João P E Machado

Isabelle Capron

Rilton A De Freitas

Lazhar Benyahia

Taco Nicolai
email: taco.nicolai@univ-lemans.fr

Stabilization of Amylopectin-Pullulan Water in Water Emulsions by Interacting Protein Particles

Keywords: Water-in-water emulsion, Pickering, microgel, aqueous two phase, polysaccharide separating mixed biopolymer solution containing colloidal particles

Water in water emulsions were prepared by mixing aqueous solutions of amylopectin (AMP) and pullulan (PUL) in the presence of whey protein microgels (MG). Attractive interaction between the MG was introduced by decreasing the pH from neutral towards their isoelectric pH either by adding HCl while stirring or progressively in-situ by adding glucono-δ-lactone (GDL).

Decreasing the pH led to a change in the preference of the MG from the PUL phase to the AMP phase and to adsorption of the MG at the interface. The morphology of the emulsions was observed using confocal laser scanning microscopy. The morphology and stability of the emulsions depended strongly on the pH and differed when AMP droplets were dispersed in the PUL phase or vice versa. In some cases, stable weak emulsion gels were formed that flowed when tilted. In others, droplets remained dispersed in a liquid phase stabilized by a gelled interface layer of MG. The interaction between the MG was further modulated by adding small amounts of anionic polysaccharides that formed complexes with the MG below pH 5.6. This was found to influence the partitioning of the MG between the phases, as well as the stability and morphology of the emulsions.

Introduction

Thermodynamically incompatible mixtures of aqueous soluble polymers give rise to aqueous two-phase systems (Frith, 2010;Gonzalez Ortiz et al., 2020), which are common in food products that contain different biopolymers that form distinct phases on mesoscopic scales. When solutions of two incompatible polymers are mechanically mixed above a certain concentration, they form water in water (W/W) emulsions with one polymer phase dispersed as droplets in a continuous phase rich in the other polymer. W/W emulsions have properties that differentiate them from conventional oil/water (O/W) emulsions. One of the most important differences is that since both phases are aqueous solutions, the interfacial tension in W/W emulsions is orders of magnitude lower and becomes zero at the critical point (Firoozmand et al., 2009;[START_REF] Scholten | Interfacial tension of a decomposed biopolymer mixture[END_REF]. Another feature of W/W interfaces is that their width is on the same length scale as the correlation length of the polymers in the phases (Nicolai & Murray, 2017). These characteristics inhibit the use of molecular surfactants as stabilizers for W/W emulsions. However, the addition of solid particles has been shown to stabilize W/W emulsions in some cases [START_REF] Dickinson | Particle-based stabilization of water-in-water emulsions containing mixed biopolymers[END_REF][START_REF] Esquena | Water-in-water (W/W) emulsions[END_REF]Nicolai & Murray, 2017;[START_REF] Sarkar | Sustainable food-grade Pickering emulsions stabilized by plant-based particles[END_REF].

Particles adsorb at the interface, thus procuring steric hindrance against droplet coalescence, known as the Pickering effect. The driving force of interfacial particle adsorption was first described for oil-water interfaces [START_REF] Aveyard | Aspects of the stabilisation of emulsions by solid particles: Effects of line tension and monolayer curvature energy[END_REF][START_REF] Levine | Stabilisation of emulsion droplets by fine powders[END_REF] and later applied to W/W emulsions [START_REF] Balakrishnan | Particles trapped at the droplet interface in water-in-water emulsions[END_REF]. The free energy gained by adsorption (ΔG) of a spherical particle depends on the particle radius (R) and the contact angle (θ) between the particle and the interface:

Δ = - (1 -|cos )|) (1)
The contact angle is determined by the difference between the interfacial tension of the particle with phase A (γPA) and B (γPB) and that between the phases (γAB):

cos( ) = ( ) (2)
From Eq. 2, it follows that |cos (θ)| < 1 only if γAB > |γPA-γPB|, implying that if γAB < |γPA-γPB| the particles do not adsorb at the interface. The most favorable condition for adsorption is when γPA = γPB, i.e. when the particles partition equally between the phases.

Recently, we showed that the partition of particles between two polysaccharide phases can be controlled by adding small amounts of a third polysaccharide that partitions between the phases and does not have a specific interaction with the particles (Machado et al., 2021). The reason is that the presence of the third polysaccharide within a phase modifies the interfacial tension of particles with that phase. In this manner, particles could be induced to adsorb at the interface in situations where they did not adsorb in the absence of the third polysaccharide. Equal partition could be achieved by fine-tuning the concentration of the third polysaccharide, and it was shown that in that case, the particles remained adsorbed at the interface upon dilution down to very close to the binodal. It was found, however, in that study as well as in others (Gonzalez-Jordan et al., 2018;Nguyen et al., 2013) that the adsorption of particles at the interface is not a sufficient condition to inhibit the coalescence of dispersed droplets. It was speculated that interaction between the particles at the interface is also required.

One type of particle that has been used in the past to stabilize W/W emulsions is whey protein microgels (MG) [START_REF] De Freitas | Stabilization of Water-in-Water Emulsions by Polysaccharide-Coated Protein Particles[END_REF]Gonzalez-Jordan et al., 2017[START_REF] Dickinson | Particle-based stabilization of water-in-water emulsions containing mixed biopolymers[END_REF]Hazt et al., 2020;Nguyen et al., 2013Nguyen et al., , 2015)). At neutral pH these protein particles are negatively charged and repel each other. However, when the net charge density of the proteins is reduced by reducing the pH towards their isoionic point (IP=5.0 (Kharlamova et al., 2016)) hydrogen bonding and hydrophobic interactions drive aggregation of the particles [START_REF] Schmitt | Internal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment[END_REF]. Gonzalez-Jordan et al., (2017) investigated the effect of adding MG on the stability of W/W emulsions formed by mixing dextran (DEX) and poly(ethylene) oxide (PEO) at different pH. Between pH 6.0 and pH 3.5, the microgels aggregated at a rate that depended on the pH. Closer to the IP, large clusters of MG were formed rapidly during mixing that migrated to the interface and extended into the dextran phase. Further from the IP, aggregation was slower, allowing the formation of a continuous protein layer at the droplet interface that subsequently gelled. However, MG at the interface of different droplets could also bind to each other, leading to clustering of droplets.

Flocculation of protein microgels close to the IP can be avoided by adding polysaccharides (Peinado et al., 2010;[START_REF] Santipanichwong | Core-shell biopolymer nanoparticles produced by electrostatic deposition of beet pectin onto heat-denatured β-lactoglobulin aggregates[END_REF]. It was shown that that anionic (κ-carrageenan), cationic (chitosan) and neutral (xyloglucan) polysaccharides formed complexes with MG in a pH range close to the IP, which adsorbed at the W/W interface in DEX/PEO emulsions, but did not bind to each other so that the droplets remained individually dispersed [START_REF] De Freitas | Stabilization of Water-in-Water Emulsions by Polysaccharide-Coated Protein Particles[END_REF][START_REF] Khemissi | Exploiting Complex Formation between Polysaccharides and Protein Microgels To Influence Particle Stabilization of W/W Emulsions[END_REF]. Thus, we may conclude that the addition of polysaccharides influences the behavior of protein microgels in this W/W emulsion at all pH, but differently when electrostatic repulsion between the polysaccharides and the microgels is strong compared to when electrostatic attraction leads to complexation. In both cases, the interfacial tension between the phases and the particles is influenced by the presence of polysaccharides, but when complexes are formed the nature of the particles themselves is modified and, therefore, their interaction with each other.

Here we present an investigation of W/W emulsions formed by mixing two neutral foodgrade polysaccharides: amylopectin (AMP) and pullulan (PUL), in the presence of whey protein microgels. AMP is a highly branched polysaccharide present in starch [START_REF] Copeland | Form and functionality of starch[END_REF][START_REF] Tester | Starch -Composition, fine structure and architecture[END_REF], whereas PUL is a linear polysaccharide obtained from a yeast-like fungus (Nishinari et al., 1991;[START_REF] Singh | Pullulan: Microbial sources, production and applications[END_REF]. We recently investigated this system at neutral pH and found that the MG was strongly partitioned to the PUL phase and only adsorbed at the interface at high PUL and AMP concentrations (Machado et al., 2021). However, as mentioned above, after adding small amounts of a third polysaccharide, the MG adsorbed at the interface even very close to the binodal. Unfortunately, the adsorption of the MG did not significantly improve the stability against coalescence, which we suggested was due to a lack of sufficiently strong attractive interaction between the MG at the interface. Therefore, the objective of the present study was to evaluate the effect of introducing attraction between whey protein microgels (MG) by decreasing the pH. The interaction was further modulated by adding anionic polysaccharides that form complexes with the MG. We will show that these emulsions can be rendered stable by introducing attractive interactions between the particles in a controlled manner. The pH was decreased either by adding the required amount of HCl under stirring or in-situ by adding glucono-δ-lactone (GDL) that slowly degrades, releasing H + . The method of pH decrease and the amount of added polysaccharide on the structure and stability of the W/W were found to be very important. It will be demonstrated that very stable emulsions can be obtained by in-situ pH decrease and fine-tuning the amount of added polysaccharide.

