Functional Analysis of Injectable Substance Treatment on Surgically Injured Rabbit Vocal Folds.

OBJECTIVES: The objective of this study was to evaluate the efficacy of immediate injection treatments of dexamethasone, hyaluronic acid (HA)/gelatin (Ge) hydrogel and glycol-chitosan solution on the phonatory function of rabbit larynges at 42 days after surgical injury of the vocal folds, piloting a novel ex vivo phonatory functional analysis protocol. METHODS: A modified microflap procedure was performed on the left vocal fold of 12 rabbits to induce an acute injury. Animals were randomized into one of four treatment groups with 0.1 mL injections of dexamethasone, HA/Ge hydrogel, glycol-chitosan or saline as control. The left mid vocal fold lamina propria was injected immediately following injury. The right vocal fold served as an uninjured control. Larynges were harvested at Day 42 after injection, then were subjected to airflow-bench evaluation. Acoustic, aerodynamic and laryngeal high-speed videoendoscopy (HSV) analyses were performed. HSV segments of the vibrating vocal folds were rated by three expert laryngologists. Six parameters related to vocal fold vibratory characteristics were evaluated on a Likert scale. RESULTS: The fundamental frequency, one possible surrogate of vocal fold stiffness and scarring, was lower in the dexamethasone and HA/Ge hydrogel treatment groups compared to that of the saline control (411.52±11.63 Hz). The lowest fundamental frequency value was observed in the dexamethasone group (348.79±14.99 Hz). Expert visual ratings of the HSV segments indicated an overall positive outcome in the dexamethasone treatment group, though the impacts were below statistical significance. CONCLUSION: Dexamethasone injections might be used as an adjunctive option for iatrogenic vocal fold scarring. An increased sample size, histological correlate, and experimental method improvements will be needed to confirm this finding. Results suggested a promising use of HSV and acoustic analysis techniques to identify and monitor post-surgical vocal fold repair and scarring, providing a useful tool for future studies of vocal fold scar treatments.

Cryotherapy Modifies Extracellular Matrix Expression of Vocal Fold in Rat Models.

OBJECTIVES: Vocal fold (VF) scarring is the major cause of voice disorders. Cryotherapy is an effective anti-scarring therapy for skin lesions. The aim of this study was to explore the anti-scarring potential of cryotherapy in vocal folds. METHODS: The extracellular matrix (ECM) mRNA expression of cryotherapy on normal VF tissue and the histologic results of cryotherapy on vocal fold healing were studied. Fifteen rats were introduced cryotherapy on the normal VF bilaterally and were harvested for real-time polymerase chain reaction (RT-PCR) analysis for collagen I, collagen III, TGFß1, decorin, fibronectin and HAS1 at 1 day, 3 days and 7 days. Ten rats were unilaterally injured by stripping lamina propria and immediately treated with or without cryotherapy and were harvested at 2 months for histological and immunohistochemical analysis. RESULTS: Regenerative effect of cryotherapy was validated of ECM gene expression. Histological and immunohistochemical analysis showed significantly increased hyaluronan, decreased collagen, and increased decorin deposition in injury-cryotherapy cohort compared with injury control cohort and normal control cohort. CONCLUSIONS: Cryotherapy may provide an optimal environment for vocal fold tissue regeneration. The results of the present investigation suggest that cryotherapy has therapeutic potential in prevention and treatment of vocal fold scarring.

Intracordal injection therapy for vocal fold scarring: Steroid versus basic fibroblast growth factor.

Objectives: Vocal fold scarring is caused by replacement of vocal fold mucosa with fibrous tissue due to repeated inflammation or trauma. It can lead to severe dysphonia. It is currently treated conservatively and with phonosurgery and intracordal injections. Intracordal injection of steroid or basic fibroblast growth factor (bFGF) has been recently found to be useful for treating vocal fold scarring that does not respond to voice therapy. Methods: This retrospective study involved the administration of steroid injection and bFGF injection bilaterally under local anesthesia in 16 patients each. Laboratory measurements of voice parameters were performed before and 3-6 months after injection. Results: In the steroid injection group, the Voice Handicap Index (VHI) score significantly improved from 57.1 to 40.5, total Grade, Roughness, Breathiness, Asthenia, Strain (tGRBAS) score significantly improved from 4.2 to 2.6, and mean speech fundamental frequency (SFF) increased from 192.5 to 211.4 dB, but there was no improvement in maximum phonation time (MPT) and mean airflow rate (MFR). In the bFGF injection group, significant improvements in the VHI score (from 53.3 to 35.7), MPT (from 16.9 to 21.8 s) and MFR (from 314.6 to 210.5 ml/s) were seen; however, the tGRBAS score did not improve. In addition, the SFF significantly decreased from 178.1 to 160.5 Hz. Conclusion: These results suggest that both steroid and bFGF injections are effective for treating vocal fold scarring, with steroids improving voice quality and bFGF improving glottic closure, thereby contributing to improvements in VHI scores. Level of Evidence: 4.

