Distribution of collagen in the lamina propria of the human vocal fold

OBJECTIVES: To describe the arrangement of collagen fibers in the lamina propria of the human vocal fold. STUDY DESIGN: Analysis of the lamina propria of the vocal fold obtained from human cadavers. METHODS: The Picrosirius-polarization method was used to visualize collagen fibers. RESULTS: Analysis of 20 human vocal folds by the Picrosirius-polarization method permitted the visualization of two fiber populations arranged in three layers in the lamina propria: two layers of thick, strongly birefringent collagen fibers (collagen type I), one immediately below the epithelium and another more dense layer in the deep region superficially to the vocal muscle, penetrating between muscle fibers. The third layer consisted of fine, weakly birefringent fibers (collagen type III) located between the two layers of thick fibers. In addition, the collagen fibers in the lamina propria showed an intertwined network arrangement in the form of a "wicker basket." CONCLUSIONS: This basket-like configuration better explains how the vocal fold is able to stretch even though it contains nonstretchable fibers and to modulate the frequency of the voice under the action of the intrinsic musculature of the larynx. Segmental areas of disarray of the basket-like structure of the collagen layers were systematically observed in older patients. Thus, it is possible that vocal alterations occurring in the elderly might be the result of a loss of histoarchitectural arrangement of the collagen system and its relationship with the lamina propria and underlying musculature.     

Immuno-scanning electron microscopy of collagen types I and III in human vocal fold lamina propria

OBJECTIVES: This study was undertaken to identify the types of collagen fibrils in the extracellular matrix of the human vocal fold lamina propria. METHODS: Human vocal folds were obtained from 3 autopsy cases less than 65 years of age. The vocal fold specimens were labeled by primary antibodies of anti-type I and anti-type III collagens, and then by secondary antibody conjugated with 15 nm colloidal gold. The specimens were observed with a scanning electron microscope. Secondary electron imaging and backscatter electron imaging of high-resolution field emission scanning electron microscopy were used to detect gold particles indicating immunolabeling. RESULTS: Type III collagen-labeling gold particles were abundant on the fibrils constructing collagenous fibers, whereas type I collagen-labeling gold particles were sparsely present on fibrils in collagenous fibers. A few reticular fibers were labeled by both collagen type I and collagen type III. CONCLUSIONS: The results suggest that collagen type I coexists with collagen type III in fibrils of both collagenous fibers and reticular fibers.     

Histological study of acute vocal fold injury in a rat model

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.     

自体脂肪间充质干细胞移植促进声带修复再生的实验研究

目的 探讨自体脂肪间充质干细胞移植入损伤声带后的生长分布及对声带固有层及其主要细胞外基质变化的影响特点.方法 对34只实验用兔(68侧声带)的53侧声带进行全麻支撑喉镜下声带锐性损伤.分离培养、鉴定及定向诱导20只实验用兔脂肪间充质干细胞.将自体脂肪间充质干细胞植入20侧损伤声带后,示踪观察脂肪间充质干细胞在声带固有层内的生长分布;同时以单纯支架(胶原或透明质酸凝胶)植入18侧受损声带(胶原10侧,透明质酸8侧)及单纯损伤15侧声带作为对照组.术后15 d～12个月时采用HE染色、Masson染色、Alcian Blue染色及免疫组化染色观察声带形态学变化及声带固有层胶原、透明质酸、纤维连接蛋白的含量及分布变化.结果 脂肪间充质干细胞呈梭形贴壁生长,具有多向分化潜能,植入损伤兔声带后持续分布于声带固有层.损伤声带在脂肪间充质于细胞植入6个月时形态接近正常,12个月时部分组织学结构类似正常;声带细胞外基质中的胶原于植入3个月内含量有增高趋势,无序分布于固有层,后逐渐减少至正常水平附近,12个月时部分分布略显不规则;透明质酸于植入40 d含量亦有增高趋势,分布于固有层各层,6个月含量减至正常水平附近,局限于固有层浅中层;纤维连接蛋白于植入后始终在固有层内散在分布,含量在40 d内有增高趋势,后逐渐减少,12个月时含量接近正常.单纯声带损伤3个月后局部开始出现瘢痕挛缩,以胶原纤维为主的大量纤维组织增生,12个月时仍紊乱分布于声带固有层各层.单纯支架植入组变化介于二者之间.结论 脂肪间充质干细胞植入兔损伤声带后具有促进声带细胞外基质分泌、合理分布及部分有序化排列的功能,具有促进声带修复再生的作用.     

