Paracrine Effects Of Adipose-Derived Stromal/Stem Cells And Stromal Vascular Fraction In An In Vitro Fibrogenesis Model Of Human Vocal Fold Scarring

Paracrine Effects Of Adipose-Derived Stromal/Stem Cells And Stromal Vascular Fraction In An In Vitro Fibrogenesis Model Of Human Vocal Fold Scarring | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short report Paracrine Effects Of Adipose-Derived Stromal/Stem Cells And Stromal Vascular Fraction In An In Vitro Fibrogenesis Model Of Human Vocal Fold Scarring Mélanie Velier, Alexia MATTEI, Stéphanie SIMONCINI, Jérémy MAGALON, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-20745/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Vocal folds (VF) scarring leads to severe dysphonia which negatively impacts daily life of patients. Current therapeutic options are limited due in large part to the high complexity of the micro-structure of the VF. Innovative therapies derived from adipose tissue such as stromal vascular fraction (SVF) or adipose derived stromal/ stem cells (ASC) are currently being evaluated in this indication and paracrine anti-fibrotic effects are considered as predominant mechanisms. Methods: The paracrine anti-fibrotic effects of SVF and ASC from healthy donors were tested in an innovative in vitro fibrogenesis model employing human VF fiboblasts (hVFF) and the principles of macromolecular crowding (MMC). Biosynthesis of collogen and alpha-smooth-muscle actin (αSMA) expression in hVFF were quantified after five days of indirect coculture with ASC or SVF using silver stain, western blot and RT-qPCR analysis. Results: Fibrogenesis was promoted by addition of transforming growth factor beta 1 (TGFβ1) combined with MMC characterized by an enhanced deposition of fibrillar collagens and the acquisition of a myofibroblast phenotype (overexpression of αSMA). Adipose-derived therapies led to a reduction in the αSMA expression and the collagen content was lower in hVFF co-cultivated with SVF. Discussion: ASC and SVF promoted significant prevention of fibrosis in an in vitro fibrogenesis model through paracrine mechanisms, supporting further development of adipose-derived cellular therapies in VF scarring. Stem Cell & Developmental Cell Biology adipose derived stromal cells fibrosis macromolecular crowding vocal fold scarring Figures Figure 1 Figure 2 Introduction Vocal fold (VF) scar is one of the most challenging benign laryngeal pathologies [ 1 ]. Depending on severity and extent, it can result in a range of causes both physical and psychological disability, especially for persons with a high vocal demand, such as teachers, singers or call center agents. VF microstructure is complex [ 2 ], particularly due to its foliated organization allowing optimal vibration. The proportions and the organization of the extracellular matrix (ECM) components largely determine the mechanical properties of the VF. VF scarring is most often the result of laryngeal microsurgery, but it may also be found congenitally (sulcus vocalis) oracquired following a trauma or chronic inflammatory phenomena. Thus, the normal microstructure is replaced by fibrous tissue, with an excessive and disorganized ECM inducing vibration disorder [ 1 ]. Compared to normal fibroblasts, VF scar tissue fibroblasts produce a larger amount of collagen, proliferate more rapidly and overexpress α-smooth-muscle actin (αSMA), also known as actin alpha 2 (ACTA2), a myofibroblast differentiation marker [ 3 , 4 ]. Despite recent advances (medialization, scar resection, microflap, injection of hyaluronic acid, etc.) [ 5 – 8 ], current therapies are often disappointing since they do not restore a normal ECM distribution. Other more experimental treatments such as administration of basic Fibroblast Growth Factor (bFGF) [ 9 , 10 ] or Hepatocyte Growth Factor (HGF) [ 11 – 14 ] have shown encouraging effects in vitro or in vivo on animals. However, only one phase I/II clinical trial assessed the safety and effectiveness of intracordal injection of a recombinant human HGF in patients with vocal fold scar or sulcus [ 15 ]. The results of this first-in-man clinical trial indicated no serious adverse events and encouraging clinical results. Thus, the identification of innovative strategies able to improve the mechanical vibrational properties of VF remains an important clinical challenge. Mesenchymal stromal cells (MSC), which were first described in bone marrow to support hematopoiesis [ 16 ], are multipotent stem cells that can be isolated from a variety of tissues but adipose tissue remains a very attractive source due to its abundance and accessibility. Autologous adipose-derived Stromal Vascular Fraction (SVF) is a heterogeneous population of cells obtained in a few hours after enzymatic digestion of adipose tissue that does not require an expansion step. SVF is also an advantageous source of adipose derived stromal cells (ASC) because of its fluidity and ease to inject. Besides, SVF brings the synergistic effect of its various cellular subpopulations, including ASC but also endothelial progenitor cells combining vasculogenic, anti-fibrotic and anti-inflammatory properties. The exact mechanism of action of ASC and SVF are not fully elucidated but it is now admitted that the major effect rests on paracrine activity through a large type of secreted molecules [ 17 – 19 ]. To facilitate research on this field, Graupp et al. described an in vitro fibrogenesis model using human vocal fold fibroblasts (hVFF) and the principles of macromolecular crowding (MMC) [ 20 ]. MMC promotes fibrogenesis after the addition of transforming growth factor-beta 1 (TGF-β1) to a medium containing inert macromolecules. The aim of this experimental study was to assess the in vitro paracrine potential of SVF cells and ASC on scarred hVFF in this innovative model. Materials And Methods Donors ASC and SVF cells from healthy donors (n = 4) were obtained from adipose tissue surgical residues following liposuction for aesthetic purposes. All patients provided informed consent for the scientific use of surgical residues. Cell culture Immortalized human vocal fold fibroblasts (hVFF) were obtained from the lab of Prof. Susan Thibeault (University of Wisconsin, Madison, USA) [ 21 ]. Dulbecco´s modified Eagle´s medium (DMEM) (Life Technologies, Waltham, Massachusetts) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Vienna, Austria) and 100 µg/mL Normocin (Invivogen, San Diego, California, USA) was used as standard medium (SM). Adipose tissue collection was conducted under conscious sedation with local anesthesia. SVF was obtained in a Cell Therapy Unit using the automated processing Celution 800/CRS system (Cytori Therapeutics Inc., San Diego, California, USA) as previously described [ 22 ]. Collected lipoaspirate was washed and enzymatically digested to obtain SVF in accordance with the European Regulations and Good Manufacturing Practices (GMP) for ‘Advanced Therapy Medicinal Products’. To isolate the ASC, SVF was then plated into a T75 cell culture flask in a culture medium consisting of DMEM supplemented with 50% Ham's F-12 Nutrient Mix (Thermo Scientific, Waltham, Massachusetts, USA ), 10% FCS, GlutaMAX (100X, Thermo Scientific), Gentamicin and Penicillin G (both Panpharma, Luitré, France), Fungizone (Bristol-Meyers Squibb, New York, NY, USA). Adherent cells were grown under standard cell culture conditions (5% CO 2 , 37 °C, humidified), detached with Trypsin-EDTA and passed to extend the culture. For co-culture experiments, hVFF from passages 13–20 were seeded into cell culture plates (15.000 cells/cm 2 ). The following day, SM was switched to FCS-free SM for another 24 h. Cells were then allocated to different treatment groups. For the control group DMEM enriched with 0.5% FCS, 100 µg/mL Normocin and 100 nM of L-ascorbic acid 2-phosphate was used [ 23 ]. Myofibroblast differentiation was induced by adding of 5 ng/mL TGF-β1 [ 24 ] combined with “macromolecular crowding” (MMC) by adding a mixture of 37.5 mg/mL 70 kDa Ficoll (Fc) with 25 mg/mL 400 kDa Fc (both Sigma-Aldrich) [ 25 , 26 ]. To assess possible anti-fibrotic effects, three conditions were tested in co-culture with hVFF: (i) recombinant HGF (50 ng/mL, Sigma-Aldrich), (ii) paracrine effect of thawed SVF and (iii) paracrine effect of thawed ASC from passages 3–5. After 5 days of incubation, samples were further processed. Reverse Transcription-qPCR (RT-qPCR) RNA isolation of hVFF was performed using the QIAZOL Lysis Reagent (Qiagen, Hilden, Germany) and total mRNA was isolated with the miRNeasy Mini Kit (Qiagen) according to the manufacturer´s instructions. Purified RNA was eluated in RNAse-free water and concentration was determined using the NanoDrop 2000c spectrophotometer (Thermo Scientific). Reverse transcription (RT), as well as RT quantitative PCR (RT-qPCR), was performed