Formulation and characterization of a porous, elastomeric biomaterial for vocal fold tissue engineering research.

OBJECTIVE: Biomaterials able to mimic the mechanical properties of vocal fold tissue may be particularly useful for furnishing a 3-dimensional microenvironment allowing for in vitro investigation of cell and molecular responses to vibration. Motivated by the dearth of biomaterials available for use in an in vitro model for vocal fold tissue, we investigated polyether polyurethane (PEU) matrices, which are porous, mechanically tunable biomaterials that are inexpensive and require only standard laboratory equipment for fabrication. METHODS: Rheology, dynamic mechanical analysis, and scanning electron microscopy were performed on PEU matrices at 5%, 10%, and 20% w/v mass concentrations. RESULTS: For 5%, 10%, and 20% w/v concentrations, shear storage moduli were 2 kPa, 3.4 kPa, and 6 kPa, respectively, with shear loss moduli being 0.2 kPa, 0.38 kPa, and 0.62 kPa, respectively. Storage moduli responded to applied frequency as a linear function. Mercury intrusion porosimetry revealed that all 3 mass concentrations of PEU have a similar overall percentage porosity but differ in pore architecture. CONCLUSION: Twenty-µm diameter pores are ideal for cell seeding, and a range of mechanical properties indicates that the lower [corrected] mass concentration PEU formulations are best suited for mimicking the viscoelastic properties of vocal fold tissue for in vitro research.

Adhesion of a monolayer of fibroblast cells to fibronectin under sonic vibrations in a bioreactor.

OBJECTIVES: We examined cell adhesion to a surface under vibrational forces approximating those of phonation. METHODS: A monolayer of human fibroblast cells was seeded on a fibronectin-coated glass coverslip, which was attached to either the rotating part or the stationary part of a rheometer-bioreactor. The temperature, humidity, carbon dioxide level, nutrients, and cell seeding density were controlled. The cell density was on the order of 1,000 to 5,000 cells per square millimeter. Target stresses above 1 kPa at an oscillatory frequency of 100 Hz were chosen to reflect conditions of vocal fold tissue vibration. RESULTS: Fibronectin coating provided enough adhesion to support at least 2 kPa of oscillating stress, but only about 0.1 kPa of steady rotational shear. For stresses exceeding those limits, the cells were not able to adhere to the thin film of fibronectin. CONCLUSIONS: Cells will adhere to a planar surface under stresses typical of phonation, which provide a more stringent test than adherence in a 3-dimensional matrix. The density of cell seeding on the coverslip played a role in cell-extracellular matrix adhesion, in that the cells adhered to each other more than to the fibronectin coating when the cells were nearly confluent.

DETERMINING MOTOR INERTIA OF A STRESS-CONTROLLED RHEOMETER.

Viscoelastic measurements made with a stress-controlled rheometer are affected by system inertia. Of all contributors to system inertia, motor inertia is the largest. Its value is usually determined empirically and precision is rarely if ever specified. Inertia uncertainty has negligible effects on rheologic measurements below the coupled motor/plate/sample resonant frequency. But above the resonant frequency, G' values of soft viscoelastic materials such as dispersions, gels, biomaterials, and non-Newtonian polymers, err quadratically due to inertia uncertainty. In the present investigation, valid rheologic measurements were achieved near and above the coupled resonant frequency for a non-Newtonian reference material. At these elevated frequencies, accuracy in motor inertia is critical. Here we compare two methods for determining motor-inertia accurately. For the first (commercially-used) phase method, frequency responses of standard fluids were measured. Phase between G' and G" was analyzed at 5-70 Hz for motor inertia values of 50-150% of the manufacturer's nominal value. For a newly-devised two-plate method (10 mm and 60 mm parallel plates), dynamic measurements of a non-Newtonian standard were collected. Using a linear equation of motion with inertia, viscosity, and elasticity coefficients, G' expressions for both plates were equated and motor inertia was determined to be accurate (by comparison to the phase method) with a precision of ± 3%. The newly developed two-plate method had advantages of expressly eliminating dependence on gap, was explicitly derived from basic principles, quantified the error, and required fewer experiments than the commercially used phase method.

Cell viability viscoelastic measurement in a rheometer used to stress and engineer tissues at low sonic frequencies.

Effects of vibration on human vocal fold extracellular matrix composition and the resultant tissue viscoelastic properties are difficult to study in vivo. Therefore, an in vitro bioreactor, simulating the in vivo physiological environment, was explored. A stress-controlled commercial rheometer was used to administer shear vibrations to living tissues at stresses and frequencies corresponding to male phonation, while simultaneously measuring tissue viscoelastic properties. Tissue environment was evaluated and adjustments made in order to sustain cell life for short term experimentation up to 6 h. Cell nutrient medium evaporation, osmolality, pH, and cell viability of cells cultured in three-dimensional synthetic scaffolds were quantified under comparably challenging environments to the rheometer bioreactor for 4 or 6 h. The functionality of the rheometer bioreactor was demonstrated by applying three vibration regimes to cell-seeded three-dimensional substrates for 2 h. Resulting strain was quantified throughout the test period. Rheologic data and cell viability are reported for each condition, and future improvements are discussed.