Materials and methods

Materials

Amylopectin (from maize, batch: SLBP9703V), alginate (alginic acid sodium salt , batch:

MKBZ5563V) (ALG), and κ-carrageenan (KC) (batch: BCBX5072) were purchased from Sigma-Aldrich. Pullulan and low-methylated pectin (PEC) from lemon peel were kindly provided by Hayashibara co and Cargill, respectively. KC and AMP were purified before use. An aqueous KC solution at 0.5 wt% was dialyzed using 13.5 kDa dialysis bags first against 0.1 mol L -1 NaCl and then against ultrapure water (Milli-Q) for 2 days, regularly refreshing the outside water to remove residual K + . Subsequently, the solution was freeze-dried. An AMP solution at 5 wt% was prepared in a mixture of dimethyl sulphoxide (DMSO) and water 95:5 v/v. The solution was centrifuged, and AMP in the supernatant was precipitated by adding 3 volumes of ethanol. The precipitate was rinsed with acetone and diethyl ether and dried for 2 days under vacuum at 30 °C. Both PEC and ALG solutions were centrifuged to remove insoluble aggregates. The polysaccharides were characterized by size-exclusion chromatography with on-line light scattering detection, and the Whey protein isolate (BiPRO ®) was procured from Davisco Foods International, Inc (Le Sueur, MN, USA). Size exclusion chromatography showed that the sample contained 65% β-lg and 20% α-lac. The remaining 15% consisted of other whey proteins and a small amount of caseins. Protein microgels (MG) were obtained and characterized as described in detail by [START_REF] Phan-Xuan | On the crucial importance of the ph for the formation and self-stabilization of protein microgels and strands[END_REF]. Briefly, 4 wt% WPI in aqueous solution at pH = 5.90 was heated for 15 h at 80 °C. After this treatment, approximately 65 % of the WPI had formed microgels. The MG were analyzed by static and dynamic light scattering from which it was found that the average hydrodynamic radius was 120 nm, the radius of gyration was 125 nm, and the molar mass was 3.3 × 10 8 g/mol. The remaining 35 % was composed of small strand-like aggregates formed by heating that do not have W/W interfacial properties (Nguyen et al., 2013).

The pH of all polymer solutions was set to 7.0 before use by dropwise addition of NaOH or HCl (0.1 and 0.01 mol L -1 ). Emulsions were prepared by mixing stock solutions of the various ingredients in the required amounts using a vortex mixer. No effect on the order of mixing was observed. Two emulsions were investigated in detail according to the phase diagram reported elsewhere (Machado et al., 2021) and reproduced as fig. S1 in the supplementary information.

One composed of a dispersed phase containing 1.4 wt% PUL in a continuous phase of 7.8 wt% AMP (P/A) and one composed of a dispersed phase containing 1.2 wt% AMP in a continuous phase of 5.1 wt% PUL (A/P). The amount of PUL in the AMP phase was negligible, but the phase diagram showed that the AMP phase contained approximately 0.8 wt% PUL. The volume fraction of the dispersed phase was in both cases 13 %. These mixtures were situated on the same tie-line, and therefore, the interfacial tension between the PUL and AMP phases was the same. Unless otherwise specified, the concentration of total added protein was 0.4 wt%. The pH was decreased either by adding dropwise an HCl solution while stirring with a vortex or by adding a fresh solution of GDL. Hydrolysis of GDL leads to the release of H + and to the formation of gluconate ions. The pH of the samples was measured as function of time after addition of GDL in parallel with the microscopic observations. The final pH of an aqueous solution of GDL depends only on the initial GDL concentration, as long as it is significantly above the pKa =3.8 of GDL, and the rate of dissociation is linear until approximately half of the GDL is dissociated [START_REF] Liu | Effect of Kappa carrageenan on acid-induced gelation of whey protein aggregates. Part I: Potentiometric titration, rheology and turbidity[END_REF].

At room temperature, it takes approximately 24h for the GDL to degrade fully.

Methods

A Zeiss LSM800 (Carl Zeiss Microscopy GmbH) adapted with a water objective was used to obtain confocal laser scanning microscopy (CLSM) images. Two water objectives (HC×PL APO 63× and HC×PL APO 25×) were used. The samples were inserted in acrylic wells plates or in a hermetically-sealed concave slide covered with a glass slip to prevent the water from evaporating for long time observations. Rhodamine B at approximately 5 mg.L -1 was used to label the MG physically. Excess rhodamine partitioned preferentially to the AMP phase. The rhodamine B was excited at 580 nm and detected in a range between 580 and 800 nm.

Results

We will first show the effect of decreasing the pH on AMP/PUL emulsions containing 0.4 wt% MG and then the influence of adding different amounts of anionic polysaccharides. In both cases, we will compare reducing the pH by adding an aliquot of a concentrated HCl solution while stirring under a vortex with reducing the pH in-situ under quiescent conditions by adding GDL.

Fluorescent labelling of the MG allowed us to visualize the distribution of the MG between the phases and the interface using confocal laser scanning microscopy.

Effect of pH on the microstructure of AMP/PUL emulsions in the presence of MG

pH reduction with HCL while stirring

As was reported by Machado et al. ( 2021 coalesce, but were bound to each other forming large clusters or even a weak space spanning network. In the latter case, the emulsions remained visually homogeneous during at least one week, but flowed when tilted, implying that the network was strong enough to resist the effect of buoyancy of the droplets, but not of gravity on the macroscopic sample. We note that aggregation of the MG in pure AMP and PUL phases was observed in the same pH range as in the emulsions.

It is remarkable that simply neutralizing the negative charges on the proteins causes a shift in preference of the MG from being in contact with PUL to being in contact with AMP.

According to Eq. 4, it is the decrease of the interfacial tension between MG and AMP relative to that between MG and PUL which drives the MG to the interface. The difference decreases when the pH is decreased to pH 5, but further decrease of the pH increases the difference again, this time with γPUL-MG > γAMP-MG, until at pH ≤ 3.5 the MG no longer adsorb at the interface. These observations resemble those reported for W/W formed by mixing DEX and PEO, mentioned in the introduction (Gonzalez-Jordan et al., 2017). However, for that system the MG adsorbed at the interface already at pH 7.0 and the excess MG remained in the DEX phase down to pH 3.5 even though a shift in the partitioning towards the PEO phase with decreasing pH was found for native proteins. In P/A emulsions, the droplets are filled with MG at pH 7.0, and no MG were observed at the interface. Probably due to the higher local concentration of MG inside the droplets, the aggregation started at pH 5.9 instead of pH 5.5 for A/P emulsions, see Fig. 2. At pH ≥ 5.8 and pH ≤ 3.5 macroscopic phase separation occurred within one day, see Fig. S2 of the supplementary information.. At pH 5.6 the droplets were non-spherical as for A/P emulsions at pH 5.5 showing that MG formed a gelled layer at the droplet interface, results not shown. However, the gelled interface was not enough to prevent coalescence. Careful observation showed that excess MG (mostly in the form of aggregates) partitioned to the continuous AMP phase at pH < 5.6. Between pH 5.6 and 4.0, no clear layer of MG at the droplet interface was observed, but the MG formed a weak network in the AMP phase that trapped the PUL droplets for at least 1 week so that the emulsion remained macroscopically homogeneous, see Fig. S2 of the supplementary information,. Although the network was strong enough to inhibit creaming of the PUL droplets, the emulsion still freely flowed when tilted.

in-situ pH reduction with GDL under quiescent conditions

By adding GDL to the emulsions, it was possible to observe how the microstructure changes while the pH decreases in-situ. Video 1 of the supplementary information shows the evolution of an A/P emulsion. Images taken from the video at different pH are shown in Fig. S3 of the supplementary information. As the pH decreased, the MG, which were situated in the dispersed phase, first started to aggregate at pH close to 5.5 and formed small clusters. With decreasing pH, the clusters grew and migrated towards the interface, where they remained irreversibly adsorbed. No transfer of MG to the AMP phase was observed at lower pH, which apparently requires mechanical mixing. As the pH decreased further towards the final value of pH 3.7, the AMP droplets covered with MG clusters flocculated, see fig. 3. If less GDL was added so that the final pH was 5.0, macroscopic phase separation into continuous AMP and PUL phases occurred before a stable MG layer was formed around the AMP droplets, with MG clusters dispersed in the PUL phase, see fig. 3. This observation shows that stabilization below pH 5.6

necesitates that the pH is reduced very rapidly, e.g. by adding HCl, so that phase separation has no time to develop at higher pH during slow reduction of the pH.

The evolution of a P/A emulsion after the addition of GDL was very different, see video 2 of the supplementary information. In this case the MG situated in the dispersed phase accumulated at the interface below pH 5.9 without forming clusters. Again no transfer of MG from the PUL to the AMP phase was observed. At the final pH 5.0 the layer of MG stabilized PUL droplets for at least one week, see fig. 3. The droplets were relatively small with diameters less than 10 µm, and no sedimentation was observed after a week. The different behavior of P/A emulsions compared to A/P emulsions can be explained by the fact that in P/A emulsions, the MG are concentrated within the droplets and can therefore adsorb more easily at the interface that is nearby. In addition, the viscosity of the AMP phase was found to be ten times higher than that of the PUL phase, which slows down coalescence when AMP is the continuous phase.

Figure 3. CLSM image of A/P and P/A emulsions with 0.4 wt% MG one week after adding GDL.

