Elastic fibers in orthopedics: Form and function in tendons and ligaments, clinical implications, and future directions.

Elastic fibers are an essential component of the extracellular matrix of connective tissues. The focus of both clinical management and scientific investigation of elastic fiber disorders has centered on the cardiovascular manifestations due to their significant impact on morbidity and mortality. As such, the current understanding of the orthopedic conditions experienced by these patients is limited. The musculoskeletal implications of more subtle elastic fiber abnormalities, whether due to allelic variants or age-related tissue degeneration, are also not well understood. Recent advances have begun to uncover the effects of elastic fiber deficiency on tendon and ligament biomechanics; future research must further elucidate mechanisms governing the role of elastic fibers in these tissues. The identification of population-based genetic variations in elastic fibers will also be essential. Minoxidil administration, modulation of protein expression with micro-RNA molecules, and direct injection of recombinant elastic fiber precursors have demonstrated promise for therapeutic intervention, but further work is required prior to consideration for orthopedic clinical application. This review provides an overview of the role of elastic fibers in musculoskeletal tissue, summarizes current knowledge of the orthopedic manifestations of elastic fiber abnormalities, and identifies opportunities for future investigation and clinical application.

Bilaminar Structure of the Human Optic Nerve Sheath.

PURPOSE/AIM: We aimed to characterize the connective tissue microanatomy, elastin abundance, and fiber orientation in the human optic nerve sheath, also known as the optic nerve dura mater, for correlation with its biomechanical properties. MATERIALS AND METHODS: Seven whole human orbits aged 4-93 years, and five isolated human optic nerve sheaths aged 26-75 years were formalin fixed, paraffin embedded, coronally sectioned, stained by Masson trichrome and van Gieson's elastin methods, and analyzed quantitatively for elastin fiber abundance and orientation. Elastin area fraction was defined as area stained for elastin divided by total area. RESULTS: While unilaminar in children, the adult ON sheath exhibited distinct inner and outer layers. Collagen was denser and more compact in the inner layer. Elastin area fraction was significantly greater at 6.0 ± 0.4% (standard error of mean) in the inner than outer layer at 3.6 ± 0.4% (P < 10-5). Elastin fibers had three predominant orientations: longitudinal, diagonal, and circumferential. Of circumferential fibers, 63 ± 4.7% were in the inner and 37 ± 4.7% in the outer layer (P < 10-4). Longitudinal and diagonal fibers were uniformly distributed in both layers. Elastin density and sheath thickness increased significantly with age (P < .01). CONCLUSIONS: The adult human optic nerve sheath is bilaminar, with each layer containing elastin fibers oriented in multiple directions consistent with isotropic properties. Differences in laminar elastin density and orientation may reflect greater tensile loading in the inner than in the outer layer.

The vocal organ of hummingbirds shows convergence with songbirds.

How sound is generated in the hummingbird syrinx is largely unknown despite their complex vocal behavior. To fill this gap, syrinx anatomy of four North American hummingbird species were investigated by histological dissection and contrast-enhanced microCT imaging, as well as measurement of vocalizations in a heliox atmosphere. The placement of the hummingbird syrinx is uniquely located in the neck rather than inside the thorax as in other birds, while the internal structure is bipartite with songbird-like anatomical features, including multiple pairs of intrinsic muscles, a robust tympanum and several accessory cartilages. Lateral labia and medial tympaniform membranes consist of an extracellular matrix containing hyaluronic acid, collagen fibers, but few elastic fibers. Their upper vocal tract, including the trachea, is shorter than predicted for their body size. There are between-species differences in syrinx measurements, despite similar overall morphology. In heliox, fundamental frequency is unchanged while upper-harmonic spectral content decrease in amplitude, indicating that syringeal sounds are produced by airflow-induced labia and membrane vibration. Our findings predict that hummingbirds have fine control of labia and membrane position in the syrinx; adaptations that set them apart from closely related swifts, yet shows convergence in their vocal organs with those of oscines.

How flight feathers stick together to form a continuous morphing wing.

