hyaluronic acid hydrogel for rejuvenation of geriatric larynx

Accepted Manuscript Full length article Injectable basic fibroblast growth factor-loaded alginate/hyaluronic acid hydrogel for rejuvenation of geriatric larynx Young Hwan Choi, Sae Hyun Kim, In Gul Kim, Jin Ho Lee, Seong Keun Kwon PII: DOI: Reference:

S1742-7061(19)30171-0 https://doi.org/10.1016/j.actbio.2019.03.005 ACTBIO 5987

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

14 November 2018 28 February 2019 4 March 2019

Please cite this article as: Choi, Y.H., Kim, S.H., Kim, I.G., Lee, J.H., Kwon, S.K., Injectable basic fibroblast growth factor-loaded alginate/hyaluronic acid hydrogel for rejuvenation of geriatric larynx, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.03.005

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Injectable basic fibroblast growth factor-loaded alginate/hyaluronic acid hydrogel for rejuvenation of geriatric larynx Young Hwan Choi a, b, Sae Hyun Kim c, In Gul Kim a, Jin Ho Lee c *, Seong Keun Kwon a, d*

a

Department of Otorhinolaryngology-Head and Neck, Seoul National University Hospital, Seoul 03080, Republic of Korea b

School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Republic of Korea c

Department of Advanced Materials and Chemical Engineering, Hannam University, Daejeon 34054, Republic of Korea d

Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea

*

To whom correspondence should be addressed:

Jin Ho Lee, Ph.D. Addresses: Department of Advanced Materials and Chemical Engineering, Hannam University, 1646 Yuseong Daero, Yuseong Gu, Daejeon 34054, The Republic of Korea Tel: +82-42-629-8859; fax: +82-42-629-8854 E-mail address: [email protected]

Seong Keun Kwon, M.D., Ph.D. Address: Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University Hospital, 101, Daehak-ro, Jongno-gu, Seoul, 03080, The Republic of Korea Tel: +82-2-2072-2286; fax: +82-2-745-2387 E-mail: [email protected]

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Abstract Increase in the geriatric population has led to an increase in the number of elderly patients with laryngeal atrophy and dysfunction. Symptoms of voice change, dysphagia, and aspiration pneumonia negatively influence patient’s health status, quality of life, and life span. Injection laryngoplasty used to treat laryngeal dysfunctions does not recover intrinsic functions of the larynx. Thus, we fabricated an injectable basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/hyaluronic acid (HA) hydrogel for inducing rejuvenation of geriatric laryngeal muscles. Optimal in situ-forming bFGF–loaded ALG/HA hydrogel for injection laryngoplasty was prepared and the release profile of bFGF was analyzed. For in vivo analysis, the bFGF–loaded ALG/HA hydrogel was injected into the laryngeal muscles of 18-month-old Sprague-Dawley rats. The rejuvenation efficacy of bFGF–loaded ALG/HA hydrogel in geriatric laryngeal muscle tissues 4- and 12-weeks post-injection was evaluated by quantitative polymerase chain reaction (qPCR), histology, immune-fluorescence staining and functionality analysis. The bFGF-loaded ALG/HA hydrogel induced an increase in the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis. Administration of the bFGF-loaded ALG/HA hydrogel caused successful glottal gap closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for laryngoplasty aimed at rejuvenating geriatric larynx.

Statement of Significance

In this manuscript, optimal in situ-forming bFGF–loaded ALG/HA hydrogel for injection laryngoplasty was prepared and the release profile of bFGF was analyzed. Herein, we introduced the materials and methods of injection laryngoplasty for geriatric rat experiment. In addition, we studied effects of bFGF-loaded ALG/HA hydrogel on the therapeutic rejuvenation

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of geriatric rat larynx. The bFGF-loaded ALG/HA hydrogel induced an increase in the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis. Furthermore, our functional analysis through the high-speed camera setup demonstrated that the administration of the bFGF-loaded ALG/HA hydrogel induced successful glottal gap closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for injection laryngoplasty with therapeutic effects.

Keywords: Geriatric larynx, Injection laryngoplasty, Basic fibroblast growth factor, Alginate, Hyaluronic acid, Rejuvenation

1. Introduction

Injection laryngoplasty is an endoscopic surgical procedure that has been employed in clinical practice for several decades. It is successfully used in patients with unilateral vocal fold paralysis to manage glottic insufficiency [1]. Although injection laryngoplasty is a valuable treatment modality for alleviating the symptoms of laryngeal dysfunction, problems related to regeneration of laryngeal tissues and restoration of intrinsic laryngeal functions remain unresolved. Increase in the geriatric population around the world has led to an increase in the number of elderly patients suffering from laryngeal disorders [2]. Symptoms of laryngeal disorders

