Thermoresponsive Gel Embedded with Adipose Stem-Cell-Derived

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Thermoresponsive Gel Embedding Adipose Stem CellDerived Extracellular Vesicles Promotes Esophageal Fistula Healing in a Thermo-Actuated Delivery Strategy Amanda K. A. Silva, Silvana Perretta, Guillaume Perrod, Laetitia Pidial, Véronique Lindner, Florent Carn, Shony Lemieux, Damien Alloyeau, Imane Boucenna, Philippe Menasché, Bernard Dallemagne, Florence Gazeau, Claire Wilhelm, Christophe Cellier, Olivier Clément, and Gabriel Rahmi ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b00117 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 20, 2018

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Thermoresponsive Gel Embedding Adipose Stem Cell-Derived

Extracellular

Vesicles

Promotes

Esophageal Fistula Healing in a Thermo-Actuated Delivery Strategy Amanda K. A. Silva1‡*, Silvana Perretta2‡, Guillaume Perrod3, Laetitia Pidial3, Véronique Lindner4, Florent Carn1, Shony Lemieux1, Damien Alloyeau5, Imane Boucenna1, Philippe Menasché6, Bernard Dallemagne2, Florence Gazeau1, Claire Wilhelm1, Christophe Cellier8, Olivier Clément3,7, Gabriel Rahmi3,8* 1. Laboratoire Matières et Systèmes Complexes (MSC), UMR 7057 CNRS, Université Sorbonne Paris Cité (USPC), Université Paris-Diderot, 10 rue Alice Domon et Léonie Duquet, 75205 Paris cedex 13, France. 2. Department of Digestive and Endocrine Surgery, Hôpital Civil de Strasbourg, and Institut de Recherche contre les Cancers de l’Appareil Digestif (IRCAD), Strasbourg, France. IHU, Minimally Invasive Hybrid Surgical Institute, Strasbourg, France. 3. Laboratoire Imagerie de l’Angiogénèse, Plateforme d'Imagerie du Petit Animal, PARCC, INSERM U970, Université Sorbonne Paris Cité (USPC), Université Paris Descartes, 56 rue Leblanc, 75015, 75015, Paris, France.

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4. Department of Pathology, Hôpital Hautepierre, 1, Avenue Molière, 67098 Strasbourg, France. 5. Laboratoire Matériaux et Phénomènes Quantiques (MPQ), UMR 7162 CNRS/Université Paris - Diderot, 10 rue Alice Domon et Léonie Duquet, 75205 Paris cedex 13, France. 6. Department of Cardiovascular Surgery, Hôpital Européen Georges Pompidou; Paris Cardiovascular Research Center, INSERM U970, Université Paris Descartes, Paris, 75015 France. 7. Department of Radiology, Hôpital Européen Georges Pompidou, Assistance Publique Hôpitaux de Paris, Université Paris Descartes, 20 rue Leblanc, 75015, Paris, France. 8. Gastroenterology and Endoscopy Department, Hôpital Européen Georges Pompidou, Assistance Publique Hôpitaux de Paris, Université Paris Descartes, 20 rue Leblanc, 75015, Paris, France. ‡These authors contributed equally * Author to whom correspondence should be addressed: [email protected], [email protected]

Keywords: extracellular vesicles, mesenchymal stem cells, minimally-invasive local delivery, digestive fistulas, thermoresponsive hydrogels


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ABSTRACT

Extracellular vesicles (EVs) are increasingly envisioned to be the next-generation of biological proregenerative nanotherapeutic agents, as already demonstrated for heart, kidney, liver, lung injury, brain and skin regeneration. Herein, we explore another potential EV therapeutic application, for fistula healing, together with a local minimally-invasive delivery strategy. Allogenic extracellular vesicles (EVs) from adipose tissue-derived stromal cells (ASCs) are administered in a porcine fistula model through a thermoresponsive pluronic F127 (PF-127) gel, injected locally at 4°C and gelling at body temperature to retain EVs in the entire fistula tract. Complete fistula healing is reported to be 100% for the gel + EVs group, 67% for the gel group and 0% for the control, supporting a therapeutic use of Pluronic F-127 gel alone or combined with EVs. However, only the combination of gel and EVs results in a statistically significant (i) reduction of fibrosis, (ii) decline of inflammatory response, (iii) decrease in the density of myofibroblasts and (iv) increase of angiogenesis. Overall, we demonstrate that ASC-EV delivery into a PF-127 gel represents a successful local minimallyinvasive strategy to induce a therapeutic effect in a swine fistula model. Our study brings prospects in EV administration strategies and in the management of postoperative fistulas.


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Extracellular vesicles (EVs) are sophisticated biological nanocarriers produced by the own organism to carry out information transfer and biomolecule delivery tasks. The term EV encompasses exosomes, microvesicles and apoptotic bodies, which are particles in the 405000 nm range released by cells in a constitutive or inducible manner,1-4 carrying lipids, proteins, mRNAs and miRNAs.5-7 EVs derived from mesenchymal stem cells (MSC) are competent to (i) induce a pro-angiogenic effect (e.g. bone marrow MSC EVs);8 (ii) inhibit cell apoptosis (e.g. bone marrow and umbilical cord MSC EVs);9,10 (iii) promote cell proliferation (e.g. umbilical cord MSC EVs);11 (iv) reduce inflammation (e.g. embryonic stem cell-derived MSC EVs)12 and (v) decrease fibrosis (e.g. embryonic stem cell-derived, liver stem cellderived and umbilical cord MSC EVs).12-14 This body of evidence gave rise to a paradigm shift in which EVs appear as an advanced biogenic nanomaterial to replace stem cells administration in regenerative medicine. Indeed, EVs represent an alternative to cell therapy mitigating risks of uncontrolled cell replication, undesired cell differentiation or vasculature occlusion.15,16 Besides, EVs offer easier storage as well as extended shelf-life advantages and the immune-privileged status of MSC EVs

17

15

may allow them to be exploited in an

allogeneic setting.16,18,19 Previous studies showed the beneficial effect of EVs as regenerative nanoconveyors in the treatment of heart (e.g. embryonic stem cell-derived MSC EVs), kidney (e.g. bone marrow MSC EVs), liver (e.g. umbilical cord MSC EVs), brain (e.g. bone marrow MSC EVs) and skin (e.g. umbilical cord MSC EVs) injuries.11,14,20-22 EVs may also bring prospects in the management of other diseases for which current strategies are limited and ineffective. This is the case of fistulas,23-26 consisting in abnormal communication between two organs or organ and skin.27 Fistulas are a major health problem related to Crohn's disease or secondary to surgery, cancer therapy or trauma,28-30 affecting millions of people worldwide with a mortality rate of 6–33%.31-33 Current surgical, 4 ACS Paragon Plus Environment

