Rat Vocal Folds Research Articles

Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds

Following injury, pathologically activated vocal fold fibroblasts (VFFs) can engage in disordered extracellular matrix (ECM) remodeling, leading to VF fibrosis and impaired voice function. Given the importance of scar VFFs to phenotypically appropriate in vitro modeling of VF fibrosis, we pursued detailed characterization of scar VFFs obtained from surgically injured rat VF mucosae, compared with those obtained from experimentally naïve, age-matched tissue. Scar VFFs initially exhibited a myofibroblast phenotype characterized by increased proliferation, increased Col1a1 transcription and collagen, type I synthesis, increased Acta2 transcription and α-smooth muscle actin synthesis, and enhanced contractile function. These features were most distinct at passage 1 (P1); we observed a coalescence of the scar and naïve VFF phenotypes at later passages. An empirical Bayes statistical analysis of the P1 cell transcriptome identified 421 genes that were differentially expressed by scar, compared with naïve, VFFs. These genes were primarily associated with the wound response, ECM regulation, and cell proliferation. Follow-up comparison of P1 scar VFFs and their in vivo tissue source showed substantial transcriptomic differences. Finally, P1 scar VFFs responded to treatment with hepatocyte growth factor and transforming growth factor-β3, two biologics with reported therapeutic value. Despite the practical limitations inherent to working with early passage cells, this experimental model is easily implemented in any suitably equipped laboratory and has the potential to improve the applicability of preclinical VF fibrosis research.

Distribution and characteristics of slow-cycling cells in rat vocal folds.

Stem cells are known to proliferate at a slow rate in adult organs, and thus slow-cycling cells exhibiting pluripotency are considered tissue-specific stem cells in some organs. Slow-cycling cells in the vocal fold (VF) have not been well documented. Here we sought to clarify the distributions and characteristics of slow-cycling cells in rat VFs. We applied double-labeling technique to detect the distribution of slow-cycling cells. We injected the exogenous proliferation marker 5-bromo-2'-deoxyuridine (BrdU) into Sprague-Dawley rats. After a chasing period, VFs were immunostained with antibodies to BrdU and the second endogenous proliferation marker, Ki-67. BrdU (+) Ki-67(+) cells were regarded as slow-cycling cells and counted by VF regions. To reveal slow-cycling cells' characteristic, their immunophenotypes were histologically investigated and their kinetics in injured VFs were evaluated. Most slow-cycling cells were detected in the basal layer of the epithelium. Slow-cycling cells in the epithelium displayed a low positive ratio of E-cadherin and CK5 and a high positive ratio of vimentin and CD31 as compared with the other epithelial cells. The expression of S100A4 was low in slow-cycling cells of the lamina propria and the macula flava. FGFR1, HAS1, HAS2, and HAS3 were not detected in the slow-cycling cells. A time-dependent reduction of slow-cycling cells was observed in injured VFs. Most slow-cycling cells resided in the epithelium, exhibiting various phenotypes in a relatively undifferentiated condition, and they are suspected to contribute to the tissue repair of the injured VFs. N/A.

Treatment of Acute Vocal Fold Injury With Platelet-Rich Plasma

Platelet-rich plasma (PRP) is a reliable and has low side-effect profile and has beneficial effects on wound healing. Its investigatory effects on wound-healing process were shown on various tissues. The aim of the present study was to evaluate effectiveness of PRP application on scar tissue of acute vocal fold injury. Twenty-four Wistar rats were used in the study. The entire layer of the lamina propria down to the thyroarytenoid muscle of 10 subjects was unilaterally injured by with a microscissor. Gelfoam-absorbed PRP was applied on the injured area for 10minutes. Control group consisted of rats unilaterally injured using a microscissor, and gelfoam with normal saline was applied on the injured area. Following sacrifice, the larynxes were carefully dissected and removed for histopathologic examination. After excised larynx experiments, serial sections were prepared from vocal fold. Hematoxylin eosin and immunohistochemical staining were done for epithelial growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR1), and vascular endothelial growth factor (VEGF) staining for histopathologic examinations. There was not a significant difference between the two groups for lymphocyte. Although collagen and VEGF were higher in the study group, there was not a significant difference between the groups (P>0.05). There was a significant difference between control and study groups for EGFR and FGFR1(P<0.05). PRP has beneficial effects on wound healing. PRP accelerates epithelization of injured rat vocal folds by inducing EGFR secretion. PRP is an autogenous, reliable, low side-effect profile, easily harvested material. PRP may be useful to prevent scar formation.