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such as voice change [3, 4], dysphagia [5], and aspiration pneumonia negatively influence the patient’s health status, quality of life, and life span [6]. Geriatric larynx shows significant structural and histological changes in the composition of the extracellular matrix (ECM) of the vocal folds, laryngeal muscles, cartilage, and nerves [7-9]. Structurally, a normal vocal fold is made up of three layers differing in the composition of the ECM and hence, each of the three layers has different viscoelastic properties. With aging, there is an increase in the number of collagen fibers and a decrease in the number of elastic fibers in the ECM, resulting in higher rigidity that consequently affects the vibration properties of the vocal fold [10]. In addition, a decrease in the concentration of hyaluronic acid reduces the viscosity of the vocal fold [11]. Several studies have aimed to identify the therapeutic effects of exogenously administered growth factors in aged vocal folds based on their ability to alter the composition of the ECM [12, 13]. Although laryngeal disorders relate to histological alterations of the vocal fold, the most common cause of laryngeal dysfunction in elderly patients is the atrophy of the vocal fold resulting in incomplete closure of the glottis [5]. The intrinsic laryngeal muscles responsible for controlling phonation are classified into three functional groups: adductors, abductors, and tonics [14]. Among these, the adductor muscles contribute to the closure of the glottis. The thyroarytenoid muscle plays a crucial role in inducing glottal closure and creating stable sounds. Progressive decline of skeletal muscle mass with advancing age results in loss of muscle strength [15]. Accordingly, progressive atrophy and loss of muscle mass occur in the laryngeal muscles with aging. Atrophy of the thyroarytenoid muscle causes unstable vibration of the vocal folds and incomplete glottal closure [16]. For decades, several materials including Teflon, calcium hydroxyapatite (CaHA), silicone, collagen, and autologous fat have been used to ameliorate glottic insufficiency in clinical trials

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[17]. Regeneration of laryngeal tissues and restoration of intrinsic laryngeal functions are challenging problems that have yet to be addressed by existing injectable materials. Previously, we reported several studies related to the scope of using diverse materials for injection laryngoplasty, including polycaprolactone, Pluronic F127, poly(lactic-co-glycolic acid) (PLGA), and adipose-derived ECM [18-23]. Given the studies of the past along with our previous studies, ideal substance for laryngoplasty is injectable, easy to prepare and easy to use, cost-effective, biocompatible and therapeutic agent for restoration of intrinsic laryngeal functions. In the present study, we fabricated a basic fibroblast growth factor (bFGF)-loaded alginate/hyaluronic acid hydrogel as a cost-effective, biocompatible, and easy-to-prepare injectable material. Further, we studied its effects on the therapeutic rejuvenation of geriatric larynx. The prepared materials were injected into aged-rat larynx using a laryngoscope. Histological assessments were performed at 4 and 12 weeks post-injection. Myogenic response was investigated using quantitative polymerase chain reaction (qPCR) at 4 weeks postinjection. Functionality test was performed to evaluate the potential of glottal gap closure using high-speed camera setup at 12 weeks post-injection.

2. Materials and Methods

2. 1. Materials Purified sodium alginate (ALG, medium viscosity, Sigma Aldrich, USA) [24], and hyaluronic acid (HA, molecular weight: 3,000 kDa, medical grade, SK-Bioland, Republic of Korea) were used in the present experiment. Calcium sulfate (CaSO4; Oriental Chemical Industry, Republic of Korea) was used as a cross-linking agent without further purification.

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Basic fibroblast growth factor was selected (bFGF, human source; R & D Systems, USA) as a bioactive molecule for rejuvenation of geriatric laryngeal muscles. 2. 2. Preparation of injectable and in situ gelation of ALG/HA hydrogels Based on the results of our previously published in vitro study (results of mechanical test, stability, and cell culture) [25], ALG/HA mixture at mixing ratio of 7:3 (w/w) was selected to evaluate the potential of the bFGF-loaded ALG/HA hydrogel on the rejuvenation of geriatric larynx. ALG/HA (in the ratio 7:3 (w/w)) was dissolved in phosphate-buffered saline (PBS, pH ~ 7.4) to provide a polymer concentration of 1 % w/v. Fabrication of the injectable and in situ gelation of ALG/HA hydrogel were achieved by regulating the concentration of CaSO4 that controlled the gelation rate and uniformity of the ALG/HA hydrogel. Accordingly, ALG/HA solutions were mixed with an equal volume of CaSO4 suspension (final concentration of CaSO4: 0.2 % w/v, 0.3 % w/v, 0.4 % w/v, and 0.5 % w/v in PBS).

2. 3. Rheological properties of ALG/HA hydrogel The concentration of CaSO4 that induced gelation of the ALG/HA hydrogel was identified by rheological analysis using a rheometer (DHR-3, TA Instrument, USA) equipped with coneand-plate geometry (cone angle, 2° ; cone diameter, 40 mm). The gap distance was fixed at 65 µm and the temperature was maintained at 25 °C using a thermostat. Syringes loaded with CaSO4 and ALG/HA solutions at respective concentrations were fixed to the syringe connecter and 60 µL of the mixed solution was injected into the plate. A solvent trap was used to prevent sample drying during the course of measurement. Oscillatory measurements were conducted to obtain the storage modulus (G’) and loss modulus (G’’) of ALG/HA hydrogel (strain, 90 %; frequency, 1 Hz). Gelation points were determined as the point of intersection of the storage modulus (G’) and loss modulus (G’’).

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For further in vitro and in vivo study, we selected 0.4 w/v% of CaSO4. bFGF-loaded hydrogel was prepared by mixing bFGF (reconstituted in PBS) diluted in CaSO4 suspension with ALG/HA solutions, such that the final concentration of bFGF was 1 µg/mL for in vitro study and 1 µg/20 µL for in vivo study, in a manner similar to the method described above. For animal studies, ALG/HA and bFGF/CaSO4 solutions were filtrated using a syringe filter (0.45 µm pore size, Minisart®, Sartorius, Germany).

