Hyaluronic acid-based microgels and microgel networks for vocal fold regeneration

Vocal fold scarring disrupts the viscoelastic properties of the lamina propria that are critical for normal phonation. There is a clinical need for the development of advanced biomaterials that approximate the mechanical properties of the lamina propria for in vivo vocal fold regeneration. We have developed hyaluronic acid (HA)-based microgels and cross-linked microgel networks with tunable degradation and mechanical properties. HA microgels were prepared by cross-linking HA derivatives carrying hydrazide (HAADH) and aldehyde (HAALD) functionalities within the inverse emulsion droplets. Alternatively, poly(ethylene glycol) dialdehyde (PEGDiALD) was employed in place of HAALD. Microgels based on HAADH/HAALD are more resistant to enzymatic degradation than those generated from HAADH/PEGDiALD. In vitro cytotoxicity studies using vocal fold fibroblasts indicate that microgels synthesized from HAADH/HAALD are essentially nontoxic, whereas microgels derived from HAADH/PEGDiALD exhibit certain adverse effects on the cultured cells at high concentration (> or =2 mg/mL). These microgels exhibit residual functional groups that can be used as reactive handles for covalent conjugation of therapeutic molecules. The presence of residual functional groups also allows for subsequent cross-linking of the microgels with other reactive polymers, giving rise to doubly cross-linked networks (DXNs) with tunable viscoelasticity. Mechanical measurements using a torsional wave apparatus indicate that HA-based DXNs exhibit elastic moduli that are similar to those of vocal fold lamina propria at frequencies close to the range of human phonation. These HA-based microgel systems are promising candidates for the treatment of vocal fold scarring, not just as biocompatible filler materials, but as smart entities that can repair focal defects, smooth the vocal fold margin, and potentially soften and dissolve scar tissue.     

Vocal fold tissue failure: preliminary data and constitutive modeling

In human voice production (phonation), linear small-amplitude vocal fold oscillation occurs only under restricted conditions. Physiologically, phonation more often involves large-amplitude oscillation associated with tissue stresses and strains beyond their linear viscoelastic limits, particularly in the lamina propria extracellular matrix (ECM). This study reports some preliminary measurements of tissue deformation and failure response of the vocal fold ECM under large-strain shear The primary goal was to formulate and test a novel constitutive model for vocal fold tissue failure, based on a standard-linear cohesive-zone (SL-CZ) approach. Tissue specimens of the sheep vocal fold mucosa were subjected to torsional deformation in vitro, at constant strain rates corresponding to twist rates of 0.01, 0.1, and 1.0 rad/s. The vocal fold ECM demonstrated nonlinear stress-strain and rate-dependent failure response with a failure strain as low as 0.40 rad. A finite-element implementation of the SL-CZ model was capable of capturing the rate dependence in these preliminary data, demonstrating the model's potential for describing tissue failure. Further studies with additional tissue specimens and model improvements are needed to better understand vocal fold tissue failure.     

Homotypic variation of canine flexor tendons: implications for the design of experimental studies in animal models

Water, collagen and glycosamimoglycan contents, cross-sectional area, stiffness and elastic modulus were carefully quantitated in flexor digitorum superficialis tendons from mature canines. From these data the within- and between-animal variability was estimated and used to demonstrate sample size calculations for both two-group and paired (within-animal) study designs. The estimated between-dog variance was typically 50% or less of the total variance for the parameters investigated. In other words, the correlation among the tendons within an animal for most measures was not strong. Therefore, for some variables (e.g., elastic modulus) in this animal and tendon model, there is no appreciable gain in statistical power by using a paired study design. A two-group design could be used, but any within-animal correlation must be accounted for in the analysis. For other variables such as collagen content, a paired design would gain substantial power.     

Optical measurements of vocal fold tensile properties: implications for phonatory mechanics

In voice research, in vitro tensile stretch experiments of vocal fold tissues are commonly employed to determine the tissue biomechanical properties. In the standard stretch-release protocol, tissue deformation is computed from displacements applied to sutures inserted through the thyroid and arytenoid cartilages, with the cartilages assumed to be rigid. Here, a non-contact optical method was employed to determine the actual tissue deformation of vocal fold lamina propria specimens from three excised human larynges in uniaxial tensile tests. Specimen deformation was found to consist not only of deformation of the tissue itself, but also deformation of the cartilages, as well as suture alignment and tightening. Stress-stretch curves of a representative load cycle were characterized by an incompressible Ogden model. The initial longitudinal elastic modulus was found to be considerably higher if determined based on optical displacement measurements than typical values reported in the literature. The present findings could change the understanding of the mechanics underlying vocal fold vibration. Given the high longitudinal elastic modulus the lamina propria appeared to demonstrate a substantial level of anisotropy. Consequently, transverse shear could play a significant role in vocal fold vibration, and fundamental frequencies of phonation should be predicted by beam theories accounting for such effects.     

