In Vitro Investigation of Vocal Fold Cellular Response to Variations in Hydrogel Porosity and Elasticity.

Tissue regeneration is intricately influenced by the dynamic interplay between the physical attributes of tissue engineering scaffolds and the resulting biological responses. A tunable microporous hydrogel system was engineered using gelatin methacryloyl (GelMA) and polyethylene glycol diacrylate (PEGDA), with polyethylene glycol (PEG) serving as a porogen. Through systematic variation of PEGDA molecular weights, hydrogels with varying mechanical and architectural properties were obtained. The objective of the present study was to elucidate the impact of substrate mechanics and architecture on the immunological and reparative activities of vocal fold tissues. Mechanical characterization of the hydrogels was performed using tensile strength measurements and rheometry. Their morphological properties were investigated using scanning electron microscopy (SEM) and confocal microscopy. A series of biological assays were conducted. Cellular morphology, differentiation, and collagen synthesis of human vocal fold fibroblasts (hVFFs) were evaluated using immunostaining. Fibroblast proliferation was studied using the WST-1 assay, and cell migration was investigated via the Boyden chamber assay. Macrophage polarization and secretions were also examined using immunostaining and ELISA. The results revealed that increasing the molecular weight of PEGDA from 700 Da to 10,000 Da resulted in decreased hydrogel stiffness, from 62.6 to 8.8 kPa, and increased pore dimensions from approximately 64.9 to 137.4 µm. Biological evaluations revealed that hydrogels with a higher stiffness promoted fibroblast proliferation and spreading, albeit with an increased propensity for fibrosis, as indicated by a surge in myofibroblast differentiation and collagen synthesis. In contrast, hydrogels with greater molecular weights had a softer matrix with expanded pores, enhancing cellular migration and promoting an M2 macrophage phenotype conducive to tissue healing. The findings show that the hydrogels formulated with a PEGDA molecular weight of 6000 Da are best among the hydrogels considered for vocal fold repair. The microporous hydrogels could be tuned to serve in other tissue engineering applications.

Rayleigh wave propagation method for the characterization of a thin layer of biomaterials.

An experimental method based on Rayleigh wave propagation was developed for quantifying the frequency-dependent viscoelastic properties of a small volume of expensive biomaterials over a broad frequency range. Synthetic silicone rubber and gelatin materials were fabricated and tested to evaluate the proposed method. Planar harmonic Rayleigh waves at different frequencies, from 80 to 4000 Hz, were launched on the surface of a sample composed of a substrate with known material properties coated with a thin layer of the soft material to be characterized. A transfer function method was used to obtain the complex Rayleigh wavenumber. An inverse wave propagation problem was solved and a complex nonlinear dispersion equation was obtained. The complex shear and elastic moduli of the sample materials were then calculated through the numerical solution of the obtained dispersion equation using the measured wavenumbers. The results were in good agreement with those of a previous independent study. The proposed method was found to be reliable and cost effective for the measurement of viscoelastic properties of a thin layer of expensive biomaterials, such as phonosurgical biomaterials, over a wide frequency range.

Experimental methods for the characterization of the frequency-dependent viscoelastic properties of soft materials.

A characterization method based on Rayleigh wave propagation was developed for the quantification of the frequency-dependent viscoelastic properties of soft materials at high frequencies; i.e., up to 4 kHz. Planar harmonic surface waves were produced on the surface of silicone rubber samples. The phase and amplitude of the propagating waves were measured at different locations along the propagation direction, which allowed the calculation of the complex Rayleigh wavenumbers at each excitation frequency using a transfer function method. An inverse wave propagation problem was then solved to obtain the complex shear/elastic moduli from the measured wavenumbers. In a separate, related investigation, dynamic indentation tests using atomic force microscopy (AFM) were performed at frequencies up to 300 Hz. No systematic verification study is available for the AFM-based method, which can be used when the dimensions of the test samples are too small for other existing testing methods. The results obtained from the Rayleigh wave propagation and AFM-based indentation methods were compared with those from a well-established method, which involves the generation of standing longitudinal compression waves in rod-shaped test specimens. The results were cross validated and qualitatively confirmed theoretical expectations presented in the literature for the frequency-dependence of polymers.