In vivo hamster cheek pouch subepithelial ablation, biomaterial injection, and localization: pilot study.

SIGNIFICANCE: The creation of subepithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable biomaterials toward a clinically viable therapy for vocal fold scarring. AIM: We aim to prove that subepithelial voids can be created in a live animal model and that the ablation process does not engender additional scar formation. We demonstrate localization and long-term retention of an injectable biomaterial within subepithelial voids. APPROACH: A benchtop nonlinear microscope was used to create subepithelial voids within healthy and scarred cheek pouches of four Syrian hamsters. A model biomaterial, polyethylene glycol tagged with rhodamine dye, was then injected into these voids using a custom injection setup. Follow-up imaging studies at 1- and 2-week time points were performed using the same benchtop nonlinear microscope. Subsequent histology assessed void morphology and biomaterial retention. RESULTS: Focused ultrashort pulses can be used to create large subepithelial voids in vivo. Our analysis suggests that the ablation process does not introduce any scar formation. Moreover, these studies indicate localization, and, more importantly, long-term retention of the model biomaterial injected into these voids. Both nonlinear microscopy and histological examination indicate the presence of biomaterial-filled voids in healthy and scarred cheek pouches 2 weeks postoperation. CONCLUSIONS: We successfully demonstrated subepithelial void formation, biomaterial injection, and biomaterial retention in a live animal model. This pilot study is an important step toward clinical acceptance of a new type of therapy for vocal fold scarring. Future long-term studies on large animals will utilize a miniaturized surgical probe to further assess the clinical viability of such a therapy.

Asiatic Acid Improves Extracellular Matrix Remodeling in Vocal Fold Scarring Via SMAD7 Activation.

OBJECTIVES/HYPOTHESIS: Vocal fold (VF) fibroblasts are the central target for developing new strategies for the treatment of VF scarring and fibrosis. Asiatic acid (AA) is a triterpenoid derivate with antifibrotic properties. However, the effect of AA in VF scarring is poorly understood. The objective of this study was to investigate the potential application of AA as a therapeutic treatment in VF scarring. STUDY DESIGN: Xxxxx. METHODS: The functional expression of SMAD7 was knocked down with recombinant adenoviruses and adeno-associated viruses carrying shRNAs in the in vitro and in vivo models, which were constructed to investigate AA's antifibrotic function. The expression of collagens and SMADs in cultured human and rabbit cell lines and animal models was evaluated with quantitative reverse transcription polymerase chain reaction and immunohistochemistry labeling, respectively. Cell migration capacity and contraction in VF fibroblast cell lines were also evaluated. RESULTS: AA downregulated the downstream fibrotic activation in a dose-dependent manner. Meanwhile, AA attenuated VF scarring/fibrosis by reducing collagen deposition. Furthermore, the antifibrotic effects of AA were associated with the upregulation of SMAD7. In contrast, knockdown of SMAD7 inhibited the effect of AA on transforming growth factor-beta-1 (TGF-ß1) stimulation, which suggests a central role for SMAD7 in AA-induced antifibrotic activities during VF fibrosis. CONCLUSION: We concluded that AA, which is a novel therapeutic candidate for preventing VF scarring/fibrosis, might exert its antifibrotic effect via the TGF-ß1/SMAD signaling pathway. LEVEL OF EVIDENCE: NA Laryngoscope, 132:1237-1244, 2022.

The Role of Cytokines in Modulating Vocal Fold Fibrosis: A Contemporary Review.

