El Hadi Saidane
email: el-hadi.saidane@uha.fr

Daniel Scida

Marie-José Pac

Rezak Ayad

Mode-I interlaminar fracture toughness of flax, glass and hybrid flax-glass fibre woven composites: failure mechanism evaluation using Acoustic Emission analysis

Keywords: Flax fibre, hybrid woven composites, interlaminar fracture toughness, delamination, acoustic emission

The mode-I interlaminar fracture toughness of flax, glass and hybrid flax-glass fibre woven composites was studied by using a DCB test. The acoustic emission signals recorded during the tests and scanning electron microscope images were used to analyse the damage mechanism of each composite. The crack initiation for the flax-fibre laminate needs the highest energy (1079 versus 945 for hybrid flax-glass fibre and 923 J/m² for glass-fibre laminates). The morphology of the flax fibres, short and bonded together in bundles to manufacture the twill fabric, allows the creation of a larger amount of fibre bridging as the origin of this highest energy. Furthermore, hybridisation of glass fibres with flax fibres in an appropriate combination offers an interesting solution when the toughness of glass fibre composites needs to be increased. More interesting is the considerable advantage of the composite structure weight reduction due to the low flax fibre density.

Introduction

For the past two decades, natural fibre-reinforced composites have been used in many industrial applications, especially in the automotive and construction sectors [START_REF] Yan | Can Plant-Based Natural Flax Replace Basalt and E-Glass for Fiber-Reinforced Polymer Tubular Energy Absorbers? A Comparative Study on Quasi-Static Axial Crushing[END_REF][START_REF] Yan | Crashworthiness characteristics of flax fibre reinforced epoxy tubes for energy absorption application[END_REF][START_REF] Huang | Reinforced concrete beams strengthened with externally bonded natural flax FRP plates[END_REF][START_REF] Ramesh | Plant fibre based bio-composites: Sustainable and renewable green materials[END_REF][START_REF] Yan | Effect of triggering and polyurethane foam-filler on axial crushing of natural flax/epoxy composite tubes[END_REF][START_REF] Yan | Experimental study of flax FRP tube encased coir fibre reinforced concrete composite column[END_REF][START_REF] Yan | Flax fibre and its composites -A review Composites : Part B[END_REF]. For example, the plant-based natural flax fibre shows great potential to be a suitable replacement for mineral-based basalt and synthetic glass fibres for crushable energy absorber application, as highlighted by Yan et al. [START_REF] Yan | Can Plant-Based Natural Flax Replace Basalt and E-Glass for Fiber-Reinforced Polymer Tubular Energy Absorbers? A Comparative Study on Quasi-Static Axial Crushing[END_REF][START_REF] Yan | Crashworthiness characteristics of flax fibre reinforced epoxy tubes for energy absorption application[END_REF].

