Differentiation potential of the cells in the macula flava of the human vocal fold mucosa

The latest research suggests cells in the maculae flavae located at both ends of the lamina propria of the human vocal fold mucosa have stemness. This study investigated the differentiation potential of the cells in the maculae flavae of the human vocal fold mucosa. Four normal human adult vocal folds from surgical specimens were used. After extraction of the anterior maculae flavae located at the anterior end of the lamina propria of the human vocal fold mucosa under microscope, the maculae flavae were minced, cultured and proliferated in mesenchymal stem cell growth medium and morphological features were assessed. Cell surface markers were detected using flow cytometry. Cell differentiation into adipogenic, chondrogenic and osteogenic lineages was performed. Cell’s differentiation potential was assessed using a human pluripotent stem cell functional identification kit and immunohistochemistry. Subcultured cells formed a colony-forming unit. Subcultured cells expressed CD90, CD105 and CD73 and lacked expression of CD45, CD34, CD11b, CD19 and HLA-DR. They differentiated into adipogenic, chondrogenic and osteogenic lineages. Consequently, the cell features in the maculae flavae meet the minimal criteria defining mesenchymal stromal cells. In addition, subcultured cells differentiated into ectoderm, mesoderm and endoderm and expressed stage-specific embryonic antigen 3 (SSEA-3). The results of this study are consistent with the hypothesis that the cells in the maculae flavae in the lamina propria of the human vocal fold mucosa are putative stem cells.     

Microstructure of the vocal fold in elderly humans

Significant changes in the voice occur after the age of 50 years. Changes in the structure of the vocal fold (VF) can interfere with the voice. The aim of this study is to investigate the structure of the VF of elderly people that may contribute to the tendency of the human voice to deteriorate. Larynges were obtained from eight embalmed cadavers aged 72-98 years. The middle portion of each vocal fold was removed and placed in 4% buffered formalin. Tissue blocks were then processed and embedded in wax. Four to six micron coronal sections were cut and stained with Haematoxylin and Eosin (H&E), Masson's Trichrome (MTS), Elastin van Gieson (EVG), Herovici (HERO), picrosirius and CD31 immunohistochemical marker in order to study the collagen fibers, elastic fibers and microvasculature of the VF. The maturity of collagen fibers within the VF were noted to increase from the superficial to the deep layer of the lamina propria (LP). Contrary to current literature, the amount of elastic fibers was sparse in the superficial layer of the LP in the vocal tissue of elderly cadavers. Numerous cross-sectioned blood vessels were seen in the lamina propria near the free edge, and near the superior and inferior surfaces of the VF. The presence of lymphatic vessels was confirmed in the VF of elderly subjects. This study revealed that the collagenous component of the deep layer of the VF LP was made up of mature fibers whilst immature collagen fibers made up the superficial layer of the LP. There was a notable scarcity of elastic fibers in the superficial layer of the LP. Lymphatics were seen and were orientated differently in the geriatric vocal folds.     

A rich lymphatic network exists in the inferior surface of the vocal cord

Conservation laryngeal surgery is an increasingly available alternative for the treatment of laryngeal cancer. Understanding anatomy of laryngeal lymph drainage is essential for clinicians to diagnose, grade and surgically manage the laryngeal cancer. Although the lymphatic drainage of the larynx has been extensively studied, few studies revealed the relationship of the lymphatic drainage between various parts of the larynx. The distribution of lymphatic vessels in the inferior surface of the vocal cord also remains unclear. The aim of this study was to investigate the communication of the lymphatic networks between the vocal cord, the supraglottic and subglottic parts of the larynx, paying special attention to the lymphatic drainage of the inferior surface of the vocal cord. Eighteen larynx specimens from 18 fresh fetal cadavers were manually injected with prassion blue solution into the mucosal or submucosal layer of the larynx in order to reveal the lymphatic vessels in the inner larynx. We found that a rich lymphatic network is present in the inferior surface of the vocal cord, and the lymphatic networks in the superior and inferior surfaces of the vocal cord appear as two different patterns. These findings provide an anatomical basis for the design of a partial or conservation laryngeal surgery, particularly when considering the precise resection margin.     