Paradoxical movement of rat vocal folds following recurrent laryngeal nerve injury

Objective: To establish a rat model with paradoxical vocal fold movement to understand the detailed etiology and physiology of laryngeal synkinesis by evaluating vocal fold movement and by electromyography. Methods: Adult Wistar rats were used. The recurrent laryngeal nerve was transected, anastomosed, and the anastomotic portion was placed in a silicone tube. At 2, 4, and 10 weeks after the treatment (n = 30), we performed laryngoscopy and electromyography. The vocal fold movement was recorded, the hemiglottal area was measured, and the Δarea was calculated by subtracting the area during expiration from that during inhalation. We evaluated the ratio of the Δarea on the treated side to that of the normal side. After laryngoscopy, electromyography of the thyroarytenoid and posterior cricoarytenoid muscles was performed. Results: The mean Δareas were 1.5 ± 3.4%, 2.3 ± 21.5%, and 0.7 ± 31.8% at 2, 4, and 10 weeks after anastomosis, respectively. Eighteen of 18 rats indicated synkinetic reinnervation at 4 and 10 weeks. Regarding vocal fold mobility, 8 of 18 animals showed paradoxical movement, and 5 of 18 exhibited impaired mobility. Conclusion: We have established an animal model of paradoxical movement following recurrent laryngeal nerve injury. This model may be useful in studying laryngeal synkinesis.

Biocompatibility and efficacy of collagen/gelatin sponge scaffold with sustained release of basic fibroblast growth factor on vocal fold fibroblasts in 3-dimensional culture.

Treatment of vocal fold scarring remains challenging. We have previously reported the therapeutic effects of local injection of basic fibroblast growth factor (bFGF) in animal models and humans. A novel collagen/gelatin sponge (CGS) is capable of sustained release of bFGF, which compensates for its quick absorption in vivo, avoiding multiple injections. This study aimed to evaluate the biocompatibility and efficacy of the CGS in rat vocal fold fibroblasts prior to human trials. Fibroblasts extracted from Sprague-Dawley rat vocal folds were seeded onto a CGS and then cultivated with bFGF at concentrations of 0, 10, and 100 ng/mL. Vocal fold fibroblast morphology, adhesion, proliferation, and gene expression were measured under these 3-dimensional conditions. Cells adhered to the CGS from day 1. Although no significant differences in cell morphology were detected, cell proliferation was accelerated by bFGF administration. Expression of endogenous bFGF and hepatocyte growth factor was significantly up-regulated at 10 ng/mL bFGF. The expression of procollagen I and procollagen III was significantly suppressed, whereas HAS-1 and HAS-2 were up-regulated at 10 and 100 ng/mL bFGF. The collagen/gelatin sponge is biocompatible with vocal fold fibroblasts and may be useful as a bFGF drug delivery system for the treatment of scarred vocal folds.

An In Vivo Study of Composite Microgels Based on Hyaluronic Acid and Gelatin for the Reconstruction of Surgically Injured Rat Vocal Folds

PURPOSE The objective of this study was to investigate local injection with a hierarchically microstructured hyaluronic acid-gelatin (HA-Ge) hydrogel for the treatment of acute vocal fold injury using a rat model. METHOD Vocal fold stripping was performed unilaterally in 108 Sprague-Dawley rats. A volume of 25 μl saline (placebo controls), HA-bulk, or HA-Ge hydrogel was injected into the lamina propria (LP) 5 days after surgery. The vocal folds were harvested at 3, 14, and 28 days after injection and analyzed using hematoxylin and eosin staining and immunohistochemistry staining for macrophages, myofibroblasts, elastin, collagen type I, and collagen type III. RESULTS The macrophage count was statistically significantly lower in the HA-Ge group than in the saline group (p &lt; .05) at Day 28. Results suggested that the HA-Ge injection did not induce inflammatory or rejection response. Myofibroblast counts and elastin were statistically insignificant across treatment groups at all time points. Increased elastin deposition was qualitatively observed in both HA groups from Day 3 to Day 28, and not in the saline group. Significantly more elastin was observed in the HA-bulk group than in the uninjured group at Day 28. Significantly more collagen type I was observed in the HA-bulk and HA-Ge groups than in the saline group (p &lt; .05) at Day 28. The collagen type I concentration in the HA-Ge and saline groups was found to be comparable to that in the uninjured controls at Day 28. The concentration of collagen type III in all treatment groups was similar to that in uninjured controls at Day 28. CONCLUSION Local HA-Ge and HA-bulk injections for acute injured vocal folds were biocompatible and did not induce adverse response.