Huang et al. [START_REF] Huang | Reinforced concrete beams strengthened with externally bonded natural flax FRP plates[END_REF] studied the possibility of the using flax fabric reinforced epoxy composite plates as external strengthening material for reinforced concrete beams. The results showed that the flax fabric reinforced polymer composite can be used to retrofit or strengthen deficiently-designed concrete structures as an environmentally-friendly external reinforcement material. In addition to their ecofriendly aspect and biodegradability [START_REF] Mohanty | Biofibres, biodegradable polymers and biocomposites: An overview[END_REF], natural fibres present high specific properties [START_REF] Pickering | A review of recent developments in natural fibre composites and their mechanical performance[END_REF], due to their low density [START_REF] Le Duigou | Replacement of Glass / Unsaturated Polyester Composites By Flax / PLLA Biocomposites : Is It Justified ?[END_REF][START_REF] Lefeuvre | A study of the yearly reproducibility of flax fiber tensile properties[END_REF], and an interesting damping behaviour [START_REF] Cheour | Effect of water ageing on the mechanical and damping properties of flax-fibre reinforced composite materials[END_REF]. Therefore numerous comprehensive studies have been conducted on natural fibre composites [START_REF] Sanjay | Characterization and Properties of Natural Fiber Polymer Composites: A Comprehensive Review[END_REF][START_REF] Väisänen | A review on new bio-based constituents for natural fiberpolymer composites[END_REF][START_REF] Fortea-Verdejo | Plant fibre-reinforced polymers: where do we stand in terms of tensile properties?[END_REF][START_REF] Li | An overview of structural-functional-integrated composites based on the hierarchical microstructures of plant fibers[END_REF][START_REF] Merotte | Flax and hemp nonwoven composites: The contribution of interfacial bonding to improving tensile properties[END_REF][START_REF] Fragassa | Experimental evaluation of static and dynamic properties of low styrene emission vinylester laminates reinforced by natural fibres[END_REF]. However, the development of these green materials may be limited because of the low interfacial properties between hydrophilic fibres and the hydrophobic matrix, which leads to delamination between the composite plies, thus lowering the mechanical properties [START_REF] Bensadoun | Interlaminar fracture toughness of flax-epoxy composites[END_REF]. In addition, delamination may also occur due to the presence of residual stresses during the manufacturing process or in a changing environment because natural fibres are temperature and moisture sensitive. Accordingly, knowledge of the interlaminar properties of these materials is essential for the design of composite structures, in particular when they are composed of long continuous unidirectional fibres or woven fabrics. Within this context, some authors have analysed the interlaminar fracture properties of natural fibre composites [START_REF] Bensadoun | Interlaminar fracture toughness of flax-epoxy composites[END_REF][START_REF] Pinto | Improving the strength and service life of jute/epoxy laminar composites for structural applications[END_REF][START_REF] Ravandi | The effects of through-thethickness stitching on the Mode I interlaminar fracture toughness of flax/epoxy composite laminates[END_REF][START_REF] Zhang | Tensile and interfacial properties of unidirectional flax / glass fiber reinforced hybrid composites[END_REF][START_REF] Khan | Fracture behaviour of bamboo fiber reinforced epoxy composites[END_REF][START_REF] Almansour | Effect of water absorption on Mode I interlaminar fracture toughness of flax/basalt reinforced vinyl ester hybrid composites[END_REF][START_REF] Almansour | Investigation into Mode II interlaminar fracture toughness characteristics of flax/basalt reinforced vinyl ester hybrid composites[END_REF][START_REF] Chen | Interlaminar toughening in flax fiber-reinforced composites interleaved with carbon nanotube buckypaper[END_REF][START_REF] Le Duigou | Macroscopic analysis of interfacial properties of flax/PLLA biocomposites[END_REF]. For example, Bensadoun et al. [START_REF] Bensadoun | Interlaminar fracture toughness of flax-epoxy composites[END_REF] studied the influence of fibre architectures on the mode-I and mode-II interlaminar fracture toughness of flax fibre epoxy composites. They compared the fracture toughness of plain weave, twill weave and quasi-unidirectional architectures. The results showed that the flax composites exhibited a high interlaminar fracture toughness which was related to several energy absorbing mechanisms such as the crack branching and fibre bridging. By comparing the different fibre architectures, they showed that the yarn crimp and irregular surface of the layers could generate more energy consumption by forcing the crack to follow an irregular delamination path. In a similar study, Pinto et al. [START_REF] Pinto | Improving the strength and service life of jute/epoxy laminar composites for structural applications[END_REF] demonstrated that jute fibre woven composites showed a greater initiation fracture toughness than unidirectional ones because of fibre bridging and increasing interaction in the intra-ply. Ravandi et al. [START_REF] Ravandi | The effects of through-thethickness stitching on the Mode I interlaminar fracture toughness of flax/epoxy composite laminates[END_REF] studied the effects of through-the-thickness stitching using natural fibres on the interlaminar fracture toughness and tensile properties of flax fibre/epoxy composites. They found that the fracture toughness of the laminates was more improved by flax yarn stitches compared to cotton thread stitches. Furthermore, the interlaminar fracture toughness of woven flax composites was significantly higher than that of glass fibre composites. The hybridisation effect on the interlaminar fracture properties of unidirectional flax/glass fibre reinforced hybrid composites was investigated by Zhang et al. [START_REF] Zhang | Tensile and interfacial properties of unidirectional flax / glass fiber reinforced hybrid composites[END_REF] who found that the interlaminar shear strength and the interlaminar fracture toughness of hybrid flax/glass fibre composites were higher than those of glass fibre ones. This was explained by the rough surface of flax fibres, which led to more fibre bridging between flax fibres, flax yarn and glass fibres.

