3D structure of the macula flava in the human vocal fold

OBJECTIVE: To investigate the 3D structure of the macula flava in the human vocal fold. MATERIAL AND METHODS: The 3D structure of the macula flava in the human vocal fold was investigated by means of scanning electron microscopy using a chemical digestion method (modified sodium hydroxide maceration method) and the results compared with those obtained using light microscopy. RESULTS: The macula flava of the human vocal fold comprised a dense mass of cells and extracellular matrices. Many cells were stellate in shape and possessed cytoplasmic processes. Extracellular matrices were composed of collagenous, reticular and elastic fibers, and ground substances (hyaluronic acid). Many fibers ran in various directions. The stellate cells were surrounded by these extracellular matrices. Amorphous materials were attached to the surface of the stellate cells. Extracellular matrices in the macula flava extended to those in the lamina propria of the vocal fold mucosa. CONCLUSION: Anterior and posterior maculae flavae in the human vocal fold mucosa are postulated to be involved in the metabolism of extracellular matrices, which are essential to the viscoelasticity of the lamina propria (the vibrating tissue) of the human adult vocal fold mucosa.     

Fibroblasts in human vocal fold mucosa.

Fibroblasts in two portions of the vocal fold mucosa, the macula flava (MF) and Reinke's space (RS), were investigated by means of transmission electron microscopy. Five adult and five newborn larynges from autopsy cases were subjected to study. Both in adults and newborns, fibroblasts in the MF tended to be stellate in shape, to have a small nucleus/cytoplasm (N/C) ratio, and to have a well developed rough endoplasmic reticulum (rER) and Golgi apparatus (GA). They were active in producing collagenous and elastic fibres. Most fibroblasts in RS were oval in newborns and spindle-shaped in adults. They had a large N/C ratio and less developed rER and GA, indicating that they were inactive in producing fibres. We postulate that many fibroblasts in the newborn and infant MF contribute to the development of the vocal ligament, many of those in the adult MF participate in the metabolism of the vocal ligament, and many of those in RS of both age groups are activated when the vocal fold tissue is injured.     

Roles of vitamin A and macula flava in maintaining vocal folds

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

Fibroblasts in Human Vocal Fold Mucosa

Fibroblasts in two portions of the vocal fold mucosa, the macula flava (MF) and Reinke's space (RS), were investigated by means of transmission electron microscopy. Five adult and five newborn larynges from autopsy cases were subjected to study. Both in adults and newborns, fibroblasts in the MF tended to be stellate in shape, to have a small nucleus/cytoplasm (N/C) ratio, and to have a well developed rough endoplasmic reticulum (rER) and Golgi apparatus (GA). They were active in producing collagenous and elastic fibres. Most fibroblasts in RS were oval in newborns and spindle-shaped in adults. They had a large N/C ratio and less developed rER and GA, indicating that they were inactive in producing fibres. We postulate that many fibroblasts in the newborn and infant MF contribute to the development of the vocal ligament, many of those in the adult MF participate in the metabolism of the vocal ligament, and many of those in RS of both age groups are activated when the vocal fold tissue is injured.     

Morphological and functional changes of human vocal fold fibroblasts with hepatocyte growth factor

Fibroblasts produce extracellular matrix and play an important role in wound healing and scarring. Hepatocyte growth factor (HGF) has strong antifibrotic activity, and has been suggested to have therapeutic potential for treatment of fibrotic diseases. In the present in vitro study, morphological and functional changes of human vocal fold fibroblasts with HGF were examined by transmission electron microscopy and enzyme-linked immunosorbent assay to help clarify the potential use of HGF in the prevention or treatment of vocal fold scarring. The HGF stimulated the production of hyaluronic acid (HA) and decreased the production of collagen type I from the fibroblasts in Reinke's space (FbRS), whereas fibronectin production was not affected. The HGF also changed the shape of the FbRS from an oval shape toward a spindle and stellate shape, and developed Golgi apparatus (GA) and rough endoplasmic reticulum (rER) in the FbRS. The fibroblasts in the macula flava (FbMF) presented with much more production of HA and collagen type I than did FbRS, and were more frequently formed in a stellate shape with well-developed GA and rER. The HGF decreased the production of collagen type I from the FbMF, but barely affected the FbMF in terms of the shape of the cells, the development of GA and rER, or the production of HA. These results were interpreted to suggest that the FbMF are not as susceptible to HGF as are FbRS. On the contrary, HGF appeared to activate the FbRS and modify the function. The increased HA and decreased collagen type I production from the FbRS suggest that HGF may be useful in the prevention or treatment of fibrotic vocal fold scarring.     

