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![Rheological characterization of various vocal fold injectables. Reprinted with permission from [47]. Copyright 2007 Wiley. Viscoelasticity of hyaluronan and nonhyaluronan-based vocal fold injectables: implications for mucosal versus muscle use. Shear stress applied by a parallel plate rheometer evaluated the viscoelastic properties of four vocal fold injectables prior to injection. Cadaveric vocal fold mucosa data are present for comparison. Data include mean elastic shear moduli (a), mean viscous shear moduli (b), and mean dynamic viscosities (c).](publication/223136582/figure/fig4/AS:1132187785142272@1646946011169/Rheological-characterization-of-various-vocal-fold-injectables-Reprinted-with-permission.jpg)
Rheological characterization of various vocal fold injectables. Reprinted with permission from [47]. Copyright 2007 Wiley. Viscoelasticity of hyaluronan and nonhyaluronan-based vocal fold injectables: implications for mucosal versus muscle use. Shear stress applied by a parallel plate rheometer evaluated the viscoelastic properties of four vocal fold injectables prior to injection. Cadaveric vocal fold mucosa data are present for comparison. Data include mean elastic shear moduli (a), mean viscous shear moduli (b), and mean dynamic viscosities (c).
Source publication
Vocal folds are anatomically and biomechanically unique, thus complicating the design and implementation of tissue engineering strategies for repair and regeneration. Integration of an enhanced understanding of tissue biomechanics, wound healing dynamics and innovative gel-based therapeutics has generated enthusiasm for the notion that an efficacio...
Citations
... The development, maintenance, and regeneration following injury of the VFs are heavily influenced by the phonation-induced mechanical stimulation [25,28]. As a result, various in vitro systems have been developed to closely mimic the mechanical environment of the VFs while incorporating relevant biomaterials, biochemical cues, and cells in a controlled experimental manner. ...
The vocal folds (VFs) are constantly exposed to mechanical stimulation leading to changes in biomechanical properties, structure, and composition. The development of long-term strategies for VF treatment depends on the characterization of related cells, biomaterials, or engineered tissues in a controlled mechanical environment. Our aim was to design, develop, and characterize a scalable and high-throughput platform that mimics the mechanical microenvironment of the VFs in vitro. The platform consists of a 24-well plate fitted with a flexible membrane atop a waveguide equipped with piezoelectric speakers which allows for cells to be exposed to various phonatory stimuli. The displacements of the flexible membrane were characterized via Laser Doppler Vibrometry (LDV). Human VF fibroblasts and mesenchymal stem cells were seeded, exposed to various vibratory regimes, and the expression of pro-fibrotic and pro-inflammatory genes was analyzed. Compared to current bioreactor designs, the platform developed in this study can incorporate commercial assay formats ranging from 6- to 96-well plates which represents a significant improvement in scalability. This platform is modular and allows for tunable frequency regimes.
... As biomaterials are injected into the biological system, immune cells such as leukocytes detect them as foreign particles. The different chemistry and exterior characteristics of hosts can trigger an immune response [69]. During this immune response, pro-inflammatory cytokines such as TNF-α, IL-6, IL-8, IL-1β and MCP-1 will be liberated whereas antiinflammatory cytokines such as IL-10 and IL-12 play crucial roles in quantifying the efficiency of eliminating harmful materials while providing protection to host [67,70]. ...
Vocal fold injection is a preferred treatment in glottic insufficiency because it is relatively quick and cost-saving. However, researchers have yet to discover the ideal biomaterial with properties suitable for human vocal fold application. The current systematic review employing PRISMA guidelines summarizes and discusses the available evidence related to outcome measures used to characterize novel biomaterials in the development phase. The literature search of related articles published within January 2010 to March 2021 was conducted using Scopus, Web of Science (WoS), Google Scholar and PubMed databases. The search identified 6240 potentially relevant records, which were screened and appraised to include 15 relevant articles based on the inclusion and exclusion criteria. The current study highlights that the characterization methods were inconsistent throughout the different studies. While rheologic outcome measures (viscosity, elasticity and shear) were most widely utilized, there appear to be no target or reference values. Outcome measures such as cellular response and biodegradation should be prioritized as they could mitigate the clinical drawbacks of currently available biomaterials. The review suggests future studies to prioritize characterization of the viscoelasticity (to improve voice outcomes), inflammatory response (to reduce side effects) and biodegradation (to improve longevity) profiles of newly developed biomaterials.