Immunomodulatory Microgels Support Proregenerative Macrophage Activation and Attenuate Fibroblast Collagen Synthesis.

Scars composed of fibrous connective tissues are natural consequences of injury upon incisional wound healing in soft tissues.  Hydrogels that feature a sustained presentation of immunomodulatory cytokines are known to modulate wound healing. However, existing immunomodulatory hydrogels lack interconnected micropores to promote cell ingrowth. Other limitations include invasive delivery procedures and harsh synthesis conditions that are incompatible with drug molecules. Here, hybrid nanocomposite microgels containing interleukin-10 (IL-10) are reported to modulate tissue macrophage phenotype during wound healing. The intercalation of laponite nanoparticles in the polymer network yields microgels with tissue-mimetic elasticity (Young's modulus in the range of 2-6 kPa) and allows the sustained release of IL-10 to promote the differentiation of macrophages toward proregenerative phenotypes. The porous interstitial spaces between microgels promote fibroblast proliferation and fast trafficking (an average speed of ≈14.4 µm h-1 ). The incorporation of hyaluronic acid further enhances macrophage infiltration. The coculture of macrophages and fibroblasts treated with transforming growth factor-beta 1 resulted in a twofold reduction in collagen-I production for microgels releasing IL-10 compared to the IL-10 free group. The new microgels show potential toward regenerative healing by harnessing the antifibrotic behavior of host macrophages.

Injectable, Pore-Forming, Perfusable Double-Network Hydrogels Resilient to Extreme Biomechanical Stimulations.

Biological tissues hinge on blood perfusion and mechanical toughness to function. Injectable hydrogels that possess both high permeability and toughness have profound impacts on regenerative medicine but remain a long-standing challenge. To address this issue, injectable, pore-forming double-network hydrogels are fabricated by orchestrating stepwise gelation and phase separation processes. The interconnected pores of the resulting hydrogels enable direct medium perfusion through organ-sized matrices. The hydrogels are amenable to cell encapsulation and delivery while promoting cell proliferation and spreading. They are also pore insensitive, tough, and fatigue resistant. When tested in biomimetic perfusion bioreactors, the hydrogels maintain physical integrity under prolonged, high-frequency biomechanical stimulations (>6000 000 cycles at 120 Hz). The excellent biomechanical performance suggests the great potential of the new injectable hydrogel technology for repairing mechanically dynamic tissues, such as vocal folds, and other applications, such as tissue engineering, biofabrication, organs-on-chips, drug delivery, and disease modeling.

Efficacy of Aerosol Reduction Measures for Dental Aerosol Generating Procedures.

Aerosol particles generated by dental procedures could facilitate the transmission of infectious diseases and contain carcinogen particles. Such particles can penetrate common surgical masks and reach the lungs, leading to increased risk for dental care professionals. However, the risk of inhaling contaminated aerosol and the effectiveness of aerosol reduction measures in dental offices remain unclear. The present study aimed to quantify aerosols produced by drilling and scaling procedures and to evaluate present recommendations for aerosol reduction. The concentration of aerosol particles released from the mock scaling and drilling procedures on dental mannequin were measured using a TSI Optical Particle Sizer (OPS 3330) during 15-min sessions carried out in a single-patient examination room. Using a drilling procedure as the aerosol source, the aerosol reduction performance of two types of high-volume evacuators (HVEs) and a commercial off-the-shelf air purifier was evaluated in a simulated clinical setting. Using either HVEs or the air purifier individually reduced the aerosol accumulated over the course of a 15-minutes drilling procedure at a reduction rate of 94.8 to 97.6%. Using both measures simultaneously raised the reduction rate to 99.6%. The results show that existing HVEs can effectively reduce aerosol concentration generated by a drilling procedure and can be further improved by using an air purifier. Following current regulatory guidelines can ensure a low risk of inhaling contaminated aerosol for dentists, assistants, and patients.

Liquid-infused microstructured bioadhesives halt non-compressible hemorrhage.