Effect of adding anionic polysaccharides

pH reduction with HCL while stirring

Fig. 4 shows the morphology of A/P emulsions at different pH in the presence of 0.05 wt% alginate (ALG). In this case, the MG adsorbed at the interface already at pH 7.0, as was reported elsewhere (Machado et al., 2021). The microstructure remained the same down to pH 5.6, but the MG partitioned more strongly to the AMP phase with decreasing pH. However, between pH 5.5 and pH 5.0 the fraction of MG that adsorbed rapidly at the interface decreased, and the excess MG partitioned more strongly to the PUL phase, i.e. the opposite of what happened without adding ALG. In addition, no aggregation of MG was observed. This behavior can be explained by the fact that ALG prefers the PUL phase, considering that ALG complexes with the MG below pH 5.6. We note that formation of complexes between MG and κ-carrageenan (KC) [START_REF] Khemissi | Exploiting Complex Formation between Polysaccharides and Protein Microgels To Influence Particle Stabilization of W/W Emulsions[END_REF]PEC (Machado et al., 2021) was demonstrated to occur below pH 5.6.

The effect of adding ALG on the stability of the MG as a function of the pH was the same in pure AMP and PUL phases as in the emulsions. Complexation also explains why the MG did not form large clusters in this pH range. However, at pH ≤ 4.80, aggregation of the MG was observed, which was perhaps induced by the bridging of the MG by ALG chains. At pH ≥ 5.6, the droplets coalesced, and macroscopic phase separation was observed within one day. Between pH 5.5 and 5.0, more MG migrated to the interface during ageing forming a gelled layer. Stable more or less deformed droplets were formed that sedimented into a compact bottom layer. The droplets were much larger than in fresh emulsions implying that they coalesced before the accumulation and the structuration of MG/ALG complexes at the interface was sufficient to inhibit further coalescence. We investigated this process in more detail after setting the pH to 5.0 and found that coalescence slowed down after 4h, but the dense suspension of sedimented droplets continued to coarsen very slowly, see Fig. 5. Clearly, in this pH range the complexes were formed in the bulk phases and only slowly adsorbed at the W/W interface. Most likely, the complexes at the interface adhered to each other strongly slowing down further coalescence.

One might expect that if the complexes were formed separately in the continuous phase before mixing with the dispersed phase, they could migrate more quickly towards the interface after emulsification. However, when the complexes were allowed to form in the pure PUL phase for 24 h at pH 5.0 before mixing with the AMP phase, they did not adsorb at the interface, and the emulsions destabilized within 24 h after preparation. Fig. 6 shows the effect of varying the ALG concentration at pH 5.0. In the presence of less ALG (0.03 wt%), the complexes aggregated rapidly and adhered to the interface in the form of clusters, as was found in the absence of ALG. However, excess clusters of complexes remained in the PUL phase even when as little as 0.05 wt% ALG was added, whereas excess pure MG clusters partitioned to the AMP phase below pH 5.6. It shows that complexation even with little ALG was sufficient to render the MG compatible with PUL. When more than 0.05 wt% ALG was added, smaller droplets were observed after one week of standing, and the MG layer appeared more strongly gelled. Apparently, the attraction between the MG increased again with high ALG concentrations, perhaps caused by bridging of ALG between the MG. Similar behaviors were observed when ALG was substituted by PEC or KC, see figures S4 and S5 of the supplementary information. The microstructure of P/A emulsions with 0.05 wt% ALG at different pH is shown in Fig. 7. For this emulsion, the MG at pH 7.0 partitioned to the AMP phase, and no interfacial layer was observed. We suggested elsewhere that this was due to the higher concentration of the ALG in the PUL phase when the volume fraction of the latter is smaller (Machado et al., 2021). When complexes are formed below pH < 5.6, a layer of MG is clearly visible around the PUL droplets, which decreased in size with decreasing pH. At pH 5.0 and lower, excess MG partitioned to the PUL phase. The droplets remained stable for at least one week, but stuck to each other, forming a weak network or large flocs that sedimented. The effect of the ALG concentration on P/A emulsions at pH 5.0 is shown in fig. 8. Below 0.05 wt% the behavior was similar to that without ALG suggesting that complexation with ALG was insufficient to inhibit aggregation of the MG. At higher ALG concentrations the behavior was similar to that at 0.05 wt% with the formation of stable PUL droplets that associated into larger clusters or a weak gel.

pH reduction under quiescent conditions

In the presence of 0.05 wt% ALG, KC or PEC, the MG were situated at the interface of A/P emulsions already at pH 7.0, and when the pH was reduced to pH 5.0 in-situ under quiescent conditions they remained there, see video 3 of the supplementary information. Images taken from the video at different pH are shown in fig. S6 in the supplementary information. This means that complexes are formed below pH 5.6 with the MG already situated at the interface. In order to reduce the time needed to reach pH values were complexation occurred, GDL was added to solution prepared at pH 5.7. However, this did not inhibit coalescence during the decrease of the pH so that large droplets were formed that sedimented into a dense layer, see Fig. 9. The size of the AMP droplets depended somewhat on the type of polysaccharide that was added. There appears to be no major difference between the structure of A/P emulsions whether it was set at pH 5.0 using HCl or using GDL. A few measurements at different MG and ALG concentrations were done, see Fig. 11.

At 0.2 wt% MG, similar stable PUL droplets were formed, but at 0.1 wt% MG, macroscopic phase separation occurred, indicating that the MG concentration in the interfacial layer was insufficient to inhibit coalescence. There was little effect of ALG on the size and stability of the PUL droplets when its concentration was low. However, at 0.05 wt% ALG the emulsion was not stable, and macroscopic phase separation was observed even though the MG did absorb at the interface when the pH was decreased in-situ. Apparently, the attraction between the complexes at the interface containing more ALG was not strong enough to avoid coalescence.

Discussion

The present investigation shows that subtle changes in the strength of the interaction between the particles can have a strong effect on the morphology and stability of W/W emulsions.

Decreasing the pH below 5.6 caused the MG to adsorb at the W/W interface, but also caused attractive interaction between the MG. If the attraction is too strong, it leads to clustering of the MG before forming a smooth layer at the interface. This can lead to the formation of a weak gel that traps the dispersed phase, but does not inhibit the emulsion from flowing easily when tilted.

Interestingly, the MG partitioned to the the AMP phase when the pH was set to 5.0 or below, whereas at higher pH they partitioned to the PUL phase. The reason for this inversion is not clear to us at this moment. An important observation is, however, that if the pH is decreased without mechanical mixing, the MG did not migrate from the PUL phase to the AMP phase, but remained trapped at the interface. For this reason, the behavior of the emulsions was very different when the pH was reduced with or without shaking, in particular, that of the P/A emulsions. When pH of P/A emulsions was decreased in-situ to pH 5.0, PUL droplets with diameters smaller than 10 µm were stabilized by a smooth interfacial gel layer of MG. Remarkably these droplets did not bind to each other to form larger flocs, but remained well dispersed in solution for at least one week. Preliminary measurements have shown that this system is stable to dilution to below the binodal and even to increasing the pH to 7. More research is currently being done to establish in more detail the properties of this interesting food-grade W/W emulsion.

The strength of the interaction between the MG at a given pH can be modified by adding small amounts of anionic polysaccharides that form complexes with the MG. Complexation can inhibit clustering of the MG and reduces the strength of the attraction between the MG at the interface. Consequently, droplets with smooth interfaces were formed at pH values where largescale flocculation of MG occurred in the absence of polysaccharides. The effect of adding anionic polysaccharides was similar for ALG, KC, and PEC, but it depended strongly on the concentration. At high concentrations is can reduce the attraction between the MG to such an extent that the layer no longer inhibits coalescence.

Here we did not investigate the effect of adding salt, but it is evident that the strength of the interactions between MG can also be modulated by adding salt, as was shown by Gonzalez-Jordan et al. (2017). In this manner, it should be possible to introduce attractive interaction between the MG at pH > 5.6. Another possible extension to this work is to use fractal protein aggregates instead of MG. A comparison between the effect of adding MG and fractals on the stability of W/W emulsions was reported by Gonzalez-Jordan et al., 2016. Fractals have a lower density, and therefore less protein is required to cover the interface. In addition, the strength of the interaction between fractals and their interaction with anionic polysaccharides is different.

Clearly, much more research is needed in order to fully understand and exploit particle stabilization of W/W emulsions.

Conclusion

The stability and morphology of W/W emulsions can be controlled by adding MG particles and tuning the interaction between the particles. The partition of protein microgels in AMP-PUL emulsions depends on the pH and changes from preferentially to the PUL phase at pH > 5.0 to preferentially to the AMP phase at pH < 5.0. The change in the interfacial tension between the MG and the phases causes the MG to adsorb at the W/W interface below pH 5.6. Decreasing the pH towards the pI reduces electrostatic repulsion between the MG and leads to aggregation of the MG at the interface and in the AMP phase causing the formation of stable weak emulsion gels that flow when tilted. If the pH is decreased in-situ without shaking, the MG cannot migrate to the AMP phase but remain trapped at the W/W interface. In this case, weak emulsion gels are formed in A/P emulsions, whereas in P/A emulsions a strong gelled MG layer is formed around the AMP droplets that remain freely dispersed in the PUL phase for at least a week. Addition of small amounts of anionic polysaccharides can be used to modulate the interfacial tension of the particles with the phases as well as the interaction between the particles.

