Vocal Fold Regeneration Research Articles

Immunocytochemistry assessment of vocal fold regeneration after cell-based implant in rabbits.

Cell-based outer vocal fold replacement (COVR) offers a potential treatment for severe vocal fold scarring or cancer reconstruction. Previous work in rabbits using human adipose-derived stem cells (ASC) in fibrin suggested that a hybrid structure emerged within 2 months, containing both implanted and host cells. This project uses immunocytochemistry to better define the phenotypic fate of implanted cells and features of the extracellular environment. Immunocytochemistry was performed on sections collected from rabbits 2 months after COVR implantation or scar surgery. Cellular targets included human leukocyte antigen (HLA), CD31, and smooth muscle actin (SMA). HLA was present in all implanted sections and was used to identify human cells. In adjacent sections, HLA-positive cells were identified expressing CD31. SMA was not identified in the same cells as HLA. These markers were also present in injured vocal folds not receiving COVR. SMA protein content did not differ according to treatment. Implanted human ASC persist in rabbit vocal folds. Some appear to express CD31, an endothelial marker. Smooth muscle actin, a marker of myofibroblast phenotype, was present in all sections regardless of treatment, and was not identified in hASC. Host cells also infiltrate the structure, producing a hybrid host-graft vocal fold.

Trends in Injectable Biomaterials for Vocal Fold Regeneration and Long-Term Augmentation.

Human vocal folds (VF), a pair of small, soft tissues in the larynx, have a layered mucosal structure with unique mechanical strength to support high-level tissue deformation by phonation. Severe pathological changes to VF have causes including surgery, trauma, age-related atrophy, and radiation, and lead to partial or complete communication loss and difficulty in breathing and swallowing. VF glottal insufficiency requires injectable VF biomaterials such as hyaluronan, calcium hydroxyapatite, and autologous fat to augment VF functions. Although these biomaterials provide an effective short-term solution, significant variations in patient response and requirements of repeat reinjection remain notable challenges in clinical practice. Tissue engineering strategies have been actively explored in the search of an injectable biomaterial that possesses the capacity to match native tissue's material properties while promoting permanent tissue regeneration. This review aims to assess the current status of biomaterial development in VF tissue engineering. The focus will be on examining state-of-the-art techniques including modification with bioactive molecules, cell encapsulation, composite materials, as well as, in situ crosslinking with click chemistry. We will discuss potential opportunities that can further leverage these engineering techniques in the advancement of VF injectable biomaterials.

Unraveling the Relevance of Tissue-Specific Decellularized Extracellular Matrix Hydrogels for Vocal Fold Regenerative Biomaterials: A Comprehensive Proteomic and In Vitro Study.

Decellularized extracellular matrix (dECM) is a promising material for tissue engineering applications. Tissue-specific dECM is often seen as a favorable material that recapitulates a native-like microenvironment for cellular remodeling. However, the minute quantity of dECM derivable from small organs like the vocal fold (VF) hampers manufacturing scalability. Small intestinal submucosa (SIS), a commercial product with proven regenerative capacity, may be a viable option for VF applications. This study aims to compare dECM hydrogels derived from SIS or VF tissue with respect to protein content and functionality using mass spectrometry-based proteomics and in vitro studies. Proteomic analysis reveals that VF and SIS dECM share 75% of core matrisome proteins. Although VF dECM proteins have greater overlap with native VF, SIS dECM shows less cross-sample variability. Following decellularization, significant reductions of soluble collagen (61%), elastin (81%), and hyaluronan (44%) are noted in VF dECM. SIS dECM contains comparable elastin and hyaluronan but 67% greater soluble collagen than VF dECM. Cells deposit more neo-collagen on SIS than VF-dECM hydrogels, whereas neo-elastin (~50 μg/scaffold) and neo-hyaluronan (~ 6 μg/scaffold) are comparable between the two hydrogels. Overall, SIS dECM possesses reasonably similar proteomic profile and regenerative capacity to VF dECM. SIS dECM is considered a promising alternative for dECM-derived biomaterials for VF regeneration.

The life-cycle and restoration of the human vocal fold.