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biomaterial, drug-based or cell therapy approaches only provide poor and variable healing rates. On the one side, biomaterials as fibrin glue and porcine plug are applied locally for fistula therapy, but they are quite limited by abscess formation25 and spontaneous expulsion,24 respectively. On the other side, current pharmacological approaches relying on systemic administration of antibiotics and immunosuppressors/immunomodulators are limited and ineffective resulting in side effects and unsatisfactory therapeutic outcome.27,34,35 Concerning cell therapy, controversial success has been reported in fistula management. Some studies suggest cell therapy safety and efficacy,36 while others report fistula relapse37 or fail to prove a statistically significant effect.38 The investigation of EVs for fistula management, as an alternative to cell therapy, remains totally unexplored. A key issue related to EV therapy concerns the administration strategy. Recent findings demonstrate that systemically administered EVs have a short half-life,39-41 pointing out the importance of local delivery to avoid rapid EV clearance and improve EV retention in the site of interest.42,43 This retention attempt may be clearly reinforced by the choice of a vehicle/biomaterial to embed EVs.44,45 Research on EV association to biomaterials is in its very early steps. Only some papers have reported preliminary work, such as MSC exosomes embedded into chitosan hydrogels for skin wound healing;46 or platelet-rich plasma exosomes loaded into sodium alginate hydrogel for treating skin defects.47 Herein, we investigated a thermo-actuated strategy to deliver stem cell-derived EVs for digestive fistula management in a minimally-invasive procedure. We propose a local therapy based on the combination of stem cell-derived EVs with a thermoresponsive hydrogel. The thermoresponsive hydrogel is expected to cope with the difficulties of EV local delivery, promoting an occlusive effect, retaining EVs in the fistula tract and preventing EV wash-out by fistula secretions, while enabling the filling of the entire fistula tract despite its size and irregular morphology. In this attempt, we investigated EV administration directly into the 5 ACS Paragon Plus Environment

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fistula lumen embedded in a FDA-approved thermoresponsive biocompatible polymer. Adipose tissue stromal cell (ASC)-derived EVs embedded in a Pluronic F-127 (PF-127, also known as Poloxamer 407) thermoresponsive gel were injected in the liquid state through a catheter gelling in situ at body temperature (Figure 1). All in vivo testing has been performed in a clinically-relevant digestive fistula porcine model. Additionally, we carried out in vitro characterization of EVs and PF-127 gel, and evaluated the therapeutic potential of ASCderived EVs on endothelial cells and primary porcine oesophageal cells.

Results and discussion EVs were produced by porcine ASC under serum starvation and isolated as previously described.48 Size and morphology of EVs were first investigated by nanoparticle tracking analysis (NTA) data, indicating a broad EV size distribution (Figures 2A and Supplementary Table 1). Mean size and mode were 175 ± 4 nm and 138 nm ± 12 nm, respectively. According to in situ liquid-cell transmission electron microscopy (LCTEM)49 experiments performed in RPMI media, EVs also displayed a broad size distribution. Mean size and mode were 213 nm and 138 nm, respectively. Concerning morphology, LCTEM experiments showed that EVs displayed a round to irregular shape (Figure 2B). We next characterized the rheological properties of PF-127 gel embedding EVs. Figures 2C and Supplementary Figure 1 show dynamic rheological data (G’ and G”) as a function of temperature for PF-127 solution at a weight fraction of 20%. The rheology data clearly showed that the increase of temperature induced a sol-gel transition at temperature near 20°C, which corresponds to the cross-over of G’ and G”. This result is in accordance with those previously reported in the literature.50 When temperature was below the sol-gel transition temperature of the PF-127 solution, both G’ and G” were very weak, but with G” > G’, 6 ACS Paragon Plus Environment

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illustrating a liquid-like behavior. Above the transition temperature, both G’ and G” reached a plateau at higher almost identical levels, signature of a viscoelastic gel. Importantly, the solgel transition temperature was kept unchanged near the value of 20°C for the mixture containing EVs. Only a slight increase of the G’ value was detected due to the EVs incorporation. The sample containing both PF-127 and 1.3x1011 EVs/ml displayed a slight increase for G’ value in comparison to PF-127. Overall, the presence of EVs almost not affected the rheological behavior of PF-127 solution. A micromagnetophoresis investigation following EV magnetic loading, as reported in a previous paper,51 was performed as a stability/integrity test in order to check if EVs preserved their inner content in PF-127 gel. EVs were loaded with magnetic nanoprobes by previous internalization of iron oxide nanoparticles in parental ASC cells. Micromagnetophoresis experiments carried out for magnetic EVs in PBS or incubated overnight with PF-127 gel at 20% at 37°C attested magnetophoretic mobility of EVs to the micromagnet tip in both cases (Supplementary Figure 2). Non-magnetic EVs (having lost their integrity) do not display magnetophoretic mobility, neither single magnetic nanoprobes not enclosed into EVs due to Brownian motion. Considering this, micromagnetophoresis experiments indicated that EVs preserved their inner content in PF-127 gel, suggesting their integrity. We further characterized EVs in PF-127 gel biomaterial at 37°C by dynamic light scattering (DLS), NTA and confocal microscopy analyses. The micellization of PF-127 was investigated by DLS in PBS media at 37 °C (Supplementary Figure 3). For PF-127 concentration superior to 0.01 wt.%, the scattered intensity increased with PF-127 concentration and the normalized electric field autocorrelation functions (g(1)(q,t) depicted a mono exponential decay corresponding to an hydrodynamic diameter (DH) of 27 ± 2.0 nm. For PF-127 concentration ≤ 0.005 wt.%, the evolution of the scattered intensity with PF-127 concentration was minute and no significant correlation could be detected in the time variation of the scattered intensity 7 ACS Paragon Plus Environment