The work reported in this paper aims at evaluating the mode-I interlaminar fracture toughness of flax, glass and hybrid flax-glass fibre reinforced epoxy (FFRE, GFRE and HFRE) laminates. The choice of flax and glass fibres, which have comparable mechanical performances, aims at analysing the effect of their hybridisation on the interlaminar fracture properties of the HFRE composite and enables studying the delamination between flax and glass fibre layers. For this purpose, three twill weave fabric laminates (FFRE, GFRE and HFRE with a single stacking sequence) were manufactured by a compression moulding process. Then, the double cantilever beam (DCB) tests were conducted to evaluate the mode-I interlaminar fracture toughness properties. In addition, the DCB tests were monitored using the acoustic emission (AE) technique to identify the different damage mechanisms of these composites. This identification was made with an unsupervised method based on a statistical multivariable analysis (k-means algorithm) using Noesis software. Several previous studies used this method to assess the damage failures of natural fibre composites during different mechanical tests, in particular tensile tests [START_REF] Saidane | Damage mechanisms assessment of hybrid flaxglass fibre composites using acoustic emission[END_REF][START_REF] Monti | Mechanical behaviour and damage mechanisms analysis of a flax-fibre reinforced composite by acoustic emission[END_REF][START_REF] Assarar | Acoustic emission characterization of damage in short hemp-fiber-reinforced polypropylene composites[END_REF], cyclic and fatigue tests [START_REF] Haggui | Static and fatigue characterization of flax fiber reinforced thermoplastic composites by acoustic emission[END_REF]. However, to the best of our knowledge, this method was not yet employed to evaluate the damage mechanisms of natural fibre composites during DCB tests. In addition, scanning electron microscopy (SEM) was performed to observe and understand the different damage mechanisms previously identified by the AE results.

Experimental methods

Materials and manufacturing process

The fabric weave used for the laminated composites was a 2/2 twill weave. The flax fabric, based on untwisted yarns and provided by Depestele Group, had an areal weight of 350 g/m 2 and a fibre density of 1450 kg/m 3 . The linear mass of the warp and weft yarns of the dry fabric was the same and equal to 300 Tex. The flax fibres were bonded together by a specific binder to form a flat roving and allow weaving. The glass fabric, supplied by Sicomin Company, had an areal weight of 300 g/m 2 and a fibre density of 2450 kg/m 3 . The yarns distribution in the warp and weft directions was the same and equal to 50%. The matrix used for the laminated composites was an SR 1500 epoxy resin associated with SD 2503 hardener marketed by Sicomin Company. The mixing ratio of resin and hardener was 100:33 by weight. FFRE, GFRE and HFRE composites with a single stacking sequence were manufactured by compression moulding. 450 mm × 300 mm laminate plates were prepared with a different number of layers as summarised in Table 1. These numbers were chosen to obtain approximately the same and minimum 4 mm thickness for all composites. The advantage of a single stacking sequence is to allow the study of the delamination between the flax and glass fibres layers in the middle plane of the HFRE composite. First, flax and glass fabrics were manually pre-impregnated with the resin system. Next, the impregnated layers were placed one over the other following the same orientation with resin layers. During the layup process, a 12 µm thick Teflon insert of thickness was inserted at the midplane of the laminate to form a precrack, i.e. an initiation site for delamination (Figure 1a). The whole assembly was carefully placed between two steel platens covered with Teflon paper. Laminates were then cured in a compression moulding machine (SATIM model) under 5-bar pressure at 35°C for 3 hours, following the supplier's recommendations. Finally, the laminate plates were cut and shaped in rectangular form (20 mm × 170 mm) according to ASTM D5528-13 standard [START_REF]Standard Test Method for Mode I Interlaminar Fracture Toughness of Unidirectional Fiber-Reinforced Polymer Matrix Composites[END_REF] by using a diamond saw blade. The thickness and fibre volume fraction of different composites are also given in Table 1.

The fibre volume fractions (V f ) of FFRE, GFRE and HFRE composites were experimentally determined by the same procedure detailed in Saidane et al [START_REF] Saidane | Damage mechanisms assessment of hybrid flaxglass fibre composites using acoustic emission[END_REF].

