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Objectives/hypothesis: Vocal fold scarring, which causes severe hoarseness, is intractable. The optimal treatment for vocal fold scarring has not been established; therefore, prevention of scarring is important. The aim of this study was to clarify the effectiveness of basic fibroblast growth factor (bFGF) for prevention of postsurgical vocal fold scarring.Study Design: Prospective animal experiments with controls.Method: The vocal folds of Sprague-Dawley rats were injured unilaterally or bilaterally after local application of a 10 μL solution of bFGF. Larynges ware harvested for histological and immunohistochemical examination 2 months postoperation and for quantitative real-time polymerase chain reaction (qRT-PCR) analysis 1 week postoperation.Results: Histological examination showed significantly increased hyaluronic acid and decreased deposition of dense collagen in the bFGF-treated group at 100 ng/10 μL compared with the sham-treated group. Immunohistochemical examination showed significantly decreased collagen type III deposition in the bFGF-treated group at 100 ng/10 μL compared with the sham-treated group. qRT-PCR revealed that hyaluronan synthase 2 (Has2), Has3, and hepatocyte growth factor were upregulated in bFGF-treated groups compared with sham-treated group.Conclusion: The current results suggest that local application of bFGF at the time of injury has the potential to prevent vocal fold scarring. Preventive injection of bFGF could be applied at the time of phonomicrosurgery to avoid postoperative scar formation.Level Of Evidence: N/A. Laryngoscope, 2016 127:E67-E74, 2017. [ABSTRACT FROM AUTHOR]

Objectives/hypothesis: Vocal fold scar remains a therapeutic challenge. Mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. Nevertheless, few in vivo studies have directly compared various sources of MSCs. The aim of this study was to investigate the therapeutic potential of adipose-derived stromal cells (ASCs) in comparison with bone marrow-derived stromal cells (BMSCs) for vocal fold regeneration.Study Design: Prospective animal experiments with controls.Methods: Two months after stripping of the lamina propria, 18 beagles were divided into four implantation groups: atelocollagen alone (collagen group), atelocollagen with BMSCs (BMSC-collagen), atelocollagen with ASCs (ASC-collagen), or a sham-treated group. One or 6 months after implantation, vibratory and histological examinations were performed.Results: Mucosal vibration was significantly improved in both of the MSC-implanted groups compared with the sham-treated group, whereas only the ASC-collagen group showed a significantly smaller glottal gap than the collagen group. Moreover, in the ASC-collagen group, a significant reduction of collagen density was observed compared to the sham-treated group, and there was a trend for better restoration of hyaluronic acid (HA). Implanted MSCs were detected 1 month postimplantation; however, none survived 6 months postimplantation.Conclusions: Although implantation of an atelocollagen sponge and ASCs into vocal fold scars induced vibratory recovery comparable to that of BMSCs, ASCs might have more potential in terms of restoration of HA and suppression of excessive collagen deposition.Level Of Evidence: NA Laryngoscope, 126:1143-1150, 2016. [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: Vocal fold injury can cause intractable scarring resulting in dysphonia. Mesenchymal stem cells (MSCs) have great therapeutic potential in wound healing. They continuously circulate in the peripheral blood and migrate into wound sites where they induce regenerative effects. However, their roles in vocal fold wound healing are poorly understood because few MSCs exist in the peripheral blood and there is no specific marker to identify them. The present study evaluates how intravenously injected MSCs affect vocal fold wound healing using Green Fluorescent Protein (GFP) -labeled MSCs. Study Design: Prospective study using animal model. Methods: GFP-labeled MSCs were obtained from femurs of GFP transgenic Sprague-Dawley rats and incubated in culture. Sprague-Dawley rats underwent intravenous injection of GFP-labeled MSCs (1.0 × 106 cells) immediately after vocal fold injury. Histological examination was performed. Results: Injected MSCs were distributed throughout the vocal fold wound site from day 1 up to day 56. These vocal folds showed increased hepatocyte growth factor (HGF)-positive cells within the wound and improved wound healing compared with sham-treated folds. Conclusion: Circulating MSCs can migrate to vocal fold wound sites and upregulate the expression of HGF during wound healing; thus, they are considered to play a significant role in wound healing within the vocal folds. Laryngoscope, 2012 [ABSTRACT FROM AUTHOR]