Non-compressible hemorrhage is an unmet clinical challenge that accounts for high mortality in trauma. Rapid pressurized blood flows under hemorrhage impair the function and integrity of hemostatic agents and the adhesion of bioadhesive sealants. Here, we report the design and performance of bioinspired microstructured bioadhesives, formed with a macroporous tough xerogel infused with functional liquids. The xerogel can rapidly absorb interfacial fluids such as whole blood and promote blood clotting, while the infused liquids facilitate interfacial bonding, sealing, and antibacterial function. Their synergy enables the bioadhesives to form tough adhesion on ex vivo human and porcine tissues and diverse engineered surfaces without the need for compression, as well as on-demand instant removal and storage stability. We demonstrate a significantly improved hemostatic efficacy and biocompatibility in rats and pigs compared to non-structured counterparts and commercial products. This work opens new avenues for the development of bioadhesives and hemostatic sealants.

Polymeric Microspheres Containing Human Vocal Fold Fibroblasts for Vocal Fold Regeneration.

OBJECTIVE: Most acellular injectable biomaterials for vocal fold (VF) wound treatment have limited regenerative potential due to their fast enzymatic degradation and limited recruitment of native cells postinjection. The injection of cells as therapeutic treatment often results in apoptosis due to stresses within the needle and the immune response of the host. Degradable microspheres may improve treatment effectiveness by increasing cell residence time, shielding cells during injection, and offering early protection against the immune system response. The objective of the present study was to investigate the potential of human VF fibroblasts encapsulated in polymeric microspheres as an injectable therapeutic treatment in vitro. METHODS: Alginate, alginate-poly-L-lysine, and alginate-chitosan microspheres were fabricated using electrospraying and characterized in terms of biocompatibility, swelling, and mechanical properties as well as cytokine production. RESULTS: Alginate microspheres were found to have the most desirable properties for VF regeneration. They were resistant to mechanical challenges. They were found to have a stiffness similar to that reported for native VF-lamina propria. They were found to be biocompatible and increased the proliferation of fibroblasts. Human VF fibroblasts encapsulated in alginate microspheres induced the production of interleukin (IL)-8 and IL-4 at 24 hours. CONCLUSION: The alginate microspheres fabricated in this study were found to offer potential advantages, as cell delivery tool. This study highlights the importance of combining biomaterials and cells to expedite the wound-healing process through cytokine production. Future work is aimed to further analysis of the wound-healing properties the microspheres. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1828-1834, 2021.

Ionotronic Tough Adhesives with Intrinsic Multifunctionality.

Ionotronic hydrogels find wide applications in flexible electronics, wearable/implantable devices, soft robotics, and human-machine interfaces. Their performance and practical translation have been bottlenecked by poor adhesiveness, limited mechanical properties, and the lack of biological functions. The remedies are often associated with complex formulations and sophisticated processing. Here, we report a rational design and facile synthesis of ionotronic tough adhesives (i-TAs), which have excellent mechanical, physical, electrical, and biological properties and promise high scalability and translational potential. They consist of an interpenetrating network with high-density amine groups and highly mobile chains, which enable intrinsic adhesiveness, self-healing, ionic stability, cytocompatibility, and antimicrobial functions. The i-TAs in both pristine and swollen states possess high toughness, stretchability, and strong adhesion to diverse substrates such as tissues and elastomers. The superior mechanical performance is achieved simultaneously with high ionic conductivity and stability in electrolyte solutions. We further demonstrate the use of i-TAs as wearable devices, strain sensors, and sensory sealants. This work is expected to open avenues for new ionotronics with novel functions and stimulate the development and translation of ionotronics.

Needle-free injection: Dental infiltration anesthesia.