Variable feather overlap enables birds to morph their wings, unlike aircraft. They accomplish this feat by means of elastic compliance of connective tissue, which passively redistributes the overlapping flight feathers when the skeleton moves to morph the wing planform. Distinctive microstructures form "directional Velcro," such that when adjacent feathers slide apart during extension, thousands of lobate cilia on the underlapping feathers lock probabilistically with hooked rami of overlapping feathers to prevent gaps. These structures unlock automatically during flexion. Using a feathered biohybrid aerial robot, we demonstrate how both passive mechanisms make morphing wings robust to turbulence. We found that the hooked microstructures fasten feathers across bird species except silent fliers, whose feathers also lack the associated Velcro-like noise. These findings could inspire innovative directional fasteners and morphing aircraft.

Stereological analysis of elastic fibers of the corpus cavernosum of rats during the aging process.

PURPOSE: To evaluate changes in the quantity of elastic fibers in the corpora cavernosa of rats during the natural aging process, and to assess the degree of this change by determining volumetric density (Vv) at different ages via stereological analysis. METHODS: Forty-eight rats, raised under similar conditions, were subjected to the natural aging process and divided into four groups (G1 to G4), according to age at the time of penectomy (6, 9, 12, and 24 months, respectively). Histological sections of the middle segment of the penis were stained with Weigert's resorcin-fuchsin, and the volumetric density (Vv) of elastic fibers of the corpora cavernosa were determined via stereological analysis. RESULTS: There were no statistically significant differences in Vv among groups G1, G2, and G3. These three groups were therefore considered as a single group. The mean Vv of this group showed a statistically significant reduction compared to that of G4 (0.16 vs. 0.11, p<0.05). CONCLUSION: Natural aging in rats was responsible for a reduction in volumetric density of elastic fibers of the corpora cavernosa (approximately 30% decrease in Vv) during senescence.

Elastic tissue forces mask muscle fiber forces underlying muscle spindle Ia afferent firing rates in stretch of relaxed rat muscle.

Stretches of relaxed cat and rat muscle elicit similar history-dependent muscle spindle Ia firing rates that resemble history-dependent forces seen in single activated muscle fibers ( Nichols and Cope, 2004). Owing to thixotropy, whole musculotendon forces and muscle spindle firing rates are history dependent during stretch of relaxed cat muscle, where both muscle force and muscle spindle firing rates are elevated in the first stretch in a series of stretch-shorten cycles ( Blum et al., 2017). By contrast, rat musculotendon exhibits only mild thixotropy, such that the measured forces when stretched cannot explain history-dependent muscle spindle firing rates in the same way ( Haftel et al., 2004). We hypothesized that history-dependent muscle spindle firing rates elicited in stretch of relaxed rat muscle mirror history-dependent muscle fiber forces, which are masked at the level of whole musculotendon force by extracellular tissue force. We removed estimated extracellular tissue force contributions from recorded musculotendon force using an exponentially elastic tissue model. We then showed that the remaining estimated muscle fiber force resembles history-dependent muscle spindle firing rates recorded simultaneously. These forces also resemble history-dependent forces recorded in stretch of single activated fibers that are attributed to muscle cross-bridge mechanisms ( Campbell and Moss, 2000). Our results suggest that history-dependent muscle spindle firing in both rats and cats arise from history-dependent forces owing to thixotropy in muscle fibers.

Non-wet kingfisher flying in the rain: The water-repellent mechanism of elastic feathers.

HYPOTHESIS: Flying in the rain presents a greater challenge for smaller animals such as kingfishers, compared with aircraft in the same situation. Regardless, kingfishers have developed advanced water repellency as reflected in the hydrophobicity and elasticity of their feathers. Therefore, it is possible to confirm that the elastic superhydrophobic surface can enhance the water repellency of the surface by experimental and theoretical analysis. EXPERIMENTS: A simplified device simulating droplet impact on a kingfisher feather was configured for comparison. Moreover, the dynamic behavior of droplets (with varying Weber numbers-2 ≤ We ≤ 42) impinging on the elastic and rigid substrate was analyzed, such as spreading, retraction, lift-off, the secondary droplet, and contact time with a high-speed camera. FINDINGS: The elastic substrate significantly affected the retraction and lift-off of the droplet-that is, an earlier and more efficient morphological rearrangement of the droplet-reducing the contact time by up to 8.3% (17 < We ≤ 32). The combination of elasticity and hydrophobicity is a new bioinspired strategy that provides an insight into one of the mechanisms by which birds flying in the rain cannot be bedewed while guiding the design of water-repellent surfaces.