as previously described [ 27 ]. Primer sequences are provided in Table 1. Relative quantification of all mRNAs of interest was performed based on the 2 −ΔΔC T method [ 28 ]. Pepsin digestion, sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain Supernatants and cell layers from hVFF seeded in 24-well-plates were harvested separately and pepsinized as previously described [ 29 ]. Briefly, 50 µl of a pepsin stock solution (1 mg/mL dissolved in 1N HCL) were added to 500 µL of supernatants, while a pepsin digestion solution (25% pepsin stock solution, 0.005% Triton X-100 in ddH 2 O) was added to cell layers. All samples were incubated for 2 h followed by neutralization with 1 N NaOH. SDS-PAGE was performed under non-reducing conditions using 3–8% precast Criterion XT Tris-Acetate gels and XT Tricine running buffer (both Bio-Rad, Hercules, California, USA); electrophoresis was run for 70 min at 200 V. Vitro-Col, human collagen I solution served as collagen standard (0.16 µg/lane). Gels were subsequently stained with the SilverQuest™ Silver Staining kit (Thermo Scientific) according to the manufacturer´s protocol. Gel images were acquired using Quantity One software (Bio-Rad) and densitometric analysis of bands was performed using Image Lab Software, Version 5.2. Western blot Proteins were extracted from hVFF cell layers seeded in 6-well-plates and subjected to SDS-PAGE using 4–12% Criterion XT Bis-Tris Gels (Bio-Rad). SDS-PAGE was run at 200 V for 60 min, followed by electroblotting of proteins (90 min at 0.5 A and 4 °C) onto Nitrocellulose membranes (Bio-Rad). Immuno-detection was carried out in Tris-buffered saline supplemented with 0.1% Tween-20 (Carl Roth, Karlsruhe, Germany) and 5% milk. Membranes were incubated overnight at 4 °C with primary antibodies for detection of ACTA2 (#A5228, Sigma Aldrich, 1:1000) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #2118C, Cell Signaling, Danvers, Massachusetts, USA, 1:5000). Subsequently, blots were incubated with secondary antibody (Goat Anti-Mouse 1:5000 for ACTA2; Goat Anti-Rabbit 1:5000 for GAPDH, both abcam, Cambridge, UK). Detection was conducted using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc Touch Imaging System (bio-rad), densitometric analysis was performed using Image Lab Software, Version 5.2. Results Validation of the MMC/TGF-β1 model We first validated the myofibroblast differentiation induced by TGF-β1 combined with MMC (MMC/TGF-β1) through the assessment of ACTA2 and Collagen Iα1 (COL1A1) contents in hVFF. Results from RT-qPCR analysis revealed that mRNA expression of ACTA2 and COL1A1 were significantly up-regulated under MMC/TGF-β1 condition (p = 0.019 and p = 0.002 respectively) (Fig. 1 A and 2 A) compared to the negative condition (untreated fibroblasts). Western blot and silver stain analysis performed on hVFF protein lysates were in line with the previous data: we observed a significant elevation of the ACTA2/GADPH ratio (p = 0.024) and of fibrillar collagen contents (p = 0.0004) in the MMC/TGF-β1 condition compared to the untreated condition (Fig. 1 B and 1 C). Altogether, these data validated the myofibroblast phenotype acquisition of immortalized hVFF after MMC/TGF-β1 treatment. Co-culture The expression of the myofibroblast marker ACTA2 and collagen content of hVFF co-cultured with ASC or SVF or treated with HGF for 5 days were assessed. The results revealed a reduction in the mRNA level of ACTA2 on hVFF when they were co-cultured with ASC and SVF (p = 0.043 and p = 0.028 respectively) (Fig. 1 A). Western blot analysis confirmed these results highlighting a tendency to reduce the level of ACTA2 in hVFF co-cultured with both ASC and SVF (p = 0.061 and p = 0.083 respectively) (Fig. 1 B and 1 C). Figure 2 represents the expression of mRNA of COL1A1 and fibrillar collagen in protein lysates of hVFF. We observed no significant reduction in the COL1A1 mRNA expression (Fig. 2 A) in both ASC and SVF co-culture conditions whereas silver stain analysis revealed a significant reduction of fibrillar collagen content in hVFF after co-culture with SVF (p = 0.006) (Fig. 2 B and 2 C). Of note, no significant reduction of ACTA2 or collagen levels were obtained when hVFF were treated with HGF (Figs. 1 and 2 ). Discussion Vocal fold scarring remains one of the most difficult conditions to treat among benign VF diseases and adipose-derived cell therapies represent hope for these patients. The therapeutic potential of MSC has largely been attributed to paracrine activity [ 30 , 31 ]. In this study, we demonstrated that ASC and SVF promoted significant prevention of fibrosis in an in vitro model through paracrine mechanisms. Previously, a similar study explored whether ASC have therapeutic potential for treating vocal fold scarring through the effects of HGF on scar fibroblasts in an indirect co-culture model [ 4 ]. They showed that co-culture with ASC led to a significant decrease of ACTA2 expression in fibroblasts comforting our data. However, they also demonstrated a reduction in collagen production in the ASC co-culture conditions whereas, in our study, we did not observe significant effect of ASC on the fibrillar collagen content. Compared with ASC, SVF can be real time obtained in a sufficient quantity without in vitro culture, reducing drastically the delay of preparation. Yasuda et al . reported that injection of SVF had a protective effect in an acute kidney injury in vivo model by secreting renoprotective molecules, such as HGF and VEGF [ 32 ]. We can hypothesize that in the present study, SVF and ASC reduced the ACTA2 and collagen expression in hVFF partly by secreting those growth factors transferred through the co-culture system. Only conditions with SVF showed a reduction in both ACTA2 and collagen contents which might be attributed to the higher secretion of soluble mediators by SVF. In line with this, the secretome of ASC and SVF has been compared recently and revealed that SVF released a greater variety of cytokines or soluble protein at a significantly higher amounts as compared with ASC [ 33 ]. Several in vivo studies on animals reported significant improvements in scarred vocal folds after injection of ASC (immediately after injury or at a longer interval) in both histological and macroscopic aspects of the vocal folds [ 34 ] with an excellent tolerance of the procedure. A first clinical trial of SVF injection which enrolled 8 patients was recently reported by our group [ 35 ] suggesting a clear improvement in the majority of the vocal parameters obtained at 12 months. However, the etiology of vocal fold scarring is very heterogenous so a better understanding of the mechanisms underlying the efficacy of ASC and SVF is mandatory. In addition, identification of in vitro potency assay able to anticipate the anti-fibrotic potential of the cell therapy product could allow a better selection of patients who can benefit from these innovative therapies. Based on our results, it appears that adipose derived-cellular therapies are interesting candidates for reducing myofibroblast phenotype acquisition. Nevertheless, global approaches to further characterize ASC and SVF anti-fibrotic potential and studies on a large scale are further needed to better characterize the mechanisms of action. Abbreviations VF Vocal fold ECM extracellular matrix αSMA alpha-smooth-muscle actin ACTA2 actin alpha 2 bFGF basic Fibroblasts Growth Factor HGF Hepatocyte Growth Factor MSC Mesenchymal stromal cells SVF Stromal Vascular Fraction ASC adipose derived stromal cells hVFF human vocal fold fibroblasts MMC macromolecular crowding TGF-β1 transforming growth factor beta 1 DMEM Dulbecco´s modified Eagle´s medium FCS fetal calf serum SM standard medium GMP Good Manufacturing Practices RT Reverse Transcription RT-qPCR Reverse Transcription quantitative Polymerase Chain Reaction GAPDH glyceraldehyde-3-phosphate dehydrogenase SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis VEGF vascular endothelial growth factor COL1A1 Collagen 1 alpha 1 Declarations Ethics approval and consent to participate All patients provided informed consent for the scientific use of surgical residues. Consent for publication: Not applicable Funding This work was supported by PHC Amadeus Campus 2017 (grant number FR05/2017). Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Author’s contributions: Conceptualization : AM, AG, FS, MG, TG Methology: MV, AM, JM, AG, FS, MG, TG Funding acquisition: AM, AG, FS, MG, TG Investigation: MV, AM, SS, LG, LA, TG Data interpretation : MV, AM, SS, JM, AG, FDG, FS, MG, TG Original draft : MV, AM Revise the manuscript : JM, FDG, FS, MG, TG All authors read and approved the final manuscript. Acknowledgments: Not applicable References Hantzakos A, Dikkers FG, Giovanni A, Benninger MS, Remacle M, Sjögren EV, et al. Vocal fold scars: a common classification proposal by the American Laryngological Association and European Laryngological Society. 