This study aimed to develop an optimal Needle-Free Liquid Jet Injection (NFLJI) technique for dental infiltration anesthesia and evaluate its clinical safety and feasibility. The fluid dynamics of NFLJI in the dentoalveolar region were investigated using soft tissue phantoms supported by rigid glass. NFLJIs were performed at different incident angles and recorded using a high-speed camera. Accordingly, an optimal NFLJI for infiltration anesthesia was developed and validated on cadavers, then assessed in two pilot Randomized Controlled Trials (RCT): one for validating the safety of optimal NFLJI technique, the other for evaluating its feasibility and safety. High-speed videos showed that perpendicular NFLJIs induced significantly more regurgitation than oblique NFLJIs, which was confirmed in cadavers. Clinical trials revealed that perpendicular NFLJIs induced a high risk of bleeding (83.3%) and laceration (83.3%), whereas oblique NFLJIs induced a low risk of bleeding (33.3%) and laceration (16.7%). Moreover, the preliminary success rates of oblique NFLJIs and needle injections were both 83.3%. The recruitment took 3-5 weeks with a rate of 100%. Oblique NFLJIs could be a promising approach for dental infiltration anesthesia, causing minimal drug regurgitation with a relatively low risk of complication. The pilot RCTs confirmed the feasibility for conducting a non-inferiority RCT.

Triggered micropore-forming bioprinting of porous viscoelastic hydrogels.

Cell-laden scaffolds of architecture and mechanics that mimic those of the host tissues are important for a wide range of biomedical applications but remain challenging to bioprint. To address these challenges, we report a new method called triggered micropore-forming bioprinting. The approach can yield cell-laden scaffolds of defined architecture and interconnected pores over a range of sizes, encompassing that of many cell types. The viscoelasticity of the bioprinted scaffold can match that of biological tissues and be tuned independently of porosity and stiffness. The bioprinted scaffold also exhibits superior mechanical robustness despite high porosity. The bioprinting method and the resulting scaffolds support cell spreading, migration, and proliferation. The potential of the 3D bioprinting system is demonstrated for vocal fold tissue engineering and as an in vitro cancer model. Other possible applications are foreseen for tissue repair, regenerative medicine, organ-on-chip, drug screening, organ transplantation, and disease modeling.

Investigation of surgical adhesives for vocal fold wound closure.

OBJECTIVES: Surgical adhesives are increasingly used for vocal fold microsurgery to assist wound closure and reduce the risks of scar formation. Currently used vocal fold adhesives such as fibrin glue, however, have thus far not been found to promote wound closure or reduce scarring. The objectives of this study were to investigate the mechanical strength and the cytotoxicity of three commercially available adhesives (Glubran 2, GEM, Viareggio, Italy; BioGlue, CryoLife, Kennesaw, GA; and Tisseel, Baxter Healthcare, Deerfield, IL) for vocal fold wound closure. METHODS: Shear and tension tests were performed on 150 porcine larynges. The cytotoxicity of the adhesives to immortalized human vocal fold fibroblasts was investigated using neutral red uptake assays. RESULTS: The average shear adhesive strength for Tisseel, BioGlue, and Glubran 2 was 13.86 ± 5.03 kilopascal (kPa), 40.92 ± 17.94 kPa, and 68.79 ± 13.29 kPa, respectively. The tensile adhesive strength for Tisseel, BioGlue, and Glubran 2 was 10.70 ± 6.42 kPa, 34.27 ± 12.59 kPa, and 46.67 ± 12.13 kPa, respectively. The vocal fold cell viabilities in extracts of Tisseel, BioGlue, and Glubran 2 were 99.27%, 43.05%, and 1.79%, respectively. CONCLUSION: There was a clear tradeoff between adhesive strength and toxicity. The maximum failure strength in shear or tension of the three surgical adhesives ranked from strongest to the weakest was: 1) Glubran 2, 2) BioGlue, and 3) Tisseel. Tisseel was found to be the least toxic of the three adhesives, whereas Glubran 2 was the most toxic. LEVEL OF EVIDENCE: NA Laryngoscope, 129:2139-2146, 2019.

Determination of superior surface strains and stresses, and vocal fold contact pressure in a synthetic larynx model using digital image correlation.

Stresses and strains within the vocal fold tissue may play a critical role in voice fatigue, in tissue damage and resulting voice disorders, and in tissue healing. In this study, experiments were performed to determine mechanical fields on the superior surface of a self-oscillating physical model of the human vocal folds using a three-dimensional digital image correlation method. Digital images obtained using a high-speed camera together with a mirror system were used to measure displacement fields, from which strains, strain rates, and stresses on the superior surface of the model vocal folds were computed. The dependence of these variables on flow rate was established. A Hertzian impact model was used to estimate the contact pressure on the medial surface from superior surface strains. A tensile stress dominated state was observed on the superior surface, including during collision between the model folds. Collision between the model vocal folds limits the medial-lateral stress levels on the superior surface, in conjunction with compressive stress or contact pressure on the medial surface.