Mode-I interlaminar fracture toughness testing

The mode-I interlaminar fracture toughness (G IC ) and the resistance curve (R-curve) were evaluated by the DCB tests in accordance with ASTM D5528-13 standard [START_REF]Standard Test Method for Mode I Interlaminar Fracture Toughness of Unidirectional Fiber-Reinforced Polymer Matrix Composites[END_REF]. They were conducted on an Instron 3382 universal machine with a 5 kN capacity load cell. To produce steady crack growth, a cross-head speed of 5 mm/min was used. For each composite, five specimens were tested. The average value and the standard deviation were then reported. The specimen geometry and its nominal dimensions are depicted in Figure 1a. The initial delamination length, a 0 , of all samples was 50 mm. A pair of piano hinge tabs was bonded to the end of each specimen, as shown in Figure 1a. Before bonding the hinges, surfaces of both sides were lightly scrubbed with sandpaper and wiped clean with acetone. After surface preparation, the hinges were aligned parallel with the specimen and with each other, and bonded using a Penloc GTI glue (Adheko MMA-3295). Clamps were used to hold them in position for 48 h until the adhesive was fully cured. To make the detection of delamination onset easier, one side edge of each specimen was painted with white lacquer. The use of the AE technique enabled clear identification of the initiation of the crack when the cumulative number of AE hits significantly increases (Figure 2a).

The strain energy release rate G IC is given by Modified Beam Theory (MBT), as follows [START_REF]Standard Test Method for Mode I Interlaminar Fracture Toughness of Unidirectional Fiber-Reinforced Polymer Matrix Composites[END_REF]:

( ) 3 2 IC P G b a δ = + ∆ ( 1
)
where P is the load, δ is the load point displacement, b is the specimen width and a is the delamination length. The crack length correction factor, ∆, was experimentally obtained by plotting the cube root of compliance, C 1/3 , as a function of the delamination length (Figure 2b). The compliance C is the ratio of the load point displacement to the applied load, δ/P. To evaluate the factor, ∆, note that four samples with different initial delamination lengths, 30, 50, 60 and 70 mm, were used, and the compliance was determined for each sample.

Acoustic emission

To obtain information about damage evolution in the tested specimens, AE was continuously monitored during the delamination tests. AE measurements were conducted using two-channel data acquisition system, with a sampling rate of 5 MHz and 40 dB pre-amplification, produced by Mistras Group Company. This system was equipped with a PCI acquisition card connected to the microcomputer. AE signals were recorded by two resonant Micro-80 piezoelectric sensors with a frequency range of 100 kHz-1 MHz and a resonance peak of around 300 kHz. The sensors were placed on the upper surface of the test specimen with a spacing of 80 mm, as shown in Figure 1a. To provide a good acoustic coupling, a silicone grease was employed as a coupling agent between the sensors and the sample surface. Before the DCB tests, a pencil lead break procedure was used to optimise the distance between the two AE sensors. Then, the AE acquisition system was calibrated to record the signals located between the two sensors using the AE localisation method. The quality of the measured AE data mainly depends on the chosen waveform system timing parameters. These parameters, namely, PDT (peak definition time), HDT (hit definition time) and HLT (hit lockout time), enable the selection of the event characteristics. For the composite material tests, the values of the timing parameters employed were: PDT = 30 µs, HDT = 200 µs and HLT = 300 µs. The main parameters measured on the AE event parameters are depicted in an example of AE waveform in Figure 1b.

Results and discussion

Mode-I interlaminar fracture properties

Load-displacement curves

The load-displacement curves of the mode-I DCB tests of FFRE, HFRE and GFRE composites are shown in Figure 3. For the three composites, the curves present an initial linear region corresponding to linear elastic behaviour followed by à non-linear region in which the initiation and propagation of the cracks occur. The latter depends on the type of composite. Delamination initiation was visually observed and detected by the first change in the slope of the AE cumulative hits number (Figure 2a).

For the FFRE, after the initiation of cracks close to 39N, the load slowly increases with increasing displacement due to fibre bridging and, then, a steady crack appears. The GFRE composite exhibits the highest load, around 47 N. After the maximum load of 52 N, the load sharply drops with increasing displacement until the composite fails. The drop of this maximum corresponds to delamination onset at the precrack tip, i.e. the initiation and growth of the cracks in the laminate. In the case of HFRE composite, crack initiation is near 42N, an intermediate value between those of FFRE and GFRE. Moreover, a large amount of load drop occurs due to crack stop during the displacement increase.