OBJECTIVES: Vocal fold (VF) scarring and laryngeal stenosis are a significant clinical challenge. Excessive scar formation causes low voice quality or even life-threatening obstructions. Cytokines are thought to modulate multiple steps of the establishment of VF fibrosis, but there is no systematic report regarding their role in modulating VF fibrosis. This review aims to investigate the role of cytokines in modulating vocal fold fibrosis. STUDY DESIGN: Literature review. METHODS: This review searched for all relevant peer publications in English for the period 2009 to 2019 in the PubMed database using search terms: "laryngeal stenosis," "vocal fold scarring," and "cytokines." A thorough investigation of the methods and results of the reviewed studies was performed. RESULTS: Comprehensive research in various studies, including analyses of prostaglandin E2 (PGE2), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), transforming growth factor-ß3 (TGF-ß3), and interleukin-10 (IL-10), supports cytokine therapy for VF scarring and laryngeal stenosis to some extent. A few clinical studies on this topic support the conclusion that HGF and bFGF can be selected as effective drugs, and no serious side effects were found. CONCLUSIONS: This review describes the potential of cytokines for modulating the process of VF fibrogenesis, although cytokines are still an unproven treatment method. As no ideal drugs exist, cytokines may be considered the candidate treatment for preventing VF fibrogenesis. Laryngoscope, 131:139-145, 2021.

Characterizing Vocal Fold Injury Recovery in a Rabbit Model With Three-Dimensional Virtual Histology.

OBJECTIVES/HYPOTHESIS: In animal studies of vocal fold scarring and treatment, imaging-based evaluation is most often conducted by tissue slicing and histological staining. Given variation in anatomy, injury type, severity, and sacrifice timepoints, planar histological sections provide limited spatiotemporal details of tissue repair. Three-dimensional (3D) virtual histology may provide additional contextual spatial information, enhancing objective interpretation. The study's aim was to evaluate the suitability of magnetic resonance imaging (MRI), microscale computed tomography (CT), and nonlinear laser-scanning microscopy (NM) as virtual histology approaches for rabbit studies of vocal fold scarring. METHODS: A unilateral injury was created using microcup forceps in the left vocal fold of three New Zealand White rabbits. Animals were sacrificed at 3, 10, and 39 days postinjury. ex vivo imaging of excised larynges was performed with MRI, CT, and NM modalities. RESULTS: The MRI modality allowed visualization of injury location and morphological internal features with 100-µm spatial resolution. The CT modality provided a view of the injury defect surface with 12-µm spatial resolution. The NM modality with optical clearing resolved second-harmonic generation signal of collagen fibers and two-photon autofluorescence in vocal fold lamina propria, muscle, and surrounding cartilage structures at submicrometer spatial scales. CONCLUSIONS: Features of vocal fold injury and wound healing were observed with MRI, CT, and NM. The MRI and CT modalities provided contextual spatial information and dissection guidance, whereas NM resolved extracellular matrix structure. The results serve as a proof of concept to motivate incorporation of 3D virtual histology techniques in future vocal fold injury animal studies. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1578-1587, 2021.

Endoscopic Sealing With a Polyglycolic Acid Sheet for Restoration of Vocal Fold Mucosa in Dogs.

OBJECTIVES/HYPOTHESIS: Voice outcomes of cordectomy for early glottic cancer are often poor due to vocal fold scarring and tissue defects. Improvements in this aspect could make cordectomy a more acceptable treatment option than radiotherapy. We hypothesized that a polyglycolic acid (PGA) sheet could be used to cover vocal fold defects. The present study aimed to prevent vocal fold scarring after cordectomy using the PGA sheet. STUDY DESIGN: Animal experiment. METHODS: Nine male beagles were divided into three groups including a control group (n = 3). Following cordectomy, the vocal fold defect was covered with the PGA sheet plus fibrin glue (PGA group; n = 3) or with the PGA sheet plus fibrin glue containing basic fibroblast growth factor (bFGF; the PGA-bFGF group, n = 3). Vocal folds were chronologically observed, and larynges were removed 6 months after surgery. Mucosal amplitude was measured using a high-speed camera, and histological analysis was performed. RESULTS: The re-epithelialization process was delayed in the PGA and PGA-bFGF groups compared with the control group. The mucosal amplitude was significantly more normalized and the thickness ratio significantly higher in the PGA and PGA-bFGF groups compared with the control group. The PGA-bFGF group had the highest elastic fiber density, followed by the PGA group and then the control group, with a significant difference between the PGA-bFGF and control groups. CONCLUSIONS: The PGA sheet plus fibrin glue could serve as an effective regenerative scaffold for reconstructing vocal fold morphology and function after cordectomy, with the potential benefit of establishing an endoscopic sealing method for vocal fold defects. LEVEL OF EVIDENCE: NA Laryngoscope, 130:E436-E443, 2020.