Vocal Fold Fibroblast Response to Growth Factor Treatment is Age Dependent: Results From an In Vitro Study

Vocal fold (VF) fibroblasts (VFFs) are the central target in developing new strategies for treatment of VF injury and scarring. Nevertheless, only little is known about the basic biological characteristics of these cells. The aim of this study was to explore the impact of age of VFFs on the response to external growth factor stimulation. Invitro cell study using a rat model. VFFs were extracted from young and aged rat VF 3 months after establishing unilateral injury. Resulting scar fibroblasts (SFs) and normal fibroblasts (NFs) were subsequently cultured separately with or without the addition of hepatocyte growth factor (HGF). After 24 and 72 hours, the production of hyaluronic acid (HA) was examined in the supernatant culture media using enzyme-linked immunosorbent assay. Only cultured SF and NF from young animals could be stimulated significantly in the production of HA by HGF. Within these, average percentage increase was higher in NF compared with SF. The response of VFFs in cell culture to growth factors stimulation is highly depending on the age of the animals. This is another step in a nearer characterization of scar VFF and could furthermore be an important point when estimating the success of an intervention. Age-depending effects must be considered as an important factor in developing possible therapeutic agents for VF scarring.

Temporal and spatial expression of high‐mobility group box 1 in surgically injured rat vocal folds

High-mobility group box 1 (HMGB1) protein has been identified as a principal instigator of injury-induced inflammation in many organ systems. Physiologically, HMGB1 binds to chromatin in cell nucleus. Upon injury, cells release HMGB1 to extracellular milieu, triggering a destructive inflammatory response. Neutralizing or removing HMGB1 has been shown to control inflammation. Unfortunately, the role of HMGB1 in laryngeal inflammation and healing has yet to be defined. The purpose of this study was to determine spatial and temporal patterns of HMGB1 expression in surgically injured rat vocal folds up to 2 weeks after injury. Prospective animal study. Bilateral vocal fold injury was performed on 70 Sprague-Dawley rats. An additional 14 rats served as uninjured controls. Animals were sacrificed at 1 day, 3 days, 5 days, 1 week, and 2 weeks following surgery. Immunohistochemistry staining and enzyme-linked immunosorbent assay (ELISA) were performed to determine the spatial distribution and temporal expression, respectively, of HMGB1 in vocal fold tissue. Hematoxylin-and-eosin staining for cell counting was performed to evaluate cell infiltration. Cell number peaked significantly 5 days after injury. HMGB1 was positively stained in the nuclear, cytoplasmic, and extracellular compartments from days 1 to 7 after injury, whereas a strict nuclear staining was observed in uninjured controls and week 2 animals. Staining results were corroborated by ELISA. Spatial and temporal changes of HMGB1 expression were shown in injured vocal fold tissue, indicating this protein may be one of the principal drivers of inflammation and healing response to surgical injury in the larynx.

Recruitment patterns of side population cells during wound healing in rat vocal folds

Despite big advances in understanding the mechanisms of wound healing in vocal fold injury, it still remains unclear which are the decisive factors that lead to a complete restoration or to scarring. Among several other factors, stem cells are believed to play an important role in vocal fold restoration. Side population (SP) cells are considered to contain high numbers of stem cells and have gained great interest in the tissue engineering community. The aim of the following study was to investigate the recruitment pattern of SP cells in a rat vocal fold injury model. Experimental animal study. Unilateral vocal fold scarring was performed in Sprague Dawley rats. Larynges were harvested 1, 3, 5, 7, 14, 21, and 35 days after initial injury and were examined immunohistochemically for the presence of SP cells. This was done in coronal sections of the posterior and anterior macula flava as well as in the midportion of the vocal fold investigating the lamina propria. The number of SP cells peaked significantly after 7 days in the midportion of injured vocal folds, with a return to pre-injury levels after 14 days. No increase was detected throughout the observed time in the contralateral side. The number of SP cells increased slightly but not significantly in both anterior and posterior macula flava. Our findings suggest that SP cells may play an important role in early vocal fold wound healing and may serve as a possible therapeutic target.

