Chemically or surgically induced thyroid dysfunction altered gut microbiota in rat models.

Thyroid hormones are essential for the regulation of energy homeostasis and metabolic processes. However, the relationship between thyroid function and host gut microbial communities is not properly understood. To determine whether and how gut microbiota is associated with thyroid function, metagenomics analysis of the bacterial population in fecal samples of rat models of hyperthyroidism (induced by levothyroxine) and hypothyroidism (induced by propylthiouracil or thyroidectomy) was conducted through 16S rRNA gene sequencing. Our results revealed that all thyroid dysfunction models were definitely established and gut microbial composition varied according to different thyroid functional status. The relative abundance of Ruminococcus was significantly higher in the hyperthyroidism group (HE) vs both the normal and hypothyroidism groups (HO) while S24-7 was significantly higher in the HO group. The population of Prevotellaceae and Prevotella were significantly lower in the HO group vs the normal. Firmicutes and Oscillospira were significantly higher in the SHO (surgery-induced hypothyroidism) group, while Prevotellaceae and Prevotella showed lower abundance in the SHO group than the SHAM group. Present results suggest that thyroid functions may have the potential to influence the profile of gut microbiota and could be used as foundation to investigate interaction mechanism between thyroid and gut microbiome.

Treatment of chemically induced oral ulcer using adipose-derived mesenchymal stem cell sheet.

BACKGROUND: This study investigated the effects of mesenchymal stem cell (MSC) sheet transplantation on healing of chemically induced oral ulceration in a rabbit animal model. METHODS: Oral mucosal ulcers were induced by topical application of filter paper soaked with 70% acetic acid to the anterior gingiva and buccal mucosa of 12 New Zealand white rabbits. The animals were randomly assigned to two groups: with (treatment group, n = 6) or without (control group, n = 6) cell sheets applied to ulcers. Gross findings were sequentially evaluated, and histologic examination was performed on day 7. RESULTS: Based on gross inspection, ulceration resolved before day 5 in the treatment group; however, in the control group, healing was incomplete on day 7. In the treatment group, the total area of the ulcer decreased significantly from day 2 to day 5 (P < 0.001) and from day 5 to day 7 (P = 0.020), whereas the area decreased significantly from day 5 to day 7 in the control group (P < 0.001). Histologic and immunofluorescence examination revealed full-thickness mucosa healing and complete basal cell coverage in the treatment group; in contrast, only partial healing was observed on day 7 in the control group. CONCLUSIONS: Cell sheet technology using MSC can be an alternative treatment for oral ulcerations in that it can decrease healing time without invasive properties.

Restoration of Homeostasis in the Tracheal Mucosa After Thyroid Surgery in a Rat Model.

BACKGROUND/AIM: This study aimed to investigate the process of homeostatic restoration in the tracheal mucosa (TM) after thyroid surgery. MATERIALS AND METHODS: Fifty-four rats were divided into normal controls (NC) and three experimental groups: (i) flap elevation (FE), (ii) thyroid exposure (TE), and (iii) thyroid isthmusectomy (TI). Expression of mRNA and proteins of key factors regulating homeostasis were evaluated in the TM obtained 3, 7, and 21 days after thyroid surgery. RESULTS: Increased mRNA expression of transforming growth factor-ß1 (TGF-ß1), hypoxia-inducible factor-1α (HIF-1α), and matrix metalloproteinase-9 (MMP-9) were observed 21 days after thyroid surgery in all experimental groups compared to that of NC group. CONCLUSION: Thyroid surgery leads to an actual increase of TGF-ß1, HIF-1α, and MMP-9 expression in the TM. This increased expression of key regulators of homeostatic restoration in the TM lasts for a considerable period of time after surgery, especially if the extent of surgery increased.

Effects of allergic rhinitis on the progression and recovery of acute otitis media in a mouse model.