Disarrangement of collagen fibers in Reinke's edema

Objective/Hypothesis: To describe the arrangement of collagen fibers in the superficial layer of the lamina propria of the vocal folds with Reinke' edema. Study Design: Cross sectional analysis of the lamina propria of the vocal folds with Reinke's edema (RE). Method: The picrosirius polarization method was used to study the arrangement of collagen fiber. Findings of collagen disarrangement were categorized semiquantitatively and correlated with RE severity, age, cigarette smoking and duration of dysphonia. Results: Analysis of 20 specimens of vocal folds with RE showed that the intertwined network of collagen fibers resembling a wicker‐basket normally observed in vocal folds was disarranged in RE. The collagen fibers were loosely arranged, fragmented and intermixed with varying amounts of myxoid stroma. Moderate and large areas of disarrangement (90% of cases) predominated. Collagen fiber arrangement in the region underneath the epithelium was better preserved when compared with fibers in the deeper region of the superficial layer of the lamina propria. There was a statistical difference in collagen disarrangement between grade II and grade III severity (P = .007) that appeared to be due to the large areas of disarrangement observed in 73% of patients with grade III severity and in 44% of grade II severity. Age was the only variable correlated with collagen fiber disarrangement (r = 0.47, P = .037). Conclusion: Our findings suggest that the flexible framework which maintains the uniformity of the lamina propria was lost in RE caused by the disarrangement of the collagen fibers.     

Characterization of vocal fold scarring in a canine model

OBJECTIVE: The objective was to assess the histological and viscoelastic shear tissue properties of the scarred vocal fold lamina propria at 2 and 6 months postoperatively in a canine model. STUDY DESIGN: Experimental, nonrandomized prospective study. METHODS: Six canine larynges were injured using a vocal fold stripping procedure. At 2 and 6 months postoperatively, histological analyses of the scarred and control lamina propria samples were completed for collagen, procollagen, elastin, and hyaluronic acid. RESULTS: In canines killed at 2 months, scarred tissue samples contained increased procollagen and decreased elastin. Elastin fibers in the scarred lamina propria were characteristically tangled and disorganized. In canines killed at 6 months, scarred tissue samples showed decreased elastin and increased collagen. Collagen fibers formed thick, disorganized bundles, and elastin fibers were disorganized throughout the entire scarred vocal fold lamina propria. Viscoelastic shear tissue measurements revealed increased stiffness and viscosity in one of three cases at 2 months and in all three cases at 6 months, indicating increased stiffness and resistance to shear flow during oscillatory shear deformation for scarred tissue samples. No differences were observed between the two postoperative times. CONCLUSIONS: Results indicated that viscoelastic tissue changes may take place before scar maturation in the scarred vocal fold lamina propria and that, although abundant collagen deposition may influence viscoelastic shear tissue properties, disorganization of collagen and elastin fibers, thick bundle collagen formation, or the interplay of several of these factors might also play a contributing role.     