2. 4. Analysis of bFGF release behavior For evaluating the release rate of bFGF from the ALG/HA hydrogel, 1 mL of hydrogel was incubated with 2 mL of PBS supplemented with 1% bovine serum albumin (BSA, Sigma Aldrich, USA) for up to 35 days in a shaking incubator (temperature, 37 °C; rpm, ~50). At predetermined time intervals, the medium was collected and replaced with fresh PBS supplemented with 1% BSA. The amount of bFGF released into the medium was determined using an ELISA kit according to the manufacturer’s instructions (R&D systems, USA).

2. 5. Injection of hydrogel into geriatric rat laryngeal muscle All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC No, 160009-C2A0), Republic of Korea. Male Sprague-Dawley rats (SD rats, 10 weeks and 18 months-old) were purchased from Young Bio (Young Bio Inc, Republic of Korea). Rats were anesthetized with an intraperitoneal injection of Zoletil 50 (Tiletamine/Zolazepam, 0.6 mL/kg) and Rompun (Xylazine, 0.4 mL/kg). The preparation and assembly of injection laryngoscope is shown in Fig. 2A. Briefly, a trimmed and crooked 20 gauge spinal needle (Tae Chang Industrial Co., Republic of Korea) was fixed to a 4.0 mm, 30° rigid endoscope (Richards,

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Knittlingen, Germany). A 27 gauge spinal needle mounted with Hamilton syringe (Hamilton® syringe, luer tip 702LT, volume 25 µL) was inserted into the 20 gauge spinal needle. The assembled endoscope with an LED based light source (Mediview, UMT-511, Australia) was mounted with a digital camera (Nikon Coolpix 4500, Japan). The anesthetized 18 months-old SD rats were fixed using a custom-made frame (Fig. 2B). Experimental animals were divided into five groups: Control, non-treated 18 months-old SD rat; Sham, PBS injected 18 monthsold SD rat; ALG/HA, hydrogel injected 18 months-old SD rat; GF+ALG/HA, bFGF-loaded hydrogel injected 18 months-old SD rat; Positive control, non-treated young 10 weeks–old SD rat. Accordingly, the prepared material was injected (each injection 20 µL/rat) into the right vocal folds of the rats (each group, n = 12) using a Laryngoscope aided by live imaging (Fig. 2C, D). Unilateral injection was performed instead of bilateral injections to avoid vocal fold edema and airway obstruction which can lead to dyspnea and death of animals.

2. 6. Real-time PCR analysis Four weeks post-injection, three rats from each group were euthanized and the larynges were harvested for isolation of laryngeal muscles. The isolated laryngeal muscle samples were immediately transferred into 1.8 mL Eppendorf tubes embedded in dry ice and later stored at −80 ºC until further use. Total RNA was isolated from homogenized muscle tissue using Trizol™ reagent according to the manufacturer’s protocol. RNA was reverse transcribed into cDNA using LeGene Premium Express First Strand cDNA Synthesis System (LeGene Biosciences, USA). Real-time PCR assays were performed using Power SYBR® Green PCR Master mix (Applied Biosystems, USA) and ABI StepOnePlus™ Real-time PCR System (Applied Biosystems, USA). Myogenic response of the laryngeal muscle was analyzed by measuring the expression level of myogenic-related genes: myogenic differentiation 1

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(MyoD1), myogenic factor 5 (Myf5), Myogenin, and myogenic regulatory factor4 (Mrf4). The following gene-specific primers were used in the present study: MyoD1: forward, 5’ATTGAAGGTCTGCAGGCTCT-3’, and reverse, 5’-CCGCTGTAATCCATCATGCC-3’; Myf5:

forward,

5’-AGTCTTCAGGAGCTGCTGAG-3’,

TCCGATCCACTATGCTGGAC-3’;

Myogenin:

and

reverse,

forward,

5’5’-

CATCCAGTACATTGAGCGCC-3’, and reverse, 5’-GCGAGCAAATGATCTCCTGG-3’; Mrf4:

forward,

5’-GTACCCTATCCCCTTGCCAG-3’,

and

reverse,

5’-

CTGCTTTCCGACGATCTGTG-3’. The housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as an internal control.

2. 7. Histological evaluation of laryngeal muscle tissues Four and twelve weeks post-injection, three rats from each group were euthanized and the larynges were isolated and fixed in 4 % paraformaldehyde solution (4% PFA, Biosolution Co., Republic of Korea). The samples were dehydrated, embedded in paraffin blocks, and sectioned at a thickness of 3 µm using a microtome. Sections were then deparaffinized, rehydrated, and used for Hematoxylin and Eosin (H&E) staining, Masson’s trichrome (MT) staining, and immunofluorescence staining. H&E and MT stained tissue sections were observed under an optical microscope. For immunofluorescence staining, tissue sections were subjected to antigen retrieval, permeabilized, blocked, and were incubated with the respective primary antibody overnight at 4 ºC. The primary antibodies used in the present study included anti-laminin (ab11575), anti-fast myosin skeletal heavy chain (ab51263), anti-PAX7 (ab199010), monoclonal anti-CD31 (MA5-13188), and anti-alpha smooth muscle actin (ab5694). After incubation, sections were washed and incubated with the respective secondary antibody, Alexa Fluor 488 goat anti-mouse, Alexa Fluor 594 goat anti-mouse, Alexa Fluor 488

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goat anti-rabbit, and Alexa Fluor 594 goat anti-rabbit (Invitrogen, Thermo Fisher Scientific, USA). The nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). Histological sections were analyzed using Image J Software.

