In Vivo Engineering of the Vocal Fold ECM With Injectable HA Hydrogels—Late Effects on Tissue Repair and Biomechanics in a Rabbit Model
Introduction
Normal vocal fold vibration is dependent on tissue composition and viscoelasticity. When composition of the extracellular matrix (ECM) of the vocal fold cover (ie, lamina propria—superficial and middle layers) is altered, vocal fold vibratory function can be severely disrupted because of alterations in tissue viscoelasticity.1, 2, 3, 4 The dysphonias that result are generally difficult to treat effectively with current surgical paradigms and available biomaterials. Treatment failures have been ascribed to poor understanding of pathologic processes in the ECM, as well as suboptimal materials that may negatively affect vocal fold biomechanical properties. Accordingly, there is a clinical need for improved understanding of the pathophysiology of disrupted ECM and the development of advanced biomaterials that appreciate the biomechanical properties of the lamina propria. The long-term aim of our laboratory's efforts was to engineer injectable products that promote wound repair and induce tissue regeneration, for treatment of scarring and other ECM defects of the lamina propria.
To date, we have optimized a hyaluronic acid (HA) hydrogel Extracel, for prophylaxic use as a means of improving wound healing. Extracel is gelatin-DTPH, a thiolated derivative of gelatin that is covalently co-cross-linked with Carbylan-S, using poly ethylene glycol diacrylate (PEGDA) as the thiol-reactive cross-linker. Extracel and Carbylan-S have been developed by the Center for Therapeutic Biomaterials at the University of Utah. The hydrogel has been shown to have viscoelastic properties similar to those of human vocal fold mucosa in vitro.5, 6 In vivo, Extracel has been shown to improve wound healing in a prophylactic rabbit wound healing model6, 7 as measured by histological outcomes and protein and gene expression. Furthermore, it has been shown to be biocompatible8 and noninflammatory.8 Rheologically, vocal folds treated at the time of injury with Extracel have been shown to have similar biomechanical properties (tissue elasticity and viscosity) to normal tissue and less stiffer and less viscous vocal fold tissues compared with saline-treated injured vocal folds.6, 7, 9
To date, all in vivo investigations of the efficacy of Extracel have been completed with a 21-day end point. The aim of this study was to evaluate the long-term (6 months) transcript and rheological changes in vocal fold tissue after the intentional vocal fold research, in response to the prophylaxic treatment with Extracel compared with saline-treated controls, to determine if improvements seen at the early time point continue into the chronic injury phase.
Section snippets
HA-gelatin hydrogel
The engineered injectable chemically modified HA-gelatin hydrogel, Extracel (Sentrx Surgical, Inc., Salt Lake City, UT; now Carbylan BioSurgery, Palo Alto, CA) was developed in conjunction with the Center for Therapeutic Biomaterials at The University of Utah. Extracel was prepared by mixing a 1.5% solution of Carbylan-S in phosphate buffered saline (PBS; pH 7.4) with a 4.0% (v/v) solution of PEGDA and PBS (pH 7.4) according to a volume ratio of 4 to 1. Thiolated gelatin (gelatin-DTPH) was
Results
Gene transcript levels for fibronectin, fibromodulin, TGF-β1, procollagen I, HA synthase, and hyaluronidase were measured for five rabbit vocal folds for each condition. Results are shown in Figure 1. When compared with saline-treated vocal folds, at 6-month postsurgery, significantly less transcript levels were measured for fibronectin (P = 0.0001), fibromodulin (P = 0.000), TGF-β1 (P = 0.0011), procollagen (P = 0.0005), and HA synthase (P = 0.000). A nonsignificant difference was measured for
Discussion
Scarring of the vocal folds after surgical resection can have adverse effects on voice production, and efforts have been made to minimize the impact of surgery using microsurgical techniques.11 Prophylaxis of the wound healing process at the time of surgery with HA, an endogenous glycosaminoglycan that has been shown to aid in the healing process through to 6 months after injury, may be an approach for minimizing scar tissue formation during the acute phase of wound healing while maintaining
Conclusions
Prophylactic injection of an HA hydrogel, Extracel at the time of surgical resection was evaluated in rabbit vocal folds. Extracel-treated vocal folds had viscous moduli and elastic shear moduli values that were significantly lower than saline-treated scarred tissue. The improved viscoelastic properties of the vocal folds measured at 6 months posttreatment appear to be due to improved healing with less fibrosis of the ECM. These finds demonstrated that the early benefits of the HA injections,
Acknowledgments
NIH Grant R01 DC4336 from the National Institute of Deafness and other Communicative Disorders funded this work.
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