Vocal Fold Regeneration Research Articles

Adipose stromal cells improve healing of vocal fold scar: Morphological and functional evidences.

Adipose derived stromal cells (ASCs) are abundant and easy to prepare. Such cells may be useful for treating severe vocal disturbance caused by acute vocal fold scars. Prospective animal experiments with controls. Twenty New-Zealand white rabbits were used in the present study. We evaluated vocal fold healing, with or without injection of autologous ASCs, after acute scarring. A defined lesion was created and the ASCs were immediately injected. Vocal fold regeneration was evaluated histomorphometrically and via viscoelastic analysis using an electrodynamic shaker. Six weeks after ASC injection, vocal folds exhibited significantly less inflammation than control folds (P < 0.005). In addition, hypertrophy of the lamina propria and fibrosis were significantly reduced upon ASC injection (P < 0.02). The decrease in viscoelastic parameters was less important in the ASC injected group compared to the noninjected group (P = 0.08). Injection of autologous ASCs improved vocal fold healing in our preclinical model. Further studies are needed, but this method may be useful in humans. NA. Laryngoscope, 126:E278-E285, 2016.

Outcome of regenerative therapy for age-related vocal fold atrophy with basic fibroblast growth factor.

Age-related vocal fold atrophy has become a significant voice disorder as the elderly population grows. However, several therapeutic challenges have limited attempts to improve voice quality. We reported that basic fibroblast growth factor (bFGF) stimulates fibroblasts to produce extracellular matrices such as hyaluronic acid in the lamina propria, leading to a regeneration of pliable vocal folds in animal models. The aim of this study was to determine the efficacy of bFGF for the treatment of age-related vocal fold atrophy. Prospective study. Six patients with age-related vocal fold atrophy underwent injection of bFGF in their vocal folds. Vocal outcomes and stroboscopic examinations were evaluated 1, 3, and 6 months after the injection. The outcome measures included the Voice Handicap Index-10 (VHI-10), GRBAS (grade, roughness, breathiness, asthenia, strain) scale, maximum phonation time (MPT), the amplitude perturbation quotient (APQ), and the pitch perturbation quotient (PPQ). The VHI-10 was significantly improved 6 months after bFGF injection. The GRBAS scale, MPT, APQ, and PPQ were also improved. Stroboscopic examinations showed significant improvement of glottic closure and better mucosal wave. This is the first study to evaluate the regenerative effects of bFGF injection for the treatment of age-related vocal fold atrophy using the VHI-10. Injection of bFGF significantly improved VHI-10 scores and glottal insufficiency for at least 6 months. 4. Laryngoscope, 126:1844-1848, 2016.

Stem cell approaches for vocal fold regeneration.

Current interventions in the management of vocal fold (VF) dysfunction focus on conservative and surgical approaches. However, the complex structure and precise biomechanical properties of the human VF mean that these strategies have their limitations in clinical practice and in some cases offer inadequate levels of success. Regenerative medicine is an exciting development in this field and has the potential to further enhance VF recovery beyond conventional treatments. Our aim in this review is to discuss advances in the field of regenerative medicine; that is, advances in the process of replacing, engineering, or regenerating the VF through utilization of stem cells, with the intention of restoring normal VF structure and function. English literature (1946-2015) review. We conducted a systematic review of MEDLINE for cases and studies of VF tissue engineering utilizing stem cells. The three main approaches by which regenerative medicine is currently applied to VF regeneration include cell therapy, scaffold development, and utilization of growth factors. Exciting advances have been made in stem cell biology in recent years, including use of induced pluripotent stem cells. We expect such advances to be translated into the field in the forthcoming years. Laryngoscope, 126:1865-1870, 2016.

Adipose-Derived Mesenchymal Stem Cells in the Regeneration of Vocal Folds: A Study on a Chronic Vocal Fold Scar.