OBJECTIVES: The objective of this study was to evaluate the effects of allergic rhinitis (AR) on the development, progression, and recovery of acute otitis media (OM) in an animal model and investigate the secondary effects of bacterial infection. METHODS: BALB/c mice were divided into four groups: AR + OM, AR, OM, and control groups. AR + OM and AR groups were sensitized with ovalbumin (OVA) and alum and then challenged intranasally with OVA. Phosphate-buffered saline (PBS) was administered to the OM and control groups the same number of times. After AR induction, OM was induced by surgical inoculation of non-typeable Haemophilus influenza (NTHi) into the middle ear (ME) cavity of the mice in the AR + OM and OM groups. PBS was injected into the bulla in the AR and control groups. Each group was subdivided into sets of six mice, one for each of the four time points (0, 2, 7, and 10 days post-bacterial inoculation), at which point the mice were euthanized and ME and nasal cavity mucosa were obtained and evaluated. The occurrence of OM and the ME mucosa thickness were evaluated and compared among the four groups. Tissue expression of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) in infected ME mucosa was assessed by immunohistochemical staining. We also investigated IgE, IL-4, and IL-5 in the nasal mucosa. RESULTS: Most of the ears showed OM on post-inoculation day 2 in both AR + OM and OM groups. In the AR + OM group, 58.3% of ears still had OM on post-inoculation day 10, while only 16.7% of the OM group had OM. The ME mucosa of all groups increased, and the AR + OM group exhibited the thickest mucosa. The OM group showed peak thickness on post-inoculation day 2 and then decreased, whereas the ME mucosa thickness of the AR + OM group continued to increase to day 7. In the OM group, the expression of IL-1ß, IL-6, and TNF-α in the ME also increased significantly, peaking on post-inoculation day 2, and then gradually decreased. In the AR + OM group, the expression of these proteins increased until day 7 and then decreased. The IgE and Th2 response (IL-4 and IL-5) cytokines were expressed at higher levels in the AR + OM and AR groups than in the OM and control groups. CONCLUSION: The inflammatory reaction to NTHi was more intense and lasted longer in the allergic group, which indicates that AR affects the progression and subsequent recovery of acute bacterial OM.

Changes in Tracheal Respiratory Mucosa After Thyroidectomy: A Rat Model.

BACKGROUND/AIM: This study aimed to investigate changes in the tracheal mucosa after thyroidectomy, that can be a cause of post-thyroidectomy discomfort. MATERIALS AND METHODS: Forty rats were divided into normal controls and 3 surgical groups: (i) thyroid isthmectomy with cauterization, (ii) isthmectomy by a cold instrument without hemostasis, and (iii) sham (exposure of the trachea and thyroid gland without thyroidectomy by dissection through pretracheal fascia). Animals were euthanized at 1 and 4 weeks. Mucosal edema and glandular hyperplasia were measured. Mucin production and basal cell activities were evaluated by mucin 5AC (MUC5AC) and keratin 5 (KRT5) using immunofluorescence staining. RESULTS: Larger mucosal areas were observed in all surgical groups at 1 and 4 weeks. More submucosal glandular hyperplasia was noted in the group with isthmectomy without hemostasis. MUC5AC and KRT5 expressions were significantly higher in the surgical groups. CONCLUSION: The tracheal mucosa may change after surgery, which could explain postoperative discomfort after thyroidectomy.

A New Therapeutic Approach Using a Calcilytic (AXT914) for Postsurgical Hypoparathyroidism in Female Rats.

Postsurgical hypoparathyroidism is the most common complication of thyroid surgery. Conventional therapy with high-dose calcium and vitamin D can correct hypocalcemia but can increase the risk of hypercalciuria, renal stones, or ectopic calcification. The aim of the present study was to investigate the efficacy of a calcium-sensing receptor antagonist, also called a calcilytic (AXT914), in rat models of postsurgical hypoparathyroidism. Two postsurgical hypoparathyroidism rat models were made by hemi-parathyroidectomy or total parathyroidectomy with autotransplantation in 10-week-old female Wistar rats. AXT914 or vehicle was administered orally for 2 to 3 weeks. Serum PTH, calcium, and phosphorus levels, and the urinary excretion of calcium were measured. Autotransplanted parathyroid tissues were collected and examined histologically. In the hemi-parathyroidectomy model, the oral administration of the calcilytic AXT914 (5 and 10 mg/kg) for 2 weeks increased serum PTH and calcium levels and decreased serum phosphorus levels and urinary calcium excretion. In the total parathyroidectomy with autotransplantation model, the oral administration of AXT914 (10 mg/kg) for 3 weeks increased serum PTH and calcium levels and decreased serum phosphorus levels. The serum PTH and calcium levels increased by AXT914 were maintained for 1 week, even after discontinuation of the drug. In conclusion, AXT914 increased PTH secretion in rat models of postsurgical hypoparathyroidism, thereby correcting abnormal calcium and phosphorus homeostasis. Furthermore, AXT914 improved the functional recovery of autotransplanted parathyroid tissues.