Objectives/Hypothesis: In the treatment of tumorous diseases, scarring often forms after resection or irradiation. Scarring of the buccal mucosa causes difficulty in opening the mouth and mastication, decreasing quality of life. Transforming growth factor (TGF) β3 is an isoform of TGFβ1 that is known to accelerate scarring, although it has different effects on wound healing. TGFβ3 administration into wounds has been associated with improvement in the quality of healing skin in vivo. TGFβ3 is also considered to be an important anti-scarring factor in buccal mucosa. The present study aimed to examine whether TGFβ3 is effective for prevention and treatment of buccal mucosa scarring. Study Design: Prospective study using an animal model. Methods: Thirty Sprague-Dawley rats were involved in this study. We injected 0.5 mL of TGFβ3 (0.005 lg/mL, 0.05 lg/mL, 0.5 lg/mL, 5 lg/mL) or saline was injected into the buccal submucosa. Fifteen minutes after the injection, the mucosa was removed down to the masseter muscle or orbicularis oris muscle layer using a 6-mm biopsy punch. Six weeks after the operation, the buccal mucosae were harvested after euthanasia. Morphologic and histologic examinations were performed. Results: The administration of 0.5 lg/mL TGFβ3 induced rapid re-epithelialization and suppressed scar formation. In the submucosal layer, favorable restoration of hyaluronic acid and elastin was seen in the TGFβ3-treated groups compared to the saline-treated group. Conclusions: TGFβ3 is considered to be effective for better restoration of extracellular matrices of injured buccal mucosa, suggesting a preventative effect of buccal mucosa scarring. [ABSTRACT FROM AUTHOR]

Objectives: Vocal fold scarring remains a therapeutic challenge. A new regenerative approach is needed to restore disorganized extracellular matrix. Tissue regeneration requires appropriate cells and a scaffold. Bone marrow-derived mes- enchymal stromal cells (BMSCs) are multipotent and secrete many kinds of growth factors to regenerate tissues. Ate- locollagen sponges have many large pores that permit cell entry. The present study was performed to evaluate whether implantation of an atelocollagen sponge plus BMSCs is effective for the treatment of vocal fold scarring. Methods: Twelve beagles underwent implantation of an atelocollagen sponge or of an atelocollagen sponge with autolo- gous BMSCs ( 1.0 x 106 cells) in the subepithelial pockets of scarred vocal folds. Six months after the operation, vibratory examinations and histologie examinations were performed. Results: Mucosal vibrations improved significantly for the atelocollagen sponge-implanted vocal folds. Histologie analyses revealed favorable restoration of the extracellular matrix in the lamina propria. Increased distribution of hyaluronic acid and decreased dense collagen deposition were also noted. These improvements were enhanced by implantation of BMSCs. Conclusions: Implantation of atelocollagen sponges with autologous BMSCs into scarred vocal folds significantly increased hyaluronic acid distribution and decreased dense collagen deposition in the lamina propria, leading to better mucosal vibration. [ABSTRACT FROM AUTHOR]

