[Abstract] Objective To investigate the ultrastructural changes of fibroblasts, collagen fibers, and elastic fibers in vocal cord polyp lesions. Methods Ten cases of surgically resected vocal fold polyps and three normal vocal fold controls (hypopharyngeal carcinoma with total laryngectomy and no tumor invasion of the vocal fold) were observed by transmission and scanning electron microscopy. The specimens with elastic fibers were digested with 90% formic acid and those with collagen fibers were digested with 10% aqueous NaOH solution. Results The number of fibroblasts in the lamina propria of the vocal fold polyps was increased by transmission electron microscopy, and the organelles were abundant, and a large number of endoplasmic reticulum, Golgi complex and mitochondria were visible, indicating that the fibroblasts were in an active functional state. Inflammatory cell infiltration was seen in the intrinsic layer, suggesting that the inflammatory reaction was related to the pathogenesis. The number of blood vessels in the vocal fold polyps was significantly increased. Scanning electron microscopy showed that the morphology of collagen and elastic fibers in the vocal fold polyps was changed and the fiber arrangement was disturbed. Conclusion The morphological alterations of fibroblasts, elastic fibers, and collagen fibers in vocal fold polyps may be the pathological basis of the vocal fold vocal function. [Keywords] Vocal folds; polyps; fibroblasts; collagen; elastin The vocal folds are histologically divided into three layers: epithelial layer, lamina propria, and muscular layer, of which the lamina propria plays an important role in maintaining vocal fold vibration and mucosal wave production as well as normal articulatory function. The intrinsic layer is composed of a small number of cells and extracellular matrix, which consists of fibers (collagen, elastic, reticular fibers), glycoproteins and proteoglycans, the latter two together called interstitial proteins. Under normal conditions, the extracellular matrix is mainly produced by fibroblasts, and its production and degradation are in dynamic balance. In pathological conditions, the synthesis and turnover of the matrix are dysregulated, leading to vocal disorders. In this study, we performed transmission electron microscopy and scanning electron microscopy on surgically excised vocal fold polyp specimens to observe the ultramicrohistological structure of normal vocal folds and vocal fold polyps, especially the morphological changes of fibroblasts, elastic fibers and collagen fibers. Materials and methods I. Subjects Ten cases of vocal fold polyps who were hospitalized for surgical treatment in PLA General Hospital from October 2004 to May 2005 were selected, and the surgically excised specimens were retained. The process of taking the specimens did not affect the surgical procedure and scope. There were 7 male and 3 female cases; age ranged from 24 to 55 years, with a median of 40.5 years. In addition, three cases of total laryngectomy for hypopharyngeal carcinoma were taken and their vocal folds were used as controls for normal vocal folds, with tumor tissue more than 1 cm from the normal folds. all three controls were male, aged 48-68 years, with a median of 65 years. Among the above cases, 6 specimens were observed by transmission electron microscopy, including 4 cases of vocal fold polyps (2 male and 2 female) and 2 cases of normal vocal folds; 9 cases were observed by scanning electron microscopy, including 7 cases of vocal fold polyps (5 male and 2 female) and 2 cases of normal vocal folds, and 2 specimens were observed by both transmission electron microscopy and scanning electron microscopy. Experimental methods 1. Transmission electron microscopy sample preparation and observation: surgically excised specimens were immediately fixed in 2% glutaraldehyde fixative for more than 4 h. 0.1 mol/L phosphate buffer washed for 15 min × 3 times. 1% osmium acid was fixed for 2 h at 4°C. 0.1 mol/L phosphate buffer washed for 15 min × 3 times. Dehydrate in an ethanol gradient. Epoxy resin Epson 812 embedding. 60 ℃ oven polymerization. Prepare semi-thin sections. Observe the semithin sections under light microscope, and select the parts with intact epithelial layer and lamina propria to prepare ultra-thin sections by conventional methods. The film-carrying copper mesh was retrieved and double stained with uranyl acetate solution and lead citrate solution. Transmission electron microscopy was observed (JEOL JEM-1230 type) with an accelerating voltage of 75 kV and a magnification of 6 000-30 000 times, and photographs were taken. 