Morphological changes of the vocal fold mucosa in hyperfunctional dysphonia diagnosed by psychoacoustic and videostroboscopic methods

PurposeThe aim of the study was to provide a morphological assessment of the laryngeal mucosa in patients with hyperfunctional dysphonia diagnosed by psychoacoustic and videostroboscopic methods.Material and methodsForty patients with voice quality disorders of hyperfunctional dysphonia were recruited for participation in the study. The diagnosis of dysphonia was based on the Voice Rating Scale GRBAS, and endoscopic and stroboscopic assessment of the vocal folds. Acoustic assessment was carried out using following parameters: fundamental frequency, Jitter, Shimmer, Noise to Harmonic Rate and Yanagihara (YG) scale. In order to evaluate the morphology of the vocal fold mucosa transmission electron microscopy was performed using postoperative material obtained from the larynx. Results of clinical and morphological analysis were compared with the reference group. The morphological material was obtained from patients with hypopharyngeal cancer without pathological changes of the vocal folds.ResultsThe psychoacoustic assessment using the perceptual GRBAS scale enables the appropriate diagnostics of hyperfunctional dysphonia, which was confirmed by evaluation of acoustic parameters and YG scale analysis. In 40 patients with voice quality disorders causing by hyperfunctional dysphonia, in morphological assessment of the laryngeal mucosa, 4 (10%) patients demonstrated the presence of oedema and signs of intensive dysphonia in psychoacoustic and stroboscopic examination.ConclusionsOedema of the laryngeal mucosa confirmed by stroboscopic and ultramorphological examination may coexist with hyperfunctional dysphonia. The presence of the laryngeal oedema in patients with hyperfunctional dysphonia has the negative impact on voice quality in psychoacoustic assessment with the use of the GRBAS and YG scales.     

Lamina propria of the human vocal fold: histomorphometric study of collagen fibers

Background  

Since the seminal work of M. Hirano, which defined the three-layered lamina propria of the human vocal fold, there has been confusion in the labeling of each layer. Recent studies described the composition of fibers and interstitial molecules within the lamina propria leading to various biomechanical properties. However, collagen fibers appear as the most important structure component.     

Accessing the vocal folds by transcutaneous injection

This study tested three techniques and two approaches to access the vocal fold (VF) by transcutaneous injection using a cadaver model. The three techniques include an anatomically informed geometric technique, flexible laryngoscope (FL) guidance, and planned injection with use of computed tomography (CT). The two approaches for injection include the thyrohyoid membrane (THM) and the cricothyroid membrane (CTM). The hypothesis was that the VF is more reliably reached with techniques using technological visualization and the THM and CTM provides equal access to the VF. A geometric approach to the VF was developed and tested on seven formalin‐fixed human cadavers with three injections of 0.5 ml pigmented acrylic polymer through the CTM and one through the THM. The FL‐guided technique through the THM and the CTM was tested on eight formalin‐fixed human cadavers and the CT informed technique on two subjects. All tests were inspected by dissection and results recorded. Pertinent measurements and characteristics of each technique were recorded. Data were analyzed for statistical significance for each approach and technique. A chi‐square analysis revealed that a technology guided technique was more successful than anatomical knowledge alone (X² = 6.55, P = 0.01). The null hypothesis that the VF was equally accessed through the THM and the CTM was rejected (X² = 5.33, P = 0.02). We found that technology guided visualization is better than anatomical knowledge alone in accessing the VF. We also found that the CTM approach provides more reliable access to the entire length of the VF. Clin. Anat. 23:270–276, 2010. © 2010 Wiley‐Liss, Inc.     

Influence of glycosaminoglycan identity on vocal fold fibroblast behavior

Poly(ethylene glycol) (PEG) hydrogels have recently begun to be studied for the treatment of scarred vocal fold lamina propria due, in part, to their tunable mechanical properties, resistance to fibroblast-mediated contraction, and ability to be polymerized in situ. However, pure PEG gels lack intrinsic biochemical signals to guide cell behavior and generally fail to mimic the frequency-dependent viscoelastic response critical to normal superficial lamina propria function. Recent results suggest that incorporation of viscoelastic bioactive substances, such as glycosaminoglycans (GAGs), into PEG networks may allow these gels to more closely approach the mechanical responses of normal vocal fold lamina propria while also stimulating desired vocal fold fibroblast behaviors. Although a number of vocal fold studies have examined the influence of hyaluronan (HA) on implant mechanics and vocal fold fibroblast responses, the effects of other GAG types have been relatively unexplored. This is significant, since recent studies have suggested that chondroitin sulfate C (CSC) and heparan sulfate (HS) are substantially altered in scarred lamina propria. The present study was therefore designed to evaluate the effects of CSC and HS incorporation on the mechanical response of PEG gels and vocal fold fibroblast behavior relative to HA. As with PEG-HA, the viscoelasticity of PEG-CSC and PEG-HS gels more closely approached that of the normal vocal fold lamina propria than pure PEG hydrogels. In addition, collagen I deposition and fibronectin production were significantly higher in CSC than in HA gels, and levels of the myofibroblast marker smooth muscle α-actin (SM α-actin) were greater in CSC and HS gels than in HA gels. Since collagen I, fibronectin, and SM α-actin are generally elevated in scarred lamina propria these results suggest that CSC and HS may be undesirable for vocal fold implants relative to HA. Investigation of various signaling intermediates indicated that alterations in NFκB-p50, NFκB-p65, or pERK1/2 levels may underlie the observed differences among the PEG-GAG gels.     