Adapted to roar: functional morphology of tiger and lion vocal folds

Vocal production requires active control of the respiratory system, larynx and vocal tract. Vocal sounds in mammals are produced by flow-induced vocal fold oscillation, which requires vocal fold tissue that can sustain the mechanical stress during phonation. Our understanding of the relationship between morphology and vocal function of vocal folds is very limited. Here we tested the hypothesis that vocal fold morphology and viscoelastic properties allow a prediction of fundamental frequency range of sounds that can be produced, and minimal lung pressure necessary to initiate phonation. We tested the hypothesis in lions and tigers who are well-known for producing low frequency and very loud roaring sounds that expose vocal folds to large stresses. In histological sections, we found that the Panthera vocal fold lamina propria consists of a lateral region with adipocytes embedded in a network of collagen and elastin fibers and hyaluronan. There is also a medial region that contains only fibrous proteins and hyaluronan but no fat cells. Young's moduli range between 10 and 2000 kPa for strains up to 60%. Shear moduli ranged between 0.1 and 2 kPa and differed between layers. Biomechanical and morphological data were used to make predictions of fundamental frequency and subglottal pressure ranges. Such predictions agreed well with measurements from natural phonation and phonation of excised larynges, respectively. We assume that fat shapes Panthera vocal folds into an advantageous geometry for phonation and it protects vocal folds. Its primary function is probably not to increase vocal fold mass as suggested previously. The large square-shaped Panthera vocal fold eases phonation onset and thereby extends the dynamic range of the voice.     

Non-invasive in vivo measurement of the shear modulus of human vocal fold tissue

Voice is the essential part of singing and speech communication. Voice disorders significantly affect the quality of life. The viscoelastic mechanical properties of the vocal fold mucosa determine the characteristics of the vocal folds oscillations, and thereby voice quality. In the present study, a non-invasive method was developed to determine the shear modulus of human vocal fold tissue in vivo via measurements of the mucosal wave propagation speed during phonation. Images of four human subjects' vocal folds were captured using high speed digital imaging (HSDI) and magnetic resonance imaging (MRI) for different phonation pitches, specifically fundamental frequencies between 110 and 440 Hz. The MRI images were used to obtain the morphometric dimensions of each subject's vocal folds in order to determine the pixel size in the high-speed images. The mucosal wave propagation speed was determined for each subject and at each pitch value using an automated image processing algorithm. The transverse shear modulus of the vocal fold mucosa was then calculated from a surface (Rayleigh) wave propagation dispersion equation using the measured wave speeds. It was found that the mucosal wave propagation speed and therefore the shear modulus of the vocal fold tissue were generally greater at higher pitches. The results were in good agreement with those from other studies obtained via in vitro measurements, thereby supporting the validity of the proposed measurement method. This method offers the potential for in vivo clinical assessments of vocal folds viscoelasticity from HSDI.     

Cervids with different vocal behavior demonstrate different viscoelastic properties of their vocal folds

The authors test the hypothesis that vocal fold morphology and biomechanical properties covary with species‐specific vocal function. They investigate mule deer (Odocoileus hemionus) vocal folds, building on, and extending data on a related cervid, the Rocky Mountain elk (Cervus elaphus nelsoni). The mule deer, in contrast to the elk, is a species with relatively little vocal activity in adult animals. Mule deer and elk vocal folds show the typical three components of the mammalian vocal fold (epithelium, lamina propria and thyroarytenoid muscle). The vocal fold epithelium and the lamina propria were investigated in two sets of tensile tests. First, creep rupture tests demonstrated that ultimate stress in mule deer lamina propria is of the same magnitude as in elk. Second, cyclic loading tests revealed similar elastic moduli for the vocal fold epithelium in mule deer and elk. The elastic modulus of the lamina propria is also similar between the two species in the low‐strain region, but differs at strains larger than 0.3. Sex differences in the stress–strain response, which have been reported for elk and human vocal folds, were not found for mule deer vocal folds. The laminae propriae in mule deer and elk vocal folds are comparatively large. In general, a thick and uniformly stiff lamina propria does not self‐oscillate well, even when high subglottic pressure is applied. If the less stiff vocal fold seen in elk is associated with a differentiated lamina propria it would allow the vocal fold to vibrate at high tension and high subglottic pressure. The results of this study support the hypothesis that viscoelastic properties of vocal folds varies with function and vocal behavior. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Regeneration of Vocal Fold Mucosa Using Tissue-Engineered Structures with Oral Mucosal Cells