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JAMA Otolaryngol Neck Surg [Internet]. 2020 Feb 13; Available from: ttps://jamanetwork.com/journals/jamaotolaryngology/fullarticle/2760908 Table Table 1: Primer sequences used for RT-qPCR Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-20745","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short report","associatedPublications":[],"authors":[{"id":464568,"identity":"f19b68ec-125d-4bc2-b1ce-168ba30bfd38","order_by":1,"name":"Mélanie Velier","email":"","orcid":"https://orcid.org/0000-0002-1121-8270","institution":"Assistance Publique Hopitaux de Marseille","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mélanie","middleName":"","lastName":"Velier","suffix":""},{"id":464569,"identity":"f821a062-a653-4c6b-b3ab-5f0c329ac9c6","order_by":2,"name":"Alexia 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Publique Hopitaux de Marseille","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Laurent","middleName":"","lastName":"GIRAUDO","suffix":""},{"id":464573,"identity":"1750f519-aa04-4b68-80ca-db15c9bf0ed4","order_by":6,"name":"Laurent ARNAUD","email":"","orcid":"","institution":"Assistance Publique Hopitaux de Marseille","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Laurent","middleName":"","lastName":"ARNAUD","suffix":""},{"id":464574,"identity":"edaf3710-13f3-46cc-9f43-4fbb1ef59944","order_by":7,"name":"Antoine GIOVANNI","email":"","orcid":"","institution":"Assistance Publique Hopitaux de Marseille","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Antoine","middleName":"","lastName":"GIOVANNI","suffix":""},{"id":464575,"identity":"d11c9559-cb84-4316-9159-c53529b88b03","order_by":8,"name":"Francoise DIGNAT GEORGE","email":"","orcid":"","institution":"Aix-Marseille Universite","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francoise","middleName":"DIGNAT","lastName":"GEORGE","suffix":""},{"id":464576,"identity":"f6309657-54a2-4036-81ba-93dc7a6e92b3","order_by":9,"name":"Florence SABATIER","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIie3OMWuDQBTA8Xcc6PJa1xcM5itEBO1Qkq9yQXByz9YIhevS7PpFJKMg1CXfwMVSyNShXUIDDfRCICFBa8cO94fjHQc/3gHodP8w8+l8N9Qh9ZQAsOQ428LygghFsDiSw/wLOSwSPYTzl4/P1cQBq9w0u687J8jeNg1b5YB2u0FuhFm2Dj2gKHCfBXnDOgrGbF0D3opWMuXo8RtZzBICn1DQLLWFb+9lDVPs+pgie1ksEqvaDr4FLdJBtSWmCP5GmCzUH2LfVlsEEfo9xAjZUoaupHjuDSNyU4znY0Wwk5iPJezkZGRZVf76fv8wIrPKG0WcLnLKuL73gW6u0+l0OvgBJD5Mwnqm6YgAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Florence","middleName":"","lastName":"SABATIER","suffix":""},{"id":464577,"identity":"d2689dd9-b836-4565-aaca-25342af8113d","order_by":10,"name":"Markus GUGATSCHKA","email":"","orcid":"","institution":"Medizinische Universitat Graz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Markus","middleName":"","lastName":"GUGATSCHKA","suffix":""},{"id":464578,"identity":"90843dc1-7389-4310-b5b5-07a8f4bb9518","order_by":11,"name":"Tanja GROSSMANN","email":"","orcid":"","institution":"Medizinische Universitat Graz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tanja","middleName":"","lastName":"GROSSMANN","suffix":""}],"badges":[],"createdAt":"2020-04-01 11:59:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-20745/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-20745/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":828247,"identity":"c5dbc986-3001-4a18-a21c-e1a2927fd81a","added_by":"auto","created_at":"2020-04-02 21:23:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78373,"visible":true,"origin":"","legend":"(A) mRNA expression level of ACTA2 by RT-qPCR. Data is presented as mean ± S.E.M.. Statistical analysis was performed using Mann-Whitney test. (B) The protein level of ACTA2 was examined by Western blot analysis on whole cells lysates from hVFF after co-culture with ASC (n= 4) and SVF (n=4). Representative blots for ACTA2 and GAPDH (loading control) are shown. (C) Densitometric data from ACTA2 protein levels. Data is presented as mean ± S.E.M., normalized to GAPDH protein levels and relative to the untreated condition (negative). Statistical analysis was performed using Mann-Whitney test","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-20745/v1/fig1.png"},{"id":828248,"identity":"94f32fdc-7e92-4c72-b9a2-29cce27f0c11","added_by":"auto","created_at":"2020-04-02 21:23:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38324,"visible":true,"origin":"","legend":"(A) mRNA expression level of COL1A1 by RT-qPCR. Data is presented as mean ± S.E.M.. Statistical analysis was performed using Mann-Whitney test. (B) Densitometric data from fibrillar collagen I protein levels. Data is presented as mean ± S.E.M., normalized to collagen I protein levels of alpha2 chain under crowded condition with TGF-β1, and relative to the untreated condition (negative). Statistical analysis was performed using Mann-Whitney test","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-20745/v1/fig2.png"},{"id":15666648,"identity":"0987e721-9893-4712-bed3-3fa5c31e10d8","added_by":"auto","created_at":"2021-11-18 13:37:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":462547,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-20745/v1/e4ac29d9-800c-49d5-aac7-4f95555c4c46.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eParacrine Effects Of Adipose-Derived Stromal/Stem Cells And Stromal Vascular Fraction In An \u003cem\u003eIn Vitro \u003c/em\u003eFibrogenesis Model Of Human Vocal Fold Scarring\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eVocal fold (VF) scar is one of the most challenging benign laryngeal pathologies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Depending on severity and extent, it can result in a range of causes both physical and psychological disability, especially for persons with a high vocal demand, such as teachers, singers or call center agents.\u003c/p\u003e \u003cp\u003eVF microstructure is complex [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], particularly due to its foliated organization allowing optimal vibration. The proportions and the organization of the extracellular matrix (ECM) components largely determine the mechanical properties of the VF. VF scarring is most often the result of laryngeal microsurgery, but it may also be found congenitally (sulcus vocalis) oracquired following a trauma or chronic inflammatory phenomena. Thus, the normal microstructure is replaced by fibrous tissue, with an excessive and disorganized ECM inducing vibration disorder [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Compared to normal fibroblasts, VF scar tissue fibroblasts produce a larger amount of collagen, proliferate more rapidly and overexpress α-smooth-muscle actin (αSMA), also known as actin alpha 2 (ACTA2), a myofibroblast differentiation marker [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite recent advances (medialization, scar resection, microflap, injection of hyaluronic acid, etc.) [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], current therapies are often disappointing since they do not restore a normal ECM distribution. Other more experimental treatments such as administration of basic Fibroblast Growth Factor (bFGF) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] or Hepatocyte Growth Factor (HGF) [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] have shown encouraging effects \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e or \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e on animals. However, only one phase I/II clinical trial assessed the safety and effectiveness of intracordal injection of a recombinant human HGF in patients with vocal fold scar or sulcus [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The results of this first-in-man clinical trial indicated no serious adverse events and encouraging clinical results. Thus, the identification of innovative strategies able to improve the mechanical vibrational properties of VF remains an important clinical challenge.