Fibroblasts in geriatric vocal fold mucosa

Fifty fibroblasts in the macula flava (MF) and 50 fibroblasts in the Reinke's space (RS) of geriatric vocal folds were studied by transmission electron microscopy. Five larynges from autopsy cases in the age range 74-83 years were included in the study. The results were compared with those obtained previously from younger adults. The majority of fibroblasts in RS were spindle-shaped and those in MF were stellate. This was the case with younger adults. The nucleus/cytoplasm (N/C) ratio was 0.5-2.0 in most fibroblasts in RS and MF. Again, this was in common with the findings in younger adults. The development of rough endoplasmic reticulum (rER) and Golgi apparatus (GA) in MF was less marked in the geriatric group than in adults. Glycogen particles were observed in 28 fibroblasts in MF. Lipofuscin granules were found in 12 fibroblasts of RS and 9 cells of MF. The findings suggest a decrease in activities and the presence of ageing processes in fibroblasts of geriatric vocal fold mucosa, to which geriatric changes in the vocal ligament can be attributed.     

Vascularity in the macula flava of human vocal fold as a stem cell niche

ObjectivesThere is growing evidence that the maculae flavae of the human vocal fold are a stem cell niche, which is a microenvironment nurturing tissue stem cells. This study investigated the microenvironment, especially vascularity, in the maculae flavae of the human vocal fold.MethodsThree normal human adult, three normal newborn vocal folds obtained from autopsy cases and three surgical specimens of glottic carcinoma were investigated using light and electron microscopy. For scanning electron microscopy, a chemical digestion method (modified sodium hydroxide maceration method) was used to observe the inner 3-dimensional structure of the macula flava.ResultsCapillaries ran around the anterior and posterior maculae flavae in adults and newborns. However, there was no vascularity in the maculae flavae of the vocal fold. The inner 3-dimensional electron microscopic structure of the macula flava showed there were no blood vessels in the maculae flavae of the vocal fold. Glottic carcinoma (squamous cell carcinoma) surrounded and was in contact with the macula flava, however, the carcinoma did not invade the macula flava indicating there was no vascular supply into the macula flava from the surrounding tissue.ConclusionsThere was no vascularity in the anterior and posterior maculae flavae in the human adult and newborn vocal folds. The present study is consistent with the hypothesis that the hypoxic microenvironment in the maculae flavae of the adult and newborn vocal fold as a stem cell niche is likely favorable to maintaining the stemness and undifferentiated states of the tissue stem cells in the stem cell system.     

Cell production in injured vocal folds: a rat study

OBJECTIVES: Fibroblasts are reported to play an important role in producing the extracellular matrix of the vocal fold. However, no reports have focused on how and where these cells are generated in the vocal fold after injury. To reveal the characteristics of vocal fold cell production, we investigated cell proliferation in the acute phase of wound healing. METHODS: Using a telescope for guidance, we made an incision in the middle region of the vocal fold tissue in 24 rats and performed immunohistochemical staining for vimentin, alpha-smooth muscle actin, and 5-bromo-2-deoxyuridine. RESULTS: After injury, epithelialization occurred with a peak at day 1, and fibroblasts proliferated in the lamina propria with a peak at day 3, whereas those in the macula flava did not show any increased proliferation. CONCLUSIONS: It is suggested that the fibroblasts in the macula flava have functions different from those of fibroblasts in the lamina propria and that the macula flava does not serve as a cell source for the vocal fold in response to injury.     