... 34,52,53 Through these inflammatory processes leading to increased resorption, especially in the rabbit model, the problem of volume stability seems to be unsolved for most injected materials. [56][57][58] In our experiment, inflammation might have been triggered by the non-autologous fibrinthrombin gel, but it could also have been the injection into the sensitive vocal fold system itself, as inflammation has been experienced by other authors with diverse materials. 53,[56][57][58] These inflammatory reactions indicate that new and different injectables, maybe containing locally active anti-inflammatory agents need to be sought as a substrate for the chondrocytes. ...
... [56][57][58] In our experiment, inflammation might have been triggered by the non-autologous fibrinthrombin gel, but it could also have been the injection into the sensitive vocal fold system itself, as inflammation has been experienced by other authors with diverse materials. 53,[56][57][58] These inflammatory reactions indicate that new and different injectables, maybe containing locally active anti-inflammatory agents need to be sought as a substrate for the chondrocytes. Since the inflammatory processes observed decreased between week two and four substantially, we concluded that the lack of remaining fibrin-thrombin mix in the 4 weeks larynxes (while the pellet culture was volume stable for 4 weeks) might be owed to the inflammation caused either by the carrier or the injection itself. ...
Objectives/Hypothesis
Injection laryngoplasty of materials for unilateral vocal‐fold paralysis has shown various results regarding the long‐term stability of the injected material. We evaluated a fibrin‐gel based cell suspension with autologous chondrocytes in‐vitro and in‐vivo as long‐term‐stable vocal‐fold augmentation material in an animal model.
Study Design
This study compises an in vitro cell‐culture part as well as an in vivo animal study with New Zealand White Rabbits.
Methods
In in‐vitro experiments, auricular chondrocytes harvested from 24 New Zealand White Rabbits cadavers were cultivated in pellet cultures to evaluate cartilage formation for 4 weeks using long‐term‐stable fibrin gel as carrier. Injectability and injection volume for the laryngoplasty was determined in‐vitro using harvested cadaveric larynxes. In‐vivo 24 Rabbits were biopsied for elastic cartilage of the ear and autologous P1 cells were injected lateral of one vocal cord into the paraglottic space suspended in a long‐term‐stable fibrin gel. Histologic evaluation was performed after 2, 4, 12, and 24 weeks.
Results
During 12‐week pellet culture, we found extracellular matrix formation and weight‐stable cartilage of mature appearance. In‐vivo, mature cartilage was found in two larynxes (n = 6) at 4 weeks, in four (n = 6) at 12 weeks, and in five (n = 6) at 24 weeks mostly located in the paraglottic space and sometimes with spurs into the vocalis muscle. Surrounding tissue was often infiltrated with inflammatory cells. Material tended to dislocate through the cricothyroid space into the extraglottic surrounding tissue.
Conclusions
A cell‐based approach with chondrocytes for permanent vocal‐fold augmentation has not previously been reported. We have achieved the formation of structurally mature cartilage in the paraglottic space, but this is accompanied by difficulties with dislocated material, deformation of the augmentation, and inflammation.
Level of Evidence
N/A Laryngoscope, 2020
... The use of hydrogels to treat vocal fold scarring has the potential to improve patient therapeutic outcomes compared to current clinical practices. 1,64 When developing vocal fold hydrogels, it is necessary to establish the material composition that minimizes undesirable inflammation, maintains optimum mechanical integrity, and promotes beneficial immunomodulatory effects. 48,55 Hydrogel stiffness is a tunable material property that can be harnessed to modulate the immune response. ...
... 35 TNF-α also has involvement in the stimulation of MMP secretion from Mφ and VFFs in vocal fold tissue. 64,81,82 Results from the ELISA assessment of TNF-α indicated that the presence of VFFs was associated with lower TNF-α levels compared to monoculture controls as time increased (Figure 4). This finding agreed with literature elsewhere that VFF may suppress Mφ pro-inflammatory cytokine production and regulate inflammatory activity via toll-like receptor 4 (TLR4). ...
The physical properties of a biomaterial play an essential role in regulating immune and reparative activities within the host tissue. This study aimed to evaluate the immunological impact of material stiffness of a glycol-chitosan hydrogel designed for vocal fold tissue engineering. Hydrogel stiffness was varied via the concentration of glyoxal cross-linker applied. Hydrogel mechanical properties were characterized through atomic force microscopy and shear plate rheometry. Using a transwell setup, macrophages were co-cultured with human vocal fold fibroblasts that were embedded within the hydrogel. Macrophage viability and cytokine secretion were evaluated at 3, 24, and 72 hr of culture. Flow cytometry was applied to evaluate macrophage cell surface markers after 72 hr of cell culture. Results indicated that increasing hydrogel stiffness was associated with increased anti-inflammatory activity compared to relevant controls. In addition, increased anti-inflammatory activity was observed in hydrogel co-cultures. This study highlighted the importance of hydrogel stiffness from an immunological viewpoint when designing novel vocal fold hydrogels. K E Y W O R D S fibroblast, hydrogel, immunomodulation, macrophage, stiffness
... As previously reported, biocompatible hydrogels have the ability to modulate and enhance wound healing response in injured vocal folds. 37 Due to tumor growth risk, bFGF is not always applicable for vocal fold surgery. 19 Therefore, we hypothesized that if the effect of simply injecting gelatin hydrogel microspheres is comparable to that of injection of the same microspheres but with bFGF, the usage of the material would be facilitated more clinically. ...