Optimal concentration of hepatocyte growth factor for treatment of the aged rat vocal fold

Hepatocyte growth factor (HGF) demonstrates beneficial properties in the treatment of aged vocal folds. However, the optimal concentration of HGF remains unknown. The purpose of the present study was to investigate the effects of HGF concentration on treatment of the aged rat vocal fold. Prospective animal study. Seventy-five rats were studied. The rats were divided into five groups and received serial injections of HGF in 10 μL of phosphate-buffered saline (PBS) at the following concentrations: 10 ng/10 μL, 50 ng/10 μL, 100 ng/10 μL, 200 ng/10 μL, or control (PBS only). Alcian blue staining was performed to investigate hyaluronan (HA), and immunohistochemistry was performed to investigate collagen type I and III. Gene expression of hyaluronic acid synthase (HAS)-1, -2, matrix metalloproteinases (MMP)-2, -9, and procollagen I and III were also investigated using real-time polymerase chain reaction (RT-PCR). Histologic analyses revealed increased HA and decreased collagen type I in rats receiving injections of HGF at 100 ng/10 μL. Results were supported by RT-PCR revealing upregulated expression of HAS-2, decreased expression of procollagen I, and a significant increase of MMP-9 mRNA in rats receiving HGF at 100 ng/10 μL. We report the first in vivo concentration study of HGF for treatment of the aged vocal fold. Results revealed desirable biochemical effects of HGF at 100 ng/10 μL. These data will be used to provide immediate direction to programmatic efforts aimed at examining future applications of HGF for treatment of the aged vocal fold.

Structural changes and immunohistochemical localisation of epidermal growth factor receptor in the true vocal fold of female albino rats administered anabolic, androgenic steroids, and effects of anti-androgen therapy

Anabolic steroid abuse by women is associated with a number of adverse effects, including laryngeal changes. The epidermal growth factor receptor is related to regulation of the cell life cycle. This study aimed to investigate the structural changes and immunohistochemical localisation of epidermal growth factor receptor in rat vocal folds following anabolic steroid administration, and also to assess the effect of anti-androgens. Thirty-two adult female albino rats were divided into: group I (controls), group II (receiving anabolic steroids for two months) or group III (receiving anabolic steroids plus anti-androgen for two months). Group II rat true vocal folds showed thicker epithelial layers with many mitotic figures, thicker lamina propria and thicker muscle fibres; epithelial cells were also immunohistochemically positive for epidermal growth factor receptor. Group III rats showed similar changes, but thin muscle fibres and extravasated red blood cells within the lamina propria. Anabolic steroids caused structural and immunohistochemical changes within the female rat true vocal fold. Co-administration of anti-androgens did not prevent these changes, suggesting that anti-androgens have a limited role in the management of such changes in humans.

Elasticity and stress relaxation of a very small vocal fold

Across mammals many vocal sounds are produced by airflow induced vocal fold oscillation. We tested the hypothesis that stress–strain and stress-relaxation behavior of rat vocal folds can be used to predict the fundamental frequency range of the species' vocal repertoire. In a first approximation vocal fold oscillation has been modeled by the string model but it is not known whether this concept equally applies to large and small species. The shorter the vocal fold, the more the ideal string law may underestimate normal mode frequencies. To accommodate the very small size of the tissue specimen, a custom-built miniaturized tensile test apparatus was developed. Tissue properties of 6 male rat vocal folds were measured. Rat vocal folds demonstrated the typical linear stress–strain behavior in the low strain region and an exponential stress response at strains larger than about 40%. Approximating the rat's vocal fold oscillation with the string model suggests that fundamental frequencies up to about 6 kHz can be produced, which agrees with frequencies reported for audible rat vocalization. Individual differences and time-dependent changes in the tissue properties parallel findings in other species, and are interpreted as universal features of the laryngeal sound source.