2. 8. Functional assessment of larynx using high-speed camera To evaluate the functionality of the larynx, vocal fold vibrations were examined using an excised laryngeal setup similar to our previously published study [19]. Four experimental groups were selected (Control, Sham, GF + ALG/HA, Positive control) for the functional assessment of the larynx and 3 rats in each group were analyzed. After total laryngectomy, the supraglottis (a region locating above the true vocal fold) was removed and the two arytenoid cartilages were sutured together to imitate the contact of arytenoid cartilages during the induction of phonation. Trachea inferior to the larynx was clamped to a pipe and the larynx was mounted with a custom-made support fixture (Fig. 6A). Filtered (AF20-02, SMC Co, Japan) and humidified (AR20P, SMC Co.) air was pumped through the pipe to generate vocal fold vibrations. Air pressure was monitored with a manometer (MS-15, SMC Co.). Vocal fold vibrations were recorded by a high-speed camera (OS4-V3-S1, IDT, USA) at 40,000 images per second with a spatial resolution of 60 horizontal × 256 vertical pixels. A macro lens (MicroNIKKOR 105mm f/2.8, Nikon, Japan) with 3.0x Teleconverter (Teleplus Pro 300, 3.0x DG Teleconverter, Kenko, Japan) was mounted anterior to the high-speed camera using a C-mount lens adapter. Illumination was provided by the Constellation 120E (Veritas, USA). To analyze the glottal gap area, the region of the vocal fold was specified as 28 horizontal × 104 vertical pixels using a MOTION STUDIO™ software (version: 2.12.19). One cycle of vocal fold vibration was composed of 10 serial images. Four images with maximal glottal gap area were chosen from four cycles of vocal fold vibrations and the glottal gap area was measured using

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Image J software.

2. 9. Statistical analysis Data are represented as mean ± standard deviation (SD). The statistical significance between two groups was determined using Student’s t-test. A P-value < 0.05 was considered to indicate statistically significant differences. *P < 0.05, **P < 0.01, ***P < 0.005.

3. Results

3.1 Fabrication and characterization of bFGF-loaded in situ-forming ALG/HA hydrogel Injectability and in situ gelation of the ALG/HA hydrogel was analyzed by mixing bFGF loaded-ALG/HA solution with equal volume of CaSO4 solution at different concentrations (Fig. 1A). Onset of gelation was identified as gelation points by rheological analysis (Fig. 1B-D). The ALG/HA hydrogel showed several gelation points depending on the concentration of CaSO4 solution (n = 3, concentration/gelation points (s); 0.2 % (w/v)/not found, 0.3 % (w/v)/400.0 ± 52.7 s, 0.4 % (w/v)/172.2 ± 19.1 s, and 0.5 % (w/v)/99.7 ± 5.3 s) (Fig. 1E). CaSO4 solution at a concentration of 0.4 % (w/v) was used for the following in vitro and in vivo studies. Slow gelation impairs the residence stability of hydrogel because of rapid clearance caused by dissolution in body fluids at the application site, while rapid gelation may reduce the ease of injection. To provide both residence stability and injectable property to the prepared material, CaSO4 solution at a concentration of 0.4 % (w/v) was used to cross-link the ALG/HA hydrogel within the optimal gelation time. The release profile of bFGF from the ALG/HA hydrogel showed an initial burst of bFGF

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in the first few days followed by sustained release over the next 30 days (Fig. 1F).

3.2 bFGF-loaded ALG/HA hydrogel stimulated rejuvenation and myogenic response in geriatric rat laryngeal muscles (4 weeks post-injection) To analyze the efficiency of the bFGF-loaded ALG/HA hydrogel in rejuvenating geriatric rat laryngeal muscles, the following intra-laryngeal injections were administered into the laryngeal muscle of 18 month-old SD rats using a laryngoscope: PBS (Sham, 20 µL), ALG/HA hydrogel (ALG/HA, 20 µL), bFGF (1 µg)-loaded ALG/HA hydrogel (GF + ALG/HA, 20 µL) (Fig. 2). Live imaging aided the tip of a micro needle into the laryngeal muscle for the injection of materials (Fig. 2D). Non-treated 18 months-old SD rats served as controls. Non-treated young 10 weeks-old rats were selected as the positive control group. H&E and MT staining were used for histological evaluation of laryngeal muscles isolated on day 28 post-injection of respective materials (Supplementary Fig. 1 and Fig. 3A). Significantly, reduced interstitial fibrosis and increased total myofiber cross sectional area were seen in the geriatric laryngeal muscle isolated from GF + ALG/HA and Positive control groups compared to that in the Control, Sham, and ALG/HA groups (Fig. 3B, C). On the contrary, myofiber number was similar in all groups (Fig. 3D). Cross sectional area of individual myofiber was significantly increased in the GF + ALG/HA group compared to that in the Control, Positive control, Sham, and ALG/HA groups (Fig. 3E). Myogenic effect of the bFGF-loaded ALG/HA hydrogel was evaluated by analyzing the expression of myogenic specific genes, MyoD1, Myf5, Myogenin, and Mrf4 in laryngeal muscle tissues from each of the five experimental groups isolated on day 28 post-injection of respective materials. Expression level of each gene marker was normalized to that of the reference