2, Scanning electron microscopy specimen preparation and observation: surgically excised specimens were immediately placed in 2% glutaraldehyde fixative for more than 4 h. Tissue digestion was performed to cut each fixed specimen into two parts with a thin blade to observe collagen fibers and elastic fibers, respectively. For collagen fibers, specimens were digested with 10% NaOH aqueous solution at 20-25 ℃ for 5 d. For elastic fibers, specimens were digested with 90% formic acid at 45 ℃ for 96 h. 0.1 mol/L phosphate buffer washed for more than 1 d. 2% tannic acid treatment for 2-3 h. 0.1 mol/L phosphate buffer washed for more than 1 h. 1% osmium acid post-fixation for 2 h. Serial ethanol dehydration. Isoamyl acetate treatment for 40 min. critical point drying (HITACHI HCP-2 type critical point dryer, Hitachi, Japan). Platinum plating (HITACHI E102 type ion sputterer) JEOL JSM-35C type scanning electron microscope observation with an acceleration voltage of 25 kV and magnification of 200-50,000 times, photographed. I. Transmission electron microscopic observation of normal vocal folds and vocal fold polyps 1. Epithelial layer of normal vocal folds: The vocal folds are compound squamous epithelium. It is divided into keratinized layer, granular layer, spiny layer and basal layer. The keratinized cells are flattened, and the nucleus and organelles are degenerated and disappeared. The keratinized cells are tightly connected with each other, and the upper and lower adjacent cells are interlocked and mosaic by the cytoplasm with many pseudopod-like protrusions, forming a mosaic connection, and some cells are densely mosaic connected with each other, forming a “zipper-like” structure. The cell surface has many cytoplasmic protrusions, and the protrusions of adjacent cells are in contact with each other, forming bridging connections, and there are obvious cell gaps between the protrusions. The basal cells had less cytoplasm and a larger gap between the basal cells and the spiny layer cells; bridging granules were visible between adjacent cells. There was no obvious basement membrane and semi-bridging granule connection between the basal cells and the lamina propria. There was a tendency for the cell gap to increase from superficial to deep in each layer of epithelial cells. 2, normal vocal folds of the lamina propria: the elastic fibers in the lamina propria were uniformly colored, lamellar amorphous material with irregular morphology, which could be ribbon-like, kidney-shaped, and lobulated, scattered between bundles of collagen fibers, within which dotted microfiber cross-sections were visible (Figure 1a). Intrinsic superficial elastic fibers are rarely seen, with an increased number in the intrinsic middle layer. A large number of collagen fibers were seen in the intrinsic layer, with various orientations, and parallel bundles of microfibers were seen in longitudinal sections, some curved and curved, and dotted microfibers in transverse sections (Figure 1b). From superficial to deep layers, the number of collagen fibers gradually increased. In the superficial intrinsic layer near the basal cells, thin, short, reticulately interwoven fibers could also be seen, but not in the middle and deep layers. A small number of scattered fibroblasts can also be seen in the lamina propria. The cytosol is shuttle-shaped, the nucleus is ovoid and located at one end, and the chromatin in the nucleus is evenly distributed and densely packed under the nuclear membrane, and nuclear pores are visible. Lysosomes and scattered ribosomes, Golgi complexes, endoplasmic reticulum and mitochondria were seen in the cytoplasm (Figure 1c). 3, epithelial layer of vocal cord polyps: There are two types of epithelial layer cells in vocal cord polyps, thickening and thinning, and in some cases there is thickening of the epithelial cell keratinized layer, and in the same case, there can be both changes of epithelial thickening and thinning. Intercellular bridging granule connections were abundant (Figure 2a). 4, Intrinsic layer of vocal cord polyps: epithelial basement membrane of vocal cord polyps was thickened, and denser collagen fibers were seen in the basement membrane area (Figure 2b). Inflammatory cell infiltration was seen in the intrinsic layer, including granulocytes, macrophages, lymphocytes, etc. The number of blood vessels in the intrinsic layer was significantly increased, and the vessels in the lesion were distributed throughout the intrinsic layer, with dilated vessels and large lumen, and a large number of erythrocytes were seen in some vessels, with thinning of the vascular endothelium and widening of the endothelial cell gap. A distinctive feature of the lamina propria of vocal fold polyps is the increase in the number of fibroblasts and the abundance of organelles, with a large number of endoplasmic reticulum, Golgi complex and mitochondria, indicating that fibroblasts are in an active functional state. Vocal fold polyps are lesions occurring in the lamina propria, and fibroblasts are the main cells that synthesize the extracellular matrix of the lamina propria, and their active function suggests that the extracellular matrix of vocal fold polyps is metabolically active (Figure 2c). Second, scanning electron microscopic observation of normal vocal folds and vocal fold polyps 1, collagen fibers in normal vocal folds: after 6-7 d of 10% NaOH digestion, the cellular components and elastic fibers in the vocal fold tissue were basically digested and observed under scanning electron microscopy, the outline of the epithelial layer and the outline of some blood vessels in the lamina propria could be seen. In the normal vocal folds, collagen fibers are massive, dense, and in bundles of varying thickness, each bundle consisting of fine fibers arranged in parallel with tortuous bundles that are not parallel to each other but intertwined in all directions (Figure 3a). Between the collagen fiber bundles, there are fine unbound fibers interwoven into a network. In the immediate vicinity of the epithelial layer, unbundled and tortuous coiled collagen fibers are seen, which may constitute the fibers of the basement membrane. 