Table 2 displays the values of initiation G Ic obtained from Eq. 1 for FFRE, HFRE and GFRE composites. These values were determined by measuring the load and deflection at the point at which delamination occurred. The values of initiation G Ic correspond to the initial delamination length (a 0 = 50 mm) and describe the resistance of the sample to crack formation. The FFRE composite has a higher initial interlaminar fracture toughness than the GFRE and HFRE ones (Table 2). The interlaminar fracture toughness of the FFRE composite is approximately 1.2 times more than that of the GFRE composite (1079 J/m 2 for the FFRE laminate and 923 J/m 2 for the GFRE laminate). A similar tendency has already been observed by Zhang et al. [START_REF] Zhang | Tensile and interfacial properties of unidirectional flax / glass fiber reinforced hybrid composites[END_REF] for unidirectional flax and glass fibre composites. In the same way, the FFRE laminate value was 17% greater than the GFRE laminate value, even though the fibre volume content of the FFRE composite (40%) was lower than that of the GFRE composite (50%). This difference in initiation G Ic values can be explained by the morphology and the structure of flax and glass fibres, which are fully different. Indeed, during manufacturing of flax fabrics from short flax fibres, the short individual flax fibres are bonded together in bundles to make long natural yarns, so as to weave them into 2D fabrics. Therefore, some individual flax fibres stretch out from the yarns more easily, as shown in Figure 4a. This generated a large amount of fibre bridging, leading to a higher delamination resistance for the FFRE composite. In the case of the glass fabric, the fibres are long, continuous and present a regular structure and smooth surface. Accordingly, the GFRE composite shows fewer rough fracture surfaces and no fibre bridging (Figure 4b). The yarn crimp in the woven fabric can also explain this difference in G Ic values. Indeed, Bensadoun et al. [START_REF] Bensadoun | Interlaminar fracture toughness of flax-epoxy composites[END_REF] showed that the yarn crimp and irregular surface of the layers forced the crack to follow an irregular path, hence, led to more consumption of energy. For our composites, the same results are observed since the waviness thickness of FFRE, which exhibits a larger G Ic , is higher than that of GFRE (0.20 mm vs. 0.09 mm). In addition, the G Ic value of the FFRE composite is higher than flax-epoxy woven composites with similar V f (40%), studied by Bensadoun et al. [START_REF] Bensadoun | Interlaminar fracture toughness of flax-epoxy composites[END_REF]. Indeed, they found values of G Ic equal to 607 J/m 2 and 754 J/m 2 for flax-epoxy twill composites with high and low twist yarns, respectively. This was mainly due to the twist angle of flax yarns because the G Ic initiation value decreases with increasing twist angle. In our case, flax fabric was made up of untwisted yarns. For the HFRE composite, the value of G Ic is approximately 945 J/m 2 rather close to the G Ic value of the GFRE composite despite the difference of V f between both composites (40% for HFRE vs. 50% for GFRE).

Thus, if the fracture toughness of glass fibre composites needed to be increased; hybridisation of glass fibres with flax fibres in an appropriate combination would offer an interesting solution. This solution also reduces the composite structure weight as the density obtained for the HFRE composite is 1.4

times lower than that of the GFRE composite (Table 2). This is mainly due to the flax fibre density (1450 kg/m 3 ) which is significantly lower than that of the glass (2540 kg/m 3 ). These structural properties are interesting since HFRE composite presents a density gain of about 34% against the GFRE composite and at the same time, a gain in the specific fracture toughness (the ratio of G Ic to the density of the material) of 37% (Table 2).

Resistance curves (R-curves)

In order to visually observe the crack propagation, the edge of each specimen was marked from the insert with vertical lines every 5 mm, as suggested by the ASTM standard [START_REF]Standard Test Method for Mode I Interlaminar Fracture Toughness of Unidirectional Fiber-Reinforced Polymer Matrix Composites[END_REF]. The vertical lines correspond to crack lengths of 50, 55, 60, 65, 70, 75, 80, 90 and 100 mm and the corresponding load and cross-head displacement were recorded. Then, the values of G Ic were calculated from Eq. 1 for each crack length. The obtained results are depicted as resistance curves (R-curves) in Figure 5, which displays the values of G Ic as a function of the delamination lengths of each composite. One can notice that the G Ic of the FFRE composite increases in a progressive way increasing the crack length. This was directly related to the increase in the amount of fibre bridging during the test. For the GFRE composite, a slight increase of G Ic can be seen at the beginning of crack propagation followed by a steady state because no fibre bridging occurred. The initial increase at the beginning of the test can be explained by the presence of some resin-rich regions due to the manufacturing process, in particular at the crack tip, while the steady state is because no fibre bridging occurred during the test. Furthermore, the crack has a clear and easy path along the glass fibre layer with no deviation in the path (Figure 4b). Concerning the HFRE composite, a combination of both R-curves behaviours of the FFRE and GFRE laminates is observed. For crack lengths between 50 and 65 mm, the HFRE and FFRE composites follow the same tendency with increasing G Ic values due to the increase of fibre bridging during the DCB test. So, in this region, the R-curve of the HFRE composite is governed by the flax fibres. When the crack length is over 65 mm, a difference is observed in FFRE and HFRE Rcurves behaviour. While the G IC values continue to increase for FFRE composite, they remain constant and higher for HFRE composite due to the presence of glass fibres. For crack lengths between 55 and 85 mm, the HFRE composite exhibits the largest propagation value of G Ic compared to the other composites because of the large amount of flax and glass fibre bridging (Figure 4c) and a deviation in the path of the crack which is not observed in FFRE specimens (Figure 4a). It could be due to the torn out glass fibres, which play a bridging role between the flax and glass fibre layers. The same phenomena were observed by Zhang et al. [START_REF] Zhang | Tensile and interfacial properties of unidirectional flax / glass fiber reinforced hybrid composites[END_REF] for unidirectional flax/glass fibre reinforced hybrid composites.