Effects of Amniotic Membrane Extract on the Hyperplastic Response of the Middle Ear Mucosa in a Bacterially-Induced Otitis Media Rat Model: A Preliminary Study.

OBJECTIVES: Human amniotic membrane extract (AME) is known to contain numerous bioactive factors and anti-inflammatory substances. However, the anti-inflammatory effects of AME on the middle ear (ME) mucosa are unclear. This study assessed the effects of AME on the growth of the ME mucosa in response to bacterially-induced otitis media (OM). METHODS: OM was induced by inoculating nontypeable Haemophilus influenzae (NTHi) into the ME cavity of rats. ME mucosal explants were cultured in AME concentrations of 0, 5, 10, or 50 µg/mL. The area of explant outgrowth was measured in culture and analyzed at 1, 3, 5, and 7 days after explantation. The expression of Ki-67, mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the explants was also evaluated using quantitative polymerase chain reaction (PCR) and immunocytochemistry (ICC). RESULTS: The NTHi-induced ME mucosa growth increased gradually over the 7-day culture period in all explants at different AME concentrations. There was a trend for mucosal growth inhibition at higher concentrations of AME, although the growth was not significantly different among the groups until day 5. The ME mucosal explants treated with the 50 µg/mL concentration of AME showed significantly suppressed growth on postexplantation day 7 compared with other explants on the same day. PCR and ICC staining revealed that the expression of Ki-67, MUC5AC, TNF-α, and IL-10 further decreased in the explants with higher concentrations of AME than in those with lower concentrations of AME. CONCLUSION: Our results showed that higher concentrations of AME reduced the mucosal proliferative response in bacterial OM in rats. These findings provide evidence that AME has an influence on the inflammatory and proliferative responses to NTHi infection in ME mucosa.

Influence of Vitamin D Deficiency on Progression of Experimental Otitis Media in Rats.

BACKGROUND: Vitamin D plays an important role in the immune response against infection. The purpose of the present study was to investigate the influence of vitamin D deficiency on the progression of otitis media (OM) using an experimental rat model. METHODS: Four-week-old male Sprague-Dawley rats (n=72) were divided into two groups based on their diet: a control diet group (n=36) and a vitamin D-deficient diet group (n=36). After 8 weeks of diet, experimental OM was induced by inoculation of non-typeable Haemophilus influenzae in the middle ear cavity. The rats were evaluated with otomicroscopy to determine the inflammation in the middle ear mucosa on days 1, 2, 4, 7, and 14 post-inoculation. Bullae from sacrificed rats were collected and analyzed histologically. RESULTS: The middle ear mucosa from rats with vitamin D deficiency showed a significantly higher thickness than that of controls during the course of OM. The maximum mucosal thickness was 56.0±9.1 µm in the vitamin D deficiency group, and 43.9±9.8 µm in the control group, although there was no significant difference in the tympanic membrane score between the two groups evaluated with otomicroscopy. An immunohistochemical study showed increased expression of interleukin 6 (IL-6) and tumor necrosis factor α in rats manifesting vitamin D deficiency and decreased expression of IL-10 compared with controls. CONCLUSION: Our results showed that vitamin D deficiency may exacerbate the pathophysiological changes of OM via altered cytokine production. Therefore, maintaining vitamin D status in the optimal range may be beneficial for proper management of OM.

Expression of surfactant Protein-A in the Haemophilus influenzae-induced otitis media in a rat model.