Effects of Intracordal Estradiol and Dexamethasone Injection on Wound Healing in Vocal Fold Injuries.

OBJECTIVE: The aim of this study was to investigate the effects of intracordal estradiol and dexamethasone injection on wound healing in vocal fold injuries. STUDY DESIGN: A prospective controlled animal study was carried out. SETTING: This study was conducted at a tertiary center. SUBJECTS-METHODS: Ten rabbits were randomly divided into two groups. As surgical procedure, cordotomy technique was performed in the middle third of the vocal folds bilaterally. In the first group, 0.1 mL of dexamethasone was injected into the right side, and 0.1 mL of saline was injected into the left side. In the second group, 0.1 mL of estradiol was injected into the right side, and 0.1 mL of saline was injected into the left side. Animals were sacrificed after 1 month and laryngeal specimens were evaluated histopathologically. RESULTS: No statistically significant difference was observed in terms of inflammatory response, epithelial thickness, type I and III collagen, and hyaluronic acid parameters in dexamethasone and estradiol injections compared to the saline injection. In terms of elastin level, estradiol injection demonstrated statistically higher values compared to the saline injection. Elastin level of dexamethasone injected vocal folds was not statistically different compared to the saline injection. No significant differences were observed in terms of inflammatory response, epithelial thickness, type I and III collagen, and hyaluronic acid parameters between the estradiol and dexamethasone injected vocal folds. CONCLUSION: It is thought that the effects of estradiol or dexamethasone injections may have similar effects on wound healing in vocal fold injuries. Intracordal estradiol injection has positive effects on tissue elastin levels.

The effects of cryotherapy on vocal fold healing in a rabbit model.

OBJECTIVES/HYPOTHESIS: Cryotherapy has been shown to be a scarless treatment modality for dermal lesions; however, there are limited data addressing the effect of cryotherapy on vocal fold tissue. The aim of this study was to clarify the effectiveness of cryotherapy for prevention of postsurgical vocal fold scarring. STUDY DESIGN: Prospective animal study in rabbits. METHODS: The lamina propria of 20 rabbit vocal folds was bilaterally stripped, followed by randomized unilateral cryotherapy. Five larynges were harvested for real-time polymerase chain reaction (RT-PCR) analysis at 1 day, 3 days, and 7 days postinjury. The remaining five were harvested for histologic analysis at 3 months. Images of the healing phase were recorded by laryngoscopy. Analyses of RT-PCR for cyclooxygenase (COX)-2, interleukin (IL)-6, collagen I, collagen III, matrix metallopeptidase 1 (MMP1), transforming growth factor ß (TGFß1), α smooth muscle actin (α-SMA), and hyaluronan synthase 1 (HAS1) were completed. Histological samples were completed for collagen and hyaluronic acid analysis. RESULTS: RT-PCR results revealed that higher expressions of HAS1 and MMP1 and lower expressions of COX-2, IL-6, collagen I, collagen III, TGFß1, and α-SMA were observed, and histological examination showed significantly increased hyaluronic acid, decreased deposition, and more organized configuration of collagen in injury with the cryotherapy cohort compared with the injury cohort. CONCLUSIONS: Cryotherapy can inhibit the inflammatory reaction and simulate a fetal healing environment in extracellular matrix synthesis to regenerate vocal fold tissue with less fibrosis. Histological results showed that cryotherapy achieves a mature healing result with less scar, which tends to return to normal. In summary, the findings of this study suggest that administration of cryotherapy at the time of injury has the potential to minimize vocal fold scarring. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E151-E157, 2019.

Adipose-Derived Mesenchymal Stromal Cells Persist in Tissue-Engineered Vocal Fold Replacement in Rabbits.