  ), MG did not adsorb at the interface of the emulsions at pH 7.0 at the concentrations studied here and almost fully partitioned to the PUL phase. The effect of setting the pH to lower values by adding HCl is shown in Fig.1for an emulsion of AMP droplets dispersed in a continuous PUL phase (A/P). Between pH 7.0 and 5.7 the MG remained dispersed in the PUL phase. At pH 5.6 a few MG adsorbed at the interface with the excess still in the PUL phase. At pH 5.5 most MG adsorbed at the interface, and the excess MG were situated in the AMP phase. The droplets were no longer spherical, indicating that the MG layer had formed a sufficiently strong gel to resist the driving force of the interfacial tension to render the droplets spherical. At lower pH, the MG rapidly formed large flocs inhibiting the formation of spherical AMP droplets. At pH 3.9, more or less deformed droplets could be distinguished, whereas at pH 3.5 all MG were situated within the AMP droplets, and no clear layer was observed at the interface. At pH ≤ 5.6 and pH ≥ 3.5 the emulsions quickly destabilized, forming continuous PUL and AMP phases with the MG situated in the PUL and the AMP phase, respectively. In the intermediate pH range, the AMP domains containing the MG clusters did not

Figure 1 .

 1 Figure 1. CLSM images of A/P emulsions in the presence of 0.4 wt% MG at different pH set by adding HCL as shown in the figure. The fluorescence from the labelled MG is shown in white.The top and the bottom rows correspond, respectively, to images taken just after preparation and 1 week later. All images are on the same scale (101 μm x 101 μm). Note that the images at pH 7.00, 5.60, and 3.50 were taken at the interface between the macroscopic PUL (top) and AMP (bottom) phases .

Figure 2 .

 2 Figure 2. CLSM images of P/A emulsions in the presence of 0.4 wt% MG at different pH set by adding HCl as indicated in the figure. The top and the bottom rows correspond, respectively, to images taken just after preparation and 1 week later. Note that the images at pH 7.00, 5.90, and 3.50 after one week were taken at the interface between the macroscopic PUL (top) and AMP (bottom) phases. All images are on the same scale (101 μm x 101 μm).

Figure 4 .

 4 Figure 4. CLSM images of A/P emulsions with 0.05 wt% ALG and 0.4 wt% MG at different pH set by adding HCl as indicated in the figure. The top and the bottom rows correspond, respectively, to images taken just after preparation and 1 week later. Note that the images at pH 7.0 and 5.6 after one week were taken at the interface between the macroscopic PUL (top) and AMP (bottom) phases and that the scale is not the same for the images in the top and bottom rows.

Figure 5 .

 5 Figure 5. CLSM images showing the evolution with time of A/P emulsions with 0.4 wt% MG and 0.05 wt% ALG at pH 5.0. All images are on the same scale (255 μm x 255 μm).

Figure 6 .

 6 Figure 6. CLSM images of A/P emulsions at pH 5.0 set by adding HCl with 0.4 wt% MG and different concentrations of ALG as indicated in the figure. The top and the bottom rows correspond, respectively, to images taken just after preparation and 1 week later. All images are at the same scale (101 μm × 101 μm).

Figure 7 .

 7 Figure 7. CLSM images of P/A emulsions with 0.05 wt% ALG and 0.4 wt% MG at different pH set by adding HCL as indicated in the image. The top and the bottom rows correspond, respectively, to images taken just after preparation and 1 week later. Note that the images at pH 7.0, 5.6 and 4.0 after 1 week were taken at the interface between the macroscopic PUL (top) and AMP (bottom) phases. All images are on the same scale (101 μm x 101 μm).

Figure 8 .

 8 Figure 8. CLSM images of P/A emulsions at pH 5.00 with 0.4 wt% MG and different concentrations of ALG as indicated in the figure The top and the bottom rows correspond, respectively, to images taken just after preparation and 1 week later. All images are at the same scale (101 μm × 101 μm).

Figure 9 .

 9 Figure 9. CLSM images of A/P emulsions at pH 5.0 set by adding GDL with 0.4 wt% MG and 0.05 wt% ALG (a), KC (b) or PEC (c). The images were taken one week after preparation. The scale of image (a) is 255 μm × 255 μm whereas that of (b) and (c) is 101 μm × 101 μm..

Figure 10 .

 10 Figure 10. CLSM images of A/P emulsions at pH 5.0 set by adding GDL with 0.4 wt% MG and 0.05 wt% ALG (a), KC (b), or PEC (c). The images were taken one week after preparation. All images are at the same scale (101 μm × 101 μm).

Figure 11 .

 11 Figure 11. CLSM images of P/A emulsions right after preparation (top row) and 1 week later (bottom row) with different MG:ALG ratios indicated above each column. GDL was used to reduce the pH from 7.0 to ~5.0. All images are at the same scale (101 μm × 101 μm).

Acknowledgements

This research was financed by the regional program Food for Tomorrow /Cap Aliment;

Research, Education and Innovation in Pays de la Loire, which is supported by the French Region Pays de la Loire and the European Regional Development Fund. The CLSM analyses were conducted within platforms "Matie re molle" at IMMM. Hydrocolloids, 102(November 2019), 105589. https://doi.org/10.1016/j.foodhyd.2019.105589 Machado, J. P. E., Benyahia, L., & Nicolai, T. (2021). Effect of adding a third polysaccharide on the adsorption of protein microgels at the interface of polysaccharide-based water in water emulsions. Journal of Colloid and Interface Science. https://doi.org/https://doi.org/10.1016/j.jcis.2021.06.053 Nguyen, B. T., Nicolai, T., & Benyahia, L. (2013). Stabilization of Water-in-Water Emulsions by Addition of Protein Particles. Langmuir, 29(34), 10658-10664. https://doi.org/10.1021/la402131e Nguyen, B. T., Wang, W., Saunders, B. R., Benyahia, L., & Nicolai, T. (2015). pH-responsive water-in-water pickering emulsions. Langmuir, 31(12), 3605-3611. https://doi.org/10.1021/la5049024 Nicolai, T., & Murray, B. (2017). Particle stabilized water in water emulsions. Food Hydrocolloids, 68, 157-163. https://doi.org/10.1016/j.foodhyd.2016.08.036 Nishinari, K., Kohyama, K., Williams, P. A., Phillips, G. O., Burchard, W., & Ogino, K. (1991). Solution Properties of Pullulan. Macromolecules, 24(20), 5590-5593. https://doi.org/10.1021/ma00020a017 Peinado, I., Lesmes, U., Andrés, A., & McClements, J. D. (2010). Fabrication and morphological characterization of biopolymer particles formed by electrostatic complexation of heat treated lactoferrin and anionic polysaccharides. Langmuir, 26(12),

Authors contributions

João Pedro Elias Machado: Experiments, writing original draft;

Taco Nicolai: Conceptualization, data curation, writing, reviewing, and editing.

Lazhar Benyahia: Reviewing and editing Rilton Alves de Freitas: Reviewing and editing Isabelle Capron: Reviewing and editing


domain: stat info math

text:
G Cordier

N Sigaux

J Giai

B Ibrahim

P Cresseaux
email: p.cresseaux@orange.fr

Assessing the incidence of nerve injuries according to different mandibular osteotomies including Wing osteotomies

Keywords: Bilateral Sagittal Split Osteotomy (BSSO), Chin Wing, Mini Wing, Orthognathic surgery, inferior alveolar nerve

We use currently 3 types of new mandibular osteotomies (a shortened bilateral sagittal split osteotomy (BSSO), associated or not with Chin Wing or Mini Wing). Chin osteotomy is a modified genioplasty extended along the basilar border and the angles of the mandible. These osteotomies are associated with a risk of inferior alveolar nerve (IAN) damage. We prospectively studied 113 consecutive patients operated on by the same surgeon who underwent one of the following procedures: 1) isolated BSSO, 2) BSSO with a Chin Wing, 3) BSSO with a Mini Wing, 4) isolated Chin Wing. We analyzed rates of IAN damage and impact on the patients' life by using a subjective questionnaire administered at the patient's 1 year follow up appointment between June 2018 and August 2019. Sixty-seven patients underwent isolated BSSO, 24 BSSO with Mini Wing, 13 BSSO with Chin Wing, and 9 Chin Wing only. Our analysis reveals that nerve damage is greater in the BSSO group associated with Chin Wing (50 % vs 21,6%; p= 0.006). However, there is no significant difference between nerve damage in the Mini wing group and the isolated BSSO group (27,1% vs 21,6%; p= 0.432). In the isolated Chin Wing group, the inferior alveolar nerve damage rate was 11 %. There is no significant difference between the groups with regard to the impact of the nerve damage on the patients' life. In case of BSSO with concomitant Wing osteotomy, we conclude that Mini Wing osteotomies cause less nerve related morbidity than Chin Wing.