\u003c/p\u003e \u003cp\u003eMesenchymal stromal cells (MSC), which were first described in bone marrow to support hematopoiesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], are multipotent stem cells that can be isolated from a variety of tissues but adipose tissue remains a very attractive source due to its abundance and accessibility. Autologous adipose-derived Stromal Vascular Fraction (SVF) is a heterogeneous population of cells obtained in a few hours after enzymatic digestion of adipose tissue that does not require an expansion step. SVF is also an advantageous source of adipose derived stromal cells (ASC) because of its fluidity and ease to inject. Besides, SVF brings the synergistic effect of its various cellular subpopulations, including ASC but also endothelial progenitor cells combining vasculogenic, anti-fibrotic and anti-inflammatory properties. The exact mechanism of action of ASC and SVF are not fully elucidated but it is now admitted that the major effect rests on paracrine activity through a large type of secreted molecules [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo facilitate research on this field, Graupp et al. described an \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e fibrogenesis model using human vocal fold fibroblasts (hVFF) and the principles of macromolecular crowding (MMC) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. MMC promotes fibrogenesis after the addition of transforming growth factor-beta 1 (TGF-β1) to a medium containing inert macromolecules. The aim of this experimental study was to assess the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e paracrine potential of SVF cells and ASC on scarred hVFF in this innovative model.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDonors\u003c/h2\u003e \u003cp\u003eASC and SVF cells from healthy donors (n\u0026thinsp;=\u0026thinsp;4) were obtained from adipose tissue surgical residues following liposuction for aesthetic purposes. All patients provided informed consent for the scientific use of surgical residues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eImmortalized human vocal fold fibroblasts (hVFF) were obtained from the lab of Prof. Susan Thibeault (University of Wisconsin, Madison, USA) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Dulbecco\u0026acute;s modified Eagle\u0026acute;s medium (DMEM) (Life Technologies, Waltham, Massachusetts) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Vienna, Austria) and 100\u0026nbsp;\u0026micro;g/mL Normocin (Invivogen, San Diego, California, USA) was used as standard medium (SM). Adipose tissue collection was conducted under conscious sedation with local anesthesia. SVF was obtained in a Cell Therapy Unit using the automated processing Celution 800/CRS system (Cytori Therapeutics Inc., San Diego, California, USA) as previously described [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Collected lipoaspirate was washed and enzymatically digested to obtain SVF in accordance with the European Regulations and Good Manufacturing Practices (GMP) for \u0026lsquo;Advanced Therapy Medicinal Products\u0026rsquo;. To isolate the ASC, SVF was then plated into a T75 cell culture flask in a culture medium consisting of DMEM supplemented with 50% Ham's F-12 Nutrient Mix (Thermo Scientific, Waltham, Massachusetts, USA ), 10% FCS, GlutaMAX (100X, Thermo Scientific), Gentamicin and Penicillin G (both Panpharma, Luitr\u0026eacute;, France), Fungizone (Bristol-Meyers Squibb, New York, NY, USA). Adherent cells were grown under standard cell culture conditions (5% CO\u003csub\u003e2\u003c/sub\u003e, 37\u0026nbsp;\u0026deg;C, humidified), detached with Trypsin-EDTA and passed to extend the culture. For co-culture experiments, hVFF from passages 13\u0026ndash;20 were seeded into cell culture plates (15.000 cells/cm\u003csup\u003e2\u003c/sup\u003e). The following day, SM was switched to FCS-free SM for another 24\u0026nbsp;h. Cells were then allocated to different treatment groups. For the control group DMEM enriched with 0.5% FCS, 100\u0026nbsp;\u0026micro;g/mL Normocin and 100\u0026nbsp;nM of L-ascorbic acid 2-phosphate was used [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Myofibroblast differentiation was induced by adding of 5\u0026nbsp;ng/mL TGF-β1 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] combined with \u0026ldquo;macromolecular crowding\u0026rdquo; (MMC) by adding a mixture of 37.5\u0026nbsp;mg/mL 70\u0026nbsp;kDa Ficoll (Fc) with 25\u0026nbsp;mg/mL 400\u0026nbsp;kDa Fc (both Sigma-Aldrich) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To assess possible anti-fibrotic effects, three conditions were tested in co-culture with hVFF: (i) recombinant HGF (50\u0026nbsp;ng/mL, Sigma-Aldrich), (ii) paracrine effect of thawed SVF and (iii) paracrine effect of thawed ASC from passages 3\u0026ndash;5. After 5 days of incubation, samples were further processed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eReverse Transcription-qPCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eRNA isolation of hVFF was performed using the QIAZOL Lysis Reagent (Qiagen, Hilden, Germany) and total mRNA was isolated with the miRNeasy Mini Kit (Qiagen) according to the manufacturer\u0026acute;s instructions. Purified RNA was eluated in RNAse-free water and concentration was determined using the NanoDrop 2000c spectrophotometer (Thermo Scientific). Reverse transcription (RT), as well as RT quantitative PCR (RT-qPCR), was performed as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Primer sequences are provided in Table\u0026nbsp;1. Relative quantification of all mRNAs of interest was performed based on the 2\u003csup\u003e\u0026minus;ΔΔC\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePepsin digestion, sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain\u003c/h2\u003e \u003cp\u003eSupernatants and cell layers from hVFF seeded in 24-well-plates were harvested separately and pepsinized as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, 50\u0026nbsp;\u0026micro;l of a pepsin stock solution (1\u0026nbsp;mg/mL dissolved in 1N HCL) were added to 500\u0026nbsp;\u0026micro;L of supernatants, while a pepsin digestion solution (25% pepsin stock solution, 0.005% Triton X-100 in ddH\u003csub\u003e2\u003c/sub\u003eO) was added to cell layers. All samples were incubated for 2\u0026nbsp;h followed by neutralization with 1\u0026nbsp;N NaOH.\u003c/p\u003e \u003cp\u003eSDS-PAGE was performed under non-reducing conditions using 3\u0026ndash;8% precast Criterion XT Tris-Acetate gels and XT Tricine running buffer (both Bio-Rad, Hercules, California, USA); electrophoresis was run for 70\u0026nbsp;min at 200\u0026nbsp;V. Vitro-Col, human collagen I solution served as collagen standard (0.16\u0026nbsp;\u0026micro;g/lane). Gels were subsequently stained with the SilverQuest\u0026trade; Silver Staining kit (Thermo Scientific) according to the manufacturer\u0026acute;s protocol. Gel images were acquired using Quantity One software (Bio-Rad) and densitometric analysis of bands was performed using Image Lab Software, Version 5.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eProteins were extracted from hVFF cell layers seeded in 6-well-plates and subjected to SDS-PAGE using 4\u0026ndash;12% Criterion XT Bis-Tris Gels (Bio-Rad). SDS-PAGE was run at 200\u0026nbsp;V for 60\u0026nbsp;min, followed by electroblotting of proteins (90\u0026nbsp;min at 0.5\u0026nbsp;A and 4\u0026nbsp;\u0026deg;C) onto Nitrocellulose membranes (Bio-Rad). Immuno-detection was carried out in Tris-buffered saline supplemented with 0.1% Tween-20 (Carl Roth, Karlsruhe, Germany) and 5% milk. Membranes were incubated overnight at 4\u0026nbsp;\u0026deg;C with primary antibodies for detection of ACTA2 (#A5228, Sigma Aldrich, 1:1000) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #2118C, Cell Signaling, Danvers, Massachusetts, USA, 1:5000). Subsequently, blots were incubated with secondary antibody (Goat Anti-Mouse 1:5000 for ACTA2; Goat Anti-Rabbit 1:5000 for GAPDH, both abcam, Cambridge, UK). Detection was conducted using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc Touch Imaging System (bio-rad), densitometric analysis was performed using Image Lab Software, Version 5.2.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eValidation of the MMC/TGF-β1 model\u003c/h2\u003e \u003cp\u003eWe first validated the myofibroblast differentiation induced by TGF-β1 combined with MMC (MMC/TGF-β1) through the assessment of ACTA2 and Collagen Iα1 (COL1A1) contents in hVFF. Results from RT-qPCR analysis revealed that mRNA expression of ACTA2 and COL1A1 were significantly up-regulated under MMC/TGF-β1 condition (p\u0026thinsp;=\u0026thinsp;0.019 and p\u0026thinsp;=\u0026thinsp;0.002 respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) compared to the negative condition (untreated fibroblasts). Western blot and silver stain analysis performed on hVFF protein lysates were in line with the previous data: we observed a significant elevation of the ACTA2/GADPH ratio (p\u0026thinsp;=\u0026thinsp;0.024) and of fibrillar collagen contents (p\u0026thinsp;=\u0026thinsp;0.0004) in the MMC/TGF-β1 condition compared to the untreated condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Altogether, these data validated the myofibroblast phenotype acquisition of immortalized hVFF after MMC/TGF-β1 treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCo-culture\u003c/h2\u003e \u003cp\u003eThe expression of the myofibroblast marker ACTA2 and collagen content of hVFF co-cultured with ASC or SVF or treated with HGF for 5 days were assessed. The results revealed a reduction in