The objective of this study was to investigate the expression and the role of surfactant protein A (SP-A) in the middle ear (ME) mucosa in response to bacterial infection in a rat model. Otitis media (OM) was induced by surgical inoculation of non-typeable Haemophilus influenza (NTHi) into the ME cavity of Sprague-Dawley rats. The rats were divided into an NTHi-induced OM group and a phosphate-buffered saline-injected control group. The NTHi-induced OM and control groups were subdivided into sets of 6 rats, one for each of the 6 time points (0, 1, 2, 4, 7, and 14 days post-inoculation), at which point the rats were euthanized after inoculation. The concentrations of SP-A in the ME effusion were determined by an enzyme-linked immunosorbent assay (ELISA). Tissue expression of SP-A, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in infected ME mucosa was assessed by immunohistochemical staining. For mRNA expression quantification, RNA was extracted from the ME mucosa and SP-A expression was monitored and compared between the control and OM groups using quantitative polymerase chain reaction (PCR). Expression of IL-1ß, IL-6, and TNF-α in the ME mucosa was also evaluated. SP-A expression was evaluated in the effusion of pediatric OM patients (70 ears) who received ventilation-tube insertion by ELISA. SP-A was detected in normal rat ME mucosa before bacterial inoculation. SP-A expression was up-regulated in the NTHi-induced OM group (p = 0.046). Immunohistochemical staining revealed increased SP-A expression on post-inoculation day 1, 2, and 4 in the OM group. Expression of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) in the ME also increased significantly on post-inoculation day 1, 2, and 4 in the OM group. It correlated with changes in SP-A expression. Expression of SP-A was also identified in the ME effusion of humans. SP-A exists in the ME of the rat and was up-regulated in the ME of NTHi-induced OM. Expression of IL-1ß, IL-6, and TNF-α was increased in the ME of the bacteria-induced OM in the rat model. The results suggest that SP-A may play a significant role in the early phase of OM induction and subsequent recovery from it.

Effects of excessive fibrin deposit and polylactide adhesion barrier on wound healing in thyroidectomy murine wound model.

BACKGROUND: We hypothesize that excessive fibrin formation and inflammation induced by antiadhesive material, SurgiWrap (SW), would have an adverse effect on wound healing. It was evaluated by a thyroidectomy murine wound model. METHODS: Excessive fibrin formation was induced by isthmectomy without hemostasis. Rats were allocated into isthmectomy with SurgiWrap (I+SW+), I+SW-, I-SW+, I-SW-, and isthmectomy after electrocautery for hemostasis (I+C+SW-). The SWs were placed on the superficial and visceral layers for gross and microscopic evaluation. RESULTS: Microscopic examination showed collagen deposition occurred in the I-SW- sham group and at a higher level in I+C+SW-. The collagen deposition decreased in groups without SW with time but increased in groups with SW. Use of SW produced more inflammation and more collagen deposition. The I+SW + group developed the largest area of collagen deposition at 4 weeks and more collagen deposition than the I-SW + group. CONCLUSION: The SW induced more collagen deposition increasing with time. The collagen deposition produced by SW was worsened by excessive fibrin formation and inflammation.

Role of group 3 innate lymphoid cells during experimental otitis media in a rat model.

The objective of this study was to evaluate the role of group 3 innate lymphoid cells (ILC3) in the middle ear (ME) mucosal response to bacterial infection in a rat model. To confirm the role of ILC3 in bacterially induced otitis media (OM), the serum concentrations of IL-17 and IL-22 were determined by ELISA, and the tissue expression of IL-17 and IL-22 in infected ME mucosa was assessed by immunohistochemical staining. Immunohistochemical staining of specific cell surface markers was also assessed to confirm the origin of the cells expressing IL-17 and IL-22. Twenty Sprague-Dawley rats were used in the surgically-induced animal model of OM. OM was induced by inoculation of non-typeable Haemophilus influenzae into the ME cavity of the rats. The rats were divided into four experimental groups: three infected groups and one control group. Infected groups were subdivided into sets of 5 rats, one for each of the three time points (1, 4 and 7 days post-inoculation). For determination of rat IL-17 and IL-22 levels in infected rats and control rats, infected or control ME mucosa sections were analyzed by immunohistochemistry with specific antibodies directed against IL-17 and IL-22. Immunohistochemical staining for CD3, RORγt, and NKp46 were also conducted on the samples to confirm the origin of cells expressing IL-17 and IL-22. IL-17 and IL-22 serum concentrations were significantly increased in the infected rats compared to control rats. Immunohistochemical staining revealed increased IL-17 and IL-22 expressions in all infected ME mucosae from the first day after inoculation. In addition, the results of tissue staining for the specific surface markers were negative for CD3 and NKp46, but were highly positive for RORγt. IL-17 and IL-22 revealed their association with the bacterially induced proliferative and hyperplastic responses of ME mucosa, which are characteristic features in pathogenesis of OM. Surface marker examination showed that the source cells for IL-17 and IL-22 seemed to be lymphoid tissue inducer (LTi) cells. The results suggest that LTi cells release IL-17 and IL-22, and play a significant role in both the early phase of OM induction and recovery from it.