Development of a bilayer ring system for achieving high strain in commercial rheometers.

Mechanical stimulation of cell cultures has been shown be an effective means of enhancing ECM production. ECM produced from vocal fold fibroblast cultures has the potential for therapeutic use for vocal fold repair. However, current bioreactor designs generally fail to produce physiological relevant frequency and strain values. Here we present an approach for using commercial oscillatory rheometers and an elastic ring bilayer system to produce physiologically relevant strain values at frequencies in the range of 20-100 Hz. We demonstrate the ability to target specific strain and frequency values by manipulating system parameters, and also show that it is possible to maintain high oscillatory strains for extended periods of time. Such a system could be used to mechanically stimulate cell cultures contained within gel carrier systems and has the potential to be extended to other applications requiring high strains at low frequencies.

Viscoelastic properties of three vocal-fold injectable biomaterials at low audio frequencies.

OBJECTIVES: Previous measurements of viscoelastic properties of Zyderm were to be extended to low audio frequencies, and properties of two other biomaterials not previously measured, thiolated hyaluronic acid (HA-DTPH) and Cymetra, were obtained. STUDY DESIGN: Rheologic investigation. METHODS: Oscillatory shear stress was applied to each sample using a controlled stress rheometer at frequencies between 0.01 and 100 Hz with a parallel plate apparatus. Versuscoelastic moduli were recorded at each frequency. The calculated resonance frequency of the machine and sample were then used to determine the maximum frequency at which reliable data existed. Extrapolation functions were fit to viscoelastic parameters, which predicted the properties up to 1,000 Hz. RESULTS: Frequency trends of Zyderm were similar to those previously reported, whereas magnitudes were different. The elastic moduli logarithmically increased with frequency, whereas dynamic viscosity demonstrated shear thinning, a condition of primary importance for humans to vocalize over a broad frequency range. Previous measurements were extended from 15 Hz up to 74 Hz. Differences in magnitude between a previous study and the present study were attributed to particulate orientation during testing. Cymetra was found to have nearly identical viscoelastic properties to those of bovine collagen, both in magnitude and frequency trend, with reliable measures extending up to 81 Hz. Rheologic properties of the hyaluronic acid gel were the closest match to cadaveric vocal fold mucosa in magnitude and frequency trend. CONCLUSIONS: Viscoelastic properties of Cymetra and Zyderm are nearly the same and are significantly greater than those of vocal fold mucosa. HA-DTPH possesses a good viscoelastic match to vocal fold mucosa and may be useful in future lamina propria repair.

Restructuring the vocal fold lamina propria with endoscopic microdissection.

OBJECTIVES/HYPOTHESIS: The purposes of this preclinical study were to investigate histologic and rheologic outcomes of Microendoscopy of Reinke's space (MERS)-guided minithyrotomy and to assess its instrumentation. STUDY DESIGN: Human cadaveric and in vivo animal study. METHODS: Three human cadaveric larynges were treated with MERS-guided placement of Radiesse VoiceGel and immediately evaluated histologically for biomaterial location. In the second part of this investigation, two scarred porcine larynges were treated with MERS-guided placement of HyStem-VF and rheologically evaluated 6 weeks later. Student t tests determined differences in viscoelastic properties of treated/untreated vocal folds. Sialendoscopes and microendoscopes were subjectively compared for their visualization capacity. RESULTS: MERS imaged the subepithelial area and vocal ligament, guiding both tissue dissection and biomaterial positioning. Sialendoscopes provided adequate visualization and feature incorporated working channels. Enhanced image clarity was created in a gas-filled rather than saline-filled environment, per rater judgment. Histological analysis revealed desirable biomaterial positioning with MERS. Per rheological analysis, viscoelastic properties of the MERS-treated porcine vocal folds compared to uninjured vocal folds 6 weeks following treatment did not statistically differ. CONCLUSIONS: MERS-guided laryngoplasty using sialendoscopes yielded satisfactory biomaterial positioning in the short-term and normalized rheologic tissue properties in the long-term, contributing to proof of concept for MERS in the treatment of scarring. Strengths of MERS include direct, real-time visualization of Reinke's space and an ability to manipulate surgical instruments parallel to the vocal fold edge while maintaining an intact epithelium. Future work will explore the clinical utility of MERS for addressing scarring, sulcus vocalis, and other intracordal processes.

In Vivo engineering of the vocal fold ECM with injectable HA hydrogels-late effects on tissue repair and biomechanics in a rabbit model.