Correction: Cross-Sample Validation Provides Enhanced Proteome Coverage in Rat Vocal Fold Mucosa

Table S1 was erroneously omitted from the Supporting Information section. Please view Table S1 here: Click here for additional data file.(27K, pdf) [^]

Cross-Sample Validation Provides Enhanced Proteome Coverage in Rat Vocal Fold Mucosa

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M r ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M r glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.

Effects of Nerve-Muscle Pedicle on Immobile Rat Vocal Folds in the Presence of Partial Innervation

We investigated whether implantation of an ansa cervicalis nerve (ACN)-muscle pedicle into the thyroarytenoid (TA) muscle is efficacious in the presence of partial recurrent laryngeal nerve (RLN) innervation. We studied a total of 36 rats. Twelve of the rats served as positive and negative control animals. In the remaining 24 rats, the left RLN was transected, a 1-mm piece of nerve was removed, and the stumps were abutted in silicone tubes (STs), inducing partial RLN regeneration. Twelve of the ST-treated rats underwent this procedure alone, and the other 12 rats had a nerve-muscle pedicle (NMP) implanted into the left TA muscle 5 weeks after ST treatment. At 15 weeks, reinnervation was assessed by histologic evaluation of the TA muscle and by electromyography with stimulation of the RLNs and ACNs. The muscle area, the number of nerve terminals, the number of acetylcholine receptors, and the ratio of nerve terminals to acetylcholine receptors were significantly greater (p < 0.05) in the NMP group than in the ST group. Electromyography elicited TA muscle compound action potentials upon stimulation of the RLNs and ACNs. In rats, NMP implantation is efficacious for reducing atrophic changes in the TA muscle in the presence of partial RLN innervation.

Controlled release of hepatocyte growth factor from a bovine acellular scaffold for vocal fold reconstruction

A bovine acellular scaffold was found to facilitate tissue remodeling in a rat model of vocal fold injury, whereas hepatocyte growth factor (HGF) has been shown to have an antiscarring effect in the larynx. This study examined the loading and release kinetics of HGF in vitro, and the potential of the acellular scaffold as a timed-release system for the delivery of HGF in vivo. Bilateral wounds were created in the posterior vocal folds of 20 rats, with HGF-loaded acellular scaffolds implanted into the wounds unilaterally, and scaffolds without HGF implanted into the contralateral vocal folds as control. The rats were humanely sacrificed after 3, 7, 30, and 90 days and their larynges were examined histologically and immunohistochemically. Expressions of key matrix proteins in the vocal fold coronal sections were quantified by digital image analysis. Results demonstrated a gradual, sustained release of HGF for at least 7 days in vitro, consistent with the detection of glycosaminoglycans inherent of the scaffold. In rat vocal folds implanted with HGF-loaded scaffolds, apparently fewer inflammatory cells were observed 3 days after surgery when compared to the control. The mean relative densities of collagen III and hyaluronic acid were significantly lower than those of the control 7 days after surgery. Scaffold implants were apparently degraded by 3 months in all animals, with no evidence of fibrosis or calcification. These data suggested that the bovine acellular scaffold could be promising for the exogenous delivery of select growth factors in vivo.

Analysis of hyaluronic acid concentration in rat vocal folds during estral and gravidic puerperal cycles

Hormone plays an important role in the larynx. Among other substances, vocal folds contain hyaluronic acid, which tissue concentration may vary according to hormone action. AIM: the objective of this study is to analyze hyaluronic acid concentration in the vocal folds during estral and gravidic-puerperal cycles. MATERIALS AND METHODS: Experimental study. 40 adult rats were divided into two groups. In the first group we used 20 rats to establish the concentration of hyaluronic acid during the estral cycle and in the second group, 20 animals were submitted to the same procedure but during the gravidic-puerperal cycle. RESULTS: Variations in hyaluronic acid concentration was not observed during the estral cycle. In the gravidic puerperal cycle group, an increase in hyaluronic acid concentration was observed in the puerperal subgroup. Comparing the two groups of estral and gravidic-puerperal cycles, no difference was observed. CONCLUSIONS: In comparing all subgroups of estral and gravidic-puerperal cycles, an increase in hyaluronic acid concentration was noticed only in the puerperal phase.