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(i.e. g(2)(q,τ→0) < 0.1). We analyze these results by considering that the critical micellar concentration (CMC) of PF-127 in PBS at 37 °C occurred between 0.005 wt.% and 0.01 wt.% in agreement with the PF-127 CMC of 0.009 wt.% in water at 37 °C reported in the literature.52 EVs in PBS and in PF-127 dispersion in PBS were analysed by DLS. As shown in Supplementary Figure 3, the autocorrelation function of EVs in PBS media was characterized by two relaxations. The fast mode corresponded to DH = 450 ± 120 nm while the slow mode corresponded to objects that sedimented with time (i.e. the amplitude of this relaxation decreases with time in aging condition).The mean size associated with the fast mode does not evolve with time and should correspond to individual EVs that represent the majority of the scattering objects in number. EV mean size by DLS differed from NTA one reported in Figure 2. This may be due to the biased right shift in DLS size distribution in the presence of large particle subpopulations, as reported in the literature.53 Concerning EVs in PF-127 dispersions, the Contin analysis of the autocorrelation functions at PF-127 concentrations near the CMC enabled to detect simultaneously the presence of micelles and EVs, which were well separated in size. This indicated that PF-127 micelles did not destabilize EVs and EVs did not destabilize PF-127 micelles either. The same DLS sample analysis following a 24-hour incubation at 37°C evidenced nearly equivalent size distribution results. The contribution of PF-127 micelles to the scattering increased with PF-127 concentration so that EV relaxations could not be detected for PF-127 concentrations ≥ 0.1 wt.% at an EV concentration of 4.6 x 106 EV/mL. Interestingly, PF-127 micelles were detected at a PF-127 concentration of 0.005 wt.% and EV concentration of 4.6 x 106 EV/mL, whereas no micelles were detected at the same PF-127 concentration without EVs. This may suggest a shift of the PF-127 CMC to lower concentration in the presence of EVs.


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Concerning NTA analysis, measurements rely on the tracking of the Brownian motion of nanoparticles to calculate their size. PF-127 micelles are near NTA detection limit of about 20 nm, thus mainly increasing the noise. With this set-up, we could however investigate the impact of PF-127 gel on EVs, as now presented with time series movies for Brownian motion tracking (Supplementary Movie 1) and computed size distribution histograms (Supplementary Figure 4). Brownian motion tracking was also carried out by confocal microscopy analysis of fluorescent red PKH26 labelled EVs (Supplementary Movie 2). Experiments were carried out at 37°C, which is above the sol-gel transition temperature (20°C) of the 20% PF-127 gel (Figure 2). EVs were observed as immobile dots in the 20% PF-127 gel by confocal microscopy and NTA analyses. Accordingly, the NTA device detected no track record of Brownian particles. Indeed, at a 20% gel concentration, there is dense network of packed PF127 micelles, which precludes any EV motion. Conversely, EVs in the PBS control were visualized as trackable Brownian objects with a high freedom to move in both NTA and confocal time series. When 20% PF-127 gel containing EVs was diluted by a factor 2 in PBS, the Brownian motion of EVs was partially restored, yet reduced compared to EVs in PBS control. Indeed, at a 1:2 dilution at 37°C, the dynamic viscosity of the gel is much higher than water (7 x 10-3 Pa.s). It was measured for our experiments, and was found at about 25 mPa.s and 15 Pa.s for shear rates of 20 and 0.01 s-1, respectively (Supplementary Figure 5). As a result, size distribution for EVs in the 20% PF-127 gel at 1:2 dilution was right shifted compared to EVs in PBS control in Supplementary Figure 4.

When 20% PF-127 gel

containing EVs was diluted by a factor 10 in PBS, the Brownian motion of EVs was comparable to EVs in PBS control, according to both NTA and confocal time series. Size analysis by NTA of the 20% PF-127 gel diluted by a factor 10 in PBS showed a main peak near 50-60 nm, and other peaks in the 100-300 nm range. The peak near 50-60 nm was clearly evidenced in the left tail of the gel (1:10 dilution) containing EVs. Importantly, this peak is


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much higher than the characteristic micelle peak at 20-30 nm range, suggesting that PF-127 micelles are near NTA detection limit, but they contribute to an increase in signal noise. Interestingly, the higher the gel dilution, the higher the noise. This can be explained by the fact that micelle mobility and Brownian motion increases as the concentration decreases. NTA analysis was used to evaluate PF-127 impact on EV stability at 37°C via EV number counting. For that, concentration counts for EVs in the 20% PF-127 gel at 1:10 dilution were normalized considering the gel sample at 1:10 dilution alone. NTA analysis was performed just after suspension preparation. Results were compared for EV suspensions at the same concentration in PBS and 0.3% triton X-100 (Supplementary Figure 6). Compared to the PBS condition, there was a trend towards an increase in EV concentration in the gel sample at 1:10 dilution. This may be due to a partial destabilization of EVs by the polymer or merely the interference of the noise detection despite the normalization. The influence of the noise was clearly observed in the large error bars when the analysis was carried out in the presence of PF-127 micelles. There was no statistically significant difference between EVs in PBS and EVs in the gel sample at 1:10 dilution. However, a statistically significant difference was observed when comparing EVs at the same concentration in PBS and 0.3% triton X-100. The decrease of EV concentration is expected to be the result of EV destructuration in the detergent solution. In order to check time effect on EV stability at 37°C in PBS or in the 20% PF-127 gel sample at 1:10 dilution, NTA analysis was repeated 24h later. There was no statistically significant difference in EV concentration in the gel sample at 1:10 dilution following a 24-hour incubation at 37°C. Similarly, no statistically significant difference was observed between EV counts in PBS after a 24-hour incubation at 37°C (Supplementary Figure 6).


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As a next step, we evaluated gel dissolution, as it is an important process for EV release. In order to shed light on the effect of dilution on the fate of PF-127 gel 20%, rheological analysis was performed for PF-127 at several inferior concentrations (Supplementary Figure 7). Evolution of dynamic storage modulus (G’) and loss modulus (G’’) indicated that at a 14% concentration, PF-127 displayed a transition from sol to gel at 34°C. At a 13% PF-127 concentration, the sol-gel transition temperature shifted to 39°C, which means that the polymer is in a sol state at body temperature. Accordingly, experiments carried out by NTA and confocal microscopy below this threshold, at 1:2 gel dilution (10% concentration), evidenced the Brownian motion of EVs (Supplementary movies 1 and 2), indicative of their release from the gel. A kinetic study of gel dissolution at 37°C as a function of the dilution factor was performed by the tube inversion method. A dilution series of the 20% gel in PBS from a 1:10 to a 9:10 (samples 1 to 9, Supplementary Figure 8) was prepared. For quantitative analysis, the volume of the sol fraction was measured using micropipettes. As expected, gel dissolution time length decreased as the dilution factor increased. For instance, only sample 1 featuring the highest dilution rate (1:10 representing a final concentration of 2% PF-127) was dissolved at 5h. At day 1 time point, the 3 highest diluted samples were dissolved (Samples 1, 2 and 3, featuring a final PF-127 concentration of 2, 4 and 6%, respectively). At day 4 time point, samples 4 (8% PF-127) and 5 (10% PF-127) presented no more gel. At a day 5 time point, sample 6 (12% PF-127) also dissolved. Sample 7 partially dissolved while samples 8 and 9 rather absorbed the sol fraction into the gel one. Accordingly, samples 7 to 9 were not expected to be totally dissolved as their dilution factor did not allowed to reach the concentration threshold of 13%, at which the sol-gel transition temperature is above the body temperature. As a following step, we investigated EV uptake by recipient HUVEC cells (Supplementary Figure 9). EV membrane was stained with lipophilic PKH26 dye. Confocal microscopy was 11 ACS Paragon Plus Environment