To better understand the challenges of designing therapies to treat damaged vocal fold lamina propria, it is essential to understand the biophysical and pathophysiological mechanisms involved in vocal fold development, maintenance, injury, and aging. This review critically analyses these points to try and direct future efforts and new strategies toward science-based solutions. MEDLINE, Ovid Embase, and Wed of Science databases were used to identify relevant literature. A scoping review was performed following the preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews checklist. The layered arrangement of the vocal fold, develops during early childhood and is maintained during adulthood unless injury occurs. The stellate cells of the macular flava are likely to be important in this process. The capacity for vocal fold regeneration and growth is lost during adulthood and repair results in the deposition of fibrous tissue from resident fibroblasts. With advancing age, viscoelastic tissue declines, possibly due to cell senescence. Strategies aimed at replacing fibrous tissue within the vocal folds must either stimulate resident cells or implant new cells to secrete healthy extracellular protein. Injection of basic fibroblast growth factor is the most widely reported therapy that aims to achieve this. The pathways involved in vocal fold development, maintenance and aging are incompletely understood. Improved understanding has the potential to identify new treatment targets that could potentially overcome loss of vocal fold vibratory tissue.

Regenerative Medicine and Amniotic Membrane Application for Vocal Fold Scars and Sulcus

Vocal fold scars and sulcus cause severe vocal problems, but no optimal treatment has been established. In severe cases, in which the entire lamina propria is occupied by severe fibrosis and the vibratory function is completely lost, more powerful regeneration materials should be used, or the mucosa should be totally replaced. Amniotic membrane is a scaffold material that is rich in stem cells and growth factors and has a high regenerative effect. It can be clinically applied in many fields. Several studies have already investigated the effects of amniotic membrane on the vocal fold in animal studies. These studies have shown that the amniotic membrane is effective for vocal fold regeneration. In particular, our study showed that the transplantation of an amniotic membrane to the rabbit vocal fold induced high hyaluronic acid retention and the suppression of collagen deposition after three months without rejection. We hope that the use of the amniotic membrane for vocal fold regeneration will be broadly applied in the future.

Tissue Engineering as a Promising Treatment for Glottic Insufficiency: A Review on Biomolecules and Cell-Laden Hydrogel.

Glottic insufficiency is widespread in the elderly population and occurs as a result of secondary damage or systemic disease. Tissue engineering is a viable treatment for glottic insufficiency since it aims to restore damaged nerve tissue and revitalize aging muscle. After injection into the biological system, injectable biomaterial delivers cost- and time-effectiveness while acting as a protective shield for cells and biomolecules. This article focuses on injectable biomaterials that transport cells and biomolecules in regenerated tissue, particularly adipose, muscle, and nerve tissue. We propose Wharton's Jelly mesenchymal stem cells (WJMSCs), induced pluripotent stem cells (IP-SCs), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin growth factor-1 (IGF-1) and extracellular vesicle (EV) as potential cells and macromolecules to be included into biomaterials, with some particular testing to support them as a promising translational medicine for vocal fold regeneration.

Cryotherapy Modifies Extracellular Matrix Expression of Vocal Fold in Rat Models

Vocal fold (VF) scarring is the major cause of voice disorders. Cryotherapy is an effective anti-scarring therapy for skin lesions. The aim of this study was to explore the anti-scarring potential of cryotherapy in vocal folds. The extracellular matrix (ECM) mRNA expression of cryotherapy on normal VF tissue and the histologic results of cryotherapy on vocal fold healing were studied. Fifteen rats were introduced cryotherapy on the normal VF bilaterally and were harvested for real-time polymerase chain reaction (RT-PCR) analysis for collagen I, collagen III, TGFβ1, decorin, fibronectin and HAS1 at 1 day, 3 days and 7 days. Ten rats were unilaterally injured by stripping lamina propria and immediately treated with or without cryotherapy and were harvested at 2 months for histological and immunohistochemical analysis. Regenerative effect of cryotherapy was validated of ECM gene expression. Histological and immunohistochemical analysis showed significantly increased hyaluronan, decreased collagen, and increased decorin deposition in injury-cryotherapy cohort compared with injury control cohort and normal control cohort. Cryotherapy may provide an optimal environment for vocal fold tissue regeneration. The results of the present investigation suggest that cryotherapy has therapeutic potential in prevention and treatment of vocal fold scarring.