Background. The aim of the study was to assess the histological effects of autologous infusion of adipose-derived stem cells (ADSC) on a chronic vocal fold scar in a rabbit model as compared to an untreated scar as well as in injection of hyaluronic acid. Study Design. Animal experiment. Method. We used 74 New Zealand rabbits. Sixteen of them were used as control/normal group. We created a bilateral vocal fold wound in the remaining 58 rabbits. After 18 months we separated our population into three groups. The first group served as control/scarred group. The second one was injected with hyaluronic acid in the vocal folds, and the third received an autologous adipose-derived stem cell infusion in the scarred vocal folds (ADSC group). We measured the variation of thickness of the lamina propria of the vocal folds and analyzed histopathologic changes in each group after three months. Results. The thickness of the lamina propria was significantly reduced in the group that received the ADSC injection, as compared to the normal/scarred group. The collagen deposition, the hyaluronic acid, the elastin levels, and the organization of elastic fibers tend to return to normal after the injection of ADSC. Conclusions. Autologous injection of adipose-derived stem cells on a vocal fold chronic scar enhanced the healing of the vocal folds and the reduction of the scar tissue, even when compared to other treatments.

Cogels of Hyaluronic Acid and Acellular Matrix for Cultivation of Adipose-Derived Stem Cells: Potential Application for Vocal Fold Tissue Engineering.

Stem cells based tissue engineering has been one of the potential promising therapies in the research on the repair of tissue diseases including the vocal fold. Decellularized extracellular matrix (DCM) as a promising scaffold has be used widely in tissue engineering; however, it remained to be an important issue in vocal fold regeneration. Here, we applied the hydrogels (hyaluronic acid [HA], HA-collagen [HA-Col], and HA-DCM) to determine the effects of hydrogel on the growth and differentiation of human adipose-derived stem cells (hADSCs) into superficial lamina propria fibroblasts. hADSCs were isolated and characterized by fluorescence-activated cell sorting. The results indicated that HA-DCM hydrogel enhanced cell proliferation and prolonged cell morphology significantly compared to HA and HA-Col hydrogel. Importantly, the differentiation of hADSCs into fibroblasts was also promoted by cogels of HA-Col and HA-DCM significantly. The differentiation of hADSCs towards superficial lamina propria fibroblasts was accelerated by the secretion of HGF, IL-8, and VEGF, the decorin and elastin expression, and the synthesis of chondroitin sulfate significantly. Therefore, the cogel of HA-DCM hydrogel was shown to be outstanding in apparent stimulation of hADSCs proliferation and differentiation to vocal fold fibroblasts through secretion of important growth factors and synthesis of extracellular matrix.

Identification of distinct layers within the stratified squamous epithelium of the adult human true vocal fold.

A precise molecular schema for classifying the different cell types of the normal human vocal fold epithelium is lacking. We hypothesize that the true vocal fold epithelium has a cellular architecture and organization similar to that of other stratified squamous epithelia including the skin, cornea, oral mucosa, and esophagus. In analogy to disorders of the skin and gastrointestinal tract, a molecular definition of the normal cell types within the human vocal fold epithelium and a description of their geometric relationships should serve as a foundation for characterizing cellular changes associated with metaplasia, dysplasia, and cancer. Qualitative study with adult human larynges. Histologic sections of normal human laryngeal tissue were analyzed for morphology (hematoxylin and eosin) and immunohistochemical protein expression profile, including cytokeratins (CK13 and CK14), cornified envelope proteins (involucrin), basal cells (NGFR/p75), and proliferation markers (Ki67). We demonstrated that three distinct cell strata with unique marker profiles are present within the stratified squamous epithelium of the true vocal fold. We used these definitions to establish that cell proliferation is restricted to certain cell types and layers within the epithelium. These distinct cell types are reproducible across five normal adult larynges. We have established that three layers of cells are present within the normal adult stratified squamous epithelium of the true vocal fold. Furthermore, replicating cell populations are largely restricted to the parabasal strata within the epithelium. This delineation of distinct cell populations will facilitate future studies of vocal fold regeneration and cancer. N/A.

ヒトiPS細胞を用いた声帯の組織再生

ヒトiPS細胞を用いた声帯の組織再生

培養口腔粘膜を用いた声帯粘膜再生の試み

培養口腔粘膜を用いた声帯粘膜再生の試み

Adipose-derived stem cells versus bone marrow-derived stem cells for vocal fold regeneration.