Objectives: The overarching goal of this line of research is to translate basic fibroblast growth factor (bFGF) treatment for vocal fold scarring into practical clinical use. In a previous canine investigation, we demonstrated that bFGF improves phonation threshold pressure, mucosal wave amplitude, and histologic measures in vocal folds treated after injury. In the present study, we studied the effects of bFGF on gene expression of the extracellular matrix and growth factors in rat vocal fold fibroblasts. Methods: Fibroblasts harvested from the vocal folds of 5 rats were treated with 3 concentrations of bFGF (0, 10, and 100 ng/mL). The fibroblasts were collected at 24 hours and 72 hours after bFGF administration. Quantitative polymerase chain reaction was then used to investigate the gene expression of the investigated growth factors and extracellular matrices. Results: The results revealed significantly down-regulated expression of procollagen I and significantly up-regulated expression of hyaluronic acid synthase (HAS) 2 and fibronectin in fibroblasts treated with bFGF. The administration of bFGF also resulted in the up-regulation of bFGF and hepatocyte growth factor (HGF). No changes in the expression of HAS-1, tropoelastin, or procollagen III were observed between the treatment and control conditions. Conclusions: Treatment with bFGF induces the down-regulation of procollagen I and the up-regulation of HAS-2 in vocal fold fibroblast cell cultures. These gene expression alterations to key mediators of the wound healing process may translate into potential benefits in the remediation of vocal fold injury. The up-regulation of HGF, an antifibrotic effector molecule, may demonstrate additional benefits by optimizing the wound healing environment and by accelerating the wound repair cascade. These findings may provide fuel for additional discoveries into the development of growth factor therapy for the treatment of vocal fold scar [ABSTRACT FROM AUTHOR]

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow-derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from call dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow-derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow-derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds. [ABSTRACT FROM AUTHOR]

Objectives: Inflammatory factors are key mediators of wound healing processes following injury, and their modulation may improve healing outcomes. The objective of this study was to characterize in vivo inflammatory factor and extracellular matrix (ECM) messenger RNA (mRNA) expression levels 1 hour after vocal fold injury. Methods: Five Sprague-Dawley rats were subjected to bilateral vocal fold injury, 5 rats were reserved as uninjured controls, and 1 rat was subjected to unilateral vocal fold injury and reserved for histology. Tissue was harvested 1 hour after injury. Real-time reverse transcription-polymerase chain reaction was performed to examine the mRNA expression profiles of inflammatory factors nuclear factor kappa beta (NF-κβ), interferon gamma (IFN-γ), cyclooxygenase 2 (COX-2), transforming growth factor beta isoform 1 (TGF-β1), tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1β), as well as ECM genes hyaluronic acid synthase (HAS) 1, HAS-2, procollagen 1, procollagen 3, and elastin, in the injured samples compared with the uninjured controls. Results: Injury resulted in subepithelial bleeding throughout the vocal fold. The COX-2, TNF-α, IL-1β, and HAS-1 mRNA expression levels were significantly up-regulated 1 hour after injury compared with the uninjured controls. Conclusions: Inflammatory factor and ECM gene expression changes occur in vocal fold wound sites as early as 1 hour after injury. These results should inform future efforts to attenuate vocal fold scarring via the modulation of inflammatory factors. [ABSTRACT FROM AUTHOR]

Objectives: Vitamin A plays important roles in development, growth, and regeneration. Vitamin A-storing stellate cells have been identified in several organs. The functional roles of vitamin A in the vocal folds are still unknown, although vitamin A-storing vocal fold stellate cells have been observed in the macula flava of human and rat vocal folds. The purpose of this study was to investigate the roles of vitamin A in vocal folds. Methods: Vitamin A-deficient rats were generated, and the vocal folds were examined histologically. Messenger RNA was extracted from the vocal folds and analyzed by real-time polymerase chain reaction. Results: Immunohistochemical analysis of normal vocal folds revealed expression of retinoic acid receptor a in vocal fold stellate cells. The cells in the macula flava of vitamin A-deficient rats showed a larger nucleus/cytoplasm ratio than did those of vitamin A-sufficient rats, but messenger RNA expression of major extracellular matrix components in the macula flava of vitamin A-deficient rats did not present a remarkable change except for procollagen type I. Expression of hyaluronic acid, collagen types I and III, and elastin did not show a significant change in vitamin A-deficient rat vocal folds. Conclusions: These results indicate that vitamin A is not essential to maintaining the extracellular matrix of normal adult vocal folds, although vocal fold stellate cells participate in vitamin A storage. [ABSTRACT FROM AUTHOR]