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housekeeping gene (Gapdh). Expression of MyoD1 and Myf5 was slightly higher in the GF + ALG/HA group compared to that in the Control, Positive control, Sham, and ALG/HA groups (Fig. 3F, G). There was slightly increased expression levels of Myogenin in the GF + ALG/HA group and Positive control groups compared to that in the Control, Sham and ALG/HA groups (Fig. 3H). Meanwhile, the expression of Mrf4 was slightly higher in the SHAM group compared to that in the other groups (Fig. 3I). Results related to the expression of MyoD1, Myf5 and Myogenin suggests that there appears to have been a stimulation of myogenic response by the bFGF-loaded ALG/HA hydrogel in geriatric rat laryngeal muscle. However, none of the differences in expression levels between groups reached statistical significance for any gene analyzed.

3.3 bFGF-loaded ALG/HA hydrogel stimulated rejuvenation and hypertrophy in geriatric rat laryngeal muscles (12 weeks post-injection) H&E, MT, and immunostaining with muscle tissue-related antibodies were used for histological evaluation of laryngeal muscle tissues isolated on day 84 post-injection of respective materials (Fig. 4, Fig. 5, and supplementary Fig. 1). Interstitial fibrosis and morphological change were analyzed by staining the fibrous connective tissue, muscle fibers, basement membrane, and myosin heavy chain of laryngeal muscle with Masson’s trichrome staining kit and antibodies against laminin and fast myosin heavy chain (Fig. 4A, B). Consistent with the results of 4 week post-injection studies, significantly reduced interstitial fibrosis and increased total myofiber cross sectional area were seen in the geriatric laryngeal muscle isolated from GF + ALG/HA and Positive control groups compared to that of the Control, SHAM, and ALG/HA groups (Fig. 4C, D). In contrast, myofiber number was similar in all the four groups except in the Positive control group (Fig. 4E). Cross sectional area of individual

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myofiber was significantly increased in the GF + ALG/HA group compared to that in the Control, Positive control, Sham, and ALG/HA groups (Fig. 4F). Interestingly, significantly higher myofiber number with lower cross sectional area of individual myofiber was seen in the Positive control group compared to that in the GF +ALG/HA group. Newly formed immature muscle fibers exhibit centrally located nuclei [26]. In the present study, significant difference was not observed in the number of myofiber with centrally located nuclei among all the five groups (Fig. 4G). Taken together, these results suggest the ability of injected bFGF-loaded ALG/HA hydrogel to rejuvenate geriatric rat laryngeal muscles by inducing hypertrophy of individual muscle fiber rather than by increasing the myofiber number. Satellite cells (muscle stem cells) play a crucial role in the regeneration and radial growth of skeletal muscle fibers [27-29]. These cells reside in the basal membrane surrounding myofibers. The ability of the bFGF-loaded ALG/HA hydrogel to stimulate and activate satellite cells was analyzed by counting the number of PAX7-positive nuclei (satellite cell marker) in the basal lamina (Fig. 5A). The number of satellite cells was significantly higher in the GF + ALG/HA and Positive control groups compared to that in the Control, SHAM, or ALG/HA groups (Fig. 5D). Further, the number of CD31-positive micro-vessels and α-SMA-positive arterioles were detected to analyze the effect of the bFGF-loaded ALG/HA hydrogel on angiogenesis (Fig. 5B, C). The number of CD31-positive micro-vessels was significantly increased in the GF + ALG/HA group compared to that in the other groups. There was no significant difference in the number of α-SMA-positive arterioles among all the five groups (Fig. 5E, F).

3.4 bFGF-loaded ALG/HA hydrogel reduced the glottal gap area in geriatric larynx (12

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weeks post-injection) The ability of the bFGF-loaded ALG/HA hydrogel to reduce the glottal gap area in geriatric larynx was evaluated by analyzing vocal fold vibrations using an excised laryngeal setup (Fig. 6A-C). Four experimental groups were selected (Control, Sham, GF + ALG/HA, Positive control) for the functional assessment of the larynx. The area of each maximal glottal gap as seen in serial photographs was measured (Fig. 6D, E and Supplementary video 1-4). Our results indicate successful induction of glottal gap closure by the bFGF-loaded ALG/HA hydrogel similar to that in the Positive control group (Fig. 6F). The glottal gap area was significantly reduced in the GF + ALG/HA and Positive control groups compared to that in the Control and Sham groups. In addition, the bFGF-loaded ALG/HA hydrogel induced regular vocal fold waves with significant reduction in the glottal gap (Supplementary Fig. 2).