Roles of hepatocyte growth factor and transforming growth factor beta1 in production of extracellular matrix by canine vocal fold fibroblasts

BACKGROUND/OBJECTIVES: When the lamina propria of the vocal fold is replaced by fibrosis after wound healing, it is difficult to restore an appropriate viscoelasticity of the vocal fold. To treat fibrotic scarring, material that reduces collagen deposition and increases soft amorphous substances, such as hyaluronic acid, is required. The potential use of hepatocyte growth factor (HGF) is intriguing. In this study, the authors examined canine vocal fold fibroblasts to determine how HGF contributes to the production of extracellular matrix. More specifically, the authors describe how the productions of hyaluronic acid, collagen type I, and fibronectin are associated with administration of HGF and transforming growth factor beta1. STUDY DESIGN: In vitro. METHODS: Fibroblasts were collected from the lamina propria of the vocal folds of five Beagles and were cultured with and without HGF or transforming growth factor beta1. The productions of hyaluronic acid, collagen type I, and fibronectin in supernatants culture were examined using ELISA. RESULTS: Hepatocyte growth factor stimulated hyaluronic acid production, reduced collagen type I production, and did not affect fibronectin production, while transforming growth factor beta1 stimulated the productions of all components. CONCLUSIONS: Collagen type I appears to be a major contributor in creation of fibrosis, and excessive fibronectin may stiffen the tissue. Since HGF reduced collagen type I production from fibroblasts and increased hyaluronic acid, HGF is considered to have therapeutic potential in prevention and treatment of the fibrosis of the vocal fold.     

Comparative Histology and Vibration of the Vocal Folds: Implications for Experimental Studies in Microlaryngeal Surgery

Objectives/Hypothesis To determine the most suitable animal model for experimental studies on vocal fold surgery and function by a histological comparison of the microflap surgical plane and laryngeal videostroboscopy (LVS) in different species of animals. A second goal was to determine how the layered vocal fold structure in humans and three different animal species affects surgical dissection within the lamina propria. Study Design Prospective laboratory. Methods Three larynges each from dogs, monkeys, and pigs were compared with three ex vivo human larynges. Microflap surgery was performed on one vocal fold from each larynx. Both the operated and nonoperated vocal folds were examined histologically using stains specific for elastin, mature collagen, and ground substance. Based on the histological results, LVS was performed on two dogs and two pigs after first performing a tracheotomy for ventilation and airflow through the glottis. Arytenoid adduction sutures were placed to facilitate vocal fold adduction. Results The distributions of the collagen and elastin fibers were found to differ among the species with concentrations varying within species. Unlike the human vocal fold, which has a higher elastin concentration in the deeper layers of the lamina propria, both the pig and the dog had a thin band of elastin concentrated just deep to the basement membrane zone in the superficial layer. Just deep to this thin band, the collagen and the elastin were less concentrated. The monkey vocal fold had a very thin mucosal layer with less elastin throughout the mucosa. The microflap dissections in each of the dog, pig, and human vocal folds were similar, being located within that portion of the superficial lamina propria where the elastin and mature collagen are less concentrated. The microflap plane in the monkey vocal fold was more deeply located near the vocalis fibers. Despite the differences in elastin concentration, the microflap plane in both the dog and the pig was found to be similar to that in humans. The dog anatomy was much more suitable for microsuspension laryngoscopy and stroboscopic examination. The dog vocal folds vibrated in a similar fashion to human vocal folds with mucosal waves and vertical phase differences, features not seen in the pig vocal folds. Conclusions Based on both the histological and stroboscopic results, the dog was believed to be a more suitable animal model for studies on vocal fold surgery, acknowledging that no animal's laryngeal anatomy is identical to that of the human. The dog LVS model presented allows for longitudinal laryngeal studies requiring repeated examinations at multiple time periods with histological correlation applied at sacrifice.     

Collagen fiber and versican distribution within the lamina propria of fetal vocal folds

OBJECTIVES: To analyze the presence and distribution of collagen fibers and versican in human vocal fold lamina propria of fetal larynges. STUDY DESIGN: Cross sectional analysis of cadaveric vocal folds of human fetuses. METHODS: Seven fetal larynges obtained from 28- to 36-week-old fetuses were analyzed with the Picrosirius-polarization method, immunohistochemistry, and image analysis. RESULTS: Collagen fibers within the lamina propria exhibited a monolaminar distribution pattern and spatial arrangement in "wicker basket." Versican distribution was larger in the superficial and intermediate layers when compared to the deep layer. CONCLUSION: Our findings suggest that collagen and versican distribution and arrangement within the lamina propria in the developing fetus are important for vocalization at birth.     