the mRNA level of ACTA2 on hVFF when they were co-cultured with ASC and SVF (p\u0026thinsp;=\u0026thinsp;0.043 and p\u0026thinsp;=\u0026thinsp;0.028 respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Western blot analysis confirmed these results highlighting a tendency to reduce the level of ACTA2 in hVFF co-cultured with both ASC and SVF (p\u0026thinsp;=\u0026thinsp;0.061 and p\u0026thinsp;=\u0026thinsp;0.083 respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e represents the expression of mRNA of COL1A1 and fibrillar collagen in protein lysates of hVFF. We observed no significant reduction in the COL1A1 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) in both ASC and SVF co-culture conditions whereas silver stain analysis revealed a significant reduction of fibrillar collagen content in hVFF after co-culture with SVF (p\u0026thinsp;=\u0026thinsp;0.006) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Of note, no significant reduction of ACTA2 or collagen levels were obtained when hVFF were treated with HGF (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eVocal fold scarring remains one of the most difficult conditions to treat among benign VF diseases and adipose-derived cell therapies represent hope for these patients. The therapeutic potential of MSC has largely been attributed to paracrine activity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, we demonstrated that ASC and SVF promoted significant prevention of fibrosis in an \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e model through paracrine mechanisms.\u003c/p\u003e \u003cp\u003ePreviously, a similar study explored whether ASC have therapeutic potential for treating vocal fold scarring through the effects of HGF on scar fibroblasts in an indirect co-culture model [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. They showed that co-culture with ASC led to a significant decrease of ACTA2 expression in fibroblasts comforting our data. However, they also demonstrated a reduction in collagen production in the ASC co-culture conditions whereas, in our study, we did not observe significant effect of ASC on the fibrillar collagen content.\u003c/p\u003e \u003cp\u003eCompared with ASC, SVF can be real time obtained in a sufficient quantity without \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e culture, reducing drastically the delay of preparation. Yasuda \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eet al\u003c/span\u003e. reported that injection of SVF had a protective effect in an acute kidney injury \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e model by secreting renoprotective molecules, such as HGF and VEGF [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We can hypothesize that in the present study, SVF and ASC reduced the ACTA2 and collagen expression in hVFF partly by secreting those growth factors transferred through the co-culture system. Only conditions with SVF showed a reduction in both ACTA2 and collagen contents which might be attributed to the higher secretion of soluble mediators by SVF. In line with this, the secretome of ASC and SVF has been compared recently and revealed that SVF released a greater variety of cytokines or soluble protein at a significantly higher amounts as compared with ASC [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e studies on animals reported significant improvements in scarred vocal folds after injection of ASC (immediately after injury or at a longer interval) in both histological and macroscopic aspects of the vocal folds [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] with an excellent tolerance of the procedure. A first clinical trial of SVF injection which enrolled 8 patients was recently reported by our group [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] suggesting a clear improvement in the majority of the vocal parameters obtained at 12 months. However, the etiology of vocal fold scarring is very heterogenous so a better understanding of the mechanisms underlying the efficacy of ASC and SVF is mandatory. In addition, identification of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e potency assay able to anticipate the anti-fibrotic potential of the cell therapy product could allow a better selection of patients who can benefit from these innovative therapies.\u003c/p\u003e \u003cp\u003eBased on our results, it appears that adipose derived-cellular therapies are interesting candidates for reducing myofibroblast phenotype acquisition. Nevertheless, global approaches to further characterize ASC and SVF anti-fibrotic potential and studies on a large scale are further needed to better characterize the mechanisms of action.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVocal fold\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eαSMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealpha-smooth-muscle actin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACTA2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eactin alpha 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ebFGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebasic Fibroblasts Growth Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHepatocyte Growth Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMesenchymal stromal cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSVF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStromal Vascular Fraction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadipose derived stromal cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ehVFF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman vocal fold fibroblasts\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emacromolecular crowding\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTGF-β1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransforming growth factor beta 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026acute;s modified Eagle\u0026acute;s medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFCS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal calf serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGood Manufacturing Practices\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReverse Transcription\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReverse Transcription quantitative Polymerase Chain Reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS-PAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium dodecylsulphate-polyacrylamide gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVEGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evascular endothelial growth factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOL1A1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCollagen 1 alpha 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients provided informed consent for the scientific use of surgical residues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication: \u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by PHC Amadeus Campus 2017 (grant number FR05/2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization : AM, AG, FS,\u0026nbsp;MG, TG\u003c/p\u003e\n\u003cp\u003eMethology: MV, AM, JM, AG, FS, MG, TG\u003c/p\u003e\n\u003cp\u003eFunding acquisition:\u0026nbsp;AM, AG, FS,\u0026nbsp;MG, TG\u003c/p\u003e\n\u003cp\u003eInvestigation: MV, AM, SS, LG, LA, TG\u003c/p\u003e\n\u003cp\u003eData interpretation :\u0026nbsp;MV, AM, SS, JM, AG, FDG, FS, MG, TG\u003c/p\u003e\n\u003cp\u003eOriginal draft : MV, AM\u003c/p\u003e\n\u003cp\u003eRevise the manuscript : JM, FDG, FS, MG, TG\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments: \u003c/strong\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHantzakos A, Dikkers FG, Giovanni A, Benninger MS, Remacle M, Sj\u0026ouml;gren EV, et al. 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J Tissue Eng Regen Med. 2018 Apr;12(4):1031\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eFriedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976 Sep;4(5):267\u0026ndash;74.\u003c/li\u003e\n\u003cli\u003eLiang X, Ding Y, Zhang Y, Tse H-F, Lian Q. Paracrine Mechanisms of Mesenchymal Stem Cell-Based Therapy: Current Status and Perspectives. Cell Transplant. 2014 Sep;23(9):1045\u0026ndash;59.\u003c/li\u003e\n\u003cli\u003eSpees JL, Lee RH, Gregory CA. Mechanisms of mesenchymal stem/stromal cell function. Stem Cell Res Ther. 2016 31;7(1):125.\u003c/li\u003e\n\u003cli\u003eDykstra JA, Facile T, Patrick RJ, Francis KR, Milanovich S, Weimer JM, et al. Concise Review: Fat and Furious: Harnessing the Full Potential of Adipose-Derived Stromal Vascular Fraction. Stem Cells Transl Med. 2017;6(4):1096\u0026ndash;108.\u003c/li\u003e\n\u003cli\u003eGraupp M, Gruber H-J, Weiss G, Kiesler K, Bachna-Rotter S, Friedrich G, et al. Establishing principles of macromolecular crowding for in vitro fibrosis research of the vocal fold lamina propria: MMC for Vocal Fold Fibrosis Research. The Laryngoscope. 