Introduction

Two types of mandibular osteotomies are most commonly used: Bilateral Sagittal Split osteotomy (BSSO) and genioplasty. We have however chosen to develop another approach, for a couple of years, by using a shortened BSSO and two variants of genioplasty: the Chin Wing and the Mini Wing. The Chin Wing was first introduced by Dr. Triaca in 2009 [START_REF] Triaca | Chin wing osteotomy for the correction of hyper-divergent skeletal class III deformity: technical modification[END_REF][START_REF] Triaca | Mandibula wing osteotomy for correction of the mandibular plane: A case report[END_REF][START_REF] Pouzoulet | Chin wing: Technical note[END_REF]. It is a modified genioplasty extending along the basilar border of the mandible up to the angles. Dr. Cresseaux has described a simplified version of the Chin Wing, known as the Mini Wing [START_REF] Cordier | Mini Wing osteotomy: a variant of Chin Wing osteotomy[END_REF]. The primary indication for Wing osteotomies is retrogenia. We perform a shortened bilateral sagittal split osteotomy [START_REF] Cordier | The intermediate length BSSO: Finding the balance between the classical and short designs[END_REF] when a concomitant sagittal mandibular movement is required in order to incorporate Wing osteotomies in all our orthodontic-surgical protocols. The rate of inferior alveolar nerve (IAN) damage in classic BSSO ranges from 8 % to 40 % [START_REF] Mcleod | Nerve injury associated with orthognathic surgery. Part 2: inferior alveolar nerve[END_REF][START_REF] Iannetti | Our experience in complications of orthognathic surgery: a retrospective study on 3236 patients[END_REF][START_REF] Agostino | Complications of orthognathic surgery: the inferior alveolar nerve[END_REF][START_REF] Mensink | Neurosensory disturbances one year after bilateral sagittal split osteotomy of the mandibula performed with separators: a multi-centre prospective study[END_REF][START_REF] Westermark | Inferior alveolar nerve function after sagittal split osteotomy of the mandible: correlation with degree of intraoperative nerve encounter and other variables in 496 operations[END_REF][START_REF] Panula | Incidence of complications and problems related to orthognathic surgery: a review of 655 patients[END_REF][START_REF] Essick | Facial altered sensation and sensory impairment after orthognathic surgery[END_REF][START_REF] Macintosh | Experience with the sagittal osteotomy of the mandibular ramus: a 13year review[END_REF]. There are practically no studies focusing on IAN damage specifically in Wing osteotomies. Only one study conducted by Tabrizi et al, found an IAN damage of 20 % one year post-operation, on 10 patients having had a Chin Wing surgery [START_REF] Tabrizi | Evaluation of mandibular wing osteotomy in obstructive sleep apnea cases with retrognathia[END_REF]. There is a risk of IAN damage in Wing surgeries, due to the orientation of the osteotomy line. Our aim was to evaluate the incidence of IAN damage in the different mandibular osteotomies including Wing osteotomies that we performed.

Materials and methods

We conducted a prospective case series study performed in a single center between June 2018 and August 2019. All patients were operated on 1 year earlier by the same surgeon (P. Cresseaux). The preoperative assessment included a cone beam computed tomography (CBCT) to control the position of the inferior alveolar nerve (IAN). The vertical position of the IAN, its distance from the basilar rim, and distance from buccal cortical plate of the mandible were assessed on coronal sections of the CBCT. We use 3 different techniques and their combinations: modified BSSO/Chin Wing/Mini Wing (fig. 2). All surgeries were performed under general anesthesia with subperiosteal infiltration with 1% adrenaline solution. The BSSO line uses a shortened version (5) allowing for the association of the Chin Wing as described by Triaca (1,2), or Mini Wing (4). The surgery was followed by systematic clinic visits at one week, one month. A clinical and radiologic assessment was performed at the one-year postoperative consultation (pictures of the patient, frontal and lateral cephalogram, panoramic radiograph and CBCT).

All patients were recruited in the study during their 1 post-operative follow up appointment. During this visit, patients were given a satisfaction questionnaire. This questionnaire evaluated the functional and aesthetic results as well as postoperative complications. The complications related domain of the questionnaire focused mainly on nerve disorders Nerve damage was assessed via the following three questions: Do you currently have:

-A loss of sensitivity of the lower right lip? -A loss of sensitivity of the lower left lip? -If that is the case, would you say that this loss of sensitivity has an impact on your everyday life? Answer choices were:

-Absent (0); -Low (1); -Medium (2); -Important (3); -Complete (4). The questionnaire was given with a consent form in accordance with our institution's ethical committee requirements.

Patients were separated in four based on the type of mandible surgery that they had undergone: 1) isolated BSSO 2) Mini Wing + BSSO 3) Chin Wing + BSSO 4) isolated Chin Wing. Some surgeries were combined with a maxillary surgery. We performed a comparative analysis of IAN damage within groups 1 to 3. Group 4 was not comparable to the others as BSSO itself increases nerve damage.

Data is presented using descriptive statistics. Fisher's exact test was used to determine whether significant differences in proportions of IAN damage and quality of life outcomes were observed amongst the first three groups. Alpha risk was set at 5%. R Software version 3.5.0 was used for all analyzes.

Results

113 consecutive patients have answered our questionnaire. Sixty-seven had an isolated BSSO, 24 an BSSO with Mini Wing, 13 had an BSSO with classical Chin Wing, and 9 had an isolated Chin Wing. All patients agreed to answer the questionnaire. The first three group had comparable baseline characteristics. The fourth group had a younger median age and had had a shorter median time to evaluation. All of the patients' follow up appointments took place at least 6 months after their surgery (tab. 1).

Nerve damage was analyzed for each hemi-mandible separately without specifying the side. We therefore considered 134 osteotomies the isolated BBSO group, 48 in the Mini Wing + BSSO group, and 24 in the Chin Wing + BSSO group. Rates of IAN damage are shown in table 2. A higher rate of IAN damage was noted in BSSO with wing osteotomies. The Chin Wing + BSSO group shows a significant increase in nerve damage one year after surgery compared to the isolated BSSO control group (50% vs 21,6%; p= 0.006). However, there is no significant increase in nerve damage when comparing the Mini Wing + BSSO group to the control group (27,1% vs 21,6%; p = 0,432). With regard to the evaluation of nerve damage depending on different levels of severity, similar results were found: a significant increase in IAN damage in the Chin Wing + BSSO group in comparison with the control group (p<0,001) as opposed to the Mini-Wing + BSSO group (p=0,14) (tab. 3). The fourth group shows an IAN nerve damage rate of 11 % (for 18 hemi-mandibles). This confirms that the Chin Wing osteotomy is in and of itself associated to a baseline rate of iatrogenic IAN damage. Despite greater nerve damage in our BSSO group associated with Wing osteotomies, there was no significant difference in the impact on our patients' life (tab. 4).

Discussion

Our study demonstrates an increased rate of IAN damage when performing a Wing osteotomy associated with a BSSO in comparison to isolated BSSO. However, this increase is not significant for the Mini Wing unlike in the Chin Wing group. The Mini Wing seems to be associated to less iatrogenic nerve damage than the Chin Wing. However, the Chin Wing increased IAN damage rate does not seem to have a significant negative impact on the patient's life, and therefore be considered acceptable by patient.

Very few studies specifically focus on nerve damage in isolated genioplasty and existing studies show nerve damage rates that are close to zero [START_REF] Troulis | Extended genioplasty: long-term cephalometric, morphometric and sensory results[END_REF][START_REF] Deshpande | Osseous genioplasty: A case series[END_REF][START_REF] Hohl | Macrogenia: a study of treatment results, with surgical recommendations[END_REF]. Our study reveals that the isolated Chin Wing presents an IAN damage rate of 11 % for 9 patients (i.e. 18 hemimandibles). Chin Wing therefore increases the risk of nerve damage compared to a classic genioplasty.

As far as the isolated BSSO is concerned, we find an IAN nerve damage rate of 21,6 %. Our results are globally similar to the rates of nerve damage in the general orthognathic surgery literature, which range from 8 % to 40 % [START_REF] Mcleod | Nerve injury associated with orthognathic surgery. Part 2: inferior alveolar nerve[END_REF][START_REF] Iannetti | Our experience in complications of orthognathic surgery: a retrospective study on 3236 patients[END_REF][START_REF] Agostino | Complications of orthognathic surgery: the inferior alveolar nerve[END_REF][START_REF] Mensink | Neurosensory disturbances one year after bilateral sagittal split osteotomy of the mandibula performed with separators: a multi-centre prospective study[END_REF][START_REF] Westermark | Inferior alveolar nerve function after sagittal split osteotomy of the mandible: correlation with degree of intraoperative nerve encounter and other variables in 496 operations[END_REF][START_REF] Panula | Incidence of complications and problems related to orthognathic surgery: a review of 655 patients[END_REF][START_REF] Essick | Facial altered sensation and sensory impairment after orthognathic surgery[END_REF][START_REF] Macintosh | Experience with the sagittal osteotomy of the mandibular ramus: a 13year review[END_REF]. This rate of nerve damage varies according to many parameters: the age of the patient, the experience of the surgeon and the type of BSSO. Our shortened BSSO consequently does not present an additional risk of nerve damage in comparison with classic BSSO.