Atomic force microscopy investigation of vocal fold collagen

OBJECTIVES: Collagen is an important constituent of the vocal fold extracellular matrix and is necessary for providing tensile strength and maintaining tissue geometry. Traditional investigations of vocal fold collagen using light and electron microscopy do not provide information on the organization and mechanical properties of collagen in native topographic state. The primary objective of this study was to use Atomic Force Microscopy (AFM) to examine the surface characteristics and organization of collagen in the deep layer of the lamina propria at nanoscale resolution. STUDY DESIGN: Experimental in vitro design. METHODS: Freshly dissected porcine vocal folds were mounted on AFM discs and imaged under contact and tapping mode to obtain information on topographic distribution of collagen. RESULTS: AFM imaging of the deep layer of the lamina propria revealed dense, abundant collagen fibers with a characteristic banding pattern. The distribution of collagen was heterogeneous, with both hydrophilic and hydrophobic regions within a sample. CONCLUSIONS: AFM offers a useful tool to obtain topographic information about biologic samples at nanoscale resolution with minimal sample preparation. Mapping the topography and mechanical properties of vocal fold collagen is necessary for designing rheologically compatible bioimplants for the treatment of dysphonia resulting from vocal fold scarring and bowing.     

Therapeutic potential of growth factors for aging voice

OBJECTIVES/HYPOTHESIS: It has been reported that in aged vocal folds, dense collagen deposition takes place and hyaluronic acid decreases in the lamina propria, which are thought to contribute to the vocal problems occurring with age (presbyphonia). To restore aged vocal folds to their younger state, it seems crucial to address these age-related lamina propria changes. Intervention that might increase hyaluronic acid and decrease collagen would appear to be a potentially useful approach. The present study examined the effects of growth factors on aged fibroblasts in terms of the production of hyaluronic acid and collagen type I. STUDY DESIGN: In vitro study using animal model. METHODS: Fibroblasts were harvested from young and aged rat vocal folds and cultured with or without hepatocyte growth factor and/or basic fibroblast growth factor at different concentrations. Subsequently, the production of hyaluronic acid and collagen type I was examined in the supernatant culture media using enzyme-linked immunosorbent assay. RESULTS: Aged fibroblasts produced less hyaluronic acid than younger fibroblasts. When aged and young fibroblasts were cultured with basic fibroblast growth factor, hyaluronic acid production increased and collagen type I production decreased regardless of the concentration, whereas the effects of hepatocyte growth factor appeared to vary with concentration. The basic fibroblast growth factor also was associated with stimulation of growth of aged fibroblasts. CONCLUSION: The present results suggest that growth factors, especially basic fibroblast growth factor, may have therapeutic potential in restoration of aged vocal fold.     

Regeneration of aged vocal folds with basic fibroblast growth factor in a rat model: a preliminary report

Aged vocal folds have been reported to have dense collagen deposition and decreased hyaluronic acid (HA) in the lamina propria. These characteristics are thought to contribute to vocal problems that occur with age (presbyphonia). To restore better viscoelasticity to aged vocal folds, an intervention that might increase HA and decrease collagen production from aged vocal fold fibroblasts would appear to be a potentially useful approach. Our previous in vitro study has revealed that basic fibroblast growth factor (bFGF) consistently stimulates HA production and decreases collagen production from aged rat vocal fold fibroblasts. The present in vivo study examined the effects of intracordal injection of bFGF into aged rats' vocal folds in terms of restoration of HA and collagen distribution in the lamina propria. We injected bFGF transorally into the lamina propria of (unilateral) vocal folds. The injection was repeated 4 times weekly, and rats were painlessly sacrificed 1 week, 1 month, and 2 months after the final injection. Histologic examination revealed that bFGF significantly increased the HA content of the lamina propria up to 2 months, but showed no effect on collagen, even after 2 months. Because it might take longer for excessive collagen to be degraded, further studies are necessary to clarify the long-term effect on collagen. A drug delivery system for bFGF also needs to be developed to maximize its effect in the future. The present study suggested at least a positive effect of bFGF in restoring the HA content in the aged vocal fold lamina propria.     