2015 Jun;125(6):E203\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eChen X, Thibeault SL. Novel Isolation and Biochemical Characterization of Immortalized Fibroblasts for Tissue Engineering Vocal Fold Lamina Propria. Tissue Eng Part C Methods. 2009 Jun;15(2):201\u0026ndash;12.\u003c/li\u003e\n\u003cli\u003eGranel B, Daumas A, Jouve E, Harl\u0026eacute; J-R, Nguyen P-S, Chabannon C, et al. Safety, tolerability and potential efficacy of injection of autologous adipose-derived stromal vascular fraction in the fingers of patients with systemic sclerosis: an open-label phase I trial. Ann Rheum Dis. 2015 Dec;74(12):2175\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eHata R-I, Senoo H. L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts. J Cell Physiol. 1989 Jan;138(1):8\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003eVyas B, Ishikawa K, Duflo S, Chen X, Thibeault SL. Inhibitory effects of hepatocyte growth factor and interleukin-6 on transforming growth factor-beta1 mediated vocal fold fibroblast-myofibroblast differentiation. Ann Otol Rhinol Laryngol. 2010 May;119(5):350\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eChen C, Peng Y, Wang Z, Fish P, Kaar J, Koepsel R, et al. The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well: Scar-in-a-Jar to assess potential antifibrotics. Br J Pharmacol. 2009 Nov;158(5):1196\u0026ndash;209.\u003c/li\u003e\n\u003cli\u003eChen C, Loe F, Blocki A, Peng Y, Raghunath M. Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies. Adv Drug Deliv Rev. 2011 Apr;63(4\u0026ndash;5):277\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eKarbiener M, Darnhofer B, Frisch M-T, Rinner B, Birner-Gruenberger R, Gugatschka M. Comparative proteomics of paired vocal fold and oral mucosa fibroblasts. J Proteomics. 2017 Feb;155:11\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u0026minus;\u0026Delta;\u0026Delta;CT Method. Methods. 2001 Dec;25(4):402\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eSteinmann B, Rao VH, Vogel A, Bruckner P, Gitzelmann R, Byers PH. Cysteine in the triple-helical domain of one allelic product of the alpha 1(I) gene of type I collagen produces a lethal form of osteogenesis imperfecta. J Biol Chem. 1984 Sep 10;259(17):11129\u0026ndash;38.\u003c/li\u003e\n\u003cli\u003eBronckaers A, Hilkens P, Martens W, Gervois P, Ratajczak J, Struys T, et al. Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis. Pharmacol Ther. 2014 Aug;143(2):181\u0026ndash;96.\u003c/li\u003e\n\u003cli\u003eNauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007 Nov 15;110(10):3499\u0026ndash;506.\u003c/li\u003e\n\u003cli\u003eYasuda K, Ozaki T, Saka Y, Yamamoto T, Gotoh M, Ito Y, et al. Autologous cell therapy for cisplatin-induced acute kidney injury by using non-expanded adipose tissue-derived cells. Cytotherapy. 2012 Oct;14(9):1089\u0026ndash;100.\u003c/li\u003e\n\u003cli\u003eHirose Y, Funahashi Y, Matsukawa Y, Majima T, Yamaguchi M, Kawabata S, et al. Comparison of trophic factors secreted from human adipose-derived stromal vascular fraction with those from adipose-derived stromal/stem cells in the same individuals. Cytotherapy. 2018;20(4):589\u0026ndash;91.\u003c/li\u003e\n\u003cli\u003eMattei A, Magalon J, Bertrand B, Philandrianos C, Veran J, Giovanni A. Cell therapy and vocal fold scarring. Eur Ann Otorhinolaryngol Head Neck Dis. 2017 Oct 1;134(5):339\u0026ndash;45.\u003c/li\u003e\n\u003cli\u003eMattei A, Bertrand B, Jouve E, Blaise T, Philandrianos C, Grimaud F, et al. Feasibility of First Injection of Autologous Adipose Tissue\u0026ndash;Derived Stromal Vascular Fraction in Human Scarred Vocal Folds: A Nonrandomized Controlled Trial. JAMA Otolaryngol Neck Surg [Internet]. 2020 Feb 13; Available from: ttps://jamanetwork.com/journals/jamaotolaryngology/fullarticle/2760908\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:16px;line-height:150%;\"\u003eTable 1: Primer sequences used for RT-qPCR\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:.0001pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style=\"font-size:16px;line-height:150%;\"\u003e\u0026nbsp;\u003cimg 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LUWXMYzXGS5ruFLx5rCbRpWsfDwsNZ1pmIUmF3fjHKE+Q852ud61i0lN+QrEZ8z+o3UzF4BTD/difxwJqC65ZlhzKE9LJ9+vhpJMnkkj7r+eUB7cHWPPz50Ayn0Fa9gq6juXzdLbtd5+/S07RHhIYzFKzyxldnVR6HBaOsGLVSSvM7envHDAOtdiPVW/HF7yAcwTRqusirYqFjzxaPslwYE+qtiabTv4YsSVGsVp+pMNDGDJdb8ma3fSGWu1sQMHAHyrwPLsnBYMrrSGbLG+8xRdjutMPMx5rDOJh/lg4z+/jk8TISNskLWKXz40mp7zEPEY2bItG9Yo1Bf3ynYZA0unqTOM0V5I4LIvKpD1A/bOCR3KoA9MTWfqZoUt+mAWvX5A3Vxnro4hRGxa1BxrYj7LUQAWkUXGqh7lLXRa5dsZUZhwWjqFfjWUZPOZRGH/CUJ5sN/ghIZt0sY4JdaPoOxNjpPbzd6eenWQicWA50urxkyV96PQ4bR0gherSF5nJqa8WMC6cTrDInJVjMKwVZVG2Xg4+rFOLjjQR1Ur2bbjoghx80lVuzhxmv7kK57N1tu34RMPyf0kZqTryphe/5g4ccAImysH5nSh3wNw5VwcW98RSpLZsH7Aw5zXdKbpYU5wQuNND50+ey4n7I6yq6fpOQ8O99BRBUxnTHvDGLuMgaXT1Bm7wzVxTu+4YmSyxz6YnW4Z9IGp6wzNsm1gmhVeGy7Nggqhm8eE5zX6H3+DWmHbNMxpXWdCZPaWd5Q3erHyZXWiRkVhwmnpxJKMdab5TKKw/wRlebDwUHFY/mGi7uATZaOzUkluV729q8fsfz5kYjHYJY3wUDmqslW6KnR5WjrBi1UkrzO3pLyIMKtjdIb1196PZJb4SjDfimCfvcFA5omtAeXzmEc6cOLzh53N1k/4Yx21ZTVPiJvx5NzLskR04dsKJLmfxFzTGXv1lf0TR3ulA4O44R/z7KJbNNwv74rBhw+syFaTuUx/ub+12ntxzLezrb+s6UzTw5xYRw8Jfn48YPdn3RwbxOXb3krvxUf7xHqiM00PxuPi+MnqSg9VwJ4R92ZCuAk6U/x0r02ywdr86sOzXcqffcpnMXTGzDp+8YL2l7/TLM0yrQ6dG2AOuTp2/nhlw4YgdZ3hky++91kEtbzRCwvD6hALIwoTTkunps5wC+naPZNzbrhFYf8JyvJg6XLhkiOXn08ePeq9HkRvMJZ9SfN21dsfj/Yfr6wxPtTtNBbCfrS4Pv7IOm6T1ip0eVo6wY9VJK8zt6S8iDCrY3SGPw0rBvtPDlgMnD3wJYviz8FH6w3NE9v15UdrA+awxiCjfh+li0hZIsXxQSkIIW6L+Li39rh38IfFm+iIazp/BoMP1v0k95OYkZ4rY5OV4vzDZ9rIcWziAJBnF93CwZeTK6x9szp5eJ3oTNPDvNiqmW5ziYzpZiFNp85kTa9ebHDSY/udvlHfpjMjJ0hht6oZ9WLoTGyWFVGtG+eFy7Z1ZW606hqXo64z9u1Swm9lRHlz0ThstSEKE05Lp7Jf9SVpDp6ax3+Esjysk3qNQbUb+xHfjbsdeL+L1bxd9fZlQdrQPImFsPMN2F5LpBXhtHSCn7LvY8c3PuVFhFkdozM27/A4J872DdRwc7EhFbiG4JhDFQSjNXtUUe7LuEMwwOtER2x+5PiKh5TcT2LGM3Xn9Bqan41j6RAI36hEnl10i4f/9p7hvo+3Wha6fQIp1ZmGh/kRPu8DvluVCi/coH4E081imm460/BghH9AGT6IabrjG/VtOjN47OrEZ1QYUJbKuVsrXRCdqcxye7i1bty9VMynbn2UFdHKutPvc/qs60x4Fe0nf/gf5Y1e3trMzhdGFCaclk5lvxp0pqqB9kzi9P4FyvKwzo4yzS+cOnwttEFuWSiN21Vvb8uZYGRbBEkshJ73rbgvXXWoQpenpRN8WUUKOjM+5UUE1o3VmepfJLoq77ZsUCg9tmterzptHz0ODiuu2HxJvLCLchWxiruM2GJOdIQfVfk1cLMaeEjuJzHXdGbDDRpH1nVZ2GClebE8u1gWkUe9/vFg8L1ffd97q/9xMBgcvLA+41G//9b0Z91+kWHUPMyRR723MCQ2fSO2rLR4rTK95sGxtov8f35bxsFq4F+ZLmN43O+71/Cf9PucaaNUnq2s8V9DletvKxswBra4mVBcXA/K6ut+v/ZaEM0ajDHrBa4sA3R2jxmgPD6/Xl953efbkrUAay8+Ysq/uvKsljcnDftVYURhwmnpVJZkz5UkyD+0f4StsuBZS1292+9/HQy+9suh0D7u7Fc1tnYbjyK837/x9ntYkSHNWIBJw6OPeLy+C45Ch9PSCalaRVp92+/73e1xKS8gsC4qMoB24UaAZG23j6J6XW3ab/UHx7vrqIp9J/orux/gsMbq6h0evUU3ceBLcwMFEc5BFffaW37E69iXb+m+a2W5jukM9z5XOTuiljTuk3rMpc6wNaLdfR0c77vW4TSq97l0AAutM6KOfRClVkPF7fxrU5DlZbGnIH89fJvASRtmLdG22XgqnWlSzYUqpDNLBHvNBfiB0BIhnVkWpDNzhTpjW/bNF9PGAp0575X/6S4m0Zn1Xu9cOrM8cBF6gdcDFhDpzLIgnZkrttF2PhhwB7Td3jZ372o7bCWJzri9GunMksBXfLVsdiekM8uCdGa+8OUgx8jv+kzAvZ/QSmecJNnb6mLx6V1eDzSduRO9UfGnXbMR8+Xj9XWr5RrRERsHg1/F9eB722HZoz6pvdfjWR0Ul+FtOWPLvMafIxBCCCGEEEIIIYQQQgghhBBCCCGEEEIIIYQQQgghhBBCCCGEEEIIIYQQQgghhBBCCCGEEEIIIYQQQgghhBBCCCGEEEKI9vSEEEKIjtiCzti/eBZCCCE6YCSdEUII0SFn0JlNIYQQoiukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6ZGY6s723t+tP64y90THzSlcIIUTMzHTmv6Io9vx5jbE3OmZe6QohhIi5h84cjori9L2/2Dwa162PvdEx80pXCCFEzPQ6s32Fjry42vaXC6Izz/f2Xroz6YwQQiwC0+vMK8pMUbzylwuiM5hj3bgz6YwQQiwC0+vMN6pMUfzwlwuiM2dIzZ1JZ4QQYhGYWme4bPbjBrMHv3AmnRFCCJFhap15h2781Q/8eeeuQ7f+ajg82tz5dlOc/XAbJXZjZ3BWnA2emsNm73AIt4th3106eke/4ThkbBbF82+QjG8I8eqkKH4fO0+bm7tfEPb0xCcKag4/hlC+YjgcvsqlK4QQ4uGZWmcgMVfbFJuBuw46g+Owz94e923vhjc+2TsDxdVzOuy5C/A7vESwufk0OO66KN6467MdBgcn5mv72F3heifn4M+L4iiTrhBCiDkwrc5sQ0q+2d+bnjmwW/c6U+qI3fJCYZgmmTT8vuBfyIGHM5zfQ/xFHHEUXAcz3tDXwF+AUwvWcCiDHWbSFUIIMQem1Zk+em9IAhfO3PJXpDOQicM9UxN0997h0569N8CJxfubLzy8weVvHI1tXHzCcecQ0xIX4ugVtacoLg7fneLACc0e3d9tb9t8iStlTYeXe5Svvb09CFySrhBCiDkwrc6cQABw4MLZ0BxinbnghgiFhjpChytu1VA2bCXNTYA2f7sojKe4R50xQgj7hc4Z4nrOI25wB8dioMox7sSh8R5APV0hhBAPz5Q608ME4guOXDgrbKsk1hlb46J0XMUO/BBMvPXP/t+f2nzGdv1JGQJiVtjLBF49kNiIl6ZAjDtxaOhMNl0hhBAPyJQ68x59N1XFFs64OlbTGbtDESi2IwfOOVx/v3P4jVsxwC7JF7scmjCUIbjkZdrj1IPK5d4H4M8xi17qkH2vuUpXCCHEgzOlzlAlhoT9ui1YpTrDW363pdbfb1ObPHQ3tv3PPgexMjV0hrsx33jp0n+aOkhnhBBi0ZhOZ3bQdUewg091hvOZ2CH093xH7Gp4dFRJgrHn5Me/kZzTmZf46zaDbPqynTpIZ4QQYtGYTmcO0XVH8PXkWGds152LWty8b/b3PRxOKQnx/ozxlLoyGq8zDMkY3XbMRcZBOiOEEIvGdDpj73ntGZ9wyr34WGf4jpidcPOk2d/v4vAfrxOd2dw8ta9gliEaOmM/j7H/XUadY9yJg1nmXmJupiuEEGIeTKUznKr49So7Z88eunUei7P+SztyW7/Z33Oxizs6fJWg1JnnA86BetCT01t0hq9Kn+HOOy7J8UW0xMHeS/iysxcZJJ0RQoh5MpXO8E3h8h+ccQZx3NAZj61pJf09JyFnP7ihUunMHpyGQ96Jokp0Zts+IuDgW9Wpg/vYQH2XRzojhBBzZCqdobS474sBik60qcKj/xrMhc0wkv6eR0IVoTvhLzGNk1veN9vc3HXqBI65w5Nx6Dnlkc4IIcSCMJXOnAyH5Y/3N3eGw+HJ5uYbHLgxYt37q1PMTwZOisKNzT2cWMf/Bnevhns9C+d5Y9p0ZnpQhjjEiX084FPwun3MLzKf/rBNGZI4POfK2QjB03SFEEI8PNO