McLeod et al's review of the literature confirms that nerve damage is greater when BSSO is associated with a genioplasty [START_REF] Mcleod | Nerve injury associated with orthognathic surgery. Part 2: inferior alveolar nerve[END_REF]. Similarly, our results demonstrate an increased nerve damage rate in BSSO associated with a Wing osteotomy. But when a BSSO is part of the surgical plan, the Mini Wing should be favored. It is simpler, faster and is associated to less iatrogenic IAN damage than the Chin Wing. This is explained by the orientation of the osteotomy line which is different in the Mini Wing and the Chin Wing (fig. 1). The IAN has an elliptical trajectory within the mandible [START_REF] Liu | Inferior alveolar canal course: a radiographic study[END_REF] and its lowest point is next to the first molar [START_REF] Hwang | Vulnerability of the inferior alveolar nerve and mental nerve during genioplasty: an anatomic study[END_REF][START_REF] De Oliveira-Santos | Assessment of variations of the mandibular canal through cone beam computed tomography[END_REF][START_REF] Sato | Rare courses of the mandibular canal in the molar regions of the human mandible: a cadaveric study[END_REF][START_REF] Massey | Determining position of the inferior alveolar nerve via anatomical dissection and micro-computed tomography in preparation for dental implants[END_REF], which corresponds to the zone of highest risk for iatrogenic damage. In the Chin Wing, the osteotomy goes all the way to the angles and is therefore made higher on the mandible. This increases the risk of nerve damage. It is therefore necessary to preoperatively detect the lowest position of the IAN via a CBCT. However, it is very difficult to compare the results of different nerve damage studies as there is no standardized test for the assessment of IAN damage. Indeed, some studies choose to conduct a subjective evaluation while others use objective testing (using criteria such as touch sensation, two-point discrimination, hot-cold, pain), or a combination of both. The subjective questionnaires have been reported both to over [START_REF] Cunningham | A comparison of questionnaire versus monofilament assessment of neurosensory deficit[END_REF][START_REF] Pratt | Labial sensory function following sagittal split osteotomy[END_REF] and under [START_REF] Blomqvist | Sensibility following sagittal split osteotomy in the mandible: a prospective clinical study[END_REF][START_REF] Coghlan | Neurological damage after sagittal split osteotomy[END_REF] estimate nerve damage compared to objective testing. We have therefore chosen to conduct a purely subjective evaluation in view of the absence of standardization. Our rational was to refocus the analysis on the patient's sensations and the repercussion of the surgery on their everyday life. We decided to limit the number of questions as much as possible in order to decrease the risk of inattentive responses and engage maximally the patient in the questionnaire. We prospectively included all patients presenting consecutively to their one-year post-operative appointment between June 2018 and August 2019. Our response rate to the questionnaire was of 100 %.

Taken together, the findings of the present paper allow us to suggest new indications for Wing osteotomies. We believe that the Mini Wing could replace the classic genioplasty in most cases, due to its relatively low rate of iatrogenic nerve damage and its improved functional and esthetic results versus genioplasty. The Mini Wing seems to be a better choice than Chin Wing when a BSSO is to be performed concomitantly. It can be associated with any classic osteotomy (Epker (27) or Obwegeser [START_REF] Trauner | The surgical correction of mandibular prognathism and retrognathia with consideration of genioplasty. I. Surgical procedures to correct mandibular prognathism and reshaping of the chin[END_REF]), as long as it is done with short proximal segments. On the other hand, it no longer seems advisable to associate a complete Chin Wing with a BSSO because of the high rate of nerve damage (50%), but also because we notice bone loss, in most cases, due to the remodeling of the posterior part of the wing (fig. 2). The isolated Chin Wing remains indicated in cases of hyperdivergent dental class I with important retrogenia and lip incompetence without need for mandibular advancement. The isolated Chin Wing seems to have less or no bone resorption at the mandibular angles when performed in a single procedure and a relatively low rate (11,1%) of IAN damage (fig. 2).

The authors declare they have no competing interest.

Illustrations

Figure 1 :Figure 2 :

 12 Figure 1: Postoperative CBCT of isolated BSSO case. Position of the Inferior Alveolar Nerve (red). Osteotomy line in the Chin Wing (yellow line) and in the Mini Wing osteotomy (blue line)

Table 1 :

 1 Characteristics of the different groups (n: number of patient)

		Isolated	Mini Wing	Chin Wing	Isolated
		BSSO	+ BSSO	+ BSSO	Chin Wing
		n= 67	n = 24	n = 13	n= 9
	Age
	Median	29.25	26.17	28.85	21
	Range	15 -58	15 -47	15 -48	13 -32
	Sex
	Female	51 (76.1%) 20 (83.3%)	7 (53.8%)	6 (66,7%)
	Male	16 (23.9%)	4 (16.7%)	6 (46.2%)	3 (33,3%)
	Days between surgery
	and evaluation
	Median	390.88	365.79	471.54	252,67
	Range	187 -681	214 -709	326 -725	205 -452

Table 2 :

 2 Comparison of nerve damage (n: number of hemi-mandibule, NA: not applicable)

			Isolated	Mini Wing +	Chin Wing +	Isolated
			BSSO	BSSO	BSSO	Chin Wing
			n=134	n=48	n=26	n=18
	Right and left	Absent	105 (78,4%)	35 (72,9%)	13 (50%)	16 (88,9%)
	nerve damage	Low	19 (14,2%)	5 (10,4%)	5 (19,2%)	(11,1%)
		Medium	(5,2%) 7	(16,7%) 8	(7,7%) 2	0
		Important	(1,5%) 2	0	(23,1%) 6	0
		Complete	1 (0,7%)	0	0	0
	Fisher's test (comparison to BSSO group)	NA	p-value = 0.141	p-value < 0,001	NA

Table 3 :

 3 Comparison of nerve damage according to different levels of severity (n: number of hemi-mandibule, NA: not applicable)

			Isolated	Mini Wing +	Chin Wing +	Isolated Chin
			BSSO	BSSO	BSSO	Wing
			n=67	n=24	n=13	n=9
	Impact on	Absent	55 (82,1%)	20 (83,3%)	9 (69,2%)	8 (88,9%)
	quality of life	Low	8 (11,9%)	N=3 (12,5%)	N=2 (15,4%)	1 (11,1%)
		Medium	(4,5%) 3	(4,2%) 1	(7,7%) 1	0
		Important	(1,5%) 1	0	(7,7%) 1	0
		Complete	0	0	0	0
	Fisher's test (comparison to BSSO group)	NA	p-value = 1	p-value = 0,297	NA

Table 4 :

 4 Comparison of the impact on the quality of life (n: number of patients, NA: not applicable)


Yara Al Chikhanie

Sébastien Bailly

Ines Amroussia

Daniel Veale

Frédéric Hérengt

Dr Samuel Verges
email: sverges@chu-grenoble.fr

Trajectories of COPD patients' response to repeated pulmonary rehabilitation programs

Keywords: chronic obstructive pulmonary disease, repeated pulmonary rehabilitation, trajectories of response, 6-min walking test

We evaluated the trajectories of response to repeated pulmonary rehabilitation (PR) in COPD patients, using the six-minute walking distance (6MWD) to determine the functional response to PR. We demonstrate that a response to repeated PR is not systematically identical, especially for non-responders to initial PR.

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) has become the third leading cause of death in the world [START_REF]Health. United States 2015 with special feature on racial and ethnic health disparities[END_REF] requiring a comprehensive intervention plan designed to improve physical and psychological condition of these patients. Pulmonary rehabilitation (PR) was designed for people with chronic respiratory disease and is recommended as a fundamental part of the COPD management plan. [START_REF] Spruit | An Official American Thoracic Society/European Respiratory Society Statement: Key Concepts and Advances in Pulmonary Rehabilitation[END_REF][START_REF] Nici | An official American Thoracic Society workshop report: the Integrated Care of The COPD Patient[END_REF] Daily symptoms, exercise performance and health status generally improve following PR. [START_REF] Ries | Pulmonary Rehabilitation[END_REF][START_REF] Mccarthy | Pulmonary rehabilitation for chronic obstructive pulmonary disease[END_REF][START_REF] Singh | An official systematic review of the European Respiratory Society/American Thoracic Society: measurement properties of field walking tests in chronic respiratory disease[END_REF] However, the benefits in exercise capacity, as well as other outcomes resulting from PR, tend to decline over the months following the completion of the intervention. [START_REF] Foglio | Is It Really Useful To Repeat Outpatient Pulmonary Rehabilitation Programs in Patients With Chronic Airway Obstruction[END_REF] There are several likely reasons for this decline in outcomes including physical deconditioning, progression of the respiratory disease and its comorbidities and intervening exacerbations. [START_REF] Foglio | Seven-year time course of lung function, symptoms, health-related quality of life, and exercise tolerance in COPD patients undergoing pulmonary rehabilitation programs[END_REF][START_REF] Spruit | Predicting Outcomes from 6-Minute Walk Distance in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Oga | Exercise Capacity Deterioration in Patients With COPD[END_REF] To mitigate this long-term decline in clinical outcomes, some clinicians prescribe repeated PR programs. A few studies have been conducted to show clinical advantages of repeated PR in patients with COPD, including a decreased rate of exacerbations, fewer hospitalizations, and improved quality of life. [START_REF] Foglio | Is It Really Useful To Repeat Outpatient Pulmonary Rehabilitation Programs in Patients With Chronic Airway Obstruction[END_REF][START_REF] Foglio | Seven-year time course of lung function, symptoms, health-related quality of life, and exercise tolerance in COPD patients undergoing pulmonary rehabilitation programs[END_REF][START_REF] Romagnoli | Repeated Pulmonary Rehabilitation in Severe and Disabled COPD Patients[END_REF] Other studies have evaluated the effect of repeated PR on the 6minute walking distance (6MWD), with small samples of patients, showing significant improvement in 6MWD following repeated PR with 12-24 months between programs [START_REF] Romagnoli | Repeated Pulmonary Rehabilitation in Severe and Disabled COPD Patients[END_REF][START_REF] Carr | Pulmonary Rehabilitation After Acute Exacerbation of Chronic Obstructive Pulmonary Disease in Patients Who Previously Completed a Pulmonary Rehabilitation Program[END_REF][START_REF] Sandoz | Magnitude of exercise capacity and quality of life improvement following repeat pulmonary rehabilitation in patients with COPD[END_REF] , but to date, no studies have described the change in responsiveness to repeated PR (between responders and non-responders).