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performed to analyze HUVEC recipient cells incubated overnight with EVs in the gel 20% at a 1:2 dilution in complete culture medium, EVs in complete culture medium or complete culture medium alone. Images acquired for recipient cells incubated with EVs in the gel at a 1:2 dilution evidenced characteristic PKH26 fluorescence emission in a dotted pattern in inner cell planes. Cell planes featuring a Hoechst nuclear staining signal were considered as inner cell planes. The dotted pattern may be attributed to EV localization in endosomal/lysosomal compartments, as we have previously reported.54,55 A similar result was observed for recipient cells incubated with EVs in complete culture medium, while no PKH26 fluorescence was detected for control cells incubated with complete control medium alone. This experiment indicated that EVs released from the gel, due to the culture medium dilution effect, were successfully uptaken by recipient cells similarly to EVs in culture medium. We next performed in vitro studies to appraise the pro-migratory properties of ASC EVs. Their effect in the migration of human umbilical vein endothelial cells (HUVECs) following a scratch was investigated. Compared to negative control, cell migration was enhanced as EV concentration increased (Figure 3A). Cell migration shifted from 17% for the negative control to 19.6%, 26% and 54.5% in the presence of EVs at 0.5x1010, 1.5x1010 and 4.5x1010 EVs/ml, respectively. However, a statistically significant difference compared to the negative control was only observed at the highest EV concentration. Cell migration in the complete medium positive control was 38.6%, which also represented a statistically significant difference compared to the negative control. Representative scratch test images for each condition are provided in Figure 3B. The straight edges observed at the initial time became irregular and sinuous at the 24-h time point, indicating the occurrence of cell migration for all conditions. Particularly, edge sinuosity markedly increased while edge gap decreased for the highest EV concentration and for the positive control, when compared to the negative control.


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Pro-survival properties of ASC EVs were evaluated in vitro by investigating their effect on the metabolic activity of primary cells from swine oesophageal epithelium under serum starvation (Figure 3C). ASC EVs were found to promote the survival of esophageal epithelial cells in serum-free medium as evidenced by an enhanced cell metabolic activity in the presence of EVs. A dose-dependent effect was observed and a statistically significant difference compared to negative control could be evidenced, even at an EV concentration of 0.5x1010 EVs/ml. At a 4.5x1010 EVs/ml concentration, cell metabolic activity was near 5-fold higher than observed for the negative control. A statistically significant 4-fold increment of cell metabolic activity was obtained for the complete medium positive control when compared to the negative control. Finally, we evaluated the therapeutic effect of ASC EVs embedded in the PF-127 gel and delivered locally in a clinically-relevant porcine model of esophageal fistula.56 This study was approved by the local ethical committee on animal experimentation (reference number: 38.2013.01.050). All animals used in the study were managed in accordance with European Community Council directives as well as French laws related to animal use and care. An overview of the in vivo experimental study is provided in Figure 4. Fistulas were created at day 0 (D0). At D30, the animals underwent clinical, endoscopic and radiological analysis and were randomized into three groups of 3 pigs each: a control untreated group (n = 6 fistulas), a group treated with PF-127 gel at 20% alone (gel group, n = 6 fistulas) and a group treated with PF-127 gel at 20% containing 1.3x1011 EVs/ml (gel + EVs group, n = 5 fistulas). PF-127 gel with and without EVs was administered at D30. A dual administration was performed by injecting the gel through both fistula internal and external orifices. A multimodal follow-up (clinical, endoscopic, radiological and histological evaluation) was then carried out at D45. According to the clinical evaluation, the weight of pigs was stable in the three groups without significant difference between D0 to D30 and D30 to D45. Clinical evaluation indicated that 13 ACS Paragon Plus Environment

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external fistula orifices were open and inflammatory in all cases (6 cases from 6) for the control group (Figures 5A, 5B and 5C). For the gel group, most of external fistula orifices (4 cases from 6) were completely non-inflammatory and closed. A small ulceration of less than one millimeter was noted for 2 fistula orifices. For the gel + EVs group, the cutaneous fistula orifices were closed in all cases (5 cases from 5) and a homogeneous non-inflammatory wound healing was observed. A statistically significant difference was observed when comparing the two gel groups, with or without EVs, to the control with regard to external fistula orifice closure and inflammatory aspect. However, the analysis of the P value, the odds ratio and its 95% confidence interval clearly discriminate a superior healing effect of the gel + EV group compared to the gel alone (Supplementary Tables 2 and 3). The endoscopic evaluation (Figures 5D and 5E) indicated that the internal orifice was open in all 6 cases for the control group. In both the gel and the gel + EVs groups, the internal orifice was close in all cases at D45. A statistically significant difference was observed when comparing the gel and the gel + EVs group to the control, both comparisons featuring similar P values, odd ratios and 95% confidence intervals (Supplementary Table 4). The radiological evaluation was carried out to investigate the occurrence of a complete fistula tract evidenced by the communication between internal and external orifices (Figure 5F). Radiological examination showed a complete fistula tract in all 6 cases in the control group. By contrast, there was a total closure of the fistula tract in all 5 cases for the gel + EVs group. For the gel group, there was a total closure of the fistula tract in 4 cases and 2 cases of blind fistula (partial tract) without abscess were observed. A statistically significant difference was observed when comparing the two gel groups, with or without EVs, to the control. However, the analysis of the P value, the odds ratio and its 95% confidence interval clearly discriminate a superior healing effect of the gel + EV group compared to the gel alone (Supplementary Table 5). 14 ACS Paragon Plus Environment