9B3AL5TRCCCGEkM4IIYToFumMEEKILpHOCCGE6JKZ6wxfP5POCCGE8MxcZ7b39sp3v4QQQvzzzFxnhBBCiAjpjBBCiC6RzgghhOgS6YwQQogukc4IIYToEumMEEKILpHOdMmn4Rf73nTJ08Nvw+GXd/y29H9HMe5d8Lr/Xv8HPB+6D1HPGm+JfabUsdvn50oP7dujTetyxh4Pvzi/gSjKWfivG+SYIkia7oSIPbGne6Q1U3p7R6wSb+Iq8eroZHjyn30a3THZYffHifu3Hrs+Rw7774O3eS/J2dHKtjSq1KUK1M68OOFciDZpLh/vh8f+DLz6gpp45Gvec597o5vO4+5IZzrkVRH+LY6HH0sgV333/+Eq+K+vG/4P+e94wChqUzOjtCQ0uOo/LAzRjzas4z9oiDBj93CSz9z7bObu6r9ukGOKIGm6EyJ21DxNn9Zs+Y//zo9c+f+CgU7l1DsN/T/qmOzg/pOtjWdCtj10u817IGNHK9syUSUucaBW5tUSzoRokeby8fy3/+9eZI//NgXcOF2tlcGi/GReOtMh7J4vqhHFDur3zfD4m/vf0idDwOrC45D/ebTufxct5+To2wV8VFHMCFpSDI+/8GAqtm3/mXRwdPQN3ecFHBrWTTK2GeW9/ScGTRcENNKdHDFoepoyrdnCvqUYfjo64n/C8IP7Pk7PfhydoKdx/zhjsoMfRJhY8n9nDIe4ZccBXG717sjZ0cq2NKrUpRaohXmNhNMQLdJcOqz2VTozKi6O+4f8fyimKruWd4Jy6GKMOg3Sme7YvuF/fOM/pHagGxtYJ/T0WxhnoMpXFb7u/+XVf/TcQyjfcGfHCZqpLRE9/eIs+YT+0Q/Ld09u3EnDutRY9KpV5pIo7+c/Z9AUQdJ0W0Wcjeruac2UnYui+GFWbvYOr5wlGIvcvLOzdyP7R+qTHcBRcfG76qVsAOzP2njP2dHKNtBMOXHJBxpvXi7hWogWaS4dO9CPT4MqB79d//Afuhc7KTnlP6BcDBZAZ/7aL9X0i5veTfHDX22+QZPwp5sv/aC31hU3/HueR8FmRGzJc1rysih+u9ZKyiWFmnWNy3cw9iKfOYsS3MN/zqApgoBGuq0izkd157RmCxSs+q+wPZf6aXETms62rc5PdgCj4tNhUVRtLuqWW3jP2dHKNtBMOXHJBbrNvFzCDZ2ZmObS0S+GL5HzplJilFOXFdTrQ386dxZAZ2bz5c2nP4bfqqa/EJygy/mGrsddbaORlOvUgVpXXPdfclWEkd2sSCw5zU+wa9ZljP1SGdsqc+395wyaIghI0m0RcT6qO6c1U/aS0ap1JNX/tSWTHQAmBbs78T5S1S238J6zo5VtIEm56ZILdJt5uYRBrDMT01w+nnMdOtWZzbOGzpwUV344Nn8WQGdQK2agMxjZRL3aIoC6/IZb+37AlW1DcVfc8F+STnHuCRpa3RKYlmuudaFoGHsDY9HMvbFJlGR6/zmDpggCGum2inhMVHdNa7YMM9J20hTIyQ7gE3uok2gfqeqWW3jP2dHKNpCk3HTJBbrNvFzCINaZiWkuKanO9IpyH8zApDttMPNiyXXm+d6er2gniGWxhij/cTixfRFmI5iqp7mMu+KG/wC6wRnvz/SblmBGmX0AsXUgvjy0sVJpbBIlmd5/zqApgoBGuq0iHhPVXdOaKds3mWjPilN/5pnsgJguimPLtL2dQapuebL3nB3tbMul3HTJBLrNvGzCINKZyWkuKanOYKRdyxKuyxXdubPkOjMqCr8L+x5nHe3ATslp8Q1/vxSFG4PhJH3scVfc8B/41HS4N8dNS1Ajs0nE1oH48rcZi5hcwCRKMr3/nEFTBAGNdFtFPCaqu6Y1UzDaYOQ1tpvTqskOADPm5xj8RrPksltu4T1nRyvbQJpywyUX6DbzcgmTSGcmprmsNHSm9woD7do4G7PrTGHOiyXXmTOE9afPF2xjD9NW6l45HUE9sGONqCtu+vc8nX2D+FEU9TWD38WVHQ/d65ClOMRCAaLL5w1jkyjJ9P5zBk0RJE23VcRjiuOuac0U9I4W67a9Yj08QVfJBOsLI5MdwA+3iv+j2kcqu+UW3nN2tLINpCk3XHKBbjMvlzCJdGZimstKrDNv0AsWxW/WvgqMT6ftVTvg79GZRePY/64i7M6dRb+zKIm64qZ/z0lxNevh8TDMAQNnvs4OrL5WFTiyjkSXRw1jkyjJ9P5zBk0RJE23VcRjiuOuac2Ud0VhPyFFX2vQkr2mok12QJ9843KBvinsI5XdcgvvOTta2ZZLuemSCQTGm5dLmFQ6MznNZSXWGZQDGcVvl21f5NYg58Z9deYVh3PPv6G///YUVxi0/z62G+8wvrAOcg8jDVuX6B3Zb6rc+++b/w0viuKEC4qRzrw7OSuuhva7kYre4RA+LoZltSiD/uCPs/iTLCg5kjlhMmbQ0y+j4myQLrM8JBd+Gnvs9ypRNLcO+Zv+HbgKP1GfGege60M5PBc72lc7TqsKHFlHosszbyyenRmbREmm958zaIogabqtIh5THHdNa6ZgluTG8odIEkNVWoIZsEsxMNnBNiasXWxXrzHCYHfSwnvOjla25VJuumQCgfHm5RImZYgWaS4rjXWznb1D9MHRysfhYunofXUGj3T4xn0g5WwHF8TeeviGE1MPOvLkqf+Mir24/hRN1UDVCB42e2jxRu1LK3shXPHb+ukoqD+pxUKDXrkgV2EePQ84p98j7wvuO9o8NrWn6rsS/0btYlbUlQx8iU2LKnAkFKS65MiyZmwSJZnef86gKYKk6baKeExx3DWtmVJ7mchbst14waiFA2dvZ2blHgZv1uFak3EnLbzn7GhlWy7lpksmEBhvXi5hUoZokeayUtXLwDZGsn4QD0aL9ULdDHSm1AEuYhmcpSQ6g+da/B7iLy+CVnAfL3jgwLI4HZ7i7ygqInQDCIgZjK9HUdAyZUwYQyyxQfPcB2P+A7aeggFGfQGVVI0j8U8gPtmNzvuBjrBuCUyrKmhUgaOmS6pLdLQlZmwSJZnef86gKYKk6baKeExx3DWt2XJR2lFZErsZkx12vIWGX4VCk3EnbbxPsmOsbWlUt0decot5uYRBGaJNmktKVS9LILtlj/emi8HOPZiBzqBjP3pFFUH7OnxHmeAgo6kzGIzYJHfncMdKobh6s/3y6AZO3oM50sd/OEYrje9v7BO4vPvbH0PQl3uUH4xNelUszqBPe9ZvV6POh2b7xn8MazgcuX4M491U98rGkfoHr26Kkw4GJYklcKiUParAUdMllbFXd8zcHf1nDZoiSDbdyRHnPIG7pjVbfkSNIliCRld/OXeyAxqXt3JY/nwcTcadtPGes6OVbWlUqUsSiNxiXi5hUIZok+aSUtXLiui979+5XyLNkVnozNVLa2aY0GAiyj05lkBTZ1AN/GIqQEt0TfEpCsN74ETFpnqQ5dr82C+e8zYOtaAo2VClQizBIFbKDpp7W/pV4igRm5TkfkReNo6M/65kJmMJHMr2GlXgqOmS8hJaf6fM3dV/3qC7B8mnOzninKcp0popL2+qF0KCJUizvpA02eHUBmsGJmAu22gydmzlPWdHK9vSqFKXJBC5xbxcwqAM0SbNJaWqlxXVq6kva3s1C8AsdMbGGCc4scfmu/7cfMbeFSA3GBC6M+A90NGNK9GKMyMN6gYOtaBjdMYMwshljjth8TcfMLbgOVrJyE+wdn74l7DLxpHxD5k57URmOB4/801s5xsLDQ7+o+K1Chw1XVJeDlJjkyjB1P7zBt09SD7dyRHnPE2R1mz5VFWf0hK0iTB2e2PSNskB/U+wm5lwt9Bk7AhaeM/Z0cK2NKpc5In9YLx5+YSrEO3SXE6qerkTtpkwVg2jn8GiaegsdMbaPHXFMjxGZzh8APaNeFSJaGDiPdRWTsuaRXb4P6i8az3oGJ2xdFHq89MZZKZ6NQaKZ8v5mJHdHO1t7+x9uwnr+6FxZPxzrPbGbVnuzbrX+hEs+XLjyugQhfX7Harm7lG05h01XRIuezeZzCVRTu+f5Ay6a5Bsuq0izhbH3dOaKduYyd8c7/U2e2/QizhLUEeK3/2Xm7t9/wHiSQ7H8eZ3eNMeTcaOoIX3nB0tbEujykWe2A/Gm5dPuArRLs3lpNKZ94UNjrb/uynO/NrPzk01bVsMHk5ntukCBtscSUQrC97Drt31hDIE2+gFAjYIiRclFlVn0NOYEQZExKTR/dsIY+Rn+6Hvyvh3uuyourAZkVhS/dsujAXMBcQ9KwiX73OZS6Oc2r+RM+iOQfLptoo452mKtGZKVH3KNbqXlRluKDvB4SLufxCfRYMmY9ekhfecHZNtS6PKRp7Yf5t5IJdwGaJlmsvGc59dwF9+veHJGXvCm1AFMc1L97nmysPpDDTCKcaRSUrVEL0Hlt7pkSf6bT/GKcXV8OjIxVsPurA687s2VCp/CfhqaO/CVT8FQo9k8/iMf2YkMPtXR/acJb/fh6nS9vEQg0XU1UG/nD3BuqpnrS5P8plLopzWvyNn0N2CjEm3VcQZT9OkNVt2T1w3O/rk14pgxqdTulx8w9xqssPLqvcGuLKKhZpm18Zk71k7JtmWRjUm8sT+8eY5Mgn7EK3TXDKiEbl9l+KdW+wpvvjBFzcXZv+/Ee9HZzrDG7Z4wAGH0xkMCelrZDv96f4MDmHoGEG/tk3BwnSXE/dn5q8z43m6Fwpj3qSW7JRfJZ2Su2Zukv+MQVMEydAq4olR3bu8pmDX3q6ssbdXrQUZkx1up433jB2tbGvDhEDp7Zwx/xLbe3t7pcgsJJ3pzHscuM32iqO+qgVjNIKhHgePNmV9jr8hCgTMvItH8bYhv9OZelBuefiXl5dFZ4QQ4h+jM52xb+4c7n2yxQX4eD6gLvRw99TWD4urNzu4e1JFwYnP0IRm77iaj2Nya2uq1C3GWwvKHVxMF/fgXTojhBALSWc6s32BI/Hzmb2Cv2PjbOR4c3PHVsNJpDMuBDzhbyQQ9Hr2w63B4rIW1LQJHElnhBBiQelMZ8JHRC+48AUf5UsS9tND+yE/iRQifofHrh3UC8IEeB0H3ey5C+mMEEIsKvfVmTfD4dAWufi/OGwv7tNw6F4I45fqzr7tbP5wPviCOxx817/zg++N2Peayyg2t4/sHZ7Rj5o+vIHPq+FeL8QbBYV8ceWMP9UKsZSx8TvRkVwJIYSYC/fVGSGEEOI2pDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIboEOiOEEEIIIYQQQgghhBBCCCGEEEIIIYQQQgghhBBCCCGEmD+93qo/E0IIIUpWfxbgejD4vutdpmC9/8diOVjzDkIIIYRjiwLhOH/s3eps9ftP/OkYXlz6GIrzde8khBBCGD2vEGSUVYkR7vjTMXx1wcln7ySEEEIY1JljTFpeX+PktXesMVlnjovLj/v7A+pMoZUzIYQQMV5nVlZehxMjWkJr6MzWhj+peN23VwB+UWe2zEUIIYRwlDrDk6/mtHLAdwP+fLZVtM8DTnQGgwH3aB59pOgUl/3srOWA93r+QgghhCClzuzj5IAnG/YGGjinZvjzouivrDyh5Bij3DvMfd555C+EEEIIEnRm6xzzFJvBcPlr0P+Ov+dQk/JVsn0nJH8GJkOmSA1sFuTPhRBCCIM6cz4YUE5GtinzAmfQlJVdHF9jdtODABU9+xXm/uVbKhGD/KTXOvaGdPZNAiGEEP8uFA3P1xd0+BMmJTj5zmP6vhkmLuf+NILzoD/6JoAQQogakc4UxUc4XIfXAQZecFKd4RvM/AiAEWYwH+B4bTOixg0hhBD/Mn5/Zr13jJPi2co6DyX0EevMxke3xEYXvjhA3M6/XblP19RvCCGE+KfxOgMoNN/r85sR3SudWY1++O9+cENMTribw1fSSO2GEEKIf5tKZ57gbLTyCH+/9zymFJXOUGZGB/2+c9lwy2P2G0174/mD81W7IYQQ4h+n0plnOPtlP5gZ2HWg1Jk1nPykeLj9mQiTGReLEEIIEVPpDNUDZ5AVt50f4M9izIETHvvdDF14DDyWzAghhBgDdeYXV7ns62TQE+6u/OFXytZ3v9qnzD7D4fuT3ccrGzjh72b4ZllNZ6g7l7ZUprfMhBBC1Knt+3Mjf9UEp6B2uPfH/LY+7vEXm5f2vbO6zphDwLsJIYQQJPo/ZyP7nWb1fbOisP9vtsoNmqJ4W2rSNT+YaV49ztnj3YQQQgjjw8D40K++tPziK+c0Pz/6b/yvffxVXH7lF2cefz4vRl+31r4P/JedHX0XhaF/dCaEEEIIIYQQQgghhBBCCCGEEEIIIYQQQgghhBBCCCGEEEIIIYQQQgghhBBCCCGEEEIIIUQX9L7y85/9vv9qKHj0djAaDfpr/nLr8/e+/SeeQO/D4GPNoUtWPw++1xNbe9b/Dvs+2te0PTk3mDkYfH/ND6B+tm+cBiynVbyZm3FYR+LQzq4kWN1TmnQa7e3ZmMrSmbG2y8oT5/jx/sdBgeTtX557cm5PBgP7n4ErPWalxP+3wO5iKUOUtHBJsjnZIY2klTVJPKl1y8DqQfxB4bUXH379Od73NXKjrMXPnIP4J/ju/p9BUfzyX6zmvz0g5762f8X5ZdVtrazzP/DwX/U8CPx32rX/VfqE/wbIGJXNMeO2euxdrt+uPPKnHrO9jDdzMw5rJA7t7EqD1T1lkm5GOyEbU1k6M7ZCbn56HVsLqSPFMEzJua2s8Ivo9l9q++5WgL1wd7FEIUomuyTZnOyQRNLOmjSe1Lol4Mkf1G9/jv6CeQAjp7Bv3RXoplKKxWRg/wv0AyrDpVWEHmoEHHB97sRlUEBYyl6L//gN1w+mM/x3pter/sK118vBwYv9PvTxerzbBqr6n+N+//NlUWytvOW/Ov1QFAMe3cytije5WQ8LEgfQwq4kWOIptasR7YRsTGPp7EDnfg1TDlBVvpsDVfTP57dP+h9w4mtMzg12wib3T9C3LC8D1ChCD93FEocITHZJsjnZIYmknTVpPKl1i4+T1FJnHo2Ky+NnPYxendAgkx/sKfXLYaL4Bxj4KoHn/5HHjZGbz6J7cg0CHoIfY1Tg1kPpzNp1gRrq/gMdQR3+48d6jz77/jznhqb60frW1bfXfp6G6UBldTPe+GYSNhNZG7uSYDlP9aSb0U7IxjSWzgzMky6dlj3+OeCBE93QJ+6OXOXJuYED1qlzfwFQ+fxZh7GAZogWLkk2JzuAeiTtrMnEk1q38Dw6Ly7fHlcdxseisA5l1xcBHpKb2IhxrPZ6vvE2GHujZLKPeVFqyHnxy5040J+5NVZ4eOH/tyh5UhSY8jyUzuwXl6vn5SiRk62B9aMROTc02dCuVx75mzWdacQb30zC5iJrYVcSLOcJxHY1op2QjWksnR2/iuuywzD1/Op7lJicGzgvDtDvVAPaSCG6iwU0Q7RwSbI52QHUI2lnTSae1LqFZ7f4vI4RVdCZ1cuwPvbTuf0DOvP4++DrffKIIiqyajH2RknwcV8TZk+pM74elJwXblAFD6gs5b7e1+J7rcfulgFq6UFx7beHVv8U183V6qwb5upJj16zuh5vfDMJm42shV1JPBlPJLarEe3t2ZjG0tmxXxQf/KkDIpf8N9mcG4DibmCeVS3QVwrRXSwgCTHZJcnmZAdQj6SdNZl4UusWnzVKZKUzGJZ6iX3rlnLxkP52nfmAzv61P5+GsXIy9kZJ8HFfE2ZPNJ8Jk35H2Kijhw9lR7WOevNwOoOUeitbRbHvLtFgkyadc0P7TNxq/Xkj3vhmEjYXWQu7kmA5TySyqxHthGxMY+nsGDS1C8PxRMxybuC4+MMBS7VrVClEd7GAJMRklySbkx1APZJ21mTiSa1bEiqdOSgK/94D6v8uDnhIf7vOcEf0Ph3kWDkZe6Mk+LivCbMn6MyLhgCi03LX9PC4vHlQnK9GPWPHvLXF6VHxM1yWu9slOTcMDdPHEVvdiDe+mYTNRdbCriRYzhOJ7GpEOyEb01g6OxrLrCtrRWOcAnJuANNjZAEVrtw1KhWiu1hAGmKySzObLRyakbS0Jo0ntW5ZqHSmOttwfQge0t+uM8+uw0bbdIyVk7E3SoKP+5owewbFeQ9golWfnn8sCje8MiH6xZEVWD0vDmo9drf8sXcTUHhuvRoqXb0U6sm5YRCV1uXY6ka88c0kbC6yFnYlwXKeSGRXI9oJ2ZjG0pmx2lwLwriEKdXIuQH0nLATvWi5awT73El3sYA0xESXJJuTHUA9knbWZOJJrVsWKnUpl0tCxcVDAr++z3yC/ffAIrqnziweAz52MkCdrsAMxjcOqylhqIzJ76Naj90pMIKijJGQS++XDc4xSHc/9LI975zb16JIlxoiq5vxxjeTsJnI2tiVBMt5IlXSzWgnZGMaS2cGIi5zYKBm2ItUq99d/rgVlXMDX93s6rhaKCoVortYQBpiokuSzckOoB5JO2sy8aTWLQuVzvyq5nKuR9n13U3xx5fD0hNGcOu1DrRkK26Tq5UIrOW9g7pYRP7CDTbpPON0ZsajzCkYFNeo+3zw8Rr+6q9i5MvHdGbt2o22BhxbPZjOfPDV9Y8/jvysiquPwBxzbj9DhxMTWd2MN76ZhM1E1sauJFjOE6mSbkY7IRvTWDoznjTrAAbftrS67rJnd3Nu9pav9buIwi/GVgrRXSy5EJNdcHSBA5MdkkjaWZPGk1q3NFQ6M4p1hismq73e+lpv9xdaQDqIWipenF8frDz+el0U/N7G7qgoLo8tS8/Q1F3HvsrfQhUjqwX03b/2Y4bdwTlu/DwIRbBv/jgKQScciUXdn93YQjO//u7u754XvyylJz7JEDSYYKlufL1EHHOW9TCxhcnRy5foDIMsOg+frYAw6IKfB9MZLtKRsLXx3Q/YN7jS990ZnnOD9ekIMLK6GW98MwmbiayNXUmwnCdSJd2MdkI2prF0ZmCGVF8MQtVwDsweulYamXOzubEz89yN10GpEN3Fkgsx2SXJ5mSHJJJ21qTxpNYtDfG6WaQzLucOTL7jyyUE1W3wAioDLh99tGPxi008dPYrj/84VzZC+uZbYFwKXUdf4PCTuvLatLgM3/THG8cuxWvbs+NPYs1nCJI5BhvDD+rmRLmAunFdbUQe+Kk+cR56Nqj6UJyjIB9KZziQY9vsYa5tbRDPqVJlX5dzbjA/Ve/K6iTe+GYSNo2slV1JsJwnUiadRDshG9NYOjNWq9++O2oO3sicG6ddf8wmiG3YNUJzcCfdxZILMdmlFhOZ7JBE0s6aNJ7UuqWhqt7VgArTuXhahstmuS0ZqG6uBweYMDi4JhQ6+RVOUgwnHs4TdMaWjy7tshSmnweYEAH0u2X4nL+AzYva6ExpY/K6yoNS6gxF1W9UYyRVvRTgPYxg5+qlDY/LttIxLMWA/TAEdvHVSIevy2Pc0ld0KquTeOObSdg0srZ2JfEknkiZdC7a27KRS2GSpbPj3G83lZxHKQQjc244VvisoDm4kw5jSUO0cclkc5JDEkk7a5rxpD6Wh6p6H4evMPHl+qr+g/OqCSwn7MuLP7u9z/aEfr7o/cSBs7fQyaM92ncMd8/9JAWDvf7nVW7YFSOMCHtUEGov9Ij9LaZ4pgUhfOLP4hi9WN+ndnAiwPZNn2WQ3LEY7ffsONsVjTtS6QyMdltOGPxWMhM8cOUFBUcfoa10zOq1W7EEmH9yTW+jsPmUw9flnBsmX6l4l1an8cZZSsImDu3sSoLlPJGQdBrt7dmYytLZgbFWPUI0t3IOHIzMuaHKe5sGl6EU4OZOOowlDdHGJcnmZIckknbWNONJfSwPVfWu5tjoVWqz7cvs295LBB6Q+2Ybt1B+YYjODTjmmzfYyaPNuSyv4SYd/ZcdOM0xdaCQoMWimdqjf4FLzlJC+KY/u/GHcwF0xubQSmfMRmhYo5o+MLX5jPWDqBBfzcHhPaAQj3+5qW5oKx0DO6wMAVL0ryFUKYe6nHPDyCIp1NLqTLxxlpKwTYeWdiXx5DyBkHQm2luzMZ2lM+NJU9TWwx4nCEbm3KKd4Q/BPjQHO3YZSxqijUuSzckOSSTtrGnGk/pYHqrqjVmM5Zfdi3sPxoOCyP5seXlgH27CwT7cFja58uVv8HnhUGWZjr4kICvOfRUTlUvnwFuoAfaT3BA+8VemSEWjQyudsRBUptp08qEpdQa5NHlB5/S9HHeD4AEy5Lux0FY65nu0kPCzuKaOP0bRh3c0Ql3OuuExWPnjdODXkkqrM/HGWUrCNh3a2pXEk/EEQtK5aG/LRi6FyZbODrSurz7Gt7ZPjap87lMvjUzd4m8T4NztGqE52BF0FUsaoo1LJpuTHDKRtLO/Hk/Ox9IQVe+RG4EzD+WkjiC3vkiWldCXI7d+JaiuMxs4VFtQpe/YnTMWlA7Gh+fPVtYx/7GRRhre+6viYEroSlrpjHlArz5vnbHfaT6DyfZGAuRm9MS2H3s9G4QFneHkzS38lO2/U6DZVePa962O70786fcebfQ+losJOTc+gA+7vd4u1NEXb7A6G2+cpSRs3aG1XUk8OU9l0tlob8kGmcbSmfEYg6w/fVSVA7/SvopEi4+7vdXeLhqOMzJ1K3//S8IH2dAc7Bp0FUsaoo1LJpuTHDKRtLO/Hk/Ox1LwGB0HBjj4a30vHsqv3srqC+SJ+Xl9zn+auPoMk/tobX4pKftwtrqMzvRwqPJY+o7dUQoMSReHjePT8N5fFQdTQnEyZXMId8YdF0FnAm7IhfpdYr1U0JnVc3s/DzyMzryON67Wr/0qwgYaaiCs7uXcMIT0/PQtNFidjbeWpSRszaG9XUk8OU8h6Xy047NhTGPpzKhyc+0VbBc9pec6pNxwQy2KzEAzCC3Lro1OYklDtHHhMcnm7Q75SFrZH8eT97EEYN4ScG8AfPBX7t/PoE56aosmywgeiuvDx+jMYxyqZ1j6jt39tIQbM+TavZCXhvf+qjhQnVi6S6Qz9u4CMvT1tZuzVxXBd2hfw7LxQfi3mmuXD/HDse+1Xw1gmu3P9l1rPD92m2pGxm3rMyeb11/L9rl27azOxhtuOpKwscMd7EriyXnySY+Jdmw2HFNZOjPeDth9jg7Kgff6R+6JQuSi/xtdd0Pj8QuAZOPaLdK/ttXmQBexpCHauLizJJu3OYyJpI39cTzjfCw8G5ZNw41PV/Y5lP310XrildXXrgUMota7pJR9+BidWcPBFwEofZu7W8/mCwDwgSZd7H4YHPd9fYrC1/zZDWvpLOTgYNWEYs7YQyrN4/x1ZhlZ7/WSwVDOrfbJhzuShG0RWSsbcp5uZ0LK01g6M9aTX3Zs9dIfe+Tcbqe7WKYiyeZkh5RW1rSIZ/nY8vtOxqpfUVt2yj58jM7Ye2gmCz3Me0vfzpvJLMf0n22rIlojrbw2/dmNS77BxkUnbt3s+xtUEQsSgjaP0hkhhFg+yj58nM7weP221zumJpS+nWxcPlt/9JpLSZANLjVeDgb9/lvnIXht+rMbxeUBP3rsFIwKVRw/oQFVkrmjdEYIIZaPsg8fpzOrbqkUDCLfcOcPazy2HMrXRBz2fZkyfNMfbnBd1XCbGVwkJ3RmkBC0eZTOCCHE8lH24eN0ZmWLDqS+bha98OFe9ebyl+d8PfLa9IcbA1sig8y4hTb7VADkiRs2DBKCNo/SGSGEWD56g8F3k5fdwcD98OlgMOBuSXljZe1gcF6MjjlJqRzJ/uBPcT346F6n5AvMEJJ1fsqYy2GR15o/3ni7soXZT/Wh543Po+Jnf23leGD/1yUEbR5XHg/Kf0ghhBDiHwOzFrcKxnlN7cesQgghxP25Lvz3Zfgama1yCSGEELODvy/687Hft98ZPeBvEoQQQvwb8INDHvfRBCGEEGKWPPIfixjsajYjhBCiG9a0MSOEEEIIIYQQQgghFo9NIYQQoiukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6RDojhBCiS6QzQgghukQ6I4QQokukM0IIIbpEOiOEEKJLpDNCCCG6pFud2d7b242PQggh/jVmojNPd/xJk/+KotiLjkIIIf417q0zO8fDK6jI8P22d4g58voSjjPh6WlxdeTPx/L0pCiuxslft2wfDpHdi2/lDK733+AUDsPj0p4vxcCfed5cFJ/8qdEbFRf1Amv66IBgeJnwFA6bL49OzuD0+zAqfObmlT935Hxl3KLot0+GMe9a3G5pyxTWZVPrjPdfXDbf+Gvw8oszpuev4emmWadKl3feTI811MR7EmOrJFLL8m5tQiYJTmHjuDz3/dHj+qqcpcvC85Ni5E89P4qbE3+6+fIYOTuNWuXcua/OvKHIGKcZoelEZ/YQ2dCfj+O/G5r01F89KE9/M2nyw5XILtuCcRVq9LA482fGDkSx+OYvjD4c4jaW+pg9leEDZ/gUDr7gyc2hcwHN3GR9Zdzi6Hf8mYcDjQm3W9oyjXW51LrieZnLYhQq9GGw5qoUSFayehMrXdj6Ikw8m96TGFskkbMs59YuZJrgFDaOy/MXf/SwCPKWLgmsjrUuZPOQGfHnfV8uN+Wjmzv31JlPLkPGS+8WMSedCVVoHtWHwjs43Ht1NCoKm9FgiFUM/tvbO/wGi3yPVNeZw6sCgWoqwoHWTTWSzPiYOWY47DzC3MsMn8KBPf/FcX9vb+/4oqz1zM1NnJucr5xbPXrc2tt7h1KwE/QUE263swVMYx3TqKXWGaw+J0dMDUMNV322cXZx/G6v/+WqbAhoFDf1ClK5PDUjUf3saO2w4T2JsU0SGcuybq1CZhKcwsaxeU6LIG/pcrCLvuWqMVS9KsraundTjN4/7fWviptMnzwf7qczeK7I8ac3e+/RMWaEZD4600MVMuagM9sXxU3fnb65YN+7g07MT2Pe4PS5ndV0BmOtHxggR+1w8ymqflG891c5HzMHhocEX5nhUzhQHge+x94+PHUnNvp/X/oEOV8ZtzR6VzDubNLtlrZMbR2IU+uKl2hfYVnu5cBNrzB0PXVVu/ctLJV8KX7/KK78pNJouqBC+TPQuJnE2CKJnGU5N9AiZCbBKWy8Nc+1yzGWLgW7mIG9+lbXGeQcKurOf/huBh3lF3NYAO6nMxhJFmeu69w78Q0xJuhLOM6EiTqD9j96xRm0q4UPyn9FUa+0mPGV678YALsFmprO/BihaOoq8h/unxW//VXOx8xBq60bPoVDP1+xmZtRlZucr5xbEj2IuvYJt9vZAqa0DjyEzpyGoUkFBPai2da2r4r/ULtCzwkSl7jPbdxMYmyTRMayrFurkJkEp7Dx1jyD6DJv6XLw7uq/zc26zkA2Xwad2b4J1XJYXLiT+XMvnXmDvryxXPZqgDndxRAFQRKdeXpMaSpOD/2Y480Qc48zbs25AeV/vB5+cfXnzXB4vPn021lxcxLXiVRnLNTg+ebT07MhfPYY2Xx0BrOXi2o4BeBQjn83NzEEt5zUdMZWXeoqMiqON48r+zM+Zk0PrbZm+HQON7klpFPkBjXA5ybna4xbPXpSde2Tb7ewhUxnHXkAnYGYJm9/JEMZgJb4FL1stM2UuCCP/iy5mcTYIomcZTk30CJkJsEpbLw1z6C6HGPpctBj91jXmVMMhILOYFTkx/TIpT+bO/fSGe6u1Xr8HvLq+G39KR5sTWdehRWtYmR9xA9/BdiJ2m43ce9aIdTw1ZU5XEXl1dQZLp6bn5ev8Dd4nI/OYDRVr/xwiBZpkCOb3DTeAwA1FcHEeHfzeaMX61ZnUHR1w6dwQF79+KIGxlq7UZ+c85VzS6InVTQTbrezBUxpHanF0w2n6bxi83e8MuQZsEV8i+Uwcak62eRmEmOLJHKW5dxAi5CZBKew8dY8g+pyjKVLRE1n+ny3NujMp+LGFwzaiF/Dnzv30hl28PFSNx50iY3i8WBrOsPjxZASYC0UeluMvmGQD45xPcTx9xfGakNV+HYqA6JJQVNnbIZEfmOI496mAfPRGVjgtwo8cIgmfBhwWXlN0JlPBd9ZHNnfkm51Bo+ibvgUDpiB5fYdjy0fv0Nucr5ybkn0pOraJ9xuZwuY0jrSvc70iiLsjlRc1Zb9jN4N6xUaRtkaU5eqk01uJjFOTiJnWdbaViEzCU5h4615JuXlGEuXiVhnti845go6U91BBc0OkObAvXSGKhC/Oodu1GYefBPIHjZ1JdaZ91fH7Puhs1ZJoBB83JjFmIwwOOXmPY4cqzJUcXG4h3Yet3RUpFhnGOoCQ1JExtcwvOucdAbPuhxMGZjyRQ6YqDCDE3QG1Yb9V0OzutWZT83SmsIBo4zmCJM0cpPzlXNLoidV1z7hdjtbwJTWke51BlU9Wd5BH/nDn5agxbCWXVRdb+oS9bmNm0mMLZLIWZZzAy1CZhKcwsZb80zKyzGWLhOxznyy/jPozLAcRe10XkFbcx+dwaOuj/Q4EzHdYd/PrAd9CccAXxPF4cYtI73DXc5ioRRn1qChF9QfhjpjZ4KGHukZKkmsM0yUsbzEMeyzg/noDCbz/sxzUtz4M4KeycTidp2BDHPVEZ7j3edudQZa7888UziM/LajXw31zwiPi7lBnXe5yfnKuSXRk6prn3C7nS3TW0e61xm0I1scxEjdYCPAWCVJdeiG55H2pi5Rn9u4mcTYIomcZTk30CJkJsEpbLw1z6S8HGPpMhHpzMsb61yDzoyqGtptn3EX7qMzaGd1ncFTc1K6jQkNO9ygL+EY4AIZDvDPeR2nN9QXXDqZwG3Gw1BWAzDajBYaGzqDUK7EKTiu+pD56MxpU2eQFX9GgnTcrjM//CrhsLbT3W2dqdsJpnA4q/fHPovfGrnJ+cq5JdGTqmufcLudLdNbRx5EZ2zhw1oaYHKo/s1UcduaR9X3pi5Rn9u8mcTYIomcZTm3diHTBJuhWtjYDAKqPBvlZd7SpSLSmRPXbVY6U87n6iPVeXKvdTM+ovKl3U3uXLsRhdszwRQl6Es4ws/xcIguAjhfZy83d7jihSuMKSPgUIbq48SqkIEaFukMQ7krlLN/nYvMR2cw86rvL6JvihxgutXvW3UGgyzzxHWAaKDVrc7A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fibrosis, macromolecular crowding, vocal fold scarring","lastPublishedDoi":"10.21203/rs.3.rs-20745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-20745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Vocal folds (VF) scarring leads to severe dysphonia which negatively impacts daily life of patients. Current therapeutic options are limited due in large part to the high complexity of the micro-structure of the VF. Innovative therapies derived from adipose tissue such as stromal vascular fraction (SVF) or adipose derived stromal/ stem cells (ASC) are currently being evaluated in this indication and paracrine anti-fibrotic effects are considered as predominant mechanisms. \u003c/p\u003e\u003cp\u003eMethods: The paracrine anti-fibrotic effects of SVF and ASC from healthy donors were tested in an innovative in vitro fibrogenesis model employing human VF fiboblasts (hVFF) and the principles of macromolecular crowding (MMC). Biosynthesis of collogen and alpha-smooth-muscle actin (αSMA) expression in hVFF were quantified after five days of indirect coculture with ASC or SVF using silver stain, western blot and RT-qPCR analysis. \u003c/p\u003e\u003cp\u003eResults: Fibrogenesis was promoted by addition of transforming growth factor beta 1 (TGFβ1) combined with MMC characterized by an enhanced deposition of fibrillar collagens and the acquisition of a myofibroblast phenotype (overexpression of αSMA). Adipose-derived therapies led to a reduction in the αSMA expression and the collagen content was lower in hVFF co-cultivated with SVF. \u003c/p\u003e\u003cp\u003eDiscussion: ASC and SVF promoted significant prevention of fibrosis in an in vitro fibrogenesis model through paracrine mechanisms, supporting further development of adipose-derived cellular therapies in VF scarring.\u003c/p\u003e","manuscriptTitle":"Paracrine Effects Of Adipose-Derived Stromal/Stem Cells And Stromal Vascular Fraction In An In Vitro Fibrogenesis Model Of Human Vocal Fold Scarring","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-04-02 21:23:53","doi":"10.21203/rs.3.rs-20745/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5d0afae7-f218-4cdc-bb15-2e85aeabfba8","owner":[],"postedDate":"April 2nd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":78484,"name":"Stem Cell \u0026 Developmental Cell Biology"}],"tags":[],"updatedAt":"2020-05-03T12:55:06+00:00","versionOfRecord":[],"versionCreatedAt":"2020-04-02 21:23:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-20745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-20745","identity":"rs-20745","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}