Vocal fold scarring presents therapeutic challenges. Recently, cell therapy with mesenchymal stromal cells has become a promising approach. The aim of this study was to compare the therapeutic potential of adipose-derived stem cells (ASC) with bone marrow-derived stem cells (BMSC) for vocal fold regeneration. Prospective animal experiments with controls. The vocal folds of Sprague-Dawley rats were unilaterally injured. Two months after injury, rats were treated with a local injection of ASC (ASC group), BMSC (BMSC group), or saline (sham-treated group). The GFP-labeled ASC and BMSC were extracted from CAG-EGFP rats. Larynges were harvested for histological and immunohistochemical examinations 1 and 3 months posttransplantation and for quantitative real-time polymerase chain reaction (PCR) 1 month posttransplantation. After 1 month, no surviving cells from the transplant were detected. Histological examination showed significantly increased hyaluronic acid (HA) and decreased dense collagen deposition in both ASC and BMSC groups compared to shams 1 and 3 months after treatment. Real-time PCR revealed that hyaluronan synthase 1 (Has1) and Has2 were upregulated in only the ASC group compared with the sham-treated group. Fibroblast growth factor 2 (basic) (Fgf2), hepatocyte growth factor (Hgf) and Has3 were upregulated in both cell transplantation groups. ASC seemed to upregulate Hgf more than did BMSC. The regenerative effects of ASC and BMSC transplantation were found to be similar for the restoration. It is suggested that ASC might have more potential because of better recovery of HA, a superior antifibrotic effect, and the upregulation of Hgf. N/A.

Transient dynamic mechanical properties of resilin-based elastomeric hydrogels.

The outstanding high-frequency properties of emerging resilin-like polypeptides (RLPs) have motivated their development for vocal fold tissue regeneration and other applications. Recombinant RLP hydrogels show efficient gelation, tunable mechanical properties, and display excellent extensibility, but little has been reported about their transient mechanical properties. In this manuscript, we describe the transient mechanical behavior of new RLP hydrogels investigated via both sinusoidal oscillatory shear deformation and uniaxial tensile testing. Oscillatory stress relaxation and creep experiments confirm that RLP-based hydrogels display significantly reduced stress relaxation and improved strain recovery compared to PEG-based control hydrogels. Uniaxial tensile testing confirms the negligible hysteresis, reversible elasticity and superior resilience (up to 98%) of hydrated RLP hydrogels, with Young's modulus values that compare favorably with those previously reported for resilin and that mimic the tensile properties of the vocal fold ligament at low strain (<15%). These studies expand our understanding of the properties of these RLP materials under a variety of conditions, and confirm the unique applicability, for mechanically demanding tissue engineering applications, of a range of RLP hydrogels.

Characterization of scarred vocal fold regeneration after the intervention of extracellular matrix and human amniotic epithelial cells transplanting

To investigate the treatment for scarred vocal folds by transplanting human amniotic epithelial cells (hAECs)and injecting collagenase as well as hyaluronic acid (HA) for the intervention of the extracellular matrix(EMC), to observe the growth, distribution of hAECs and to assess the abilities of them for scarred vocal fold regeneration. The lamina propria was injured by localized resection in thirty-eight vocal folds of twenty rabbits. hAECs were isolated from human amnion and marked by Lenti-GFP. After the formation of vocal fold scarring, hAECs were transplanted into ten vocal folds, collagenase and HA were injected into ten vocal folds, all three were injected into ten vocal folds, none were injected into eight vocal folds, and two normal vocal folds were used as control. At 1 month and 2 months after the transplanting, the survival, the distribution and the cytoactive of hAECs were examined by immunofluorescence method. Meanwhile, at 1 month, 2 months, 3 months and 6 months after the operation, HE staining was performed for histopathological research, Masson trichrome staining and immunohistochemical staining were used for collagen and fibronectin respectively. After implanted into the scarred vocal folds, hAECs could survive in vocal fold lamina propria for two months. The immunofluorescence analysis showed the cytoactive of hAECs.Six months postoperatively, compared with that in the normal vocal folds, collagen in the untreated scarred vocal folds more increased and disorderly distributed; the changes in other three groups were between the two groups above, but the group injected with all of hAECs, collagenase and HA was better than other two groups. Besides, the mean density of fibronectin in the scarred untreated control group was more significantly increased than that in the normal vocal folds; the changes in other three groups were between the two groups above, but the group injected with all of hAECs, collagenase and HA was better than other two groups. The transplanting of hAECs and the interventions of EMC by injecting collagenase as well as HA have better abilities in rabbit scarred vocal fold reparation and regeneration by promoting ECM secretion, rational distribution and part ordering arrangement.