4. Discussion In the present study, we fabricated an injectable bFGF-loaded in situ-forming ALG/HA hydrogel capable of delivering bFGF for the rejuvenation of geriatric laryngeal muscles (Fig. 1). Alginate is a natural anionic polysaccharide extracted from brown algae. It is widely used as a biomaterial in biomedical applications and tissue engineering because of its biocompatibility and cost-effectiveness [30]. In addition, alginate has easy and rapid gelation properties when mixed with divalent cationic cross-linkers such as Ca2+ [31]. In general, alginate hydrogel have been fabricated by mixing alginate solution with CaCl 2 solution. However, it is hard to control gelation rate and prepare the injectable alginate hydrogel due to the fast crosslinking. Instead, by using CaSO4 as a crosslinking agent, injectable alginate

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hydrogel could be prepared. Because, the water solubility of the CaSO4 determined the gelation rate of alginate hydrogel [25]. HA, an essential component of the ECM is a natural polysaccharide made up of repeating units of disaccharides. Its unique structural, mechanical, and physiological properties play crucial role in living organisms making it an attractive biomaterial for tissue engineering and regenerative medicine [32, 33]. In injection laryngoplasty, HA is used as an absorbable and injectable material to treat laryngeal disorders [17]. In the present study, properties of ALG and HA have been exploited to generate ALG/HA hydrogel as drug carriers in injection laryngoplasty (Fig. 1A). Optimal gelation rate and rheological properties as an injectable and in situ forming hydrogel were obtained by modulating the concentration of CaSO4, a divalent cross-linker. Rheological analysis was performed using a rotational rheometer (Fig. 1B-E). Slow gelation impairs the residence stability of hydrogel because of rapid clearance caused by dissolution in body fluids at the application site, while rapid gelation may reduce the ease of injection. To provide both residence stability and injectable property to the prepared material, CaSO4 solution at a concentration of 0.4 % (w/v) was used to cross-link the ALG/HA hydrogel within the optimal gelation time. Growth factors and cytokines have diverse roles in skeletal muscle development, growth, and regeneration [34]. During the initial stages of muscle regeneration, bFGF (also known as FGF2) promotes activation, recruitment, and proliferation of muscle satellite cells [34, 35]. In addition, bFGF is an important growth factor for angiogenesis [36, 37] and is used as a therapeutic drug for tissue regeneration. However, when injected directly, bFGF rapidly loses its biological functional performance in vivo. For effective tissue regeneration, an ideal drug delivery system is essential that retains the bioactivity of the drug and supports its controllable and sustainable release [38]. In the present study, bFGF release from in situ forming ALG/HA

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hydrogel was associated with two phases, an initial burst phase followed by a sustained release phase (Fig. 1F). This is explained by the rapid liberation of bFGF from the periphery of the hydrogel subsequent to its diffusion through the pores in the hydrogel network. Sustained release of bFGF is desirable to prolong its bioactivity in vivo. Intra-laryngeal injection of the bFGF-loaded ALG/HA hydrogel resulted in decreased interstitial fibrosis, increased cross-sectional-area of the muscle fiber, and enhanced myogenic response in geriatric laryngeal muscles (Figs. 3, 4). Non-treated young 10 weeks-old rats were used as the Positive control group to evaluate histological differences in the laryngeal muscles between young and elderly rats. Administration of the bFGF-loaded ALG/HA hydrogel induced histological and morphological changes promoting rejuvenation and transformation of geriatric laryngeal muscle similar to that of young laryngeal muscles. Both in severe myopathy and aging process, impaired muscle repair characterized by uncontrolled ECM remodeling results in the formation of fibrotic tissue in skeletal muscles [39]. Fibrosis impairs tissue function and increase the stiffness of the tissue. Unimpaired muscle regeneration requires coordination of various factors including inflammatory cells, satellite cells, growth factors, and proteolytic molecules leading to successful ECM turnover and remodeling [40]. Previous studies have investigated the role of bFGF in muscular disorders such as muscular dystrophy characterized by suppressed differentiation of skeletal muscles and excessive fibrosis of muscle tissues [41]. In the mdx mouse, deficiency of bFGF results in severe dystrophic changes. In the present study, sustained release of bFGF from the ALG/HA hydrogel led to the activation of factors associated with ECM turnover and remodeling resulting in decreased interstitial fibrosis in geriatric laryngeal muscles (Fig. 3B, Fig 4C). Newly formed immature muscle fibers are characterized by centrally positioned nuclei, whereas, in mature muscle fibers, the nuclei change their position because of centration,

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spreading, dispersion and clustering [26]. The bFGF-loaded ALG/HA hydrogel did not affect the myofiber number in geriatric laryngeal muscles compared to that of young laryngeal muscles (Fig. 3D, 4E). In addition, significant difference was not observed in the number of newly formed immature muscle fibers with centrally located nuclei among all the experimental groups (Fig. 4G). In the present study, a significant increase was seen in the cross-sectional area of individual muscle fibers that made the total cross-sectional area of the geriatric laryngeal muscle comparable to that of the young laryngeal muscle (Fig. 3C, E and Fig. 4D, F). These results indicate that the bFGF-loaded ALG/HA hydrogel stimulated the induction of myogenic response in geriatric laryngeal muscle to compensate the deficiency of muscle fibers by inducing hypertrophy of the existing individual muscle fibers. The bFGF-loaded ALG/HA hydrogel could stimulate, activate, and enhance the number of satellite cells in the laryngeal muscle tissue (Fig. 5A, D). Skeletal muscle stem cells, also known as satellite cells reside in the basal membrane surrounding the muscle fiber. Satellite cells are widely investigated for their characteristics, role, and functions since their discovery by Mauro [42, 43]. Satellite cells are essential for the growth, regeneration, and repair of skeletal muscle fibers throughout life [44, 45]. During childhood, myogenesis occurs not only as a result of increase in muscle fiber, but also because of extensive hypertrophy accompanied by activation, proliferation, and differentiation of satellite cells into newly formed myonuclei [46]. In adulthood, satellite cells are essential to compensate muscle degeneration caused by daily wear and tear under stable conditions and to induce hypertrophy [47]. With aging, the regenerative potential of skeletal muscle diminishes resulting in loss of skeletal muscle mass and strength, called sarcopenia, and is related to reduction in the number and regenerative capacity of satellite cells [48-50]. Several studies have reported the possibility of activating and rejuvenating aged satellite cells [35, 51, 52]. Satellite cells are activated by several growth