Histologic characterization of rat vocal fold scarring

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-diameter 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.     

人羊膜上皮细胞移植用于兔损伤声带组织修复的初步研究

目的 研究人羊膜上皮细胞(human amniotic epithelial cells,hAEC)移植入兔声带损伤组织内的生长分布特点,探索hAEC促进声带损伤后修复再生的潜能.方法 分离和培养hAEC,慢病毒增强型绿色荧光蛋白(1entivirus enhanced green fluorescent protein,Lenti-EGFP)基因转染作标记.建立深及声韧带的兔声带损伤模型,设hAEC移植组(13侧声带)、损伤对照组(13侧声带)及正常对照组(4侧声带).荧光显微镜下连续观察hAEC在声带内的存活、分布情况,应用HE染色和免疫组化染色分析胶原、纤维连接蛋白等主要细胞外基质在损伤后3个月时的含量及分布.结果 hAEC原代培养6 d后呈铺路石样生长,植入声带损伤组织后可在固有层内持续存活2个月,细胞呈纵向排列,有趋向性.声带损伤2个月时免疫荧光显示hAEC移植组兔肌细胞标志结蛋白荧光阳性,提示hAEC向肌细胞分化;同时Ⅲ型胶原荧光阳性,提示hAEC植入后具有分泌Ⅲ型胶原功能.光镜观察见hAEC移植后3个月兔胶原纤维密度和排序较损伤对照组改善,但未及正常;免疫组化染色示hAEC移植组纤维连接蛋白含量和分布介于损伤对照组和正常对照组之间.结论 hAEC可在异种动物声带损伤组织内持续存活、生长,并有向声带组织分化和分泌部分细胞外基质的潜能,可能促进声带损伤后的修复再生.     

人羊膜上皮细胞移植用于兔损伤声带组织修复的初步研究

目的 研究人羊膜上皮细胞(human amniotic epithelial cells,hAEC)移植入兔声带损伤组织内的生长分布特点,探索hAEC促进声带损伤后修复再生的潜能.方法 分离和培养hAEC,慢病毒增强型绿色荧光蛋白(1entivirus enhanced green fluorescent protein,Lenti-EGFP)基因转染作标记.建立深及声韧带的兔声带损伤模型,设hAEC移植组(13侧声带)、损伤对照组(13侧声带)及正常对照组(4侧声带).荧光显微镜下连续观察hAEC在声带内的存活、分布情况,应用HE染色和免疫组化染色分析胶原、纤维连接蛋白等主要细胞外基质在损伤后3个月时的含量及分布.结果 hAEC原代培养6 d后呈铺路石样生长,植入声带损伤组织后可在固有层内持续存活2个月,细胞呈纵向排列,有趋向性.声带损伤2个月时免疫荧光显示hAEC移植组兔肌细胞标志结蛋白荧光阳性,提示hAEC向肌细胞分化;同时Ⅲ型胶原荧光阳性,提示hAEC植入后具有分泌Ⅲ型胶原功能.光镜观察见hAEC移植后3个月兔胶原纤维密度和排序较损伤对照组改善,但未及正常;免疫组化染色示hAEC移植组纤维连接蛋白含量和分布介于损伤对照组和正常对照组之间.结论 hAEC可在异种动物声带损伤组织内持续存活、生长,并有向声带组织分化和分泌部分细胞外基质的潜能,可能促进声带损伤后的修复再生.