Small intestine submucosa and mesenchymal stem cells composite gel for scarless vocal fold regeneration

The purpose of this study is to demonstrate scarless vocal fold (VF) regeneration by using a composite gel composed of small intestine submucosa (SIS) and mesenchymal stem cells (MSCs). A scar was made with an electrocoagulator on both VFs in 24 rabbits, followed by injection of either MSCs, SIS, or MSCs-SIS composite gel in the right side VF, while the left side VF was left untreated. VF scars were evaluated with in vivo fluorescence live imaging system (IFLIS), endoscopy, histology, and videokymography (VKG) after eight weeks. IFLIS demonstrated that SIS enabled the MSCs to survive and be engrafted in the VF. The histological analysis showed increased hyaluronic acid accumulation and controlled collagen deposition by MSCs-SIS composite gel. VKG analysis showed more favorable vibrations of MSCs-SIS injected VF, compared to other treatment group. In conclusion, the injectable SIS supplied a niche for the MSCs to stably settle down in scarred VFs and helped to regulate ECM synthesis. The ECM remodeling underwent by the surviving MSCs eventually led to the functional improvement of the VF. The results of the present investigation suggest that SIS-MSCs composite gel is a plausible biomaterial for prolonged survival of MSCs in VFs and promotes scarless VF healing.

Favorable Vocal Fold Wound Healing Induced by Platelet-Rich Plasma Injection

ObjectivesTo introduce a new injection material for vocal fold diseases, which could be readily translated to clinical practice, we investigated the effectiveness of platelet-rich plasma (PRP) injection on the injured vocal fold in terms of histological recovery.MethodsBlood samples were drawn from New Zealand White rabbits and PRP was isolated through centrifugation and separation of the samples. Using a CO2 laser, we made a linear wound in the 24 vocal fold sides of 12 rabbits and injected each wound with PRP on one vocal fold side and normal saline (NS) on the other. Morphologic analyses were conducted at 2, 4, and 12 weeks after injection, and inflammatory response, collagen deposit, and changes in growth factors were assessed using H&E and masson trichrome (MT) staining and western blot assay.ResultsPRP was prepared in approximately 40 minutes. The mean platelet concentration was 1,315,000 platelets/mm3. In morphological analyses, decreased granulation was observed in the PRP-injected vocal folds (P<0.05). However, the irregular surface and atrophic change were not difference. Histological findings revealed significant inflammation and collagen deposition in NS-injected vocal folds, whereas the PRP-injected vocal folds exhibited less (P<0.05). However, the inflammatory reaction and fibrosis were not difference. In western blot assay, increased amounts of growth factors were observed in PRP-injected vocal folds.ConclusionInjection of injured rabbit vocal folds with PRP led to improved wound healing and fewer signs of scarring as demonstrated by decreased inflammation and collagen deposition. The increased vocal fold regeneration may be due to the growth factors associated with PRP.

Bone Marrow-Derived Clonal Mesenchymal Stem Cells as a Source of Cell Therapy for Promoting Vocal Fold Wound Healing

We investigated whether mouse bone marrow-derived clonal mesenchymal stem cells (BM-cMSCs) could promote vocal fold (VF) wound healing by using a xenograft animal model. Homogeneous BM-cMSCs isolated by a subfractionation culturing method from the bone marrow aspirates of green fluorescent protein transgenic mice were injected into the VFs of rabbits immediately after direct mechanical injury. Macroscopic, biomechanical (rheometric), histologic, immunohistochemical, and transcriptional evaluations were performed on the scarred VFs 1 to 3 months after injury. Engraftment of the implanted BM-cMSCs was determined by detection of green fluorescent protein cells in the recipient VF by confocal microscopy. The BM-cMSC-treated VFs showed improved morphological properties and viscoelasticity as compared to control VFs injected with phosphate-buffered saline solution. Histologic and immunohistochemical evaluations showed less excessive collagen deposition and increased density of glycosaminoglycans in the BM-cMSC-treated VFs as compared to the control VFs at 3 months after injury (p = 0.003 and p = 0.037, respectively). BM-cMSC transplantation led to a significant attenuation of fibronectin (p = 0.036) and transforming growth factor beta1 (p = 0.042) messenger RNA expression at 1 month after injury. Green fluorescent protein-expressing BM-cMSCs engrafted in recipient VFs were found at 1 month after implantation. BM-cMSCs appeared to survive in the injured xenogeneic VFs after transplantation for up to 1 month and favorably enhanced the wound healing of VFs after injury. We conclude that BM-cMSCs are a possible source of cell therapy for vocal fold regeneration.