OBJECTIVES:: Cell therapies using mesenchymal stromal cells (MSCs) have been proposed as a promising new tool for the treatment of vocal fold scarring. However, the mechanisms by which MSCs promote healing as well as their duration of survival within the host vocal fold have yet to be defined. The aim of this work was to assess the persistence of embedded MSCs within a tissue-engineered vocal fold mucosal replacement in a rabbit model of vocal fold injury. METHODS:: Male rabbit adipose-derived MSCs were embedded within a 3-dimensional fibrin gel, forming the cell-based outer vocal fold replacement. Four female rabbits underwent unilateral resection of vocal fold epithelium and lamina propria and reconstruction with cell-based outer vocal fold replacement implantation. Polymerase chain reaction and fluorescent in situ hybridization for the sex-determining region of the Y chromosome (SRY-II) in the sex-mismatched donor-recipient pairs sought persistent cells after 4 weeks. RESULTS:: A subset of implanted male cells was detected in the implant site at 4 weeks. Many SRY-II-negative cells were also detected at the implant site, presumably representing native female cells that migrated to the area. No SRY-II signal was detected in contralateral control vocal folds. CONCLUSIONS:: The emergent tissue after implantation of a tissue-engineered outer vocal fold replacement is derived both from initially embedded adipose-derived stromal cells and infiltrating native cells. Our results suggest this tissue-engineering approach can provide a well-integrated tissue graft with prolonged cell activity for repair of severe vocal fold scars.

Adipose-derived mesenchymal stromal cells prevented rat vocal fold scarring.

OBJECTIVES/HYPOTHESIS: This study aimed to reveal the effects of adipose-derived mesenchymal stromal cells (ASCs) on prevention of vocal fold scarring by investigating how the immediate ASCs transplantation into the injured rat vocal fold affect the levels of gene transcription and translation. STUDY DESIGN: Prospective animal experiments with controls. METHODS: ASCs harvested from green fluorescent protein transgenic rat (ASCs group) or saline (sham group) were injected into the thyroarytenoid muscle of Sprague-Dawley rats immediately after stripping the vocal fold. For histological examinations, larynges were extirpated at 3, 14, and 56 days after the injection. Quantitative real-time polymerase chain reaction (PCR) analyses were performed at 3 and 14 days after the injection. RESULTS: Transplanted ASCs were detected only in larynges at day 3. At days 14 and 56, histological examination showed significantly higher amounts of hyaluronic acid and lower deposition of collagen in the ASCs group compared to the sham group. Real-time PCR revealed that the ASCs group showed low expression of procollagen (Col)1a1, Col1a3, matrix metalloproteinase (Mmp)1 and Mmp8 in each time points. The ASCs group showed high expression of fibroblast growth factor (Fgf)2 and Hepatocyte growth factor (Hgf) compared to the sham group at day 14. CONCLUSIONS: ASCs increased expressions of Fgf2 and Hgf, and suppressed excessive collagen deposition during vocal fold wound healing. Given the fact that ASCs survived no more than 14 days, ASCs were thought to induce upregulations of growth factors' genes in surrounding cells. These results suggested that ASCs have potential to prevent vocal fold scarring. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E33-E40, 2018.

Repairing the vibratory vocal fold.

OBJECTIVE: A vibratory vocal fold replacement would introduce a new treatment paradigm for structural vocal fold diseases such as scarring and lamina propria loss. This work implants a tissue-engineered replacement for vocal fold lamina propria and epithelium in rabbits and compares histology and function to injured controls and orthotopic transplants. Hypotheses were that the cell-based implant would engraft and control the wound response, reducing fibrosis and restoring vibration. STUDY DESIGN: Translational research. METHODS: Rabbit adipose-derived mesenchymal stem cells (ASC) were embedded within a three-dimensional fibrin gel, forming the cell-based outer vocal fold replacement (COVR). Sixteen rabbits underwent unilateral resection of vocal fold epithelium and lamina propria, as well as reconstruction with one of three treatments: fibrin glue alone with healing by secondary intention, replantation of autologous resected vocal fold cover, or COVR implantation. After 4 weeks, larynges were examined histologically and with phonation. RESULTS: Fifteen rabbits survived. All tissues incorporated well after implantation. After 1 month, both graft types improved histology and vibration relative to injured controls. Extracellular matrix (ECM) of the replanted mucosa was disrupted, and ECM of the COVR implants remained immature. Immune reaction was evident when male cells were implanted into female rabbits. Best histologic and short-term vibratory outcomes were achieved with COVR implants containing male cells implanted into male rabbits. CONCLUSION: Vocal fold cover replacement with a stem cell-based tissue-engineered construct is feasible and beneficial in acute rabbit implantation. Wound-modifying behavior of the COVR implant is judged to be an important factor in preventing fibrosis. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:153-159, 2018.