Fibroblasts in Geriatric Vocal Fold Mucosa

Fifty fibroblasts in the macula flava (MF) and 50 fibroblasts in the Reinke's space (RS) of geriatric vocal folds were studied by transmission electron microscopy. Five larynges from autopsy cases in the age range 74-83 years were included in the study. The results were compared with those obtained previously from younger adults. The majority of fibroblasts in RS were spindle-shaped and those in MF were stellate. This was the case with younger adults. The nucleus/cytoplasm (N/C) ratio was 0.5-2.0 in most fibroblasts in RS and MF. Again, this was in common with the findings in younger adults. The development of rough endoplasmic reticulum (rER) and Golgi apparatus (GA) in MF was less marked in the geriatric group than in adults. Glycogen particles were observed in 28 fibroblasts in MF. Lipofuscin granules were found in 12 fibroblasts of RS and 9 cells of MF. The findings suggest a decrease in activities and the presence of ageing processes in fibroblasts of geriatric vocal fold mucosa, to which geriatric changes in the vocal ligament can be attributed.     

Histopathologic investigations of the unphonated human vocal fold mucosa

CONCLUSION: Vocal fold vibration (phonation) after birth is one of the important factors in the growth and development of the human vocal fold mucosa. OBJECTIVES: Stellate cells in the maculae flavae located at both ends of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices. Maculae flavae are also considered to be an important structure in the growth and development of the human vocal fold mucosa. Tension caused by phonation (vocal fold vibration) is hypothesized to stimulate stellate cells to accelerate production of extracellular matrices. Vocal fold mucosae unphonated since birth were investigated histologically. SUBJECTS AND METHODS: Vocal fold mucosae, which were unphonated since birth, of three younger adults (17, 24, 28 years old) were investigated by light and electron microscopy. RESULTS: Vocal fold mucosae were hypoplastic and rudimentary and did not have a vocal ligament, Reinke's space or a layered structure. The lamina propria appeared as a uniform structure. Some stellate cells in the maculae flavae showed degeneration. Not many vesicles were present at the periphery of the cytoplasm. The stellate cells synthesized fewer extracellular matrices, such as fibrous protein and glycosaminoglycan. Cytoplasmic processes of the stellate cells were short and shrinking. The stellate cells appeared to have decreased activity.     

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.     

Implantation of esterified hyaluronic acid in microdissected Reinke's space after vocal fold microsurgery: first clinical experiences

OBJECTIVE: In this pilot study are presented the first clinical experiences of the use of a resorbable bioimplant made of esterified hyaluronic acid inserted in the microdissected superficial layer of the lamina propria (SLLP), also called Reinke's space, after a flap excision procedure for a benign vocal fold lesion. Laryngeal and vocal evolution of implanted patients are depicted and discussed. STUDY DESIGN: Eleven bio-implants have been inserted in microdissected SLLP of 11 cases presenting with benign vocal fold lesions. The surgical procedure consisted of the excision of primary lesion by a microflap technique immediately followed by implantation of esterified hyaluronic acid in Reinke's space. METHODS: All patients underwent rigid laryngoscopy and a microsurgical procedure under general anesthesia. The cordal lesion was treated with cold instrumentation of Bouchayer (7 cases) or with a mixed technique using CO(2) laser (4 cases). After the classical freeing-up of Reinke's space and the creation of a mucosal flap, a few fibers of esterified hyaluronic bioimplant are gently arranged in Reinke's space before redraping the ligament and closing the cordal incision with a few drops of fibrin glue. Laryngeal and vocal assessments were performed pre- and postoperatively in all patients using videostroboscopy as well as perceptual and objective voice evaluation. All patients were followed in a longitudinal manner: between two and five postoperative evaluations were performed. The longest follow-up was 19 months and the shortest 2 months. RESULTS: All cases exhibited postsurgical improvement of the pliability of the SLLP. None of them developed an adverse scarring process. Improvement of SLLP's pliability was maintained in time in all cases. Vocal improvement was observed in all. Temporary inflammation was noted in one case. There were no serious adverse effects apparent during the follow-up period. CONCLUSION: Bio-implantation of esterified hyaluronic acid in Reinke's space is technically easy and well tolerated. All treated cases exhibited postoperative good pliability of the SLLP compared with their preoperative evaluation.     