Objectives

Scarred vocal folds result in irregular vibrations during phonation due to stiffness of the vocal fold mucosa. To date, a completely satisfactory corrective procedure has yet to be achieved. We hypothesize that a potential treatment option for this disease is to replace scarred vocal folds with organotypic mucosa. The purpose of this study is to regenerate vocal fold mucosa using a tissue-engineered structure with autologous oral mucosal cells.

Study Design

Animal experiment using eight beagles (including three controls).

Methods

A 3 mm by 3 mm specimen of canine oral mucosa was surgically excised and divided into epithelial and subepithelial tissues. Epithelial cells and fibroblasts were isolated and cultured separately. The proliferated epithelial cells were co-cultured on oriented collagen gels containing the proliferated fibroblasts for an additional two weeks. The organotypic cultured tissues were transplanted to the mucosa-deficient vocal folds. Two months after transplantation, vocal fold vibrations and morphological characteristics were observed.

Results

A tissue-engineered vocal fold mucosa, consisting of stratified epithelium and lamina propria, was successfully fabricated to closely resemble the normal layered vocal fold mucosa. Laryngeal stroboscopy revealed regular but slightly small mucosal waves at the transplanted site. Immunohistochemically, stratified epithelium expressed cytokeratin, and the distributed cells in the lamina propria expressed vimentin. Elastic Van Gieson staining revealed a decreased number of elastic fibers in the lamina propria of the transplanted site.

Conclusion

The fabricated mucosa with autologous oral mucosal cells successfully restored the vocal fold mucosa. This reconstruction technique could offer substantial clinical advantages for treating intractable diseases such as scarring of the vocal folds.     

Synthetic,Multi-Layer,Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties ¹. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone ² and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry ^3,4, clinical instrument development ⁵, laryngeal aerodynamics ^6-9, vocal fold contact pressure ¹⁰, and subglottal acoustics ¹¹ (a more comprehensive list can be found in Kniesburges et al. ¹²)Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds ¹ that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages ^3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers ¹. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.     

Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r) ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r) glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.     

Empirical measurements of biomechanical anisotropy of the human vocal fold lamina propria

The vocal folds are known to be mechanically anisotropic due to the microstructural arrangement of fibrous proteins such as collagen and elastin in the lamina propria. Even though this has been known for many years, the biomechanical anisotropic properties have rarely been experimentally studied. We propose that an indentation procedure can be used with uniaxial tension in order to obtain an estimate of the biomechanical anisotropy within a single specimen. Experiments were performed on the lamina propria of three male and three female human vocal folds dissected from excised larynges. Two experiments were conducted: each specimen was subjected to cyclic uniaxial tensile loading in the longitudinal (i.e., anterior–posterior) direction, and then to cyclic indentation loading in the transverse (i.e., medial–lateral) direction. The indentation experiment was modeled as contact on a transversely isotropic half-space using the Barnett–Lothe tensors. The longitudinal elastic modulus E _L was computed from the tensile test, and the transverse elastic modulus E _T and longitudinal shear modulus G _L were obtained by inverse analysis of the indentation force-displacement response. It was discovered that the average of E _L /E _T was 14 for the vocal ligament and 39 for the vocal fold cover specimens. Also, the average of E _L /G _L , a parameter important for models of phonation, was 28 for the vocal ligament and 54 for the vocal fold cover specimens. These measurements of anisotropy could contribute to more accurate models of fundamental frequency regulation and provide potentially better insights into the mechanics of vocal fold vibration.     