Investigation of the Viability, Adhesion, and Migration of Human Fibroblasts in a Hyaluronic Acid/Gelatin Microgel-Reinforced Composite Hydrogel for Vocal Fold Tissue Regeneration.

The potential use of a novel scaffold biomaterial consisting of cross-linked hyaluronic acid (HA)-gelatin (Ge) composite microgels is investigated for use in treating vocal fold injury and scarring. Cell adhesion integrins and kinematics of cell motion are investigated in 2D and 3D culture conditions, respectively. Human vocal fold fibroblast (hVFF) cells are seeded on HA-Ge microgels attached to a HA hydrogel thin film. The results show that hVFF cells establish effective adhesion to HA-Ge microgels through the ubiquitous expression of ß1 integrin in the cell membrane. The microgels are then encapsulated in a 3D HA hydrogel for the study of cell migration. The cells within the HA-Ge microgel-reinforced composite hydrogel (MRCH) scaffold have an average motility speed of 0.24 ± 0.08 µm min(-1) . The recorded microscopic images reveal features that are presumably associated with lobopodial and lamellipodial cell migration modes within the MRCH scaffold. Average cell speed during lobopodial migration is greater than that during lamellipodial migration. The cells move faster in the MRCH than in the HA-Ge gel without microgels. These findings support the hypothesis that HA-Ge MRCH promotes cell adhesion and migration; thereby they constitute a promising biomaterial for vocal fold repair.

An in vivo study of composite microgels based on hyaluronic acid and gelatin for the reconstruction of surgically injured rat vocal folds.

PURPOSE The objective of this study was to investigate local injection with a hierarchically microstructured hyaluronic acid-gelatin (HA-Ge) hydrogel for the treatment of acute vocal fold injury using a rat model. METHOD Vocal fold stripping was performed unilaterally in 108 Sprague-Dawley rats. A volume of 25 µl saline (placebo controls), HA-bulk, or HA-Ge hydrogel was injected into the lamina propria (LP) 5 days after surgery. The vocal folds were harvested at 3, 14, and 28 days after injection and analyzed using hematoxylin and eosin staining and immunohistochemistry staining for macrophages, myofibroblasts, elastin, collagen type I, and collagen type III. RESULTS The macrophage count was statistically significantly lower in the HA-Ge group than in the saline group (p < .05) at Day 28. Results suggested that the HA-Ge injection did not induce inflammatory or rejection response. Myofibroblast counts and elastin were statistically insignificant across treatment groups at all time points. Increased elastin deposition was qualitatively observed in both HA groups from Day 3 to Day 28, and not in the saline group. Significantly more elastin was observed in the HA-bulk group than in the uninjured group at Day 28. Significantly more collagen type I was observed in the HA-bulk and HA-Ge groups than in the saline group (p < .05) at Day 28. The collagen type I concentration in the HA-Ge and saline groups was found to be comparable to that in the uninjured controls at Day 28. The concentration of collagen type III in all treatment groups was similar to that in uninjured controls at Day 28. CONCLUSION Local HA-Ge and HA-bulk injections for acute injured vocal folds were biocompatible and did not induce adverse response.

Characterization of a hierarchical network of hyaluronic acid/gelatin composite for use as a smart injectable biomaterial.

Hybrid HA/Ge hydrogel particles are embedded in a secondary HA network to improve their structural integrity. The internal microstructure of the particles is imaged through TEM. CSLM is used to identify the location of the Ge molecules in the microgels. Through indentation tests, the Young's modulus of the individual particles is found to be 22 ± 2.5 kPa. The overall shear modulus of the composite is 75 ± 15 Pa at 1 Hz. The mechanical properties of the substrate are found to be viable for cell adhesion. The particles' diameter at pH = 8 is twice that at pH = 5. The pH sensitivity is found to be appropriate for smart drug delivery. Based on their mechanical and structural properties, HA-Ge hierarchical materials may be well suited for use as injectable biomaterials for tissue reconstruction.