OBJECTIVES: To determine if the utilization of injectable chemically modified hyaluronan (HA) derivative at the time of intentional vocal fold resection may facilitate wound repair and preserve the unique viscoelastic properties of the extracellular matrix (ECM) and lamina propria 6 months after treatment. STUDY DESIGN: Prospective, controlled animal study. METHODS: Twelve rabbit vocal folds were biopsied bilaterally, and the left side of vocal fold was treated with Extracel, an injectable, chemically modified HA derivative, and the right side of vocal fold was injected with saline as control at the time of resection. Animals were sacrificed 6 months after biopsy and injection. Outcomes measured include transcription levels for procollagen, fibronectin, fibromodulin, transforming growth factor beta one (TGF-ß1), HA synthase, and hyaluronidase, and tissue biomechanics-viscosity and elasticity. RESULTS: Extracel-treated vocal folds were found to have significantly less fibrosis than saline-treated controls. Extracel-treated vocal folds had significantly improved biomechanical properties of elasticity and viscosity. Significantly decreased levels of fibronectin, fibromodulin, TGF-ß1, procollagen I, and HA synthase were measured. CONCLUSIONS: Prophylactic in vivo manipulation of the ECM with an injectable HA hydrogel appears to induce vocal fold tissue regeneration to yield improved tissue composition and biomechanical properties at 6 months.

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.

Phonation threshold pressure predictions using viscoelastic properties up to 1,400 Hz of injectables intended for Reinke's space.

OBJECTIVES/HYPOTHESIS: Viscoelastic properties of numerous vocal fold injectables have been reported but not at speaking frequencies. For materials intended for Reinke's space, ramifications of property values are of great concern because of their impact on ease of voice onset. Our objectives were: 1) to measure viscoelastic properties of a new nonresorbing carbomer and well-known vocal fold injectables at vocalization frequencies using established and new instrumentation, and 2) to predict phonation threshold pressures using a computer model with intended placement in Reinke's space. STUDY DESIGN: Rheology and phonation threshold pressure calculations. METHODS: Injectables were evaluated with a traditional rotational rheometer and a new piezo-rotary vibrator. Using these data at vocalization frequencies, phonation threshold pressures (PTP) were calculated for each biomaterial, assuming a low dimensional model with supraglottic coupling and adjusted vocal fold length and thickness at each frequency. Results were normalized to a nominal PTP value. RESULTS: Viscoelastic data were acquired at vocalization frequencies as high as 363 to 1,400 Hz for six new carbomer hydrogels, Hylan B, and Extracel intended for vocal fold Reinke's space injection and for Cymetra (lateral injection). Reliability was confirmed with good data overlap when measuring with either rheometer. PTP predictions ranged from 0.001 to 16 times the nominal PTP value of 0.283 kPa. CONCLUSIONS: Accurate viscoelastic measurements of vocal fold injectables are now possible at physiologic frequencies. Hylan B, Extracel, and the new carbomer hydrogels should generate easy vocal onset and sustainable vocalization based on their rheologic properties if injected into Reinke's space. Applications may vary depending on desired longevity of implant. Laryngoscope, 2010.

In vivo comparison of biomimetic approaches for tissue regeneration of the scarred vocal fold.

The objective of this study was to determine if three different biomimetic approaches could facilitate tissue regeneration and improve viscoelastic properties in the scarred vocal fold lamina propria extracellular matrix (ECM). Twenty rabbit vocal folds were biopsied bilaterally; 2 months postinjury rabbits were unilaterally treated with (i) autologous fibroblasts, (ii) a semisynthetic ECM (sECM), or (iii) autologous fibroblasts encapsulated in sECM. Saline was injected as a control into the contralateral fold. Animals were sacrificed 2 months after treatment. Outcomes measured were procollagen, collagen, and fibronectin levels in the lamina propria, and tissue viscosity and elasticity across three frequency decades. All treatment groups demonstrated accelerated proliferation of the ECM. Vocal fold lamina propria treated with autologous fibroblasts were found to have significantly improved viscosity (p = 0.0077) and elasticity (p = 0.0081) compared to saline. This treatment group had significantly elevated fibronectin levels. sECM and autologous fibroblasts/sECM groups had significantly elevated levels of procollagen, collagen, and fibronectin, indicating abundant matrix production as compared to saline with viscoelastic measures that did not differ statistically from controls. The use of autologous fibroblasts led to better restoration of the vocal fold lamina propria biomechanical properties. Optimization of cell-scaffold interactions and subsequent cell behavior is necessary for utilization of scaffold and scaffold-cell approaches.

Methodology for rheological testing of engineered biomaterials at low audio frequencies.

A commercial rheometer (Bohlin CVO120) was used to mechanically test materials that approximate vocal-fold tissues. Application is to frequencies in the low audio range (20-150 Hz). Because commercial rheometers are not specifically designed for this frequency range, a primary problem is maintaining accuracy up to (and beyond) the mechanical resonance frequency of the rotating shaft assembly. A standard viscoelastic material (NIST SRM 2490) has been used to calibrate the rheometric system for an expanded frequency range. Mathematically predicted response curves are compared to measured response curves, and an error analysis is conducted to determine the accuracy to which the elastic modulus and the shear modulus can be determined in the 20-150-Hz region. Results indicate that the inertia of the rotating assembly and the gap between the plates need to be known (or determined empirically) to a high precision when the measurement frequency exceeds the resonant frequency. In addition, a phase correction is needed to account for the magnetic inertia (inductance) of the drag cup motor. Uncorrected, the measured phase can go below the theoretical limit of -pi. This can produce large errors in the viscous modulus near and above the resonance frequency. With appropriate inertia and phase corrections, +/- 10% accuracy can be obtained up to twice the resonance frequency.