Reinke's Edema: investigations on the role of MIB-1 and hepatocyte growth factor

Reinke''s edema is a benign disease of the human vocal fold, which mainly affects the sub-epithelial layer of the vocal fold. Microscopic observations show a strongly oedematous epithelium with loosened intercellular junctions, a disruption of the extracellular connections between mucosal epithelium and connective tissue, closely adherent to the thyroarytenoid muscle. Thickening of the basal layer of epithelium, known as Reinke''s space, high deposition of fibronectin and chronic inflammatory infiltration it is also visible. We analyzed, together with the hepatocyte growth factor (HGF), the expression level of MIB-1 in samples harvested from patients affected by Reinke''s edema, in order to define its biological role and consider it as a possible prognostic factor in the follow-up after surgical treatment. We observed a moderate expression of HGF in the lamina propria of the human vocal fold and in the basal membrane of the mucosal epithelium. Our finding suggests that this growth factor acts as an anti - fibrotic agent in Reinke''s space and affects the fibronectin deposition in the lamina propria. MIB-1, on the contrary, showed a weak expression in the basement membrane of the mucosal epithelium and a total absence in the lamina propria deep layer, thus suggesting that only the superficial layer is actively involved in the reparatory process with a high regenerative capacity, together with a high deposition of fibronectin. The latter is necessary for the cellular connections reconstruction, after the inflammatory infiltration.Key words: Reinke''s edema, Ki67, HGF, fibronectin, immunohistochemistry, extracellular matrix.     

Linear viscoelastic properties of the vertex model for epithelial tissues

Epithelial tissues act as barriers and, therefore, must repair themselves, respond to environmental changes and grow without compromising their integrity. Consequently, they exhibit complex viscoelastic rheological behavior where constituent cells actively tune their mechanical properties to change the overall response of the tissue, e.g., from solid-like to fluid-like. Mesoscopic mechanical properties of epithelia are commonly modeled with the vertex model. While previous studies have predominantly focused on the rheological properties of the vertex model at long time scales, we systematically studied the full dynamic range by applying small oscillatory shear and bulk deformations in both solid-like and fluid-like phases for regular hexagonal and disordered cell configurations. We found that the shear and bulk responses in the fluid and solid phases can be described by standard spring-dashpot viscoelastic models. Furthermore, the solid-fluid transition can be tuned by applying pre-deformation to the system. Our study provides insights into the mechanisms by which epithelia can regulate their rich rheological behavior.     

声带组织切片方法的比较

目的:探讨犬声带冠状位切片与水平位切片各自的特点,为声带实验提供合适的切片方法。方法:家犬4只,2只取材后行冠状位石蜡切片,2只取材后行水平位石蜡切片。通过HE染色观察声带固有层的一般组织结构,Masson三色染色观察固有层中胶原的排列情况。结果:HE染色示冠状位、水平位切片均可见声带表面被覆复层鳞状上皮,固有层内有大量排列紧密的纤维组织,纤维组织中夹杂少量腺体,固有层下方为肌层。冠状位切片可观察声带某一点冠状面固有层的情况,若观察整个声带的情况需声带连续切片;水平位切片可在一张切片中观察到前联合、声带膜部及声带突部位的固有层情况,解剖标志明显,利于定位。Masson三色染色示冠状位、水平位切片均可见固有层浅层有较细的胶原纤维束,中层有较粗的纤维束与较细的纤维束交织排列,深层纤维束排列更紧密。结论:冠状位切片可观察声带某一点冠状面固有层的整体情况,水平位切片可在一张切片中观察到前联合、膜部及声带突部位的固有层情况。     

Complex rheological properties of a water-soluble extract from the fronds of the black tree fern, Cyathea medullaris

A water-soluble extract was obtained from the fronds of a New Zealand native black tree fern (Cyathea medullaris or Mamaku in Māori). The extract exhibited complex rheological behavior. Newtonian, shear-thinning, shear-thickening, thixotropic, antithixotropic, and viscoelastic behaviors were observed depending on polymer concentration, shear rate, and shear history. The extract also displayed rod-climbing and self-siphoning properties typical of viscoelastic fluids. Such complex rheological properties have been reported in synthetic or chemically modified polymers but are less frequent in unmodified biopolymers. Although Mamaku extract obtained from the pith of the fern has been traditionally used by the Māori in New Zealand for treating wounds and diarrhea among other ailments, this material has never been characterized before. This study reports on the chemical composition of the extract and on its viscoelastic properties through rotational and oscillatory rheological measurements. Explanations of the mechanism behind the rheological properties were based on transient network models for associating polymers.     

Rheology of gastric mucin exhibits a pH-dependent sol-gel transition

Gastric mucin, a high molecular weight glycoprotein, is responsible for providing the gel-forming properties and protective function of the gastric mucus layer. Bulk rheology measurements in the linear viscoelastic regime show that gastric mucin undergoes a pH-dependent sol-gel transition from a viscoelastic solution at neutral pH to a soft viscoelastic gel in acidic conditions, with the transition occurring near pH 4. In addition to pH-dependent gelation behavior in this system, further rheological studies under nonlinear deformations reveal shear thinning and an apparent yield stress in this material which are also highly influenced by pH.     