Introduction:
Postoperative dysphonia is mostly caused by vocal fold scarring, and careful management of vocal fold surgery has been reported to reduce the risk of scar formation. However, depending on the vocal fold injury, treatment of postoperative dysphonia can be challenging.
Objective:
The goal of the current study was to develop a novel prophylactic regenerative approach for the treatment of injured vocal folds after surgery, using biodegradable gelatin hydrogel microspheres as a drug delivery system for basic fibroblast growth factor.
Methods:
Videoendoscopic laryngeal surgery was performed to create vocal fold injury in 14 rabbits. Immediately following this procedure, biodegradable gelatin hydrogel microspheres with basic fibroblast growth factor were injected in the vocal fold. Two weeks after injection, larynges were excised for evaluation of vocal fold histology and mucosal movement.
Results:
The presence of poor vibratory function was confirmed in the injured vocal folds. Histology and digital image analysis demonstrated that the injured vocal folds injected with gelatin hydrogel microspheres with basic fibroblast growth factor showed less scar formation, compared to the injured vocal folds injected with gelatin hydrogel microspheres only, or those without any injection.
Conclusion:
A prophylactic injection of basic fibroblast growth factor -containing biodegradable gelatin hydrogel microspheres demonstrates a regenerative potential for injured vocal folds in a rabbit model.
... The treatments currently employed for correcting voice defects are surgical techniques and behavioral treatment, however surgical interventions induce scar formation and produces unreliable results [129]. The field of TE involving combination of various bioactive factors, injectable hydrogel scaffolds and stem cells, efficiently helps in treatment of vocal fold defects and thus plays a major role in the regeneration of vocal fold [130,131]. HA has inherent angiogenic property and plays a key role in tissue granulation and scar remodeling [132]. It has innated rigid chain conformation having α-1,4 linkage with repeated pyrenoid ring structure. ...
... Native ECM-based biomaterials including HA and collagen have also been employed in vocal fold injection; the HA-based materials are likely the most promising materials in the field [24]. HA is a linear non-sulfated and negatively charged glycosaminoglycan, which contributes to the viscoelastic properties of vocal fold tissue [18,25] and its chemical functionality permits modification with various chemical groups that have been employed for the crosslinking of HA-based hydrogels [19]. Hylan-B, an HA-based biomaterial [26], has shown improvements in glottal closure and phonation quality in clinical trials [24]. ...
Vocal fold scar, characterized by alterations in the lamina propria extracellular matrix, disrupts normal voice quality and function. Due to a lack of satisfactory clinical treatments, there is a need for tissue engineering strategies to restore voice. Candidate biomaterials for vocal fold tissue engineering must match the unique biomechanical and viscoelastic properties of native tissue without provoking inflammation. We sought to introduce elastomeric properties to hyaluronic acid (HA)-based biomaterials by incorporating resilin-like polypeptide (RLP) into hybrid hydrogels. Physically crosslinked RLP/HA and chemically crosslinked RLP-acrylamide/thiolated HA (RLP-AM/HA-SH) hydrogels were fabricated using cytocompatible chemistries. Mechanical properties of hydrogels were assessed in vitro using oscillatory rheology. Hybrid hydrogels were injected into rabbit vocal folds and tissues were assessed using rheology and histology. A small number of animals underwent acute vocal fold injury followed by injection of RLP-AM/HA-SH hydrogel alone or as a carrier for human bone marrow mesenchymal stem cells (BM-MSCs). Rheological testing confirmed that mechanical properties of materials in vitro resembled native vocal fold tissue and that viscoelasticity of vocal fold mucosa was preserved days 5 and 21 after injection. Histological analysis revealed that hybrid hydrogels provoked only mild inflammation in vocal fold lamina propria with demonstrated safety in the airway for up to 3 weeks, confirming acute biocompatibility of crosslinking chemistries. After acute injury, RLP-AM/HA-SH gel with and without BM-MSCs did not result in adverse effects or increased inflammation. Collectively, results indicate that RLP and HA-based hybrid hydrogels are highly promising for engineering the vocal fold lamina propria.