Stereological analysis of elastic fibers of the corpus cavernosum of rats during the aging process

Abstract Purpose To evaluate changes in the quantity of elastic fibers in the corpora cavernosa of rats during the natural aging process, and to assess the degree of this change by determining volumetric density (Vv) at different ages via stereological analysis. Methods Forty-eight rats, raised under similar conditions, were subjected to the natural aging process and divided into four groups (G1 to G4), according to age at the time of penectomy (6, 9, 12, and 24 months, respectively). Histological sections of the middle segment of the penis were stained with Weigert's resorcin-fuchsin, and the volumetric density (Vv) of elastic fibers of the corpora cavernosa were determined via stereological analysis. Results There were no statistically significant differences in Vv among groups G1, G2, and G3. These three groups were therefore considered as a single group. The mean Vv of this group showed a statistically significant reduction compared to that of G4 (0.16 vs. 0.11, p<0.05). Conclusion Natural aging in rats was responsible for a reduction in volumetric density of elastic fibers of the corpora cavernosa (approximately 30% decrease in Vv) during senescence.

Micromechanics of elastic lamellae: unravelling the role of structural inhomogeneity in multi-scale arterial mechanics.

Microstructural deformation of elastic lamellae plays important roles in maintaining arterial tissue homeostasis and regulating vascular smooth muscle cell fate. Our study unravels the underlying microstructural origin that enables elastic lamellar layers to evenly distribute the stresses through the arterial wall caused by intraluminal distending pressure, a fundamental requirement for tissue and cellular function. A new experimental approach was developed to quantify the spatial organization and unfolding of elastic lamellar layers under pressurization in mouse carotid arteries by coupling physiological extension-inflation and multiphoton imaging. Tissue-level circumferential stretch was obtained from analysis of the deformation of a thick-walled cylinder. Our results show that the unfolding and extension of lamellar layers contribute simultaneously to tissue-level deformation. The inner lamellar layers are wavier and unfold more than the outer layers. This waviness gradient compensates the larger tissue circumferential stretch experienced at the inner surface, thus equalizing lamellar layer extension through the arterial wall. Discoveries from this study reveal the importance of structural inhomogeneity in maintaining tissue homeostasis through the arterial wall, and may have profound implications on vascular remodelling in aging and diseases, as well as in tissue engineering of functional blood vessels.

New insights into the viscoelastic and failure mechanical properties of the elastic fiber network of the inter-lamellar matrix in the annulus fibrosus of the disc.

The mechanical role of elastic fibers in the inter-lamellar matrix (ILM) is unknown; however, it has been suggested that they play a role in providing structural integrity to the annulus fibrosus (AF). Therefore, the aim of this study was to measure the viscoelastic and failure properties of the elastic fiber network in the ILM of ovine discs under both tension and shear directions of loading. Utilizing a technique, isolated elastic fibers within the ILM from ovine discs were stretched to 40% of their initial length at three strain rates of 0.1% s-1 (slow), 1% s-1 (medium) and 10% s-1 (fast), followed by a ramp test to failure at 10% s-1. A significant strain-rate dependent response was found, particularly at the fastest rate for phase angle and normalized stiffness (p < 0.001). The elastic fibers in the ILM demonstrated a significantly higher capability for energy absorption at slow compared to medium and fast strain rates (p < 0.001). These finding suggests that the elastic fiber network of the ILM exhibits nonlinear elastic behavior. When tested to failure, a significantly higher normalized failure force was found in tension compared to shear loading (p = 0.011), which is consistent with the orthotropic structure of elastic fibers in the ILM. The results of this study confirmed the mechanical contribution of the elastic fiber network to the ILM and the structural integrity of the AF. This research serves as a foundation for future studies to investigate the relationship between degeneration and ILM mechanical properties. STATEMENT OF SIGNIFICANCE: The mechanical role of elastic fibres in the inter-lamellar matrix (ILM) of the disc is unknown. The viscoelastic and failure properties of the elastic fibre network in the ILM in both tension and shear directions of loading was measured for the first time. We found a strain-rate dependent response for the elastic fibres in the ILM. The elastic fibres in the ILM demonstrated a significantly higher capability for energy absorption at slow compared to medium and fast strain rates. When tested to failure, a significantly higher normalized failure force was found in tension compared to shear loading, which is consistent with the orthotropic structure of elastic fibres in the ILM.

Transmural variation in elastin fiber orientation distribution in the arterial wall.