Scarless vocal fold regeneration by urine-derived stem cells and small intestinal submucosa hydrogel composites through enhancement of M2 macrophage Polarization, neovascularization and Re-epithelialization

Vocal fold (VF) scarring is a major etiology of voice disorders and a leading cause for permanent intractable dysphonia. To date, several approaches have been used to address VF injury and scarring, which included scar excision and biomaterial injection and/or implantation. Despite the considerable research progress made in this field, these have not been established as routine clinical procedures due to various restrictions. Tissue engineered scaffolds for delivering growth factors in vivo and implementation of simultaneous delivery of multi-potent cells may achieve better outcome. In this study, we have developed Urine-derived stem cells - Small intestinal submucosa hydrogel composites (USCs@SIS), which are injectable and capable of sustaining the survival and metabolism of the USCs in the scarred VF. The three-dimensional complex showed a robust ability of anti-inflammatory polarization, neovascularization, re-epithelialization and anti-fibrosis in a rabbit model for heat-injured VFs. In vitro experiment proved that the hydrogel composites could promote polarization of M2 macrophages, tube formation by human umbilical vein endothelial cells, proliferation and migration by human bronchial epithelial cells, and suppression of differentiation of human fetal lung fibroblasts and human primary fibroblast cells of VF. Application of the USCs@SIS thereby may overcome the substantial limitations of the current strategies for scarless VF regeneration.

The Effects of Amniotic Membrane Transplantation on Vocal Fold Regeneration.

Vocal fold (VF) scar and sulcus cause severe vocal problems, but optimal methods have not been established. Total replacement of the mucosa is required particularly for cases in which the whole lamina propria is occupied by severe fibrosis and vibratory function is totally lost. The amniotic membrane (AM) has been proven to have regenerative potential, as it contains stem cells and growth factors. The current study investigated the biocompatibility and effects of AM for regeneration of the VF mucosa. In vitro and in vivo studies. Vocal fold fibroblasts (VFFs) from 13 Sprague-Dawley rats were seeded on AM and subjected to histology and immunohistochemistry, and gene expressions in the VFFs on AM were examined in in vitro study. Twelve New Zealand White rabbits were used in in vivo study. VFs were stripped down and were reconstructed with AM. The regenerative effects were examined 3months later by histological examination. In vitro study indicated VFFs survived on AM and stained positively for Ki67, vimentin, and fibronectin. Gene expressions of Has1, Has2, and Hgf were significantly increased in the VFFs on AM compared with the other groups. The in vivo study indicated AM-transplanted VFs showed a significantly higher density of hyaluronic acid and lower density of collagen compared with sham VFs. The current preliminary study suggests biocompatibility and possible regenerative effects of AM for VFs. NA Laryngoscope, 132:2017-2025, 2022.

Synthetic Vocal Tracts - A Review

Synthetic vocal tracts are gadgets powered by a computer system capable of translating the brain activity into synthesized speech, by decoding the movements of muscles involved in vocalization, using advanced computer programming. New standardized method for the development of synthetic vocal tract is the 3D vocal tract model with the binary conversion. Few of the typical features considered while creating the synthetic vocal tract are fundamental frequency, perturbation measure, jitter and change in pitch. The tissue engineered larynx is the promising development in synthetic vocal tract treatment in case of patients with vocal fold repair and regeneration. The future of this interesting technology lies in using high speed video endoscopy based synthetic vocal cords. The review was done based on the articles obtained from various platforms. This review article elaborates about the principle of synthetic vocal tract. Quality of the article used was assessed using a quality assessment tool and graded as strong, moderate and weak. The aim of this study is to understand the concept of synthetic vocal tract and its significance. Synthetic vocal tract is a recently established biomedical tool that has come as a boon in treating patients with severe disabilities. Speech synthesis is evolving as a viable solution as more research is being carried out on this. To understand the full significance of this crucial technique, more research has to be carried out on this field of biomedical engineering and viable solutions need to be developed, so that this novel technique is fully utilized.

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

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. 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. 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. 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. NA Laryngoscope, 131:1828-1834, 2021.

Vocal Fold Regeneration by Growth Factors—Update—

Vocal Fold Regeneration by Growth Factors—Update—

Carbon nanotube composite hydrogels for vocal fold tissue engineering: Biocompatibility, rheology, and porosity

Porous composite hydrogels were prepared using glycol chitosan as the matrix, glyoxal as the chemical crosslinker, and carbon nanotubes (CNTs) as the fibers. Both carboxylic and hydroxylic functionalized CNTs were used. The homogeneity of CNTs dispersion was evaluated using scanning electron microscopy. Human vocal fold fibroblasts were cultured and encapsulated in the composite hydrogels with different CNT concentrations to quantify cell viability. Rheological tests were performed to determine the gelation time and the storage modulus as a function of CNT concentration. The gelation time tended to decrease for low concentrations and increase at higher concentrations, reaching a local minimum value. The storage modulus obeyed different trends depending on the functional group. The porosity of the hydrogels was found to increase by 120% when higher concentrations of carboxylic CNTs were used. A high porosity may promote cell adhesion, migration, and recruitment from the surrounding native tissue, which will be investigated in a future work aiming at applying this injectable biomaterial for vocal fold tissue regeneration.