Regulation of Wound Healing by Granulocyte-Macrophage Colony-Stimulating Factor after Vocal Fold Injury

ObjectivesVocal fold (VF) scarring remains a therapeutic challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitates epithelial wound healing, and recently, growth factor therapy has been applied to promote tissue repair. This study was undertaken to investigate the effect of GM-CSF on VF wound healing in vivo and in vitro.MethodsVF scarring was induced in New Zealand white rabbits by direct injury. Immediately thereafter, either GM-CSF or PBS was injected into the VFs of rabbits. Endoscopic, histopathological, immunohistochemical, and biomechanical evaluations of VFs were performed at 3 months post-injury. Human vocal fold fibroblasts (hVFFs) were cultured with GM-CSF. Production of type I and III collagen was examined immunocytochemically, and the synthesis of elastin and hyaluronic acids was evaluated by ELISA. The mRNA levels of genes related to ECM components and ECM production-related growth factors, such as HGF and TGF-ß1, were examined by real time RT-PCR.ResultsThe GM-CSF-treated VFs showed reduced collagen deposition in comparison to the PBS-injected controls (P<0.05). Immunohistochemical staining revealed lower amounts of type I collagen and fibronectin in the GM-CSF-treated VFs (P<0.05 and P<0.01, respectively). Viscous and elastic shear moduli of VF samples were significantly lower in the GM-CSF group than in the PBS-injected group (P<0.001 and P<0.01, respectively). Mucosal waves in the GM-CSF group showed significant improvement when compared to the PBS group (P = 0.0446). GM-CSF inhibited TGF-β1-induced collagen synthesis by hVFFs (P<0.05) and the production of hyaluronic acids increased at 72 hours post-treatment (P<0.05). The expressions of HAS-2, tropoelastin, MMP-1, HGF, and c-Met mRNA were significantly increased by GM-CSF, although at different time points (P<0.05).ConclusionThe present study shows that GM-CSF offers therapeutic potential for the remodeling of VF wounds and the promotion of VF regeneration.

Micronized acellular dermal matrix as an efficient expansion substrate and delivery vehicle of adipose‐derived stem cells for vocal fold regeneration

Cell therapy has been shown to prevent vocal fold scarring and atrophy. However, problems that include the expansion of large numbers of cells in vitro and the poor survival of transplanted cells in vivo must be solved. The aim of this study was to use micronized acellular dermal matrix (MADM) as an expansion substrate of rabbit allogeneic adipose-derived stem cells (ADSCs) and to apply the combination of the matrix and cells, ADSC-MADM, to vocal fold regeneration. Animal experiment. The proliferation of ADSCs that were cultured on the MADM was evaluated using 3-(4,5-dimethylthizazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium, and a rabbit acute vocal fold injury model was established by laser injury. Eighteen New Zealand white rabbits were randomly divided into three groups, which were injected with ADSC-MADM, ADSCs, and MADM, respectively. Morphological analysis was performed by laryngoscope, and histological analyses were indicated by hematoxylin and eosin staining, van Gieson staining, and immunofluorescence. Additionally, the in vivo survival of the ADSCs was determined by CM-Dil cell labeling. When compared with a two-dimensional culture, the MADM significantly promoted proliferation of ADSCs. Morphological and histological analyses indicated that, when compared to only using of MAMD or the nontreatment sample, the use of ADSC-MADM or only using ADSCs successfully prevent scarring and atrophy. Moreover, ADSC-MADM exhibited a better therapeutic effect than when only using ADSCs, which was probably due to the MADM significantly enhancing the survival of transplanted ADSCs. MADM could be used as an efficient expansion substrate and delivery vehicle for ADSCs in vocal fold regeneration.

Therapeutic potential of gel-based injectables for vocal fold regeneration

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 efficacious treatment for vocal fold scarring could be clinically attainable within several years. Fibroblast phenotype and gene expression are mediated by the three-dimensional mechanical and chemical microenvironment at an injury site. Thus, therapeutic approaches need to coordinate spatial and temporal aspects of the wound healing response in an injured vocal tissue to achieve an optimal clinical outcome. Successful gel-based injectables for vocal fold scarring will require a keen understanding of how the native inflammatory response sets into motion the later extracellular matrix remodeling, which in turn will determine the ultimate biomechanical properties of the tissue. We present an overview of the challenges associated with this translation as well as the proposed gel-based injectable solutions.