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factors, cytokines, and regulatory factors [34, 35, 53]. In the present study, activated satellite cells might stimulate muscle regeneration and hypertrophy in muscle fibers of geriatric larynx [28, 29, 54-56]. In addition, bFGF-loaded ALG/HA hydrogel significantly increased the number of micro-vessels in the geriatric laryngeal tissue (Fig. 5E). Interaction of the newly formed micro-vessels triggered by growth factors with satellite cells might support muscle regeneration [57, 58]. In this study, a high-speed camera was used to achieve accurate measurements of mucosal waves (Fig 6A, B). Larynx of rats were excised and mounted on a custom-made fixture to avoid minor movements that might inhibit the focus of the camera on the subject (Fig. 6C). Glottal gap area in the rat larynx was analyzed from at least 10 photographs per cycle that were taken in the present study (Fig. 6D). By decreasing the spatial resolution to 60 horizontal × 256 vertical pixels, high-frame recordings of about 40,000 frames per second was achieved. Intralaryngeal injection of the bFGF-loaded ALG/HA hydrogel led to glottal closure comparable to that of the Positive control group (Fig 6E, F and Supplementary video 1-4). Hypertrophy of the laryngeal muscle induced by bFGF-loaded ALG/HA hydrogel might have contributed to the successful glottal gap closure. In addition, kymographs of the vocal fold waves indicated regular wave pattern and reduction of glottal gap in the larynx which were injected with the bFGF-loaded ALG/HA hydrogel (GF + ALG/HA group) compared to that in the Control and Sham groups (Supplementary Fig. 2). In the present study, we fabricated a cost-effective, biocompatible, easy-to-prepare, and injectable hydrogel for injection laryngoplasty in the form of ALG/HA hydrogel. Further, bFGF was loaded into the ALG/HA hydrogel used as a delivery system to enhance its half-life and the therapeutic effects were analyzed. Although histological and functional changes beyond 12 weeks need to be assessed to determine whether the therapeutic rejuvenation is sustainable, our

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results indicate the use of the bFGF-loaded ALG/HA hydrogel as a therapeutic candidate to treat age-related laryngeal dysfunctions such as laryngeal atrophy. Satellite cells that play a crucial role in muscle regeneration and hypertrophy were also activated by the bFGF-loaded ALG/HA hydrogel. Accordingly, co-injection of satellite cells and bFGF-loaded ALG/HA hydrogel might provide synergistic effects for the rejuvenation of geriatric laryngeal muscles. Since bFGF has a short half-life, materials such as heparin-binding hydrogel or heparinmimicking hydrogel could be used as a candidate delivery system for future study. A dual-drug delivery system might also be a promising strategy. For instance, delivery of bFGF and insulin growth factor (IGF) or hepatocyte growth factor (HGF) in a time-dependent manner might be an effective strategy for activating satellite cells for rejuvenation of geriatric laryngeal muscles.

5. Conclusion Studies related to injection laryngoplasty for the treatment of laryngeal dysfunctions have been initiated since the past few decades. However, recovery of intrinsic functions of the larynx has not yet been achieved. In the present study, we fabricated an injectable in situ forming ALG/HA hydrogel loaded with bFGF for inducing rejuvenation of geriatric laryngeal muscles. Results of real-time PCR, histology, immunofluorescence staining, and functionality analysis indicated the bFGF-loaded ALG/HA hydrogel to be effective in the rejuvenation of laryngeal muscle in geriatric rats. The bFGF-loaded ALG/HA hydrogel enhanced the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, angiogenesis, and decreased interstitial fibrosis. Further, the bFGF-loaded ALG/HA hydrogel effectively induced sufficient glottal closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for injection laryngoplasty with therapeutic effects.

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Acknowledgements This work was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT, and Future Planning, Republic of Korea (NRF-2017R1A2B2003848) and the Bio & Medical Technology Development Program of the National Research Foundation funded by the Ministry of Science, ICT, and Future Planning, Republic of Korea (NRF-2017M3A9B4032446). Conflict of interest None declared. Financial disclosure Authors have nothing to disclose

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Figure Legends Fig 1. Fabrication and characteristics of basic fibroblast growth factor (bFGF)-loaded in-situ forming alginate (ALG)/hyaluronic acid (HA) hydrogels. (A) Fabrication of time-dependent cross-linked ALG/HA hydrogel. Optimal characteristics of the ALG/HA hydrogel suitable for injection and sustained release of bFGF. (B-D) Rheological analysis performed using a rheometer. Gelation points were determined as the point of intersection of the storage (G’) and the loss modulus (G’’). (E) Determination of optimal gelation rate of the ALG/HA hydrogel by modulating the concentration of CaSO4 (0.4 % w/v) for further in vitro and in vivo studies. (F) Cumulative release profile of bFGF from the ALG/HA hydrogel. The released bFGF was measured quantitatively using an ELISA kit as per the manufacturer’s instructions.