Larger studies are needed to study the response to repeated PR depending on the response profile to the first PR (i.e. in patients identified as non-responders to the first program), because program repetition has significant implications for health-care resources. There is no evidence to provide guidance as to whether repeating the program to patients previously identified as non-responders can be systematically recommended.

The objectives of the study were 1) to assess the long-term effect of PR in COPD patients, 2) to evaluate the effect of repeated PR, and 3) to determine trajectories of response following repeated PR. We hypothesize that a non-responder to a first PR may become responder to subsequent PR.

METHODS

Study design and population

We conducted a single center retrospective analysis of data from patients diagnosed with COPD admitted to a cardiopulmonary rehabilitation center (Dieulefit Sante , Drome, South Eastern France) for two programs over a 6-year period (from 2012 to 2017). The study was approved by a French independent ethics committee (ID-RCB number: 2019-A02104-53).

Data were collected from the center's electronic medical record database (Calyste ǹe, Eybens France) and from the six-minute walking test (6MWT) data recording system (BluenightAcor+, Sleepinnov, Moirans, France). Cross-analysis of the two data sources yielded 1910 COPD patients. The following factors of exclusion were applied: admitted after an acute exacerbation or hospitalization, having only one PR, having a readmission earlier than 10 months or later than 14 months post-PR1, having data from less than four 6MWT (i.e. pre-PR1, post-PR1, pre-PR2, post-PR2), having a rehabilitation duration shorter than 3 weeks 14 or longer than 5 weeks, differing amounts of oxygen supplementation during the 6MWTs. 1720 patients were excluded and 190 patients were included for analysis with at least one readmission 10-14 months post-PR1 following medical prescription (Figure 1).

Pulmonary rehabilitation

The PR program used in the center was the same over the 6-year period and was conducted according to current recommendations [START_REF] Nici | An official American Thoracic Society workshop report: the Integrated Care of The COPD Patient[END_REF] . It consisted in a 3-to-4-weeks in-patient program, with activities 5 days per week. Each day the patient attended the following sessions: 25-min aerobic training on a cycling ergometer or a treadmill (5 min warm-up, 15 min training, 5 min cooldown), 30-min low-intensity group gym session (upper and lower limbs exercises, respiratory exercises), 30-min group walk outside, and a 30-min muscle strength training session. In addition, the patients had sessions of therapeutic education concerning COPD, oxygen supply, stress management and physical activity. When judged clinically necessary, they also had psychosocial counselling.

Evaluations

The 6MWT was performed in a 40-m hallway according to current recommendations 15 with the exception that the test was performed once only. Oxygen saturation (SpO2) and heart rate (HR) were measured using a portable pulse oximeter (NONIN wrist Ox2 model 3150, Plymouth, USA, data collected every 4 seconds, the SpO2 accuracy was ± 2% and HR accuracy was ± 3 bpm). The data was sent to a Bluetooth-connected electronic tablet (Sleepinnov BlueNight Acor+ application, Moirans, France) where the following measurements were recorded: 6MWD (in meters and in percentage of theoretical walking distance), SpO2 (in %, resting and lowest values), HR (in bpm, resting and highest values), and perceived dyspnea (measured on a 10-cm visual analogic scale, at rest and end exercise). The 6MWT was conducted at the beginning (pre-PR) and at the end of the PR (post-PR) of every program for each patient.

Patients were then classified as "responders" if they gained ≥ 30 m in 6MWD (the minimal clinically significant difference, MCID [START_REF] Singh | An official systematic review of the European Respiratory Society/American Thoracic Society: measurement properties of field walking tests in chronic respiratory disease[END_REF] ) and "non-responders" if they did not. Oxygen was used during the test (pre-and post-PR) if the patient was already admitted under long term oxygen therapy prescribed by their pulmonologist. These patients also used oxygen during the PR program.

Lung function (total lung capacity -TLC-and forced expiratory volume in the first second -FEV1) was measured pre-PR by pulmonary plethysmography (Jaeger MasterScreen Body, Hoechberg, Germany), based on medical prescription only.

Other variables were reported: comorbidities, reasons for admission, physical activity questionnaire (Ricci and Gagnon) [START_REF] Maladie | Examen périodique de santé : Questionnaire de Ricci et Gagnon : Test d'auto-évaluation de l'activité[END_REF] , and the number of exercise sessions during PR.

Statistical Analysis

Descriptive statistics are presented as medians (with interquartile range) or mean (± standard deviation, SD) for the quantitative variables and frequencies (%) for the qualitative variables.

A simple imputation method was carried out in cases where the missing data were few (< 5%).

Otherwise, a multiple imputation method that was defined according to the observed missing data pattern was implemented. To compare patients according to their PR response status (responders vs. non-responders; non-responders to PR1 and PR2 vs. non-responders to PR1 and responders to PR2 vs. responders to PR1 and non-responders to PR2 vs. responders to PR1 and responders to PR2), a Chi 2 test was used for qualitative variables and a non-parametric Mann-Whitney test was used for quantitative variables. In case where more than 2 comparisons were performed, a correction for multiple tests was applied using Bonferroni approach. To identify factors associated with a probability of responding to repeated PR, a univariable analysis was performed using a logistic regression model. Variables with a p-value less than 0.10 in the univariable analysis were then included in a multivariable logistic regression model. Finally, a linear model was applied to evaluate the relation between the gain in 6MWD and the predictors.

The forward method was used in continuous 6MWD gain analysis for the selection of predictors and the Bayesian information criteria (BIC) was used to choose the best model. A threshold of 0.05 was considered significant. Statistical analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA).

RESULTS

Characteristics of the whole population

Characteristics of the 190 patients' population before starting the first PR program (pre-PR1) are given in Table 1. A flow chart of patients' inclusion is presented in Figure 1. Eighty-seven percent were readmitted to PR following the prescription by their pulmonologist of a yearly follow-up program. There was no significant difference in gender distribution. Most patients were overweight or obese and were GOLD II or III. The average walked distance pre-PR showed important functional limitation with 80% of the patients having a 6MWD < 80% predicted distance. Most patients desaturated during exercise and were severely dyspneic.

Response to PR1 and PR2

The PR program lasted for 25 ± 4 days (mean ± SD). The delay between PR1 and PR2 was 354 ± 50 days. Data collected during the 6MWT are provided in Table 2 and Figure 2. Based on the physical activity questionnaire, only 25% of the patients stayed active during the 10-14 months period between the PR programs, compared to 17% pre-PR1. The patients showed a mean significant improvement in 6MWD both post-PR1 (+65 ± 30 m) and post-PR2 (+44 ± 20 m) but the 6MWD change post-PR2 was smaller than post-PR1 (p = 0.001). 6MWD pre-PR2 was not significantly different compared to pre-PR1. SpO2, HR and dyspnea did not differ between all pre-and post-PR 6MWT. At pre-PR2, FEV1 showed a non-significant (p = 0.17) reduction compared to pre-PR1 (pre-PR1 46 ± 21% versus pre-PR2 42 ± 16% predicted). The BMI did not change over time (results not shown).

Comparison of responders and non-responders to PR1 and PR2

Post-PR1, 78% of the patients were classified as responders and 22% non-responders. Post-PR2, the percentage of non-responders was significantly higher since 33% of the patients did not achieve the 6MWD MCID of 30 m. The multivariable analysis (Table 3) indicated that being a responder to PR2 was significantly associated to being a responder to PR1. Also, the use of oxygen decreased the probability of being responders at PR2 by 40%.

Out of the 149 responders to PR1, 44 (30%) became non-responders to PR2. Out of the 41 nonresponders to PR1, 23 (56%) became responders. Hence, we described 4 classes of change in 6MWD response (Figure 3, Table 4): 55% were responders to PR1+PR2, 10% were nonresponders to neither; 23% were responders to PR1 but not to PR2; 10% were non-responders to PR1 but became responders to PR2. Two variables pre-PR1 showed significant differences between the 4 identified classes: LTOT and end-of-test dyspnea. Non-responders to PR1 who stayed non-responders to PR2 (NR1-NR2) were those under LTOT (p = 0.03) and had the highest end-of-test dyspnea (p = 0.04). On the contrary, responders to PR1 who stayed responders to PR2 (R1-R2) were those with the least LTOT (p = 0.03).

Subgroup of three repeated PRs

Out of the 190 patients having performed two PR2, 62 (33%) patients repeated PR a third time 10-14 months after PR2. Eighty-nine percent were readmitted to PR following pulmonologist's prescription of a yearly follow-up program.