2, collagen fibers of vocal fold polyps: Since vocal fold polyps occur in the superficial layer of the vocal fold lamina propria, and collagen fibers are mainly located in the deep lamina propria, fewer collagen fibers are observed in the lesion tissue. It can be observed that in vocal fold polyps, the arrangement of collagen fiber bundles is more disorganized, and the protofibers in the collagen fiber bundles lose their parallel and regular arrangement and are intertwined with each other. There were large gaps in the collagen fiber bundles (Figure 3b). Some of the collagen fiber bundles were thicker in diameter, about 10 μm. We also observed a nodular structure in the longitudinal direction of the collagen fiber bundles in the lesioned tissue (Figure 3c), and the reason for the appearance of this structure is unclear. 3. Elastic fibers in normal vocal folds: After 3-4 d of digestion with 90% formic acid at 45°C, the cellular components and collagen fibers in the vocal fold tissue were basically digested, and when observed under scanning electron microscopy, the epithelial layer, lamina propria, and muscular layer could be distinguished, but the layers of the lamina propria were indistinguishable. In the normal vocal folds, elastic fibers are abundant and dense, and differ markedly from collagen fibers in that elastic fibers do not form bundles and fibers have unequal diameters. The elastic fibers are convoluted and have a “spring-like” appearance, coiled and tortuous, but generally oriented in the same direction. There were irregular gaps between the fibers (Figure 4a). 4.Elastic fibers of vocal cord polyps: Since elastic fibers are mainly located in the middle layer of the lamina propria, elastic fibers in vocal cord polyps are sparsely and scatteredly distributed, and the number is obviously reduced. Moreover, the diameter of elastic fibers became significantly smaller, and the fibers were slender, interwoven into a network and disordered in shape (Figure 4b). Transmission electron microscopic observation of normal vocal folds a: elastic fibers in the lamina propria × 12,000; b: collagen fibers in the lamina propria × 20,000; c: fibroblasts in the lamina propria × 10,000 Figure 2 Transmission electron microscopic observation of vocal fold polyps a: epithelial cells with abundant bridging granule connections × 2,500; b: thickened basement membrane × 6,000; c: fibroblasts with abundant organelles × 12,000 Figure 3 Scanning electron microscopy of collagen fibers Observations a: large number of dense collagen fibers in normal vocal folds; b: large gaps between collagen fibers in vocal fold polyps; c: longitudinal nodularity of collagen fibers in vocal fold polyps Figure 4 Scanning electron microscopy of elastic fibers a: curled and dense elastic fibers in normal vocal folds; b: disordered arrangement of elastic fibers in vocal fold polyps Discussion Vocal fold polyps are benign diseases that occur in the lamina propria of the vocal folds and are the most common vocal disorders. There are many scholars who have performed histopathological staging of vocal fold polyps, however, the staging methods are diverse and not uniform. Jiang Jie et al [1] classified vocal fold polyps into edematous, vasodilated, vitreous and fibrous types, Chen Xuanzhu et al [2] into thrombotic, edematous, fibrous, mixed and granulomatous types, and Li Jinjan et al [3] into edematous, vasodilated, hemorrhagic and hemorrhagic thrombotic, fibrous and vitreous degenerative types. The association between these typologies and pathogenesis, recurrence rate, degree of hoarseness and disease regression is not strong. The staging of vocal fold polyps is mainly based on the pathological changes in the lamina propria. According to the cover-body theory of vocalization, the vocal folds are divided into the cover (including the epithelium and the superficial intrinsic layer), the transition (the middle and deep intrinsic layers), and the body (the muscular layer), with the cover moving over a relatively fixed body layer [4]. The passive layer is supple and elastic, without muscular contraction, and the body layer is relatively stiff, able to regulate stiffness by active contraction; it is the difference in physical-mechanical properties between the two layers that allows the vocal folds to vibrate continuously and in a controlled manner. According to the composition the lamina propria is divided into three layers: the superficial layer contains little collagen and elastin, the middle layer contains more elastic fibers and some collagen fibers, and the deep layer contains abundant collagen fibers and some elastic fibers. In addition to fibronectin, the lamina propria contains a variety of glycoproteins and proteoglycans. The layered structure of the vocal folds is the material basis for the production of mucosal waves, and the intrinsic layer plays a crucial role in vocalization. The study of its histological composition is particularly important for understanding vocal function and is also the basis for the study of voice pathology and pathophysiology. The exact etiology of vocal fold polyps is not known. However, most scholars