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Fig 2. Injection of the basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/hyaluronic acid (HA) hydrogel into geriatric rat laryngeal muscle using a Laryngoscope. Aided by live imaging, the bFGF-loaded ALG/HA hydrogel was injected into the laryngeal muscle of geriatric rat. Experimental animals were divided into five groups: Control, non-treated 18 months-old Sprague-Dawley (SD) rat; SHAM: PBS injected 18 months-old SD rat; ALG/HA: hydrogel injected 18 months-old SD rat; GF+ALG/HA: 1 µg of bFGF-loaded hydrogel injected 18 months-old SD rat; Positive control: non-treated young 10 weeks-old SD rat. (A) Injection laryngoscope fixed with 27G spinal needle syringe containing materials was prepared and assembled. (B) Anesthetized aged SD rats were fixed using a custom-made frame. (C) Intralaryngeal injection of materials using a stationary laryngoscope. (D) Live imaging aided injection of materials (white arrow = the tip of micro needle was stuck into intra-laryngeal muscle). Fig 3. Effects of the basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/hyaluronic acid (HA) hydrogel on rejuvenation and myogenic response of geriatric laryngeal muscle (4 weeks post-injection). Comparative histology and real-time polymerase chain reaction analysis of five experimental Sprague-Dawley (SD) rat groups (Control: non-treated 18 months-old SD rat, SHAM: PBS injected 18 months-old SD rat, ALG/HA: hydrogel injected 18 months-old SD rat, GF+ALG/HA: 1 µg of bFGF-loaded hydrogel injected 18 months-old SD rat, Positive control: non-treated young 10 weeks-old SD rat), 4 weeks post-injection. All histological evaluations were conducted using image J software. (A) Representative Masson’s trichrome images of laryngeal muscle (Scale bar: 100 µm). (B) Interstitial fibrosis ratio of laryngeal muscles. Fibrosis ratio was quantified by measuring the blue colored area. (C) Cross-sectional area (CSA) of the total muscle fiber. The total CSA was quantified by measuring the red colored

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area. (D) The muscle fiber number was quantified by counting individual muscle fibers in a high-magnified area. (E) CSA of a single muscle fiber. Each area of the muscle fiber was measured. (F-I) Myogenic effects of bFGF-loaded ALG/HA hydrogels were analyzed by evaluating the expression of myogenic-related gene by real-time polymerase chain reaction (qPCR) in laryngeal muscles tissues isolated from each group, 4 weeks post-injection.

Fig 4. Effects of the basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/hyaluronic acid (HA) hydrogel on rejuvenation and hypertrophy of aged laryngeal muscle (12 weeks postinjection). Histological analyses were performed on isolated samples 12 weeks post-infection. (A) Representative Masson’s trichrome images of laryngeal muscle (Scale bar: 100 µm). (B) Representative images of laminin (green) and fast-myosin heavy chain (red) staining (Scale bar: 100 µm). (C) Interstitial fibrosis ratio of laryngeal muscles. (D) Cross-sectional area of the total muscle fiber. (E) The number of muscle fiber was quantified by counting individual muscle fibers in a high-magnified area. (F) Cross-sectional area of a single muscle fiber. Each area of the muscle fiber was measured. (G) Quantification of the number of myofibers having centrally positioned nuclei.

Fig 5. Effects of the basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/hyaluronic acid (HA) hydrogel on the activation of satellite cells and angiogenesis (12 weeks postinfection). (A) Representative images of PAX7 (muscle satellite cell marker) and Laminin staining (scale bar: 100 µm). (B) Representative images of CD31 positive capillaries (green) (scale bar: 100 µm). (C) Representative images of α-smooth muscle actin positive arterioles (red) (scale bar: 200 µm). (D) Significantly enhanced number of PAX7-positive cells in the

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GF+loaded ALG/HA group compared to that in the other experimental groups. (E) Significantly enhanced number of CD31-positive cells in the GF+loaded ALG/HA group. (F) Number of α-SMA-positive vessels in the laryngeal muscle.

Fig 6. High-speed camera setup and functional analysis of larynx (12 weeks post-injection). Twelve weeks post-injection, rat larynxes from each group were isolated and analyzed for functionality. (A) Schematic image of induced phonation setup for the excised larynx. Motion of the vocal fold was recorded by a high-speed camera, and the data was transmitted to a computer (B) Actual image of the high-speed camera setup. (C) The excised larynx was fixed with custom-made fixture. (D) Serial vocal fold vibration images of each group. (E) Representative images of each maximal glottal gap from serial photographs (F) Areas of each maximal glottal gap was measured. Vocal gap area of basic fibroblast growth factor (bFGF)loaded alginate (ALG)/hyaluronic acid (HA) group was significantly reduced similar to that in the Positive control and Sham groups.

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