Vitamin A-storing stellate cells in the human newborn vocal fold

OBJECTIVES: Vocal fold stellate cells (VFSCs) in the newborn vocal fold were examined and compared with VFSCs in the adult vocal fold. METHODS: Light and electron microscopic investigation of VFSCs was carried out on 5 human newborn larynges. RESULTS: The VFSCs were distributed in human newborn maculae flavae. They were stellate or oval in shape, and possessed cytoplasmic processes. The VFSCs in some cells formed a gap junction with each other. A few lipid droplets were present in the cytoplasm, but they were much fewer than those of an adult. The nucleus-cytoplasm ratio was high, and the intracellular organelles were not very well developed. Free ribosomes were well developed in the cytoplasm. The VFSCs in some cells showed strong cytoplasm staining with periodic acid-Schiff stain and type III collagen. There were vesicles along the periphery of the cytoplasm of the VFSCs, and newly released amorphous materials were seen, but fewer were observed on the cell surface. The newborn VFSCs had started to synthesize extracellular matrices such as collagenous fibers, reticular fibers, elastic fibers, and glycosaminoglycan. CONCLUSIONS: The VFSCs in the newborn maculae flavae were immature, but had already started the synthesis of extracellular matrices essential for the viscoelastic properties of the vocal fold mucosa.     

声带息肉和Reinke水肿细胞外基质蛋白mRNA表达

目的探讨声带息肉和Reinke水肿中，声带固有层细胞外基质主要成分mRNA表达的变化，为阐明疾病发病机制提供依据。方法应用逆转录-聚合酶链反应检测手术切除标本（声带息肉12例，Reinke水肿2例，5例行全喉切除而声带未受肿瘤侵犯的声带作为正常对照），细胞外基质中与胶原纤维、弹性纤维和透明质酸有关的9种蛋白的mRNA表达。结果声带息肉与对照声带比较，胶原酶mRNA表达下降，纤连蛋白mRNA表达升高，而纤调蛋白mRNA表达下降，差异均有统计学意义（P值均〈0．05）；赖氨酰氧化酶、透明质酸合成酶在病变和对照组织mRNA表达量差异无统计学意义（P值均〉0．05）；前弹性蛋白原、透明质酸酶、弹性蛋白酶在病变组织mRNA阳性表达率低于对照组织；前胶原Ⅰ在所有组织中均无表达。声带Reinke水肿组纤连蛋白表达量接近息肉组，纤调蛋白表达量接近对照组，因样本量小未做统计学分析。结论纤连蛋白和纤调蛋白是参与声带息肉病理过程的两种细胞外基质成分。反复用声造成损伤以及声带对损伤的修复过程可能在声带息肉发病机制起重要作用。     

Macula flava in the vocal fold of human fetus

The histological analysis of fetal vocal folds (in humans) in serial horizontal sections revealed a dense cell accumulation at the anterior and posterior ends of the vocal fold. Macula flava posterior was identified in all analysed specimens and macula flava anterior in those older than 18 weeks. Maculae flavae are composed of mesenchymal immature cells which gradually originate from cartilaginous cells of the arytenoid or mesenchymal immature cells of the anterior commissure tendon. Macula flava posterior presents the continuation of the arytenoid and there is no delineation between macula flava anterior and anterior commissure tendon. Since macula flava posterior extends its protrusion to the place of the most intensive connective tissue organization (which begins already in fetal life), it has been concluded that both maculae have an important influence to the specific organization and differentiation of connective tissue in the lamina propria of the fetal vocal fold.     

Vitamin A-storing stellate cells in the human vocal fold

OBJECTIVE: Previously, we discovered cells with a star-like appearance that constantly synthesized extracellular matrices in the human adult vocal fold mucosa. These cells had no nomenclature and were thus designated "vocal fold stellate cells (VFSC)". The aim of the present study was to detect vitamin A (retinol) in the VFSC. MATERIAL AND METHODS: Five normal human adult vocal folds obtained from surgical specimens were used. Light microscopy using the gold chloride method and fluorescence microscopy were used to detect vitamin A. RESULTS: VFSC distributed in the maculae flavae of human adult vocal folds possessed lipid droplets and stored vitamin A. CONCLUSION: It is generally accepted that vitamin A, an essential liposoluble vitamin, is used as a morphogen that controls the differentiation and morphogenesis of cells. In our previous investigation and in this study VFSC were found to differ morphologically from conventional fibroblasts. VFSC represent morphologically a new category of cells in the human vocal fold.     