... The hydrophobic, ionic or hydrogen bonding inside the IPN is responsible for the improved mechanics and degradation behavior. Two examples are the IPNs formed between collagen and chitosan [29], and collagen and polyethylene oxide (PEG) [30]. In these approaches, the ECM extracted collagen is combined with diferent mass concentrations of polymers, and later this mixture is incubated at 37°C to induce the collagen polymerization. ...
... Previous investigations using injectable materials for vocal fold augmentation and repair have relied largely on chemically crosslinked hyaluronic acid (HA)-based materials due to the fact that HA is a major component of the ECM of the superficial lamina propria and because of its contribution in modulating the viscoelastic properties of vocal fold tissue. 9,12 Clinical investigations of vocal fold treatments that employ HA-based biomaterials have yielded overall improvements in glottal closure and phonation quality when injected into the thyroarytenoid muscle; unfortunately a HA product specifically for the lamina propria treatment is not yet available. Although the mechanical properties of biomaterials can be easily tailored to match those required for vocal fold tissue engineering, there is limited information available regarding transient mechanical properties upon application and release of strain and limited ability, to date, to independently control mechanical features and biological functions. ...
... In our case, the overall effect of downregulation of TGF-b by day 21 compared to day 5 across groups suggests a regenerative and remodeling transition from an early proinflammatory stage (where multiple cell types are recruited to invade into injection sites/wound bed to induce the immune reaction) to a later stage with a resolved foreign body response characterized by new tissue remodeling. 3,12 Examination of the impact of RLP-derived hydrogels on vocal fold tissue regeneration, particularly in the context of related gene expression profiles and ECM deposition, requires further in vivo studies. Future investigations will require new nontoxic, biocompatible crosslinking chemistry for in vivo hydrogel formation with the inclusion of multiple cell-instructive biological motifs and relevant cell types present in these injected hydrogel matrices. ...
Vocal folds are connective tissues housed in the larynx, which can be subjected to various injuries and traumatic stimuli that lead to aberrant tissue structural alterations and fibrotic‐induced biomechanical stiffening observed in patients with voice disorders. Much effort has been devoted to generate soft biomaterials that are injectable directly to sites of injury. To date, materials applied toward these applications have been largely focused on natural extracellular matrix (ECM)‐derived materials such as collagen, fibrin or HA; these approaches have suffered from the fact that materials are not sufficiently robust mechanically nor offer sufficient flexibility to modulate material properties for targeted injection. We have recently developed multiple resilin‐inspired elastomeric hydrogels that possess similar mechanical properties as those reported for vocal fold tissues, and that also show promising in vitro cytocompatibility and in vivo biocompatibility. Here we report studies that test the delivery of resilin‐based hydrogels via injection to the subcutaneous tissue in a wild‐type mice model; histological and genetic expression outcomes were monitored. The rapid kinetics of crosslinking enabled facile injection and ensured the rapid transition of the viscous resilin precursor solution to a solid‐like hydrogel in the subcutaneous space in vivo; the materials exhibited storage shear moduli in the range of 1000∼2000Pa when characterized via oscillatory rheology. Histological staining and gene expression profiles suggested minimal inflammatory profiles three weeks after injection, thereby demonstrating the potential suitability for site‐specific in vivo injection of these elastomeric materials. This article is protected by copyright. All rights reserved.
... The requirements of suitable biomaterials are extremely complex, because on the one hand mechanical stability for insertion into the larynx is necessary and on the other hand the vibratory ability of the vocal folds requires enormous flexibility. Hydrogels were evaluated several times regarding injection into the vocal folds [88], with materials like collagen and elastin play a key role as well as the combination with stem cells or fibroblasts from the patients' own vocal folds [89]. Stem-cell application can be performed by injection or mobilization of endogenous stem cells [87]. ...
Zusammenfassung
Die Regeneration als therapeutisches Prinzip und damit die Regenerative Medizin ist ein vielversprechender Ansatz künftig die therapeutischen Optionen der Hals-Nasen-Ohrenheilkunde um eine weitere Dimension zu erweitern. Während heute rekonstruktive chirurgische Verfahren, Medikamente und Prothesen wie bspw. das Cochlea Implantat die Funktionen defekter Gewebe im Kopf-Hals-Bereich ersetzen, sollen durch die Regenerative Medizin die defekten Gewebe und deren Funktion selbst wiederhergestellt werden. In dieser Übersichtsarbeit werden neue Entwicklungen wie das 3D-Bioprinting und dezellularisierte, natürliche Biomaterialien für regenerative Ansätze vorgestellt und durch eine Zusammenstellung aktueller präklinischer und klinischer Studien im Bereich der Regenerativen Medizin in der Hals-Nasen-Ohrenheilkunde ergänzt.