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We then performed histological and immunohistochemical analysis to go deeper in the therapeutic effect in the tissue level. Our data showed the presence of a fistula tract as well as the occurrence of necrotic areas in all control group cases (Figures 6A and Supplementary Figure 10). Conversely, all fistulas were closed in the gel + EVs group without any fistula tract. In the gel group, there was a complete closure of the fistula tract in 3 cases and a partial closure in one case. In the other 2 cases, examination showed a thin fistula tract with a small ulceration at the cutaneous side. The gel group and, to a greater extent, the gel + EVs group presented a statistically significant incidence of fistula closure compared to the control (Supplementary Table 6). Concerning inflammation (Figure 6B), histological data demonstrated an important neutrophil and macrophage infiltration reflecting an acute inflammation and necrotic areas in all control group cases. For the gel group and especially for the gel + EVs group, there was a reduction in the inflammatory infiltration, with lymphocytes and without neutrophils, reflecting a more advanced healing process. A statistically significant reduction of the inflammation compared to the control was observed only for the gel + EVs group. Regarding fibrosis (Figure 6C), the control group displayed an infiltrating fibrosis, particularly around the fistula lumen. In one case, next to the esophageal epithelium, focal re-epithelialization areas were found. In another case, a 10 mm abscess was contiguous to the tracheal wall. A reduction of fibrosis thickness was observed in the gel group and more importantly in the gel + EVs group. A statistically significant reduction of fibrosis compared to the control was observed only for the gel + EVs group. Accordingly, collagen coverage, highlighted by Masson’s Trichrome staining, indicative of wound healing was more intensively observed in the gel + EVs group when compared to the gel and control groups (Figure 6D).

Immunohistochemistry with a smooth muscle actin antibody was

investigated to assess both myofibroblasts and vessels with a muscular wall. A statistically significant reduction in the density of myofibroblasts was observed only in the gel + EVs


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group compared to the control (Figures 6E and 6F). However, a statistically significant increase in the density of muscular vessels superior to 15/per field was observed in both the gel and the gel + EVs groups compared to the control (Figures 6F and 6G). CD31+ vascularization was also assessed by immunohistochemical analysis. A statistically significant increase in the density of CD31 + vessels (reflecting the whole vascularization) superior to 30.5/per field was observed in the gel + EVs group compared to the control (Figure 6I). We evaluated herein an innovative minimally-invasive therapeutic strategy based on the delivery of EVs embedded in a thermoresponsive PF-127 gel for fistula therapy. Both EVbased fistula therapy and the thermo-actuated EV delivery strategy have never been explored before. Thermoresponsive injectable hydrogels offer the advantages of filling irregular defects and reaching low-accessible areas while bypassing surgical interventions and the related discomfort and complications.57 Due to its thermoresponsive properties, PF-127 allowed the administration of EVs in a liquid injectable carrier through a catheter, whose gelation in situ at body temperature enabled to retain EVs at the fistula site. On one hand, the integration of EVs into PF-127 gel did not alter its thermoresponsive properties accessed by rheological analysis. On the other hand, EV integration into the gel did not impact the integrity of EVs according to micromagnetophoresis experiments.

DLS data indicated that EVs did not

destabilize PF-127 micelles. According to NTA analysis, PF-127 micelles did not induce the destructuration of EVs. Conversely, EV incubation with triton-100 induced a statistically significant decrease on EV concentration. NTA and confocal time series indicated that 20% PF-127 gel entrapped EVs, as evidenced by the absence of Brownian motion. Such a trapping effect was reverted by gel dilution and subsequent dissolution, releasing EVs featuring Brownian motion. We consider that in the case of esophageal fistulas, the release process is expected to be governed by the saliva flow in the esophageal lumen, which will exert the dilution effect in the internal fistula orifice. The


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gel will then be diluted until achieving a concentration threshold at which PF-127 will change to the liquid state releasing EVs. We carried out rheological measurements and we evidenced that this threshold is 13% PF-127 concentration at body temperature. PF-127 gel dissolution kinetics depends on the dilution factor. It can vary from 5 hours to 4 days for a 1:10 (2% final concentration) and a 1:2 (10% final concentration) dilution factor, respectively. Interestingly, dynamic viscosity data indicated a shear thinning behavior of PF127 gel that may at some extent contribute in vivo to the release of EVs from the gel via viscosity decrease under the effect of esophageal peristaltic movement. The mechanism of EV release via gel dilution has already been reported for liposomes in the PF-127 gel. Bochot et al 58 showed that the release of small molecules embedded into PF-127 gel was based not only on the gel erosion by dilution in aqueous medium but also on molecule diffusion throughout the gel. Conversely, the release of liposomes embedded into gel mainly relied on gel erosion by dissolution, which may be explained by liposome difficulties in crossing the gel network due to its size (50-200 nm size range). The authors showed that PF127 gel dissolution slowed down by increasing gel concentration. Therefore, we may expect a slightly delayed EV release by shifting PF-127 concentration from 20 to 25%, for instance. The proposed thermo-actuated delivery strategy was fundamental to overcome difficulties related to fistula secretions (washing-out the therapeutic agent) and the fistula tract (sometimes irregular large defects of several centimetres).59 Our minimally invasive strategy enabled fistula therapy via percutaneous as well as endoscopic administration, retaining EVs in the fistula lumen and filling large fistula defects of about 8 cm3 in a straightforward procedure carried-out in less than 15 minutes. Concerning fistula therapeutic outcome, EV combination to PF-127 provided a major healing effect in a clinically-relevant porcine model of esophageal fistula. In brief, complete healing, 17 ACS Paragon Plus Environment

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defined as the closure of both internal and external orifices according to clinical, endoscopic and radiological evaluation, was reported to be 100% (5 cases from 5) for the gel + EVs group and 0% (0 cases from 6) for the control. Complete fistula healing was observed for 67% (4 cases from 6) of the gel group. Indeed, PF-127 features intrinsic healing properties.60 For instance, topical application of PF-127 gel (20%) has been evaluated for cutaneous wound healing in Wistar rats and resulted in a significantly increased wound closure, higher microvessel density and reduced fibrosis.61 Therefore, PF-127 may be considered more than a carrier vehicle, contributing by itself to a regenerative effect. Importantly, the healing properties of PF-127 gel were markedly enhanced by the combination with EVs. In vitro tests demonstrated that EVs displayed pro-migratory properties by improving the migration of HUVECs while inducing a pro-survival effect on primary cells from swine esophageal epithelium. In vivo tests evidenced a shift from 67% of complete fistula closure for the gel group to 100% for the gel + EVs one. Besides, only the combination of gel + EVs resulted in a statistically significant (i) reduction of fibrosis, (ii) decline of inflammatory response, (iii) decrease in the density of myofibroblasts and (iv) increase of angiogenesis attested by the higher density of CD31+ vessel according to histological and immunohistochemical findings. The present study supports a two-way perspective for fistula therapy not only based on PF127 gel associated to ASC EVs, but also based on PF-127 gel only. Although its therapeutic efficacy is more limited without embedding EVs, PF-127 gel provides by itself a therapeutic option. Importantly, PF-127 gel is prone to fulfill regulatory requirements to achieve expedited clinical investigation. The pharmaceutical grade polymer is commercially available and the gel has received FDA approval as a vessel occlusive. Therefore, PF-127 gel “off label” use for fistula therapy, as proposed herein, would clearly benefit from a straightforward regulatory pathway compared to PF-127 gel containing EVs. The clinical translation of EVbased therapy will require the development of cost-effective, standardized, robust and 18 ACS Paragon Plus Environment