The BMI of these 62 patients did not change over time (results not shown). FEV1 showed a non-significant (p = 0.12) reduction at pre-PR2 (46 ± 12%) and pre-PR3 (44 ± 13%) compared to pre-PR1 (50 ± 13%). Data from the 6MWT pre and post-PR1, PR2 and PR3 are shown in Table 5 and Figure 2. The patients started each PR with almost the same walked distance. The mean change in 6MWD following PR3 was +55 ± 58 m, which was not significantly different from neither PR2 (+52 ± 64 m, p = 0.73) nor PR1 (+73 ± 70 m; p = 0.35). The percentage of non-responders to PR based on changes in 6MWD was not significantly lower post-PR1 (20%) compared to post-PR2 (37%) and post-PR3 (31%) (p = 0.17). The change in response between each PR program is presented in Figures 4 and5. We observed that the majority of the patients showed clinically significant response to PR3 no matter their responses to PR1+PR2. Only 3 patients (5%) were non-responders to all three PR, and 37% were responders to all three PR.

DISCUSSION

The main result of this study is that among a large population of 190 COPD patients having performed 2 PRs, 54% of non-responders to PR1 become responders if PR2 was to be repeated after 10-14 months. Also, only 3% of the 62 patients having performed three PR did not show any clinically significant increase in 6MWD to any of the three programs. We were able to identify 4 different classes of repeated-PR response trajectory, showing that staying a responder or a non-responder over several PR is not a systematic trajectory, but that the patients the most likely to remain non-responders were those under LTOT and very dyspneic. In addition, this study showed that: i) the increase in 6MWD post-PR is partially lost during the following 12 months and ii) the improvement in 6MWD after each repeat PR is of smaller amplitude on average compared to PR1 despite remaining clinically significant (> 30 m MCID on average).

Repeat PR can be offered to patients because the effect of PR diminishes after 1-2 years. [START_REF] Romagnoli | Repeated Pulmonary Rehabilitation in Severe and Disabled COPD Patients[END_REF][START_REF] Carr | Pulmonary Rehabilitation After Acute Exacerbation of Chronic Obstructive Pulmonary Disease in Patients Who Previously Completed a Pulmonary Rehabilitation Program[END_REF][START_REF] Sandoz | Magnitude of exercise capacity and quality of life improvement following repeat pulmonary rehabilitation in patients with COPD[END_REF] Our results agree with these studies since we showed that the increase in 6MWD post-PR1 is partially lost during the 12 months but the walking capacity pre-PR2 did not drop below the pre-PR1 walked distance. It should be emphasized however that to alleviate the cost of repeated PR on healthcare resources and to attenuate the long-term decline in PR outcomes, other care plans should be considered in future studies, including patient education, follow-up interventions and telemedicine [START_REF] Busby | Pulmonary rehabilitation maintenance interventions: a systematic review[END_REF] .

The preservation of functional capacity over a year in these patients may illustrate an attenuation of disease progression due to PR. Indeed, FEV1 did not significantly decline from year to year, nor did BMI change. This shows a stabilization of the disease progression, possibly because the disease's increasing impact on walking capacities (higher dyspnea, etc) may have been averted. These results need to be confirmed with future studies including a control group of patients who did not take part in any PR program.

We also showed that the effect on 6MWD of PR2 was lower than the effect of PR1 but remained statistically and clinically significant (> 30 m on average). Current ATS/ERS pulmonary rehabilitation guidelines state that PR2 offers similar gains in exercise capacity to PR1 2 based on small observational studies [START_REF] Foglio | Seven-year time course of lung function, symptoms, health-related quality of life, and exercise tolerance in COPD patients undergoing pulmonary rehabilitation programs[END_REF][START_REF] Hill | Repeat pulmonary rehabilitation programs confer similar increases in functional exercise capacity to initial programs[END_REF] and two small randomized controlled trials. [START_REF] Foglio | Is It Really Useful To Repeat Outpatient Pulmonary Rehabilitation Programs in Patients With Chronic Airway Obstruction[END_REF][START_REF] Romagnoli | Repeated Pulmonary Rehabilitation in Severe and Disabled COPD Patients[END_REF] In our study, a third PR in the subgroup showed similar effect of PR3 compared to PR2, suggesting that beyond PR1, patients can expect a similar significant 6MWD response to PR. To our knowledge, we present the largest published cohort of patients with COPD undergoing a second and a third PR, which demonstrates the ongoing benefits of completing repeated PR. Although the reduced benefits may be attributed to advancing airflow obstruction over time, recent studies have demonstrated a reduced magnitude of improvement in 6MWD post-PR2 despite no overall change in spirometric values. [START_REF] Heng | Repeating pulmonary rehabilitation: Prevalence, predictors and outcomes: Benefits and predictors of repeating PR[END_REF] It is also possible that advancing age may have contributed to an overall age-related decline in 6MWD. [START_REF] Casanova | Six Minute Walk Distance Project (ALAT). The 6-min walk distance in healthy subjects: reference standards from seven countries[END_REF] However, in the National Emphysema Treatment Trial, COPD patients who had completed prior PR and had smaller improvement in 6MWD post-PR2, had no difference in age or lung function. [START_REF] Ries | The effects of pulmonary rehabilitation in the national emphysema treatment trial[END_REF] We speculate that the worsening of airflow obstruction over the years, increasing comorbidities in COPD patients [START_REF] Casanova | The 6-min walking distance: long-term follow up in patients with COPD[END_REF] , the ageing process [START_REF] Casanova | Six Minute Walk Distance Project (ALAT). The 6-min walk distance in healthy subjects: reference standards from seven countries[END_REF] , and a potential learning effect from previous PR [START_REF] Ries | The effects of pulmonary rehabilitation in the national emphysema treatment trial[END_REF] , may all contribute to the reduced outcome of repeated PR.

Most importantly, we showed that 54% of non-responders to PR1 became responders to PR2 despite that, as previously emphasized, the effect of PR2 on the 6MWD is on average lower that the effect of PR1. This is promising for patients failing to show clinically significant improvement in 6MWD after PR1 who should not avoid repeating PR since they may show larger functional benefit. Meanwhile, when comparing the classes of response to repeated PR, we found that those under LTOT and the most dyspneic, were most likely to stay nonresponders to repeated PR. Interestingly, we showed that a third PR can lead to a significant clinically response to PR in this population. While in the present study it could be hypothesized that patients with severe dyspnea during exercise and under LTOT may have difficulties to perform exercise training during PR and therefore to show significant improvement in 6MWD post-PR, others have found that these patients showed larger multidimensional benefits (including symptoms, exercise performance, health and mood status) following PR [START_REF] Spruit | Differential response to pulmonary rehabilitation in COPD: multidimensional profiling[END_REF] . Overall, the mechanisms underlying non-response to PR in patients remain to be further elucidated [START_REF] Simonÿ | Experiences in responders and non-responders to pulmonary rehabilitation among people with chronic obstructive pulmonary disease: a clinical study with convergent mixed analysis[END_REF][25] in order to optimize the effect of PR in the COPD management strategy.

A strength of our study is that it focused on a homogeneous population of COPD patients only who performed within the standardized setting of a single PR center up to three PR programs.

We are the first to describe classes of response trajectories showing that a response to repeated PR is not systematically identical. A limitation of our study was that we focused on the 6MWT only to evaluate the effect of PR. Other important outcomes of PR such as quality of life should be also considered in future studies in order to take into account the multidimensional response to PR [START_REF] Spruit | Differential response to pulmonary rehabilitation in COPD: multidimensional profiling[END_REF] . This study demonstrates that there are clinically significant benefits of repeated PR for patients with COPD, with improvements in functional exercise capacity following a second and a third PR in the majority of the patients. We validated our hypothesis that a large proportion of COPD patients non-responders to a first PR can become responders if PR were to be repeated. Further studies are needed to take into account other parameters than the 6MWT to characterize the effects of PR.

Guarantor: YAC had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, including and especially any adverse effects.

Author contributions: YAC, SB, IA, DV, FH, and SV contributed substantially to the study design, data analysis and interpretation, and the writing of the manuscript.

FIGURE LEGEND

Figure 1 .

 1 Figure 1. Flow chart showing the inclusion process for the analysis.

Figure 2 Figure 3 .

 23 Figure 2 Mean (± standard deviation) 6-min walking distance (in % predicted) pre and post

Figure 4 .

 4 Figure 4. Alluvial plot showing distinct trajectories of 62 patients having performed three

Figure 5 .

 5 Figure 5. Alluvial plot showing distinct trajectories of 62 patients having performed three

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Acknowledgments: YAC has been provided with PhD funding (CIFRE contract) from the French national association for research and technology. SB and IA are supported by the French National Research Agency in the framework of the "e-health and integrated care and trajectories medicine and MIAI artificial intelligence 'ANR-19-P3IA-0003)" Chairs of excellence from the Grenoble Alps University Foundation.

Funding information: YAC has been provided with PhD funding (CIFRE contract) from the French national association for research and technology. SB and IA are supported by the French National Research Agency in the framework of the "e-health and integrated care and trajectories medicine and MIAI artificial intelligence 'ANR-19-P3IA-0003)" Chairs of excellence from the Grenoble Alps University Foundation.

TABLES

walking distance; SpO2, pulse oxygen saturation; HR, heart rate. * The difference is significant vs NR1-NR2 (p < 0.05 with Bonferroni correction).