Objectives: Vocal fold scarring is the major cause of voice disorders after voice surgery or laryngeal trauma. The role of inflammatory factors in vocal fold wound healing and fibrosis has not been adequately investigated. Scarless wound healing has been associated with decreased inflammatory responses. To understand scar formation and develop reliable treatments, it is necessary to control extracellular matrix production and inflammation. Thus, we examined the inflammation profile and extracellular matrix production in wounded vocal folds in the acute phase of wound healing. Methods: Vocal fold stripping was performed on 30 Sprague-Dawley rats. Vocal fold tissue was collected at 5 time points (4, 8, 16, 24, and 72 hours). We examined the in vivo messenger RNA expression profile of inflammatory factors interleukin 1β, interferon γ, tumor necrosis factor α, nuclear factor κβ, transforming growth factor β, and cyclooxygenase 2, as well as hyaluronic acid synthases 1 and 2, procollagen subtypes I and III, and elastin synthase in scarred vocal folds after injury, compared to normal vocal folds, using real-time reverse transcription-polymerase chain reaction. Results: The inflammatory factors showed a time-dependent sequence of expression peaks, starting with interleukin 1β, nuclear factor κβ, tumor necrosis factor α (4 and 8 hours), and transforming growth factor β (72 hours). Interferon γ decreased at 24 hours. Correspondingly, hyaluronic acid synthase I expression peaked first (4 and 8 hours), whereas hyaluronic acid synthase 2 expression peaked at 16 hours and again at 72 hours. Procollagen I expression peaked at 72 hours, whereas procollagen III decreased from 8 to 16 hours but peaked at 72 hours. Cyclooxygenase 2 expression was elevated, whereas elastin expression remained constant. Conclusions: The results show a clear profile of vocal fold inflammation with corresponding changes in extracellular matrix production. [ABSTRACT FROM AUTHOR]

Objectives: We used an acute vocal fold injury in a rat model to characterize vocal fold wound healing by studying the expression pattern of the extracellular matrix components in the vocal fold lamina propria. Methods: Vocal fold stripping was performed unilaterally in 27 Sprague-Dawley rats. The vocal folds were harvested at 5 time points (1, 3, 5, 7, and 14 days) and histologically analyzed by Alcian blue stain, trichrome stain, and immunofluorescence with antibodies to collagen type I, collagen type III, and fibronectin. Results: Re-epithelialization occurred by day 3 and was complete by day 14. Granulation tissue was formed by day 3. Hyaluronic acid and collagen type I appeared in injured vocal folds by day 3, peaked at day 5, and thereafter decreased. Collagen type III and fibronectin appeared by day 1 and continued to be intense at all time points after day 3. Conclusions: These results suggest that the expression of these extracellular matrix components peaks in the period around days 3 to 5, and that the characteristics of wound healing in the vocal fold are similar to those in the skin in the early phases, but differ during the subsequent remodeling phase. [ABSTRACT FROM AUTHOR]

This study aimed to clarify the characteristics of rat vocal fold scarring by examining the alteration of key components in the extracellular matrix: hyaluronic acid, collagen, and fibronectin. Under monitoring with a 1.9-mm-diamcter telescope, unilateral vocal fold stripping was performed, and larynges were harvested at 2, 4, 8, and 12 weeks after operation. The vocal folds were histologically analyzed with Alcian blue stain, trichrome stain, and immunofluorescence of collagen type I, collagen type III, and fibronectin. The scarred vocal folds showed less hyaluronic acid and more collagen types I and III than did the controls at all time points. Type III was stable for 12 weeks, while type I declined until 8 weeks and thereafter remained unchanged. Fibronectin increased for 4 weeks and then decreased; it was close to the control level at 8 and 12 weeks. These results suggest that the tissue remodeling process in scarred vocal folds slows down around 2 months after wounding. [ABSTRACT FROM AUTHOR]