Regeneration of Paralyzed Vocal Fold by the Injection of Plasmid DNA Complex-Loaded Hydrogel Bulking Agent.

Various growth factor delivery systems were used in the treatment of glottal insufficiency; however, relatively little attention has been paid to a gene delivery system for aspects of active vocal fold (VF) regeneration. Herein, we present a plasmid DNA (pDNA; bFGF gene encoding) complex-loaded alginate (ALG)/hyaluronic acid (HA) mixture hydrogel dispersed with polycaprolactone (PCL) microspheres that can enhance simultaneous regeneration of VF muscle and lamina propria, as well as have a bulking effect on atrophied VF. We have demonstrated long-term efficacy of bFGF synthesized from pDNA complex-transfected cells in vitro. PCL microspheres-dispersed ALG/HA hydrogel (with or without pDNA complex loading) are injected into rabbit VFs with recurrent laryngeal nerve denervation. The PCL microspheres dispersed in the hydrogel bulking agents remain stable at the applied site, leading to constant medialization of the paralyzed VF without significant initial volume loss even after 24 weeks. A regenerative effect for collagen deposition and HA synthesis around the injected site, which are major components of VF tissue, is well confirmed in the pDNA-complex-loaded hydrogel group. Moreover, the compensation of atrophied VFs also leads to the contact of bilateral VF and the remarkable recovery of voice function in the pDNA-complex-loaded group. Based on the results, pDNA (bFGF encoding) complex-loaded hydrogel dispersed with PCL microspheres may be employed as a bioactive bulking agent for the treatment of glottal insufficiency.

The Ability of Conditioned Media From Stem Cells to Repair Vocal Fold Injuries.

This study investigated the ability of hypoxia-induced 25-fold concentrated conditioned media (hCM) from human nasal inferior turbinate-derived mesenchymal stem cells (hTMSC) to repair injured vocal folds during the early phase of the wound-healing process. The vocal fold was injured in Sprague-Dawley rats. Next, hCM from hTMSC (the hCM group) or hTMSC (the hTMSC group) were injected into the injured vocal folds. As a control, saline (the phosphate-buffered saline group) or 25-fold concentrated media (the media group) was injected in the same manner. The vocal folds were harvested for quantitative real-time polymerase chain reaction (PCR) at 1 week and 2 weeks after injury. Histologic evaluation was performed at 3 weeks postinjury. In the hCM group at 1 week after injury, PCR showed that the genes encoding hyaluronan synthase (HAS), HAS 1, and HAS 2 were significantly upregulated compared to the media and normal groups. The gene encoding procollagen III was significantly downregulated compared to the media group. Nearly identical results were obtained for the hTMSC group at 1 week after injury. Histological examination showed that the hCM group was similar to or better than the hTMSC group in collagen deposition and hyaluronic acid production. The injection of hCM into injured vocal folds produced antifibrotic effects in the early phase of wound healing. These effects were equivalent to those produced by the injection of hTMSC. These results provide a foundation for the future clinical use of hCM for vocal fold regeneration. NA Laryngoscope, 129:1867-1875, 2019.

Vocal Fold Regeneration: Current Review

Vocal Fold Regeneration: Current Review

Activation of the Wnt/β-Catenin Pathway by an Inflammatory Microenvironment Affects the Myogenic Differentiation Capacity of Human Laryngeal Mucosa Mesenchymal Stromal Cells.

Various microenvironments influence the multiple differentiation potential of mesenchymal stromal cells. For example, inflammatory microenvironment can suppress the myogenic differentiation capability of laryngeal mucosa mesenchymal stromal cells (LM-MSCs). The present study therefore sought to identify the underlying molecular mechanisms regulating these processes. We isolated a novel population of MSCs, LM-MSCs, from the laryngeal mucosa tissues. The cells were cultured in osteogenic, adipogenic, and myogenic differentiation media in the presence or absence of interleukin-1β and tumor necrosis factor α (to simulate inflammatory microenvironment). The expression of active β-catenin, p-GSK3β, and GSK3β were detected by western blot and real-time polymerase chain reaction. The myogenic differentiation of LM-MSCs in inflammatory microenvironment and the regulation by Dickkopf-1 (DKK1) were tested both in vivo and in vitro. Inflammatory microenvironment could suppress the osteogenesis, adipogenesis, and myogenesis of LM-MSCs. The Wnt/β-catenin signaling pathway was activated during myogenesis in inflammatory microenvironment. The suppressed myogenic differentiation capability of LM-MSCs in inflammatory microenvironment was reversed by DKK1. By regulating the Wnt/β-catenin signaling pathway, DKK1 can improve the myogenic differentiation of LM-MSCs in inflammatory microenvironment. Thus, the results of this study may help improve the efficacy of LM-MSCs injection therapy for vocal fold regeneration.