The complex three-dimensional elastin network is a major load-bearing extracellular matrix (ECM) component of an artery. Despite the reported anisotropic behavior of arterial elastin network, it is usually treated as an isotropic material in constitutive models. Our recent multiphoton microscopy study reported a relatively uniform elastin fiber orientation distribution in porcine thoracic aorta when imaging from the intima side (Chow et al., 2014). However it is questionable whether the fiber orientation distribution obtained from a small depth is representative of the elastin network structure in the arterial wall, especially when developing structure-based constitutive models. To date, the structural basis for the anisotropic mechanical behavior of elastin is still not fully understood. In this study, we examined the transmural variation in elastin fiber orientation distribution in porcine thoracic aorta and its association with elastin anisotropy. Using multi-photon microscopy, we observed that the elastin fibers orientation changes from a relatively uniform distribution in regions close to the luminal surface to a more circumferential distribution in regions that dominate the media, then to a longitudinal distribution in regions close to the outer media. Planar biaxial tensile test was performed to characterize the anisotropic behavior of elastin network. A new structure-based constitutive model of elastin network was developed to incorporate the transmural variation in fiber orientation distribution. The new model well captures the anisotropic mechanical behavior of elastin network under both equi- and nonequi-biaxial loading and showed improvements in both fitting and predicting capabilities when compared to a model that only considers the fiber orientation distribution from the intima side. We submit that the transmural variation in fiber orientation distribution is important in characterizing the anisotropic mechanical behavior of elastin network and should be considered in constitutive modeling of an artery.

Depth-Dependent Out-of-Plane Young's Modulus of the Human Cornea.

Purpose/Aim: Despite their importance in accurate mechanical modeling of the cornea, the depth-dependent material properties of the cornea have only been partially elucidated. In this work, we characterized the depth-dependent out-of-plane Young's modulus of the central and peripheral human cornea with high spatial resolution. MATERIALS AND METHODS: Central and peripheral corneal buttons from human donors were subjected to unconfined axial compression followed by stress relaxation for 30 min. Sequences of fluorescent micrographs of full-thickness corneal buttons were acquired throughout the experiment to enable tracking of fluorescently labeled stromal keratocyte nuclei and measurements of depth-dependent infinitesimal strains. The nominal (gross) out-of-plane Young's modulus and drained Poisson's ratio for each whole specimen was computed from the equilibrium stress and overall tissue deformation. The depth-dependent (local) out-of-plane Young's modulus was computed from the equilibrium stress and local tissue strain based on an anisotropic model (transverse isotropy). RESULTS: The out-of-plane Young's modulus of the cornea exhibited a strong dependence on in-plane location (peripheral versus central cornea), but not depth. The depth-dependent out-of-plane Young's modulus of central and peripheral specimens ranged between 72.4-102.4 kPa and 38.3-58.9 kPa. The nominal out-of-plane Young's modulus was 87 ± 41.51 kPa and 39.9 ± 15.28 kPa in the central and peripheral cornea, while the drained Poisson's ratio was 0.05 ± 0.02 and 0.07 ± 0.04. CONCLUSIONS: The out-of-plane Young's modulus of the cornea is mostly independent of depth, but not in-plane location (i.e. central vs. peripheral). These results may help inform more accurate finite element computer models of the cornea.

Crosslinked elastic fibers are necessary for low energy loss in the ascending aorta.

In the large arteries, it is believed that elastin provides the resistance to stretch at low pressure, while collagen provides the resistance to stretch at high pressure. It is also thought that elastin is responsible for the low energy loss observed with cyclic loading. These tenets are supported through experiments that alter component amounts through protease digestion, vessel remodeling, normal growth, or in different artery types. Genetic engineering provides the opportunity to revisit these tenets through the loss of expression of specific wall components. We used newborn mice lacking elastin (Eln-/-) or two key proteins (lysyl oxidase, Lox-/-, or fibulin-4, Fbln4-/-) that are necessary for the assembly of mechanically-functional elastic fibers to investigate the contributions of elastic fibers to large artery mechanics. We determined component content and organization and quantified the nonlinear and viscoelastic mechanical behavior of Eln-/-, Lox-/-, and Fbln4-/- ascending aorta and their respective controls. We confirmed that the lack of elastin, fibulin-4, or lysyl oxidase leads to absent or highly fragmented elastic fibers in the aortic wall and a 56-97% decrease in crosslinked elastin amounts. We found that the resistance to stretch at low pressure is decreased only in Eln-/- aorta, confirming the role of elastin in the nonlinear mechanical behavior of the aortic wall. Dissipated energy with cyclic loading and unloading is increased 53-387% in Eln-/-, Lox-/-, and Fbln4-/- aorta, indicating that not only elastin, but properly assembled and crosslinked elastic fibers, are necessary for low energy loss in the aorta.