Synergistic effect of laminin and mesenchymal stem cells on tracheal mucosal regeneration.

Although several studies have been successfully undertaken of tracheal reconstruction in terms of the maintaining the framework of the graft, most cases of reconstruction failure have resulted from delayed mucosal regeneration. The purposes of this study were to evaluate whether laminin-coated asymmetrically porous membrane (APM) scaffold enhances mucosal regeneration, to compare the mucosalization capability with mesenchymal stem cell (MSC) seeded APM, and to determine whether laminin coating and MSC seeding has a synergistic effect on mucosal regeneration. We reconstructed the full-thickness anterior tracheal defect of 36 New Zealand White rabbits with the APM scaffold. MSCs were isolated from the rabbit's inguinal fat. The animals were divided into 4 groups by the presence of laminin coating on APM and application of MSC [Group I, -/- (laminin/MSC); Group II, -/+; Group III, +/-; Group IV, +/+]. Endoscopy and histologic evaluation were performed and the results were compared among the groups. The results showed that ciliated columnar epithelium was regenerated earlier in groups II and III than in group I. Furthermore, the application of laminin and MSC had synergistic effects on tracheal epithelial regeneration. These results demonstrate that tracheal reconstruction by laminin-coated APM seeded with MSCs is most effective in enhancing tracheal mucosalization, and appears to be promising strategy in the regenerative treatment of tracheal defects.

Vocal Fold Augmentation with Beta Glucan Hydrogel Cross-Linked by γ Irradiation for Enhanced Duration of Effect: In Vivo Animal Study.

This study explored a novel strategy to restore the vocal gap by using cross-linked ß-glucan hydrogel by γ-irradiation. An aqueous solution of 5 wt% ß-glucan was prepared and cross-linked using (60)Co γ irradiation. Ten nude mice were injected with 0.8 mL of irradiated ß-glucan on the left back and the same volume of nonirradiated ß-glucan on the right back for comparison. The mice were sacrificed at 1 and 2 weeks after injection and histological evaluations were performed. Irradiated ß-glucan demonstrated a significantly larger volume than nonirradiated ß-glucan in the back of nude mice with less inflammatory reaction. After unilateral recurrent laryngeal nerve section in New Zealand White rabbits, irradiated and nonirradiated ß-glucan were injected into paralyzed vocal folds. Irradiated ß-glucan remained at the paralyzed vocal fold without definite inflammatory signs on endoscopy. High-speed recordings of vocal fold vibration showed decreased vocal gap in irradiated group compared to nonirradiated group. Histologically, the laryngeal epithelium and lamina propria remained intact, without inflammatory cell infiltration. Our newly developed injection material, irradiated ß-glucan, showed excellent biocompatibility and remained longer than nonirradiated ß-glucan in vivo, suggesting irradiated hydrogels as a new therapeutic approach that may be useful for the long-term treatment of vocal fold palsy.

Middle ear inflammation of rat induced by urban particles.