Clinical trial of regeneration of aged vocal folds with growth factor therapy

Aged vocal folds are characterized by atrophy of the mucosa, which causes mucosal wave deficiency and glottal insufficiency. This clinical trial examined the regenerative effects and safety considerations of basic fibroblast growth factor (bFGF) on restoration of aged vocal folds. Institutional review board-approved human clinical trial. Ten patients (6 men and 4 women; mean age, 70.1 years) were recruited in the trial. Ten micrograms of bFGF was injected into each treated vocal fold. Injection was performed unilaterally or bilaterally according to each patient's need and repeated up to seven times if necessary. Patient follow-up continued for at least 6 months. The effectiveness of the treatment was assessed by stroboscopic, acoustic, and aerodynamic measurements. All patients showed improvement of voice. Significant improvements in maximum phonation time, mean flow rate, jitter, shimmer, and noise-to-harmonic ratio lasted for at least 1 year. No allergic or long-term adverse effects were noted. This clinical trial suggests that bFGF may be effective and safe as a regenerative agent for aged vocal folds.

Controlling the Fibroblastic Differentiation of Mesenchymal Stem Cells Via the Combination of Fibrous Scaffolds and Connective Tissue Growth Factor

Controlled differentiation of multi-potent mesenchymal stem cells (MSCs) into vocal fold-specific, fibroblast-like cells in vitro is an attractive strategy for vocal fold repair and regeneration. The goal of the current study was to define experimental parameters that can be used to control the initial fibroblastic differentiation of MSCs in vitro. To this end, connective tissue growth factor (CTGF) and micro-structured, fibrous scaffolds based on poly(glycerol sebacate) (PGS) and poly(ɛ-caprolactone) (PCL) were used to create a three-dimensional, connective tissue-like microenvironment. MSCs readily attached to and elongated along the microfibers, adopting a spindle-shaped morphology during the initial 3 days of preculture in an MSC maintenance medium. The cell-laden scaffolds were subsequently cultivated in a conditioned medium containing CTGF and ascorbic acids for up to 21 days. Cell morphology, proliferation, and differentiation were analyzed collectively by quantitative PCR analyses, and biochemical and immunocytochemical assays. F-actin staining showed that MSCs maintained their fibroblastic morphology during the 3 weeks of culture. The addition of CTGF to the constructs resulted in an enhanced cell proliferation, elevated expression of fibroblast-specific protein-1, and decreased expression of mesenchymal surface epitopes without markedly triggering chondrogenesis, osteogenesis, adipogenesis, or apoptosis. At the mRNA level, CTGF supplement resulted in a decreased expression of collagen I and tissue inhibitor of metalloproteinase 1, but an increased expression of decorin and hyaluronic acid synthesase 3. At the protein level, collagen I, collagen III, sulfated glycosaminoglycan, and elastin productivity was higher in the conditioned PGS-PCL culture than in the normal culture. These findings collectively demonstrate that the fibrous mesh, when combined with defined biochemical cues, is capable of fostering MSC fibroblastic differentiation in vitro.

Adipose-Derived Mesenchymal Stem Cells in Collagen—Hyaluronic Acid Gel Composite Scaffolds for Vocal Fold Regeneration

We sought to characterize the changes in the extracellular matrix (ECM) of the lamina propria following the implantation of autologous adipose-derived mesenchymal stem cells (ADSCs) in composite scaffolds in a rabbit vocal fold wound model. The ADSCs were co-cultured with collagen or hyaluronic acid gel. Each vocal fold was injured by localized resection and injected with ADSC complexes, ADSCs, or scaffolds or left untreated. From 15 days to 12 months, the vocal fold shape and the characteristics of the ECM components were analyzed. With implantation of the ADSC complexes, the collagen content was significantly increased and had a disorderly distribution at 3 months. Subsequently, it began to decrease and reached close to normal by 12 months. The hyaluronic acid content was increased at 40 days, but it was reduced to normal levels and was limited to the superficial and middle layers of the lamina propria over the following 12 months. Fibronectin continued to be scattered in the lamina propria, at peak levels at 40 days, and then decreased over the following 12 months to reach near-normal levels. At 12 months, the vocal folds had a normal surface. ADSC complexes can play a facilitatory role in vocal fold regeneration, regulating the generation and orderly distribution of ECM.