阴道分娩对猪子宫韧带力学性能的影响

目的 研究阴道分娩对子宫骶韧带（uterosacral ligaments,USLs）和主韧带（cardinal ligaments,CLs）生物力学性能的影响,进而探讨阴道分娩对盆腔器官脱垂（pelvic organ prolapse,POP）的影响。方法 选用成年母猪（已育母猪5头,未育母猪5头）作为动物模型,通过单轴拉伸实验测量离体母猪的USLs和CLs被动力学行为,分析分娩对USLs和CLs生物力学性能的影响。结果 猪子宫韧带组织的被动力学行为呈非线性。无论分娩与否,右骶韧带的最大应力大于左骶韧带（P<0.05）;分娩后,二者力学性能差异悬殊。未育母猪左主韧带最大应力略大于右主韧带（P<0.05）;分娩后,两者差异降低（P>0.05）。USLs最大应力均大于CLs,表明USLs比CLs承受的张力更大,USLs在POP中起到关键性作用。结论 研究结果为认识USLs和CLs力学特性提供参考,可以指导更好的治疗方法的发展,如POP手术重建,也为预防POP发生提供理论基础。     

Depth-kymography: high-speed calibrated 3D imaging of human vocal fold vibration dynamics

We designed and developed a laser line-triangulation endoscope compatible with any standard high-speed camera for a complete three-dimensional profiling of human vocal fold vibration dynamics. With this novel device we are able to measure absolute values of vertical and horizontal vibration amplitudes, length and width of vocal folds as well as the opening and closing velocities from a single in vivo measurement. We have studied, for the first time, the generation and propagation of mucosal waves by locating the position of its maximum vertical position and the propagation velocity. Precise knowledge about the absolute dimensions of human vocal folds and their vibration parameters has significant importance in clinical diagnosis and treatment as well as in fundamental research in voice. The new device can be used to investigate different kinds of pathological conditions including periodic or aperiodic vibrations. Consequently, the new device has significant importance in investigating vocal fold paralysis and in phonosurgical applications.     

声带息肉中凹空样细胞和HPV的相关性

目的；通过形态学和分子生物学技术来探讨声带息肉与人类乳头状瘤病毒（ｈｕｍａｎ ｐａｐｉｌｌｏｍａ ｖｉｒｕｓ，ＨＰＶ）的关系。方法：对９９例声带息肉ＨＥ染色标本，进行形态学光镜研究；同时，随机选择其中４０例标本采用聚合酶链反应技术行ＨＰＶＤＮＡ检测。结果：９９例标本中有５５例声带息肉复层鳞状上皮棘细胞层中出现凹空样细胞，阳性率为５５．５％。     

Development of the human knee joint ligaments

ABSTRACT Background: Many studies have been published on the development of the human knee joint, but scant attention has been given to the development of the knee joint ligaments. The only elements that have received much attention are the cruciate ligaments and their relationships with the synovial membrane. Methods: We summarize our observations on the development of the knee joint ligaments in 50 serially sectioned human embryonic and fetal lower limbs (26 embryos and 24 fetuses). Results: The patellar ligament begins to form in O'Rahilly stage 20, with the muscle fibers of the quadriceps muscle being attached inferiorly to the tibial tuberosity. The cruciate ligaments (beginning with the posterior) arise from the articular interzone in O'Rahilly stage 21. Subsequently, with the organization of the Wrisberg's meniscofemoral ligament, in week 10 of development, the cruciate ligament system is completed. The lateral collateral ligament begins to form in O'Rahilly stage 23, and from its first appearance it is independent of the knee joint capsule. At this time, development of the tendon of the popliteus muscle begins. The medial collateral ligament begins to develop in week 9 of development as a condensation of the joint capsule. Two weeks later, the intra-articular pad of fat begins to form from mesenchymal tissue below the patella and between the cruciate and the patellar ligaments. With the organization of the suprapatellar bursa in week 14 of development, knee joint development is complete. Conclusions: The morphogenetic time table of the knee joint ligaments was established. Anat. Rec. 248:259-268, 1997. © 1997 Wiley-Liss, Inc.     