Physical limits to biomechanical sensing in disordered fibre networks.

Cells actively probe and respond to the stiffness of their surroundings. Since mechanosensory cells in connective tissue are surrounded by a disordered network of biopolymers, their in vivo mechanical environment can be extremely heterogeneous. Here we investigate how this heterogeneity impacts mechanosensing by modelling the cell as an idealized local stiffness sensor inside a disordered fibre network. For all types of networks we study, including experimentally-imaged collagen and fibrin architectures, we find that measurements applied at different points yield a strikingly broad range of local stiffnesses, spanning roughly two decades. We verify via simulations and scaling arguments that this broad range of local stiffnesses is a generic property of disordered fibre networks. Finally, we show that to obtain optimal, reliable estimates of global tissue stiffness, a cell must adjust its size, shape, and position to integrate multiple stiffness measurements over extended regions of space.

Chronic Treatment with Minoxidil Induces Elastic Fiber Neosynthesis and Functional Improvement in the Aorta of Aged Mice.

Normal arterial aging processes involve vascular cell dysfunction associated with wall stiffening, the latter being due to progressive elastin and elastic fiber degradation, and elastin and collagen cross-linking by advanced glycation end products (AGEs). These processes progressively lead to cardiovascular dysfunction during aging. Elastin is only synthesized during late gestation and childhood, and further degradation occurring throughout adulthood cannot be physiologically compensated by replacement of altered material. However, the ATP-dependent K+ channel opener minoxidil has been shown to stimulate elastin expression in vitro and in vivo in the aorta of young adult rats. Therefore, we have studied the effect of a 10-week chronic oral treatment with minoxidil (120 mg/L in drinking water) on the aortic structure and function in aged 24-month-old mice. Minoxidil treatment increased tropoelastin, fibulin-5, and lysyl-oxidase messenger RNA levels, reinduced a moderate expression of elastin, and lowered the levels of AGE-related molecules. This was accompanied by the formation of newly synthesized elastic fibers, which had diverse orientations in the wall. A decrease in the glycation capacity of aortic elastin was also produced by minoxidil treatment. The ascending aorta also underwent a minoxidil-induced increase in diameter and decrease in wall thickness, which partly reversed the age-associated thickening and returned the wall thickness value and strain-stress relation closer to those of younger adult animals. In conclusion, our results suggest that minoxidil presents an interesting potential for arterial remodeling in an antiaging perspective, even when treating already aged animals.

Organization of the dermal matrix impacts the biomechanical properties of skin.

BACKGROUND: Human skin has the crucial roles of maintaining homeostasis and protecting against the external environment. Skin offers protection against mechanical trauma due to the reversible deformation of its structure; these biomechanical properties are amenable to dynamic testing using noninvasive devices. OBJECTIVES: To characterize the biomechanical properties of young, black African/African-Caribbean and white Northern European skin from different anatomical sites, and to relate underlying skin architecture to biomechanical function. METHODS: Using cutometry and ballistometry, the biomechanical properties of buttock and dorsal forearm skin were determined in black African/African-Caribbean (n = 18) and white Northern European (n = 20) individuals aged 18-30 years. Skin biopsies were obtained from a subset of the volunteers (black African/African-Caribbean, n = 5; white Northern European, n = 6) and processed for histological and immunohistochemical detection of the major elastic fibre components and fibrillar collagens. RESULTS: We have determined that healthy skin from young African and white Northern European individuals has similar biomechanical properties (F3): the skin is resilient (capable of returning to its original position following deformation, R1), exhibits minimal fatigue (R4) and is highly elastic (R2, R5 and R7). At the histological level, skin with these biomechanical properties is imbued with strong interdigitation of the rete ridges at the dermoepidermal junction (DEJ) and candelabra-like arrays of elastic fibres throughout the papillary dermis. Dramatic disruption to this highly organized arrangement of elastic fibres, effacement of the rete ridges and alterations to the alignment of the fibrillar collagens is apparent in the white Northern European forearm and coincides with a marked decline in biomechanical function. CONCLUSIONS: Maintenance of skin architecture - both epidermal morphology and elastic fibre arrangement - is essential for optimal skin biomechanical properties. Disruption to underlying skin architecture, as observed in the young white Northern European forearm, compromises biomechanical function.