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reproducible methods for EV production and isolation in a scale-up perspective. In this regard, preclinical and clinical development of EVs should comply with guidelines for biological medicines. Accordingly, safety aspects regarding the supply, testing, processing, traceability, distribution and storage of EVs become critical.62 Despite these challenges, the fast-forward ongoing advances in EV field may facilitate their clinical translation in the future. Conclusions In summary, our study brings prospects for local minimally-invasive EV delivery based on a thermo-actuated administration strategy in PF-127 gel. We investigated the interaction between EVs and PF-127 micelles via nanoparticle size distribution and counting and we demonstrated no destructuration effect. Based on Brownian motion analysis, we evidenced the trapping effect of PF-127 gel on EVs (precluding their diffusion) as well as EV release process relying on gel dilution. The interest of the proposed EV delivery into PF-127 gel may stretch beyond fistula therapy being applicable for EV-based regeneration of other injury sites reaching large extents or presenting secretions, such as burning lesions, for instance. Besides enabling EV delivery, the thermoresponsive polymer PF-127 contributed to fistula healing. The gel alone or in combination with EVs may represent the next-generation therapeutic options for fistula management deserving to reach further pharmaceutical development steps towards clinical investigation.

Methods Cell culture MSC-derived EVs were prepared from ASCs isolated from the abdominal fat of 6 month-old female pig (ABCell-bio, Paris, France). In order to confirm the identity of ASC, a FACS


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analysis was performed by the supplier showing cell expression of CD 90+, CD 105+ and CD 45-, CD 31-. Cells were cultured at 37°C and 5% CO2 in minimum essential medium Eagle, alpha modification (Sigma-Aldrich, Germany) containing 10% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 U/mL streptomycin). For in vitro tests, human umbilical vein endothelial cells (HUVEC) were cultured as adherent cells at 37 °C and 5% CO2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 U/mL streptomycin). Production and isolation of EVs Pig ASCs were used at passages between 6 to 10. For EV production, ASCs were stressed in serum-free medium during 48 hours. EV isolation was carried out based on a standard literature protocol. 63 The supernatant was collected, centrifuged at 2 000 G during 10 minutes to discard the pellet (cell and apoptotic body fraction). The EV fraction in the supernatant was isolated by ultracentrifugation (100 000 G) during at least 2 hours. The pellet was then resuspended in phosphate buffer (PBS, pH: 7.4). Characterization of EV size and morphology Nanoparticle tracking analysis (NTA). Suspensions containing EVs were analyzed using a NanoSight LM10 instrument (NanoSight, Amesbury, UK). The device was equipped with a monochromatic laser beam at 532 nm, which was applied to the dilute EV suspension. A total of 10 videos of 30 seconds duration were recorded and EV Brownian movement was tracked by NTA software 3.0 version in a frame to frame basis. Two-dimensional Stokes–Einstein equation was used to compute particle size from the velocity of EV movement. The optimization of NTA post-acquisition settings was performed and these parameters were kept constant for replicate analysis. EV concentration, mean and mode were obtained as well as cumulative 10%, 50% and 90% diameter. 20 ACS Paragon Plus Environment

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In situ Transmission Electron Microscopy (TEM). In situ TEM experiments in RPMI medium were realized by exploiting a liquid-cell TEM holder commercialized by Protochips on a JEOL ARM 200F microscope used in scanning mode with a High-Angle Annular Dark-field detector (STEM – HAADF). All the experiments were performed with a 200 kV acceleration voltage. Sample preparation, STEM imaging and EV-contrast enhancement were performed as reported elsewhere.48 Preparation of PF-127 gel containing EVs PF-127 (Sigma-Aldrich, Germany) was dissolved in sterile PBS at a 25% concentration and stirred at 4 °C for 24h. After solubilization, it was filtered with a 0.2 µm filter for sterilization at 4°C. Then, ASC EVs suspended in PBS were added to the gel, whose final concentration was 20% PF-127 and 1.3x1011 vesicles/ml. The rheological properties of PF-127 gel with and without EVs was evaluated. The effect of the gel on EV integrity was assessed by a micromagnetophoresis experiment. Characterization of PF-127 gel containing EVs Rheological analysis. Rheological analysis was performed using a Physica RheoCompass MCR 302 (Anton Paar, France) equipped with a solvent trap in order to prevent solvent evaporation. The measurements were performed using a cone and plate geometry (diameter = 50 mm, cone angle = 1°). A temperature unit (Peltier plate) provided the control of temperature during analysis and the temperature dependent elastic (G’) and loss (G”) moduli were measured by applying an oscillatory shear stress at a frequency of 1 Hz, a strain of 1% and a heating rate of 5°C/min applied from 5 °C to 40 °C. The sol–gel transition temperature was defined as the point of the cross-over of the elastic and loss moduli (G’ = G”).57 Rheological analysis was performed for PF-127 in solution at weight fraction of 12, 13, 14 and 20% and mixture of PF-127 at 20% containing 1.3x1011 EVs/ml.


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Micromagnetophoresis. A micromagnetophoresis experiment was carried out to investigate the integrity of EVs according to a previously published protocol. For that, magnetic nanoparticle-loaded EVs were obtained from ASCs previously labelled using a citrated iron oxide nanoparticle suspension (5 µM in iron). The trajectories of microvesicles in magnetic field gradient created by a micromagnet were analyzed using the previously published experimental set-up.50 A glass slide/coverslip chamber featuring a 50 µm diameter nickel rod constituted the micromagnetophoresis device. A 150 mT uniform magnetic field (from a rectangular magnet) was used to magnetize the nickel microrod. The trajectories of EV suspensions introduced into the micromagnetophoresis chamber were observed by means of an optical microscope (DMIRB Leica; Leica Microsystems, Germany) using a 60× objective. The optical microscope was connected to a charge-coupled device camera and a computer. For micromagnetophoresis analysis, EVs were dispersed in PBS or incubated overnight in PF127 gel 20%. Micromagnetophoresis experiments were carried out for EV samples in PBS and EV samples in PF-127 gel 20%, diluted at a 1:1 ratio in PBS just before analysis. EVs could not be analysed when dispersed in the gel at 20% due to gelation at room temperature, which precludes EV to move. Dynamic light scattering (DLS). DLS was used to determine the hydrodynamic diameter (DH) of PF127 micelles and EVs at 37 °C in PBS media. The measurements were monitored on a NanoZS (Malvern Instrument) operating at λ = 632.8 nm (4 mW HeNe). The normalized time autocorrelation functions of the scattered intensity, g(2)(q,t), were measured at 173 °. The normalized electric field autocorrelation functions, g(1)(q,t) were derived from g(2)(q,t) through the Siegert relationship. DH was derived from the Stokes–Einstein relation. The size distributions were obtained by estimating the inverse Laplace transform of g(1)(q,t) with the Contin method. All samples were equilibrated at 37 °C during 300 s before measurements.


