribe the void. A large gas bubble created during the ablation process is visible in (a) and (b). Red arrow in (b) shows the needle entry point. (c) SHG and (d) TPF images taken at a depth of 105 μm in the healthy VF of canine #2 after ex vivo biomaterial injection. (e) Merged SHG/TPF images shown in (c) and (d). TPF signal (red) is generated from Rhodamine, while SHG signal (green) comes from collagen fibers. Scale bars are 500 μm.