OBJECTIVE: The aim of this study was to evaluate the histologic change of middle ear mucosa and the expression levels of epithelial sodium channel (ENaC) subunits and mucin production genes, after the injection of urban particulate matter (UPM) into the middle ear cavity of rats. METHODS: Fifty pathogen-free, male Sprague Dawley rats were assigned to the study. Transtympanic injection of UPM solution (300µg/ml, 50µl) was made into the middle ear cavity of rats. Rats were sacrificed at day 1 (group1); day 3 (group2); day 5 (group3); and day 14 (group4) after the procedure. The expression levels of ENaC subunits (α, ß and γ) and mucin producing genes (MUC5AC and MUC5B) were analyzed using semi-quantitative real-time reverse transcriptase-polymerase chain reaction. Thickness of middle ear mucosa was measured and analyzed. RESULT: After transtympanic injection, the thickness of middle ear mucosa increased significantly on day 1, 3 and 5 (p<0.05) and was normalized on day 14, compared to the control group. Inflammatory changes observed in the middle ear mucosa were subepithelial widening, inflammatory cell infiltration and vascular space widening on day 1, 3 and 5. These changes had reverted to normal on day 14. The level of ENaC-α expression decreased 0.60 fold on day 1 (p<0.05), but was normalized thereafter. The level of ENaC-ß and γ decreased 0.39 and 0.27 fold, respectively, on day 1, was normalized on days 3 and 5, and increased 2.30 and 2.47 fold on day 14, respectively (p<0.05). The level of MUC5AC expression increased 1.97-fold on day 1 (p<0.05) and 2.58-fold on day 5 (p<0.05), but was normalized on day 14. The level of MUC5B expression increased 5.4-fold on day 1, 3.14-fold on day 3, 3.85-fold on day 5, and 2.46-fold on day 14, respectively (p<0.05). CONCLUSION: Transtympanic injection of UPM solution into the middle ear cavity of rat induced a characteristic inflammatory response and altered gene expression related with inflammation and mucin production. These findings provide a useful clue for the understanding of how air pollutants, particularly UPM, contribute to the development of otitis media.

Evaluation of the poly(lactic-co-glycolic acid)/pluronic F127 for injection laryngoplasty in rabbits.

OBJECTIVE: Poly(lactic-co-glycolic acid) (PLGA) is an aliphatic polyester and one of the most commonly used synthetic biodegradable polymers for tissue engineering. The objectives of this study were to evaluate the biocompatibility of PLGA/Pluronic F127 in the vocal fold. STUDY DESIGN: A randomized, prospective, controlled animal study. SETTING: University laboratory. SUBJECTS AND METHODS: We used 18 New Zealand white rabbits, which were divided into 5% PLGA solution (n = 9) and 10% PLGA solution (n = 9) groups. The PLGA/Pluronic F127 solutions were injected into the rabbit vocal fold. Laryngoscopic exams were performed at 1, 4, and 8 weeks after implantation; then larynx specimens were sampled. High-speed video camera examination was performed for functional analysis of vocal mucosa vibration at 8 weeks after implantation. Also, we evaluated the amplitude of the mucosal wave from the laryngeal midline on high-speed recording. Histologic study of larynx specimen was performed at 4 and 8 weeks. RESULTS: All animals survived until the scheduled period. Laryngoscopic analysis showed that both 5% and 10% PLGA/Pluronic F127 maintained after 8 weeks after injection without significant inflammatory response. On functional analysis, high-speed camera examination revealed regular and symmetric contact of vocal fold mucosa without a distorted movement by injected PLGA/Pluronic F127. Histologically, no significant inflammation was observed in the injected vocal fold. CONCLUSION: As a vocal fold injection material, PLGA/Pluronic F127 showed a good bio-compatibility without significant inflammatory response. Further experiment will follow to elucidate its role for drug or gene delivery into the vocal fold.

Vocal fold augmentation with injectable polycaprolactone microspheres/pluronic F127 hydrogel: long-term in vivo study for the treatment of glottal insufficiency.

There is increasing demand for reconstruction of glottal insufficiency. Several injection materials have been examined for this purpose, but all had limitations, such as poor long-term durability, migration from the injection site, inflammation, granuloma formation, and interference with vocal fold vibration due to viscoelastic mismatch. Here, we developed a novel injection material, consisting of polycaprolactone (PCL) microspheres, which exhibits better viscoelasticity than conventional materials, and Pluronic F127 carrier, which decreases the migration of the injection materials. The material was injected into rabbits with glottal insufficiency and compared with the FDA-approved injection material, calcium hydroxylapatite (CaHA). Endoscopic and histological examinations indicated that PCL/Pluronic F127 remained at the injection site with no inflammatory response or granuloma formation, whereas CaHA leaked out and migrated from the injection site. Therefore, vocal fold augmentation was almost completely retained during the 12-month follow-up period in this study. Moreover, induced phonation and high-speed recording of vocal fold vibration showed decreased vocal fold gap area in the PCL/Pluronic F127 group. Our newly developed injection material, PCL/Pluronic F127, permits efficient augmentation of paralyzed vocal fold without complications, a concept that can be applied clinically, as demonstrated by the successful long-term follow-up.