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PF-127 gel dissolution kinetics. PF-127 gel dissolution kinetics at 37°C as a function of the dilution factor was evaluated by the tube inversion method.64 In order to facilitate test readout, Trypan blue dye 0.4% (Sigma-Aldrich) was added to the gel (0.004% final dye concentration). PF-127 gel at 20% was diluted in PBS at serial ratios: 100 to 1000 (sample 1); 200 to1000 (sample 2); 300 to 1000 (sample 3); 400 to 1000 (sample 4); 500 to 1000 (sample 5); 600 to 1000 (sample 6); 700 to 1000 (sample 7); 800 to 1000 (sample 8) and 900 µL to 1000µL (sample 9). In order to prepare these samples, PF-127 at 20% in the liquid form at 4°C was pipetted into 2-ml Eppendorf tubes. Following a 10-minute incubation at 37°C to allow gel transition, PBS at 37°C was gently added to tubes. Dissolution kinetics was evaluated in triplicate by the evolution of the sol volume as a function of the time. For quantitative analysis, the volume of the sol fraction was measured using micropipettes. Confocal microscopy for the characterization of EVs in PF-127 gels and EV cell uptake in vitro. Both EV suspensions and EV recipient cells were analyzed by confocal microscopy. Previously, EVs in PBS were labelled by adding the fluorescent lipophilic tracer PKH26 (Sigma-Aldrich) at 0.5 µM final concentration. PKH26 stained EVs were imaged at 37°C either in PBS, in 20% PF-127 gel or in 20% PF-127 gel diluted in PBS (1:2 and 1:10 dilution). Recipient HUVEC cell nuclei were labelled with Hoechst 33342 (Thermo Scientific) at 1 µg/ml in PBS for 15 minutes followed by 3 washing steps with PBS and addition of complete culture medium. Living HUVECs were imaged after overnight incubation with EVs in 20% PF-127 gel supplemented with complete DMEM (1:2 dilution), EVs in complete DMEM or complete DMEM alone. For the gel condition, 20% PF-127 gel containing EVs was first pipetted at 4°C onto the HUVEC layer and allowed to gel for 5 min at 37°C. Then, equal volume of complete DMEM at 37°C was gently pipetted onto the gel layer. Analyses were carried out at 37°C using an Olympus JX81/BX61 device/ Yokogawa CSU spinning disk microscope (Andor Technology plc, Belfast, Northern Ireland), equipped


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with a 60 Plan-ApoN oil objective lens (60/1.42 oil, Olympus). The PKH26 was excited at 561 nm and fluorescence emission was collected with a filter at 608 nm. For Hoechst 33342, the excitation wavelength was 405 nm and the fluorescence emission was collected with a filter at 445 nm. For the analysis of EVs in suspension, time lapse images every 0.1 s in a total of 100 images were acquired. In vitro evaluation of pro-migratory and pro-survival properties of EVs Scratch test. The quantitative migration of human umbilical vein endothelial cells (HUVECs) in monolayers following a scratch was investigated in order to assess in vitro the promigratory properties of ASC EVs. Confluent HUVEC monolayers in 24-well plates were scratched using the tip of a 1 ml pipette cone and the medium was replaced by fresh one to discard detached cells. HUVEC were incubated 24h with ASC EVs in serum-free culture medium at an EV concentration of 0.5x1010, 1.5x1010 or 4.5x1010 EVs/ml. Serum-free medium and complete medium were used as negative and positive controls, respectively. Cell monolayer images were recorded using a microscope (Leica, Germany) connected to a camera. The percentage of migration was calculated via the gap width between wound edges measured using Image J software at 0 and 24 h time points, 100% migration meaning total gap bridging at 24 h time point. Apoptosis resistance test. Pro-survival properties of ASC EVs were evaluated in vitro by investigating apoptosis resistance via an Alamar metabolic test. Primary cells from swine oesophageal epithelium were used for this experiment. Oesophageal biopsies were collected under general anaesthesia. Animals were sedated using 10mg/kg of intramuscular ketamine and 8mg/kg of intravenous propofol. Biopsies were collected using radial jaw biopsy forceps (Supplementary Figure 11), immediately placed in DMEM media containing 100 U/mL penicillin and 100 U/mL streptomycin and washed three times in this medium before cell dissociation via enzymatic digestion. Serial enzyme incubation was performed using Liberase


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(purified collagenase I and I and thermolysin), collagenase IV and trypsin. Dissociated cells were washed two times in serum-free DMEM without phenol red containing antibiotics and transferred to 96-well plates. Epithelial cells were incubated for 24h with ASC EVs in serumfree DMEM culture medium without phenol red at an EV concentration of 0.5x1010, 1.5x1010 or 4.5x1010 EVs/ml. Serum-free medium and complete medium were used as negative and positive controls, respectively.