The clinicopathologic spectrum of benign mass lesions of the vocal fold due to vocal abuse

Benign masses of the vocal fold related to phonotrauma are clinically classified into polyps, nodules, Reinke's edema, and cysts. Despite the apparent distinctiveness of the clinical nomenclature, low inter- and intraobserver diagnostic agreement has been reported. Excepting cysts, which are epithelial lined, histologic examination of the remaining lesions has shown a variety of overlapping features insufficiently specific for the clinical diagnoses. This study reviews the clinicopathologic characteristics among these benign lesions of the vocal fold. A total of 78 nonneoplastic lesions of the vocal fold were reviewed by 2 pathologists for the presence of epithelial hyperplasia, basement membrane thickening, edema, vascular proliferation, and extracellular "amyloid-like" fibrin. In 46 cases with prebiopsy stroboscopic images, 2 otolaryngologists classified each lesion as polyp, nodule, Reinke's edema, cyst, or other. They agreed in 43% (n = 20, 13 polyps, 5 nodules, 1 Reinke's edema, 1 other) and disagreed in 57% (n = 26). There was no histologic feature that reliably distinguished among the lesions. In addition, reactive stromal cell atypia was present in 14 cases. Cysts were distinctive, as all were epithelial lined. The clinicopathologic classification of benign laryngeal lesions is neither clinically reproducible nor histologically unique. Treatment will continue to be individualized based on clinical judgment.     

A self-oscillating biophysical computer model of the elongated vocal fold

A new three-dimensional model is developed to simulate the self-oscillation of the elongated vocal folds. This model allows for large deformation and longitudinal displacement. The displacement boundary condition is applied on the posterior side to represent the elongation of vocal fold length by the cricothyroid or the thyroarytenoid muscles. After this model is verified by comparing its outputs using modal analysis and principle component analysis with those of previous models and experimental studies, it is applied to simulate the vibration of elongated vocal fold. Numerical simulation showed that longitudinal elongation increases the y-direction normal stress, decreases the lateral maximum displacement, and increases the fundamental frequency. These results agree with experimental measurements from an excised larynx setup, which suggests that the proposed elongation vocal fold model could be a useful tool to investigate voice production and the control of vocal fold vibration.     

Optimal feature selection for the assessment of vocal fold disorders

Unilateral vocal fold paralysis, vocal fold polyp, and vocal fold nodules are the most common types of neurogenic and organic vocal disorders. This article aims to distinguish these types of vocal diseases into four different classes for the purpose of automatic screening. Firstly, the reconstructed signal at each wavelet packet decomposition sub-band in five levels of decomposition with mother wavelet of (db10) is used to extract the nonlinear features of self-similarity and approximate entropy. Also, wavelet packet coefficients are used to measure energy and Shannon entropy features at different spectral sub-bands. Consequently, to find a discriminant feature vector, three different methods have been applied: Davies-Bouldin (DB) criteria, genetic algorithm (GA) with the fitness functions of support vector machine's (SVM) and k-nearest neighbor's (KNN) recognition rates. Finally, obtained feature vectors have been passed on to SVM and KNN classifiers. The results show that a feature vector of length 12 obtained by the optimization method of GA with the fitness function of SVM's recognition rate fed to SVM classifier achieves the highest classification accuracy of 91%. Furthermore, nonlinear features play an important role in pathological voice classification by participating rate of approximately 67% in the optimal feature vector.     

Effects of growth factors on extracellular matrix production by vocal fold fibroblasts in 3-dimensional culture

Culturing cells in 3-dimensional (3D) systems is important in tissue engineering and in fundamental studies of cellular mechanisms that are sensitive or specific to the 3D environment. To guide the engineering of artificial vocal fold lamina propria tissue, we developed 3D cultures containing human vocal fold fibroblasts (hVFFs) dispersed in a synthetic peptide hydrogel matrix. Growth factors were added to the culture to examine their influence on extracellular matrix (ECM) synthesis, cell proliferation, and matrix contraction. The hVFF-hydrogel constructs were treated with transforming growth factor-beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF), or hepatocyte growth factor (HGF), and the culture was maintained for 21 days. TGF-beta1 induced matrix contraction and enhanced collagen and sulfated glycosaminoglycan production, bFGF effectively increased cell proliferation, and HGF stimulated synthesis of hyaluronic acid and elastin with less collagen accumulation than other conditions. Of the growth factors tested, HGF appears to be most useful for stimulating essential tissue components for restoring vocal fold pliability. The results also suggest that multiple growth factors might be employed sequentially or in combination to program the makeup of cell-hydrogel constructs for vocal fold tissue repair.