Treatment of vocal fold scarring represents a clinical challenge distinct from injury prevention. Prophylaxis, which has been shown to enhance wound repair and minimize scar formation after injury, has already been identified and has been a focus of the vocal fold scarring literature.3,10–12,25
It would be expected that the two approaches for treating vocal fold scarring—early treatment versus late treatment–would have mechanistically different biological responses. The cellular response would be phenotypically different for tissue that has been recently injured versus a chronic injury. The motivation for the present study is to investigate three approaches of tissue restoration in tissue with established ECM injury.
The functional consequence of the three treatments was measured by rheology. There were statistically significant improvements in the viscoelastic measures for the autologous fibroblast–treated group when compared to saline-treated controls. The controls had significantly elevated rheological values for both dynamic modulus and elastic shear modulus, which may be attributed to their greater levels of fibrosis noted in vocal fold scar as previously published,11,12,18,26
but contradicts collagen levels of the present study. When comparing our results with the literature for chronic injury, there are similar fits for dynamic moduli for all treatment groups. When comparing to a prophylaxic study,12
where HA hydrogels were injected immediately after injury and animals were sacrificed 21–24 days later, curve fit coefficients of G
′ and η
′ of saline controls were quite similar to saline controls of the present study as were coefficients of our sECM and their hydrogel treatment. Viscoelastic measurements for saline controls in this study were also similar to untreated vocal fold scar.18
All groups demonstrated an increase in matrix accumulation. Histological levels of procollagen 1, collagen, and fibronectin were similar to or greater than saline control for all treatment groups. Autologous fibroblast–treated vocal folds have elevated fibronectin; autologous fibroblasts/sECM has elevated collagen and fibronectin; sECM has fibronectin, collagen, and procollagen at significantly higher levels. It is still unclear what role collagen content plays in the viscoelastic function of vocal fold tissues. There have been a number of research papers in laryngology that have measured collagen levels in a chronic model, with a functional outcome measure, in attempts to determine if collagen is responsible for changes in tissue viscoelasticity. Previous work by Chhetri et al.4
in a canine model, demonstrated increased collagen levels after autologous fibroblast injections in scarred vocal folds. Increased levels of collagen were related to improved mucosal wave as measured by stroboscopy. Hertegard and coworkers5
utilized human embryonic stem cells in a rabbit acute model and found improved biomechanical properties without a concomitant change in collagen levels. Compared to normal tissue,18,26
untreated vocal fold scar, with increased viscosity and decreased elasticity in a rabbit model, has been characterized by increased procollagen and fibronectin without changes in collagen levels. Using human cadaver tissue, Chan et al.27
did not find a relationship between tensile elastic modulus of the vocal fold cover or ligament and relative collagen density.
The best biomechanical outcomes—autologous fibroblast–treated group—were characterized with increased fibronectin and the lowest collagen staining of the three experimental conditions. Increased levels of fibronectin have been reported in untreated scar26
and have been related to increased viscosity in benign lesions. Guarnieri et al.28
reported that fibronectin organizes in aggregates, intersperses in collagen, and in thin fibrils, distributes along collagen fibers. High fibronectin concentrations affected collagen fiber assembly and structure leading to drastic effects on rheological properties. The improved rheological properties in this investigation could be related to varying orientations of the proteins, augmented by the upregulation of fibronectin. Our present histological image analysis is inadequate to study such relationships and orientation. Further studies are necessary for assessing the organization and relationship between the proteins to better understand the contribution of specific proteins to ECM rheological properties. To provide an understanding of the full mechanism, it would be beneficial to measure tissue changes earlier after treatment.
Using autologous fibroblasts has led to better tissue restoration in vocal folds1,2,4
and dermal skin.29,30
Autologous fibroblasts allow for permanent engraftment. Use of autologous fibroblasts encapsulated in a scaffold would seem ideal. In this study, even though not statistically significant, the autologous fibroblast–treated/sECM-treated vocal folds had a more favorable biomechanical outcome than sECM alone. Previous research demonstrated cytocompatibility of cells in sECM,31
and improved viscoelasticity and less fibrosis in an acute wound rabbit model for the HA hydrogels were utilized in this research.12
With the addition of autologous cells, viscoelasticity was further improved. Despite collagen levels being higher, the increase in fibronectin of the fibroblast/sECM condition over the sECM condition may have compensated. Fibroblasts may very well have altered gene and protein expression in the presence of the sECM. Further, in vitro
studies are necessary to determine the effects of sECM on cell behavior. Moreover, vocal fold fibroblasts may not be an option for the clinical setting, given lack of availability; subsequently, further investigation utilizing more accessible fibroblasts, that is, dermis and/or gingival, is warranted.
In summary, findings of this study indicate that injections of autologous fibroblast into established vocal fold scar significantly improved tissue biomechanical properties closer to normal levels over the injection of autologous fibroblasts with sECM or sECM alone, as evaluated 2 months after injury. Our results also provide insight into the relative contribution of collagen, procollagen, and fibronectin to tissue biomechanics. Further investigation is necessary to understand the underlying biology of the chronic vocal fold scar, the relationship between scaffolds and cells in vitro and in vivo, and the relationship between the protein constituents of the vocal fold and rheological outcomes. Optimization of cell–scaffold interactions and subsequent cell behavior is necessary for utilization of scaffold and scaffold–cell approaches.