Microendoscopy of Reinke's space

OBJECTIVES: Contemporary surgical treatment of the superficial layer of the lamina propria or Reinke's space is most commonly performed through an incision in the overlying vocal fold epithelium. This approach may disrupt normal tissue, induce scarring, and allow extrusion of implanted materials. Previously reported external approaches to Reinke's space required either a laryngofissure or a "minithyrotomy" for access. These surgical approaches were performed without direct imaging of Reinke's space. Instruments placed below the vocal fold epithelium via this external approach were visualized through the translucent vocal fold epithelium. We designed this study to identify the feasibility of limited-access surgery of the lamina propria using microendoscopes placed into Reinke's space through an external approach. METHODS: A cadaveric human larynx was dissected, and microendoscopes were directly advanced into Reinke's space through a subepithelial puncture of the cricothyroid membrane, as well as lateral fenestration through the thyroid cartilage. RESULTS: Photodocumentation of the undersurface of vocal fold epithelium, the opposing surface of the vocal ligament, and the intervening Reinke's space was successfully accomplished. CONCLUSIONS: Advances in both microendoscopes and accompanying instrumentation permit access to the superficial layer of the lamina propria without disrupting the overlying epithelium. This approach to microendoscopy of Reinke's space may allow for more effective surgical treatment of cysts, chronic edema, vascular abnormalities, atrophy, scarring, and sulcus vocalis.     

Postnatal development of rat vocal folds

OBJECTIVES: In this study we aimed to determine the feasibility of using a rat model for the study of postnatal vocal fold (VF) development. METHODS: Eighteen male rats that were 3 days old, 3 weeks old, or 8 months old were analyzed histologically with Alcian blue stain used for detecting hyaluronic acid, elastin-van Gieson stain for elastin, Oil Red O and gold chloride stains for vitamin A-containing lipid droplets, and immunohistochemistry for vimentin (general fibroblast marker) and collagen types I and III. RESULTS: The macula flava (MF) was observed as a mass of cells that expressed vimentin intensively in the cytoplasm. The MF showed denser hyaluronic acid and collagen type I than did the midmembranous portion of the VF lamina propria. Clear developmental changes were evident in the MF and other regions. The vimentin-positive cells of the 3-day-old MF were mainly oval-shaped and had less cytoplasm, whereas those of the 8-month-old MF were spindle- and stellate-shaped and had more cytoplasm, similar to that reported in humans. Vitamin A-containing lipid droplets were limited to the 3-week-old and 8-month-old MFs and were not present in the 3-day-old VF. CONCLUSIONS: These results suggest that a rat model is useful in studying VF development and that vitamin A is related to the maturity of the VF.     

Steroid injection for Reinke's edema using fiberoptic laryngeal surgery

OBJECTIVE: Since 1990, we have performed steroid injections into the vocal fold under topical anesthesia using fiberoptic laryngeal surgery (FLS) in an outpatient clinic. The aim of this study was to retrospectively assess the usefulness of this treatment method in 44 patients with mild Reinke's edema. MATERIAL AND METHODS: Using fiberoptic monitoring of the larynx, a curved injection needle was inserted via the oral cavity and triamcinolone acetonide was injected into Reinke's space of the bilateral vocal fold. RESULTS: Remission or improvement was observed in almost all patients in terms of both patients' self-rating of hoarseness and endoscopic vocal fold findings The maximum phonation time was a mean of 9.0 s before operation and 11.4 safter operation, and this increase was significant (p < 0.01). Voice pitch also improved, from 168 to 181 Hz, in female patients, and this increase was also significant (p < 0.05). CONCLUSION: Steroid injection is considered to be useful for treating mild Reinke's edema.