Structural Fat Grafting to Improve Outcomes of Vocal Folds' Fat Augmentation: Long-term Results.

Objective Evaluating the long-term outcomes of vocal fold structural fat grafting. Study Design Case series with chart review. Setting University hospital. Subjects and Methods Seventy-nine dysphonic patients (16-82 years; 55 with unilateral laryngeal paralysis and 24 with vocal fold scarring) underwent vocal fold fat injection. Fat was harvested by low-pressure liposuction and then processed by centrifugation. Refined fat aliquots were placed in the vocal fold and paraglottic space in multiple tunnels to enhance graft neovascularization. All patients were followed for 12 months, 15 for 3 years, and 5 for 10 years with videolaryngostroboscopy, maximal phonation time (MPT) measurement, Voice Handicap Index (VHI) questionnaire, and GRBAS (grade, roughness, breathiness, asthenia, strain) perceptual evaluation. Laryngeal computed tomography (CT) and/or magnetic resonance imaging (MRI) studies were performed in 16 patients 3 to 28 months postoperatively; MRI was repeated in 5 cases 12 to 18 months after the first radiological study. Results The voice quality of all patients improved after surgery, and long-term stability was confirmed by MPT, GRBAS, and VHI ( P ranging between .004 and <.001). The results achieved 1 year postoperatively remained stable at 3 and 10 years. Videolaryn-gostroboscopy showed improved glottic closure in all patients despite a limited amount of fat resorption. CT and MRI demonstrated survival of the fat grafts in all of the 16 examined cases. Serial MRI scans showed no change in graft size over time. Conclusions The reported clinical and radiological data demonstrate that fat is an effective filler for permanent vocal fold augmentation if the refined micro-aliquots are placed in multiple tunnels.

Background Factors and Subjective Voice Symptoms in Patients with Acquired Vocal Fold Scarring and Sulcus Vocalis.

OBJECTIVE: Vocal fold scarring (VFS) and sulcus vocalis (SV) often result in severe and chronic voice disorders. This study compares subjective voice complaints as rated with the Voice Handicap Index and etiological factors for patients with VFS and SV. PATIENTS AND METHODS: Data were collected from the medical records at the Department of Otorhinolaryngology, Karolinska University Hospital, for 27 VFS patients and 27 SV patients. Descriptive background factors were compared between the groups and data were compared from the Swedish Voice Handicap Index (Sw-VHI) questionnaires. RESULTS: Previous laryngeal surgery/trauma was significantly more common for the patients with VFS. The SV group had significantly more persistent dysphonia since childhood. It was significantly more common to have a non-Germanic language origin among the SV patients. VFS and SV rated high for the total median Sw-VHI scores. The VFS group's total Sw-VHI and the three domain scores were significantly higher compared to the SV group. The physical domain showed a significantly higher score when compared to the functional and emotional domains in the SV cohort and when compared to the emotional domain in the VFS cohort. CONCLUSION: There are significant differences between the VFS group and SV group regarding etiological factors as well as the Sw-VHI. The degree and profile of VHI should be considered when selecting patients and evaluating the result of new treatments for this group of patients.

Prevention of vocal fold scarring by local application of basic fibroblast growth factor in a rat vocal fold injury model.

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.

Collagen Content Limits Optical Coherence Tomography Image Depth in Porcine Vocal Fold Tissue.

OBJECTIVE: Vocal fold scarring, a condition defined by increased collagen content, is challenging to treat without a method of noninvasively assessing vocal fold structure in vivo. The goal of this study was to observe the effects of vocal fold collagen content on optical coherence tomography imaging to develop a quantifiable marker of disease. STUDY DESIGN: Excised specimen study. SETTING: Massachusetts Eye and Ear Infirmary. SUBJECTS AND METHODS: Porcine vocal folds were injected with collagenase to remove collagen from the lamina propria. Optical coherence tomography imaging was performed preinjection and at 0, 45, 90, and 180 minutes postinjection. Mean pixel intensity (or image brightness) was extracted from images of collagenase- and control-treated hemilarynges. Texture analysis of the lamina propria at each injection site was performed to extract image contrast. Two-factor repeated measure analysis of variance and t tests were used to determine statistical significance. Picrosirius red staining was performed to confirm collagenase activity. RESULTS: Mean pixel intensity was higher at injection sites of collagenase-treated vocal folds than control vocal folds (P < .0001). Fold change in image contrast was significantly increased in collagenase-treated vocal folds than control vocal folds (P = .002). Picrosirius red staining in control specimens revealed collagen fibrils most prominent in the subepithelium and above the thyroarytenoid muscle. Specimens treated with collagenase exhibited a loss of these structures. CONCLUSION: Collagen removal from vocal fold tissue increases image brightness of underlying structures. This inverse relationship may be useful in treating vocal fold scarring in patients.

