Laryngoscopic images after injection laryngoplasty of sECM/MC hydrogels into the left paralyzed vocal fold.

<p>The sECM/MC hydrogel injection group exhibited a straight and medialized vocal fold (white arrowhead) while the control group had a curved and lateralized vocal fold (black arrowhead), which is likely due to denervation of the recurrent laryngeal nerve.</p

Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold - Fig 2

<p><b>Representative serial images of high-speed camera recording at 8 weeks (A), the asymmetric index using videokymograms (B) and the results of asymmetry index for vocal functional analysis (C)</b>. (A) Normal and symmetrical vocal contacts showed no change in the vibration of vocal mucosa in sECM/MC groups relative to the control group. (B) The maximum distance in the left denervated vocal fold (<i>a</i>) was compared to the right vocal fold (<i>b</i>) using a videokymogram to generate an asymmetry index. The asymmetry index was calculated as follows: Asymmetry index = <i>a</i> / <i>b</i>. (C) The mean asymmetry index of the sECM/MC hydrogel group (1.020 ± 0.069) and the control group (0.787 ± 0.102) are shown (<i>p</i> = 0.047 using a Mann-Whitney U test). In diseased conditions, the index deviates from the value of 1.0</p

Foamy histiocyte with intracellular fatty lobules and neo-vascularization in the injected sECM/MC hydrogels.

<p>(A) Focal capillary ingrowths (arrowhead) into the injection site. Cell aggregations with intracellular fatty lobules in the injection area (asterisk). (B) RAM 11 positive cells (dark brown) are foamy histiocytes, which increased as a function of time.</p

Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold - Fig 3

Standard hematoxylin and eosin (H&E) staining of rabbit larynx after injection laryngoplasty into the left paralyzed vocal fold (A) and the quantitative analysis of remaining volume of sECM/MC hydrogels (B). (A) Histological examination of the injected biomaterials at 8 weeks post procedure. Area of the laryngeal intrinsic muscle was smaller on the denervated side in the control group (green dotted line) than on the contralateral normal side. In the sECM/MC group, the laryngeal muscle area was compensated for by the injected sECM/MC hydrogel (brown dotted line). The injected sECM/MC hydrogel (arrowhead) induced no significant inflammatory response including neutrophils or lymphocytes aggregation in the surrounding muscle (*), lamina propria (†), or epithelium (‡). (B) Quantitative analysis of remaining sECM/MC hydrogel volume (p = 0.501 using Kruskal-Wallis test).</p

Regeneration of Paralyzed Vocal Fold by the Injection of Plasmid DNA Complex-Loaded Hydrogel Bulking Agent

Various growth factor delivery systems were used in the treatment of glottal insufficiency; however, relatively little attention has been paid to a gene delivery system for aspects of active vocal fold (VF) regeneration. Herein, we present a plasmid DNA (pDNA; bFGF gene encoding) complex-loaded alginate (ALG)/hyaluronic acid (HA) mixture hydrogel dispersed with polycaprolactone (PCL) microspheres that can enhance simultaneous regeneration of VF muscle and lamina propria, as well as have a bulking effect on atrophied VF. We have demonstrated long-term efficacy of bFGF synthesized from pDNA complex-transfected cells in vitro. PCL microspheres–dispersed ALG/HA hydrogel (with or without pDNA complex loading) are injected into rabbit VFs with recurrent laryngeal nerve denervation. The PCL microspheres dispersed in the hydrogel bulking agents remain stable at the applied site, leading to constant medialization of the paralyzed VF without significant initial volume loss even after 24 weeks. A regenerative effect for collagen deposition and HA synthesis around the injected site, which are major components of VF tissue, is well confirmed in the pDNA-complex-loaded hydrogel group. Moreover, the compensation of atrophied VFs also leads to the contact of bilateral VF and the remarkable recovery of voice function in the pDNA-complex-loaded group. Based on the results, pDNA (bFGF encoding) complex-loaded hydrogel dispersed with PCL microspheres may be employed as a bioactive bulking agent for the treatment of glottal insufficiency

Vocal Fold Augmentation with Injectable Polycaprolactone Microspheres/Pluronic F127 Hydrogel: Long-Term <i>In Vivo</i> Study for the Treatment of Glottal Insufficiency

<div><p>There is increasing demand for reconstruction of glottal insufficiency. Several injection materials have been examined for this purpose, but all had limitations, such as poor long-term durability, migration from the injection site, inflammation, granuloma formation, and interference with vocal fold vibration due to viscoelastic mismatch. Here, we developed a novel injection material, consisting of polycaprolactone (PCL) microspheres, which exhibits better viscoelasticity than conventional materials, and Pluronic F127 carrier, which decreases the migration of the injection materials. The material was injected into rabbits with glottal insufficiency and compared with the FDA-approved injection material, calcium hydroxylapatite (CaHA). Endoscopic and histological examinations indicated that PCL/Pluronic F127 remained at the injection site with no inflammatory response or granuloma formation, whereas CaHA leaked out and migrated from the injection site. Therefore, vocal fold augmentation was almost completely retained during the 12-month follow-up period in this study. Moreover, induced phonation and high-speed recording of vocal fold vibration showed decreased vocal fold gap area in the PCL/Pluronic F127 group. Our newly developed injection material, PCL/Pluronic F127, permits efficient augmentation of paralyzed vocal fold without complications, a concept that can be applied clinically, as demonstrated by the successful long-term follow-up.</p></div

Schematic showing the fabrication process of PCL microspheres by an isolated particle-melting method.

<p>The crushed PCL microparticles isolated in a Pluronic F127 gel matrix are melted at a temperature above the melting temperature of PCL, and the molten microparticles are spontaneously constrained into spherical shapes.</p

Serial endoscopic images of a larynx injected with PCL microspheres/Pluronic F127 or CaHA were taken for 1 year.

<p>From left to right, images were taken immediately after injection, 1-up period. No inflammatory reaction was observed in either group.</p

Analysis of residual volume of the injected materials.

<p>(A) Method to calculate the cross-sectional material area. (B) Comparison of the remaining volumes of injected PCL and CaHA 12 months after injection. *: P<0.01.</p

Serial vocal fold vibration images taken by a high-speed camera at a speed of 8000 frames per second.

<p>The vocal fold gap was wider in the CaHA group than in the PCL group.</p