Pulsed dye laser‐induced inflammatory response and extracellular matrix turnover in rat vocal folds and vocal fold fibroblasts

Disruption of the vocal fold extracellular matrix (ECM) can induce a profound and refractory dysphonia. Pulsed dye laser (PDL) irradiation has shown early promise as a treatment modality for disordered ECM in patients with chronic vocal fold scar; however, there are limited data addressing the mechanism by which this laser energy might induce cellular and extracellular changes in vocal fold tissues. In this study, we examined the inflammatory and ECM modulating effects of PDL irradiation on normal vocal fold tissues and cultured vocal fold fibroblasts (VFFs). We evaluated the effects of 585 nm PDL irradiation on inflammatory cytokine and collagen/collagenase gene transcription in normal rat vocal folds in vivo (3-168 hours following delivery of approximately 39.46 J/cm(2) fluence) and VFFs in vitro (3-72 hours following delivery of 4.82 or 9.64 J/cm(2) fluence). We also examined morphological vocal fold tissue changes 3 hours, 1 week, and 1 month post-irradiation. PDL irradiation altered inflammatory cytokine and procollagen/collagenase expression at the transcript level, both in vitro and in vivo. Additionally, PDL irradiation induced an inflammatory repair process in vivo that was completed by 1 month with preservation of normal tissue morphology. PDL irradiation can modulate ECM turnover in phenotypically normal vocal folds. Additional work is required to determine if these findings extend to disordered ECM, such as is seen in vocal fold scar. Lasers Surg. Med. 41:585-594, 2009. (c) 2009 Wiley-Liss, Inc.

Analysis of hyaluronic acid concentration in rat vocal folds during estral and gravidic puerperal cycles

SummaryHormone plays an important role in the larynx. Among other substances, vocal folds contain hyaluronic acid, which tissue concentration may vary according to hormone action.Aimthe objective of this study is to analyze hyaluronic acid concentration in the vocal folds during estral and gravidic-puerperal cycles.Study designExperimental study.Materials and Methods40 adult rats were divided into two groups. In the first group we used 20 rats to establish the concentration of hyaluronic acid during the estral cycle and in the second group, 20 animals were submitted to the same procedure but during the gravidic-puerperal cycle.ResultsVariations in hyaluronic acid concentration was not observed during the estral cycle. In the gravidic puerperal cycle group, an increase in hyaluronic acid concentration was observed in the puerperal subgroup. Comparing the two groups of estral and gravidic-puerperal cycles, no difference was observed.ConclusionsIn comparing all subgroups of estral and gravidic-puerperal cycles, an increase in hyaluronic acid concentration was noticed only in the puerperal phase.

Regenerative Effects of Basic Fibroblast Growth Factor on Extracellular Matrix Production in Aged Rat Vocal Folds

We investigated acute changes in extracellular matrix (ECM) gene expression and histologic changes in the deposition of collagen and hyaluronan (hyaluronic acid; HA) after basic fibroblast growth factor (bFGF) treatment of the aged rat vocal fold. For the polymerase chain reaction (PCR) experiments, we divided ten 18-month-old Sprague-Dawley rats into two groups that received serial injections of sham (saline solution) or bFGF (2 ng/microL) and euthanized them 2 weeks after the initial injection to investigate acute changes in ECM gene expression. We treated a separate group of 5 animals unilaterally and sacrificed them 4 weeks after the initial injection to investigate histologic changes in the deposition of collagen and HA. Real-time PCR revealed significantly up-regulated HA synthase (HAS)-2, HAS-3, matrix metalloproteinase (MMP)-2, and procollagen type I gene expression in the bFGF treatment group as compared to the sham treatment group. Histologic staining revealed significantly increased deposition of HA in the bFGF-treated vocal fold as compared to the sham-treated vocal fold. No differences in ECM collagen levels were observed between treatment sides. Basic fibroblast growth factor induced the up-regulation of HAS-2, HAS-3, MMP-2, and procollagen type I. Histologically, aged vocal folds treated with bFGF revealed increased deposition of HA as compared to sham-treated vocal folds.