Abstract：

Objective To investigate the survival, growth and distribution of human amniotic epithelial cells (hAEC) after injected into injured rabbit vocal folds, in addition, to assess the ability of hAEC to affect the components of lamina propria extracellular matrix (ECM) and prevent vocal fold scarring.Methods hAEC were isolated from human amnion and marked by Lenti-EGFP. Fifteen New Zealand rabbits were used for this experiment. EGFP-hAEC was injected into the left injured vocal folds in thirteen rabbits, and the contralateral thirteen vocal folds experienced an injured procedure only (" injured untreated control"), and four vocal folds were left as untreated controls. The survival, distribution, differentiation potential and secretion function of hAEC were examined by immunofluorescence method. HE staining and immunohistochemical staining were performed for the evaluation of collagen and fibronectin respectively.Results hAEC showed a cobblestone-like growth. After implanted into the injured vocal folds, hAEC could survive in vocal fold lamina propria for 2 months. The immunofluorescence analysis showed the evidence of hAEC differentiation into muscle cells as well as secretion the ECM protein. Three months postoperatively, the density of collagen was higher in the injured untreated control folds than that in the injured vocal folds injected with hAEC and the untreated controls. Besides, the content of fibronectin in the injured untreated control group was significantly increased. Conclusions hAEC survived in the vocal folds lamina propria,and had the potentiality to differentiate into vocal folds tissue and secret some ECM components. The histological improvement caused by the injected cells demonstrate that hAEC had the ability to promote the repairment and regeneration of injured vocal folds.     

Treatment of vocal fold scarring: rheological and histological measures of homologous collagen matrix

The current treatment options for dysphonia secondary to vocal fold scarring are limited. Few studies address changes in the lamina propria, which is critical to vocal fold biomechanical properties and voice production. Using rheological and histological measures of homologous collagen matrix (HCM)-injected vocal folds, we assessed HCM's potential for providing bulk and restoring biomechanical performance. Twenty rabbits underwent bilateral vocal fold scarring. After 10 weeks of scar maturation, the rabbits had unilateral injections of HCM or saline solution. Ten weeks after the injections, histological studies revealed well-defined collagen globules distributed throughout the lamina propria and underlying muscular tissue. Significantly more procollagen was observed in the HCM-treated group. No significant differences in elastic shear modulus or dynamic viscosity were found between the treatment groups. This study demonstrates that HCM is an inert, relatively stable injectate that may serve well for medialization but does not appear to improve the dynamic properties of the lamina propria.     

Advances and refinements in phonosurgery

Scientific discovery, technological advances, and improved outcomes assessment have resulted in advances and refinements in phonosurgery. Three areas of substantial evolution are phonomicrosurgery, laryngeal framework surgery, and the use of implantable materials in vocal folds. Discovery of the importance of the superficial layers of the lamina propria has led to increased use of more limited medial microflap approaches and less frequent use of the classic lateral cordotomy flap approach. Alternative approaches to managing vocal fold scarring defects have addressed the separation of body and cover and provided suitable lamina propria replacement. Approaches to sulcus vocalis have been refined to address type II (linear vergeture) and type III (focal invasive pit) sulcus, where there is loss of lamina propria, while still recognizing the common nonpathological type I (physiological) sulcus. Technological advancements such as photodynamic therapy, tuned dye lasers, and laryngeal microdebridement have augmented the armamentarium for mechanical removal of laryngeal papillomata. Careful infusion-assisted microexcision and adjunctive medical management have been refined and made more effective. Laryngeal framework surgery has embraced the development of Silastic, hydroxylapatite, expanded polytetrafluoroethylene, and titanium shims. Anatomical studies have helped to improve operative precision and safety, and have led to inventive variations in arytenoid repositioning that improve closure of the posterior subunit. Vocal fold augmentation by injection has been facilitated by innovative use of the rigid telescope and intraoperative videostroboscopy. Anatomical studies have focused on the infrafold region and rheological studies have attempted to match viscoelastic properties of injectable substances to those of vocal fold tissues. Alloplastic materials such as Teflon have been largely supplanted by newer bioimplantables such as fat, collagen, and fascia.     