believe that vocal damage caused by long-term vocal misuse or overuse plays an important role in the pathogenesis. Chronic damage to the vocal folds by vocalization disrupts the balance between production and degradation of extracellular matrix components in the lamina propria, leading to changes in the content and function of extracellular matrix components, which alters the mechanical properties of vocal fold vibration and leads to vocal disorders. In transmission electron microscopy, we noted an increase in the number of fibroblasts in the lamina propria of vocal fold polyps, an abundance of fibroblasts organelles, and the proliferation and activation of fibroblasts as the main cells producing the extracellular matrix (ECM) of the lamina propria, suggesting that the metabolism of the ECM is in an active state under pathological conditions. It has been shown that fibroblasts exposed to mechanical stress produce different levels of ECM components compared to cells not exposed to mechanical forces [5]. Since fibroblasts produce extracellular matrix proteins, attempts have been made to treat vocal cord disorders such as vocal cord scarring by transplanting cultured fibroblasts [6]. This attempt gives hope, however, whether this method can be used for treatment needs to be studied in depth. In transmission electron microscopy, we found inflammatory cell infiltration in the lesioned tissue. Inflammatory cells are associated with the development of vocal fold polyps; however, whether this is a cause or a consequence of vocal fold injury is unclear, and the role of inflammatory cells in the development of vocal fold polyps remains to be investigated. Two major fibrous proteins in the lamina propria of the vocal folds: collagen fibers and elastic fibers, play an important role in normal vocal function. Collagen fibers form the ECM scaffold that maintains the vocal fold morphology and maintains tissue tension and homogeneity of the vibratory body. The elastic fibers of the vocal folds can stretch up to twice their own length, providing tissue elasticity and allowing the vocal fold tissue to recover quickly after deformation by force [7]. Due to the combined presence of both, the vocal folds can stretch and deform within a certain range and quickly recover their original shape, and also resist external forces so that the vocal folds do not change their basic shape during rapid vibrations. After the contraction of the cricothyroid muscle elongates the vocal folds, the longitudinal elastic fibers allow the folds to quickly return to a relaxed state and rapidly restore the natural tone of the voice, which plays an important role in the vocal mechanism. Elastic fibers that curl in different directions can also resist forces from different directions [8]. Collagen and elastic fibers affect the biomechanics of the vocal folds. The pressure-stress curve of the vocal ligament has been plotted, and the length of the vocal cord becomes longer with increasing external force, and this change is nearly linear until the pressure reaches a certain level, which is mainly determined by the properties of elastic fibers; when the external force reaches a turning point, the slope of the curve increases significantly, and the external force increases greatly with little change in vocal cord length, which is mainly determined by the properties of collagen fibers [9]. In this study, we observed that the arrangement of collagen fiber bundles in the vocal fold polyp lesions was more disorganized, and the protofibers in the collagen fiber bundles lost their parallel and regular arrangement and intertwined with each other, and there were large gaps in the collagen fiber bundles; the elastic fibers were sparse, more slender than normal, and interwoven into a network. Xuanzhu Chen et al [2] observed 40 cases of vocal fold polyps with histochemical staining, and there was destruction of elastic fibers in all specimens. This morphological alteration may be the histopathological basis of vocal cord polyps causing vocal disturbances. The effect of morphological alterations of vocal fold polyps on biomechanics needs to be further investigated. In the present study, we also observed some very thick collagen fiber bundles in the lesion with large interfibrillar spaces and longitudinal nodular structure of collagen fiber bundles.Ishii et al [10] observed that normal adult collagen fibers exhibit a nodular distribution and suggested that this alteration could prevent the vocal fold tissue from deviating from its normal position during mucosal waves, maintain the laminar structure, keep the mucosal waves stable, and is an adaptation to the mucosal wave This is a change to adapt to the change of mucosal wave velocity. In this study, we investigated the ultramicropathological changes in vocal fold polyps, especially in the lamina propria, by transmission electron microscopy and scanning electron microscopy, and observed that the main cells that produce the ECM of the lamina propria, i.e., fibroblasts, were proliferating in number and active in function. changes in the number and morphology of the two main fiber components of the ECM, collagen fibers and elastic fibers, may lead to changes in the physical properties of the vocal folds as vibrating bodies, thus affecting the vocal folds’ vocal function.