Alamar Blue reagent (Invitrogen, United Kingdom) was

added directly in the wells containing cells and EVs and the test was performed according to supplier’s instructions. Wells containing EVs without cells were used as a baseline for the wells containing cells and EVs in order to exclude EV contribution to Alamar Blue reagent metabolization. Esophageal porcine fistula model Nine adult (25-30 kg) Yucatan pigs were used for the experiment. Fistula creation, therapy, radiological and endoscopic evaluation were performed under general anesthesia. Animals were sedated with 10mg/kg of intramuscular ketamine and 8mg/kg of intravenous propofol. An endotracheal tube connected to a respirator was used and continuous cardio-respiratory monitoring was performed. Anesthesia was maintained through isoflurane 2.5% inhalation. All animals were prepared with a 24-hours solid food diet before endoscopy. A combined surgical and endoscopic approach was used to create the fistula at day 0 (D0), as we described in a previous paper.51 The first step was a skin incision and tissue dissection at the cervical neck (first the right side and then left side). After esophageal wall incision under endoscopic control (Gastroscope EG-590WR Fujifilm, Tokyo, Japan), two plastic stents (10Fr T-tube, Coloplast, Humlebæk, Denmark) were inserted one after another between the skin and the esophageal lumen. More precisely, through a left and right cervicotomy, under endoscopic view, plastic catheters were introduced into the esophagus 30 cm from the dental arches bilaterally. Stents were tunneled subcutaneously, fixed to the skin and left in place for


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1 month. The mean procedure time was 43±15 minutes. Analgesia after surgery was delivered by intramuscular injection of 5 mg morphine. For each pig, 3-day antibiotic prophylaxis by intravenous injection of amoxicillin 1g/day (Clamoxyl, GSK, Brendford, UK) was carried out. Liquids were authorized on day 1 after surgery and normal feeding the days after. There was no intraoperative complication. Only one pig had vomiting with transient hematemesis at D3, but the evolution was rapidly favorable. In five pigs, partial catheter migration was observed between day 5 and day 12 and catheters were repositioned and secured by sutures. At D30, the stents were withdrawn under general anesthesia. External and internal fistula orifices were evaluated, respectively, by clinical and endoscopic evaluation. A fistulography was performed to image the fistula tract by endoscopy (Supplementary Figure 12). A catheter was introduced into the internal fistula orifice and 5 ml of a contrast agent (Xenetix 250®, Guerbet, France) was injected under fluoroscopy monitoring. Just after or in the same time, the contrast agent was injected through the external orifice using a 5 ml syringe. Fistula therapy using PF-127 gel with and without EVs At D30, the animals were randomized into three groups of 3 pigs each: a control untreated group, a group treated with PF-127 gel at 20% alone (gel group) and a group treated with PF127 gel at 20% containing 1.3x1011 EVs/ml (gel + EVs group). There were 6 fistulas created in the control group, 6 fistulas in the gel group and 5 fistulas in the gel + EVs group (1 fistula creation was not successful due to early catheter migration resulting in a single fistula tract for one animal). PF-127 gel with and without EVs was administered at D30 under general anesthesia. A dual administration was performed by injecting the gel through both fistula internal and external orifices. This procedure was carried out in less than 15 minutes. A plastic catheter inserted through the 2.8 mm operating channel of a gastroscope was used to inject the gel via the fistula internal orifice. Different endoscopic catheters were tested to allow the passage of the


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PF-127 gel through the catheter lumen without obstruction. A 6 Fr Huibregtse® guiding catheter (Cook Medical, North Carolina, USA) was found the most appropriate and was therefore selected for gel administration. Previous to gel injection, the catheter was cooled by injection of cold saline at 4 °C. Then, 4 ml of cold PF-127 at 20% containing or not 1.3x1011 EVs/ml (gel + EV group or gel group, respectively) at 4 °C was injected in liquid state through the internal fistula orifice using the endoscopic catheter. An additional 4 ml of PF127 solution at 4 °C containing or not EVs was sequentially administered through the external fistula orifice using a 5 ml cold syringe at 4 °C. PF-127 solution administration at 4 °C was monitored endoscopically and product phase transition to a gel state was observed in both internal and external fistula orifices (Supplementary Figure 13). Therapy follow-up A multimodal follow-up (clinical, endoscopic and radiologic evaluation) was carried out under general anesthesia before therapy on D30 and before animals were sacrificed on D45. Clinical evaluation. Animals were daily examined and weighed on D0, D30 and D45. Assessments of pain, behavior, food intake and external orifice fistula aspect were carried out. At D30 and D45, the external fistula orifice was evaluated by a skilled gastroenterologist to assess its opening and inflammatory aspect. External fistula orifice was considered open if a plastic catheter could be introduced into it. Inflammation was defined by the presence of an ulcerated orifice (cutaneous inspection). Endoscopic evaluation. At D30 and D45, an endoscopic evaluation under general anesthesia was also performed by a skilled gastroenterologist to investigate internal fistula orifice opening and inflammatory aspect. Similarly, catheterization of the fistula tract was systematically attempted and inflammation was defined by the presence of an ulcerated orifice.


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Radiological evaluation. At D30 and D45, radiologic evaluation (2 orthogonal incidences) was performed using a contrast agent (Xenetix® Guerbet, Villepinte, France). The most relevant incidence was kept for analysis. The diameter, length of fistula tract and presence of abscess (opacification of cavity branched along the fistula tract) were recorded. Histological analysis. At D45, just after the final endoscopic and radiological evaluation, animals were sacrificed. A large dissection around the fistula tract including cervical esophagus and the skin was performed for macroscopic evaluation and measurements. Specimens were harvested en-bloc and stored in 10% buffered formalin for at least 24 hours before they were embedded in paraffin. Four-µm sections were obtained and stained with hematoxylin-eosin or Masson’s Trichrome. After the dermal and esophageal sides were located, specific measurements were performed by a pathologist to assess the following end points: presence of a fistula tract; presence, location and maximum thickness of fibrosis around the fistula; presence and semi-quantitative analysis of inflammatory infiltrate; presence of re-epithelialization, necrosis or abscesses; and aspect of the vascularization around the fistula tract. Immunohistochemical analysis. Sections from selected paraffin blocks for each specimen were used for immunohistochemical analysis. This immunohistochemical study was realized on a Ventana Roche Benchmark XT with the same antigen retrieval process, in a EDTA citrate buffer (pH 8,3) during 30 min and via the ultraView Universal DAB Detection Kit, Ventana Roche System. Two antibodies were used: -

an anti-alpha smooth muscle actin

antibody (monoclonal mouse, 1A4, DAKO), 1/500 dilution, 20 min incubation, for the staining of the muscular layer of the vessels and of myofibroblasts; -

an

anti-

CD31/PECAM 1 antibody (monoclonal rabbit, EP78, Microm), 1/200 dilution, 32 min incubation, for the staining of the endothelial layer of the vessels (capillary and muscular vessels).


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Statistical analysis Statistical analysis was performed using GraphPad Prism software (GraphPad Software, USA). For numerical data, mainly from in vitro experiments, each condition was compared to the negative control using Mann-Whitney test. Categorical data, mainly from in vivo experiments, were expressed as number of cases per group. These data from gel group or gel + EVs group were compared to control using Chi-square test. A P-value inferior to 0.05 was considered to be significant (*P

Thermoresponsive Gel Embedded with Adipose Stem-Cell-Derived

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