Fast fluorescence laser tracking microrheometry. I: instrument development

To gain insight into cellular mechanotransduction pathways, we have developed a fluorescence laser tracking microrheometer (FLTM) to measure material rheological features on micrometer length scales using fluorescent microspheres as tracer particles. The statistical analysis of the Brownian motion of a particle quantifies the viscoelastic properties of the probe's environment, parameterized by the frequency-dependent complex shear modulus G*(ω). This FLTM has nanometer spatial resolution over a frequency range extending from 1 Hz to 50 kHz. In this work, we first describe the consecutive stages of instrument design, development, and optimization. We subsequently demonstrate the accuracy of the FLTM by reproducing satisfactorily the known rheological characteristics of purely viscous glycerol solutions and cross-linked polyacrylamide polymer networks. An upcoming companion article will illustrate the use of FLTM in studying the solid-like versus liquid-like rheological properties of fibroblast cytoskeletons in living biological samples.     

Aspects of the design and analysis of high-dimensional SNP studies for disease risk estimation

The state of readiness for high-dimensional single nucleotide polymorphism (SNP) epidemiologic association studies is described, as background for a discussion of statistical aspects of case-control study design and analysis. Specifically, the important role that multistage designs can play in the elimination of false-positive associations and in the control of study costs will be noted. Also, the trade-offs associated with using pooled DNA at early design stages for additional important cost reductions will be discussed in some detail. An odds ratio approach to relating SNP alleles to disease risk using pooled DNA will be proposed, in conjunction with a simple empirical variance estimator, based on comparisons among log-odds ratio estimators from distinct pairs of case and control pools. Simulation studies will be presented to evaluate the moderate sample size properties of such multistage designs and estimation procedures. The design of an ongoing three-stage study in the Women's Health Initiative to relate 250,000 SNPs to the risk of coronary heart disease, stroke, and breast cancer will provide illustration, and will be used to motivate the choice of simulation configurations.     

Modeling mechanical stresses as a factor in the etiology of benign vocal fold lesions

Vocal fold tissue lesions such as nodules and polyps are thought to develop in response to mechanical stress that occurs during vocal fold collision. Two computational models of vocal fold collision during voice production are used to investigate this hypothesis. A one-dimensional lumped mass model, whose parameters are derived from vocal fold tissue dimensions and material properties, predicts stress perpendicular to the direction of impact (normal stress). A previously published three-dimensional finite element model that incorporates the same dimensions and properties predicts the entire stress tensor. The hypothesis is supported by predictions from the finite element model that three components of normal stress and one component of shear stress are increased during collision in the typical location of lesions (i.e. the center of the superior medial edge of the vocal fold in the middle of the vibrating and contact region). The lumped mass model predicts that mechanical stress is negatively correlated with mucosal thickness (increased by voice warm-up and hydration), is positively correlated with driving force (proportional to voice intensity), and is affected by voice production method. These relationships are consistent with clinical observations of vocal fold lesion risk factors and have implications for improving prevention and treatment of benign vocal fold lesions.     

Videostroboscopic and morphological aspects of voice disturbances in patients with larynx atrophy and coexisting hypopharynx cancer

Vocal folds play a crucial role in voice production. The physiological vibrations of vocal folds depend on the unchanged multilayered structure of the vocal folds mucosa. Morphological changes of mucosa are the cause of voice quality disorders - dysphonia. The aim of this study was to determine the morphological base of dysphonia in patients with vocal folds atrophy. A group of 24 patients with larynx atrophy confirmed by endoscopic (VLS) and stroboscopic (VLSS) examination of the larynx was included in the study. The morphological assessment of the larynx mucosa was carried out with the use of the transmission electron microscopy (TEM). Ultramorphological examinations revealed changes in the epithelium, basal membrane and lamina propria of the vocal folds mucosa. An increased number of collagenous fibers, fibroblasts with signs of vacuolar degeneration inflammatory cells and a decreased number of blood vessels and pericytes were observed. Morphological changes found in the epithelium, basal membrane and lamina propria of the vocal folds mucosa were the cause of disorders of vocal folds vibrations registered in the stroboscopic examination of the larynx (VLSS).