Quantitative differentiation of normal and scarred tissues using second-harmonic generation microscopy.

The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p < 0.05) than control tissue (0.73 ± 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p < 0.005) fiber density (0.72 ± 0.09) compared to controls (0.18 ± 0.03). In the present study, image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as application of steroids or drugs targeting pathways involved in fibrosis. SCANNING 38:684-693, 2016. © 2016 Wiley Periodicals, Inc.

Adipose-derived stem cells versus bone marrow-derived stem cells for vocal fold regeneration.

OBJECTIVES/HYPOTHESIS: Vocal fold scarring presents therapeutic challenges. Recently, cell therapy with mesenchymal stromal cells has become a promising approach. The aim of this study was to compare the therapeutic potential of adipose-derived stem cells (ASC) with bone marrow-derived stem cells (BMSC) for vocal fold regeneration. STUDY DESIGN: Prospective animal experiments with controls. METHODS: The vocal folds of Sprague-Dawley rats were unilaterally injured. Two months after injury, rats were treated with a local injection of ASC (ASC group), BMSC (BMSC group), or saline (sham-treated group). The GFP-labeled ASC and BMSC were extracted from CAG-EGFP rats. Larynges were harvested for histological and immunohistochemical examinations 1 and 3 months posttransplantation and for quantitative real-time polymerase chain reaction (PCR) 1 month posttransplantation. RESULTS: After 1 month, no surviving cells from the transplant were detected. Histological examination showed significantly increased hyaluronic acid (HA) and decreased dense collagen deposition in both ASC and BMSC groups compared to shams 1 and 3 months after treatment. Real-time PCR revealed that hyaluronan synthase 1 (Has1) and Has2 were upregulated in only the ASC group compared with the sham-treated group. Fibroblast growth factor 2 (basic) (Fgf2), hepatocyte growth factor (Hgf) and Has3 were upregulated in both cell transplantation groups. ASC seemed to upregulate Hgf more than did BMSC. CONCLUSIONS: The regenerative effects of ASC and BMSC transplantation were found to be similar for the restoration. It is suggested that ASC might have more potential because of better recovery of HA, a superior antifibrotic effect, and the upregulation of Hgf. LEVEL OF EVIDENCE: N/A.

A rabbit vocal fold laser scarring model for testing lamina propria tissue-engineering therapies.

OBJECTIVES/HYPOTHESIS: To develop a vocal fold scarring model using an ablative laser in the rabbit as a platform for testing bioengineered therapies for missing or damaged lamina propria. STUDY DESIGN: Prospective controlled animal study. METHODS: An optimal laser energy level was first determined by assessing the depths of vocal fold injury created by a Holmium:YAG laser at various energy levels on fresh cadaveric rabbit larynges. The selected energy level was then used to create controlled unilateral injuries in vocal folds of New Zealand white rabbits, with the contralateral folds serving as uninjured controls. After 4 weeks, the larynges were harvested and subjected to excised-larynx phonation with high-speed imaging and immunohistochemical staining for collagen types I and III, elastin, and hyaluronic acid (HA) with quantitative histological analysis. RESULTS: A total of 1.8 joules produced full-thickness injury of the lamina propria without extensive muscle injury. After 4 weeks, the injured vocal folds vibrated with reduced amplitude (P = 0.036) in excised-larynx phonation compared to normal vocal folds. The injured vocal folds contained a higher relative density of collagen type I (P = 0.004), higher elastin (P = 0.022), and lower HA (P = 0.030) compared to normal controls. Collagen type III was unchanged. CONCLUSIONS: With its potential for higher precision of injury, this laser vocal fold scarring model may serve as an alternative to scarring produced by cold instruments for studying the effects of vocal fold lamina propria bioengineered therapies.