Epithelial-mesenchymal interaction mechanisms leading to the over-expression of neprilysin are involved in the UVB-induced formation of wrinkles in the skin.

In clinical studies, the formation of facial wrinkles has been closely linked to the loss of elastic properties of the skin. Repetitive UVB irradiation of animal skin at suberythemal doses significantly reduces its elastic properties, resulting in the formation of wrinkles. That also elicits a marked alteration in the three-dimensional structure of elastic fibres, which is closely associated with a subsequent reduction in the elastic properties of the skin. While UVB irradiation stimulates the activity of skin fibroblast-derived elastase in the dermis, a synthetic inhibitor specific for skin fibroblast-derived elastase as well as an extract of Zingiber officinale (L.) Rose capable of inhibiting skin fibroblast-derived elastase, but not neutrophil elastase, prevented wrinkle formation in our studies of animal and human facial skin, respectively. The close interrelationship among wrinkle formation, elastic properties and elastic fibre linearity is revealed by the effects of different concentrations of the elastase inhibitor, which indicates that enhanced elastase activity by dermal fibroblasts plays a pivotal role in the UVB wrinkling mechanism. Fortunately, we were able to identify human skin fibroblast-derived elastase as the previously known enzyme neprilysin/neutral endopeptidase. Using both a UVB-conditioned medium assay and a co-culture system, we characterized the epithelial-mesenchymal interaction between keratinocytes and fibroblasts which leads to increased expression of neprilysin at the transcriptional, translational and enzymatic levels. Our results demonstrate that interleukin-1α and granulocyte-macrophage colony-stimulating factor are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by skin fibroblasts.

Small Artery Elastin Distribution and Architecture-Focus on Three Dimensional Organization.

The distribution of ECM proteins within the walls of resistance vessels is complex both in variety of proteins and structural arrangement. In particular, elastin exists as discrete fibers varying in orientation across the adventitia and media as well as often resembling a sheet-like structure in the case of the IEL. Adding to the complexity is the tissue heterogeneity that exists in these structural arrangements. For example, small intracranial cerebral arteries lack adventitial elastin while similar sized arteries from skeletal muscle and intestinal mesentery exhibit a complex adventitial network of elastin fibers. With regard to the IEL, several vascular beds exhibit an elastin sheet with punctate holes/fenestrae while in others the IEL is discontinuous and fibrous in appearance. Importantly, these structural patterns likely sub-serve specific functional properties, including mechanosensing, control of external forces, mechanical properties of the vascular wall, cellular positioning, and communication between cells. Of further significance, these processes are altered in vascular disorders such as hypertension and diabetes mellitus where there is modification of ECM. This brief report focuses on the three-dimensional wall structure of small arteries and considers possible implications with regard to mechanosensing under physiological and pathophysiological conditions.

Submucosal Elastic Laminae of the Middle and Lower Pharynx: A Histological Study Using Elderly Cadaveric Specimens.

Although the pharyngeal wall is well known to have high elasticity, the distribution of submucosal elastic fibers has not been described. Observations of histological sections of the mid and lower pharyngeal walls from 15 elderly donated cadavers were made. We found two distinct submucosal tissue layers with a high content of elastic fibers (tentatively termed the "submucosal elastic laminae"). The inferolateral elastic lamina was restricted to the level from the upper part of the arytenoid to the lower end of the inferior cornu of the thyroid cartilage. It originated from the pharyngeal submucosa, extended laterally along the inner aspect of the thyropharyngeal muscle, and inserted into the posterior margin of the thyroid cartilage including the cornu. The posteromedial lamina extended along the supero-inferior axis from a level above the greater horn of the hyoid bone to reach the muscularis mucosae of the cervical esophagus. The inferolateral and posteromedial laminae were connected at levels below the cricoarytenoid joint. Individual variations were evident in their thicknesses (ranging from almost absent to 0.3 mm) as well as the extent of connection between them. In association with striated muscle function, the inferolateral lamina seemed to suspend the lower pharyngeal mucosa, while the posteromedial lamina seemed to provide mucosal fold forcing smoothly peristaltic conveyance of a bolus during swallowing.