Retention of Human-Induced Pluripotent Stem Cells (hiPS) With Injectable HA Hydrogels for Vocal Fold Engineering.

One prospective treatment option for vocal fold scarring is regeneration with an engineered scaffold containing induced pluripotent stem cells (iPS). In the present study, we investigated the feasibility of utilizing an injectable hyaluronic acid (HA) scaffold encapsulated with human-iPS cell (hiPS) for regeneration of vocal folds. Thirty athymic nude rats underwent unilateral vocal fold injury. Contralateral vocal folds served as uninjured controls. Hyaluronic acid hydrogel scaffold, HA hydrogel scaffold containing hiPS, and HA hydrogel scaffold containing hiPS with epidermal growth factor (EGF) were injected in both vocal folds immediately after surgery. One and 2 weeks after injection, larynges were excised for histology, immunohistochemistry, and fluorescence in situ hybridization (FISH). Presence of HA hydrogel was confirmed in vocal folds 1 and 2 weeks post injection. The FISH analysis confirmed the presence and viability of hiPS in the injected vocal folds. Histological results demonstrated that vocal folds injected with HA hydrogel scaffold containing EGF demonstrated less fibrosis than those with HA hydrogel only. Human-iPS survived in injured rat vocal folds. The HA hydrogel with hiPS and EGF ameliorated the fibrotic response. Additional work is necessary to optimize hiPS differentiation and further confirm the safety of hiPS for clinical applications.

Controlled Release of Hepatocyte Growth Factor from MPEG-b-(PCL-ran-PLLA) Diblock Copolymer for Improved Vocal Fold Regeneration.

An in situ-forming gel system comprised of diblock copolymer formed from polyethylene glycol (PEG) and polycaprolactone (PCL) {MPEG-b-(PCL-ran-PLLA)} could be used in controlled drug delivery for tissue remodeling. The purpose of this study is to demonstrate favorable vocal folds (VF) regeneration by using MPEG-b-(PCL-ran-PLLA) diblock copolymers (C97L3; CL/LA ratio 97:3) incorporating hepatocyte growth factor (HGF). Gradual release of HGF from C97L3 is detected and biochemical properties of released HGF are maintained. A scar is made with microscissors on both VFs in 32 rabbits, followed by injection of HGF-only, C97L3-only, or HGF-C97L3 composite gel in the left side VF, while the right side VF is left untreated. In vivo fluorescence live imaging system demonstrates that C97L3 enables the sustained release of injected HGF in the scarred VF for 12 weeks. The histological analysis shows increased glycosaminoglycan including hyaluronic acid accumulation and decreased collagen deposition. Videokymographic analysis shows more favorable vibrations of HGF-C97L3 treated VF mucosa, compared to other treatment groups. In conclusion, the controlled HGF release helps to regulate extracellular matrix synthesis, and leads to the eventual functional improvement of the scarred VF.

Tissue engineering-based therapeutic strategies for vocal fold repair and regeneration.

Vocal folds are soft laryngeal connective tissues with distinct layered structures and complex multicomponent matrix compositions that endow phonatory and respiratory functions. This delicate tissue is easily damaged by various environmental factors and pathological conditions, altering vocal biomechanics and causing debilitating vocal disorders that detrimentally affect the daily lives of suffering individuals. Modern techniques and advanced knowledge of regenerative medicine have led to a deeper understanding of the microstructure, microphysiology, and micropathophysiology of vocal fold tissues. State-of-the-art materials ranging from extracecullar-matrix (ECM)-derived biomaterials to synthetic polymer scaffolds have been proposed for the prevention and treatment of voice disorders including vocal fold scarring and fibrosis. This review intends to provide a thorough overview of current achievements in the field of vocal fold tissue engineering, including the fabrication of injectable biomaterials to mimic invitro cell microenvironments, novel designs of bioreactors that capture invivo tissue biomechanics, and establishment of various animal models to characterize the invivo biocompatibility of these materials. The combination of polymeric scaffolds, cell transplantation, biomechanical stimulation, and delivery of antifibrotic growth factors will lead to successful restoration of functional vocal folds and improved vocal recovery in animal models, facilitating the application of these materials and related methodologies in clinical practice.