Stellate cells in the human vocal fold

Cells have been discovered that are star-like in appearance and that actively synthesize extracellular matrices in the human adult vocal fold mucosa. These cells have no nomenclature and are thus designated as vocal fold stellate cells (VFSC) in this study. Light and electron microscopic investigation of VFSC in the human vocal fold mucosa was carried out on excised human adult larynges. A comparison between VFSC and conventional fibroblasts was made. The results are summarized as follows. 1) The VFSC are distributed in human adult maculae flavae. 2) The VFSC are irregular and stellate in shape, possessing slender cytoplasmic processes. 3) Lipid droplets are present in the cytoplasm. 4) The VFSC have a small nucleus-cytoplasm ratio and well-developed rough endoplasmic reticulum, suggesting active protein synthesis in these cells. 5) No basal lamina is present, and filaments can be seen in the cytoplasm. 6) The VFSC show strong cytoplasm staining with periodic acid-Schiff stain and type III collagen. 7) The VFSC actively synthesize collagenous fibers, including reticular fibers, as well as other extracellular matrices, such as elastic fibers and glycosaminoglycan (hyaluronic acid). 8) The VFSC, first demonstrated in this study, actively synthesize extracellular matrices in the human adult vocal fold mucosa under normal conditions. 9) The VFSC participate in the metabolism of the extracellular matrices essential for the viscoelastic properties of the lamina propria of the human adult vocal fold mucosa as a vibrating tissue.     

Age-related development of the arrangement of connective tissue fibers in the lamina propria of the human vocal fold

A scanning electron microscopic study was made on the morphological changes occurring with age in collagen and elastic fibers in the lamina propria of the human vocal fold. We obtained the specimens from 32 autopsy cases ranging from 20 gestational weeks to 22 postnatal years and submitted them to digestion treatments with 10% sodium hydroxide and 90% formic acid. The vocal folds in fetuses and neonates consisted of sparse and dense areas of collagen and elastic fibers, and the vocal ligament was not found. In subjects 5 years of age, a deep dense area was found in the anterior and posterior maculae flavae, and longitudinal fibers were noted between the maculae. A structure of superficial versus deep layers appeared in children older than 10 years of age. The layered structure of the lamina propria was complete around 17 years of age. The development of the layered structure and the maturity of the fibers appeared to reflect the complexity of phonatory function during adolescence.     

Fibronectin and adhesion molecules on canine scarred vocal folds

OBJECTIVE: To examine the expressions of fibronectin and other adhesion molecules on the scarred vocal folds in a short- and long-term animal model. STUDY DESIGN: Animal model. METHODS: Six beagles' vocal folds were stripped unilaterally and left untreated. After wounding the vocal folds were harvested from three dogs at 2 months and three dogs at 6 months. The untouched vocal fold was used as a control, and the stripped vocal fold as scarred. Subsequently, the expressions of fibronectin, cadherin, syndecan-1 and syndecan-4 on both vocal folds were examined by immunohistochemical and image analysis. RESULTS: Compared with the control vocal folds, fibronectin significantly increased in the superficial layer of the lamina propria on the scarred vocal folds at both 2 and 6 months. Co-deposition of collagen was observed only at 6 months. Syndecan-4 was significantly overexpressed at the basal layer cells of the epithelium at both 2 and 6 months. No significant expression of either cadherin or syndecan-1 was detected. CONCLUSIONS: Scar characteristics at 2 and 6 months are not identical, suggesting that a 2-month period may not be a sufficient to study vocal fold scarring. Adhesion molecules are important in reorganization of extracellular matrix during wound healing because of their binding and adhesion characteristics. The results indicate that fibronectin might be important in providing a scaffold for the deposition of other proteins such as collagen, and the binding characteristics might affect the stiffness of the scarred vocal fold. Prolonged expression of syndecan-4 may reflect the role of focal adhesion during the assembly of scar structure. Ultimately, better understanding of the histological features of the scarred vocal fold might lead to new approaches to treatment.