Damage mechanisms analysis

Acoustic emission results

In order to assess the damage mechanisms in the studied specimens during DCB tests, the AE signals were recorded and analysed by considering a statistical multi-variable analysis using Noesis software. This method was employed to separate the acoustic signals into different classes presenting the same characteristics for each clustering signal. This classification was carried out by considering the main AE parameters, i.e. amplitude, duration, number of counts, energy and rise time, as reported in the literature [START_REF] Saidane | Damage mechanisms assessment of hybrid flaxglass fibre composites using acoustic emission[END_REF][START_REF] Monti | Mechanical behaviour and damage mechanisms analysis of a flax-fibre reinforced composite by acoustic emission[END_REF][START_REF] Haggui | Static and fatigue characterization of flax fiber reinforced thermoplastic composites by acoustic emission[END_REF]. To optimise the number of acoustic signals classes, a principal component analysis (PCA) on the 2D projection planes was considered, as shown in Figure 6. For each composite, the number of classes was varied (from 2-5) and the overlap rate between the various groups of AE signals was determined [START_REF] Saidane | Damage mechanisms assessment of hybrid flaxglass fibre composites using acoustic emission[END_REF][START_REF] Assarar | Acoustic emission characterization of damage in short hemp-fiber-reinforced polypropylene composites[END_REF]. Next, the optimum number of classes was obtained by considering the minimum average value of the overlap rate between them. Three classes are obtained for the GFRE composite (Figure 6a) while four AE classes are optimum for the FFRE and HFRE composites, (Figure 6b). al. [START_REF] Mitchell | A Global Method for the Identification of Failure Modes in Fiber glass Using Acoustic Emission[END_REF], Class C can be attributed to the delamination mechanism. The last class, i.e. the Class D, detected at amplitudes up to 75 dB and high energy (>100000 aJ) for FFRE and HFRE composites is not observed for the GFRE composite for which no fibre bridging occurred during the delamination tests (Figure 4b). As a consequence, the last Class D, also observed in the literature results [START_REF] Saidane | Damage mechanisms assessment of hybrid flaxglass fibre composites using acoustic emission[END_REF][START_REF] Monti | Mechanical behaviour and damage mechanisms analysis of a flax-fibre reinforced composite by acoustic emission[END_REF][START_REF] Haggui | Static and fatigue characterization of flax fiber reinforced thermoplastic composites by acoustic emission[END_REF][START_REF] Habibi | Experimental characterization of short flax fiber mat composites: tensile and flexural properties and damage analysis using acoustic emission[END_REF], can be associated with fibre failure mechanisms.

To illustrate the damage mechanisms during DCB tests, the evolution versus time and cross-head displacement of the cumulative number of hits of each class, combined with DCB load is shown in are associated with the initiation and propagation of delamination, respectively. Region II is defined by crack initiation, corresponding to the slope change of the cumulative hits number of the matrix cracking and the maximal load. In this region and for all composites, the acoustic activity of the matrix cracking increased suddenly, accompanied by the acoustic events due to the beginning of the fibrematrix debonding and delamination. For the FFRE and HFRE composites, the signals relative to the fibre failures also appear because of failures of some fibre bridging and the generation of a large amount of fibre bridging (Figures 4a and4c). After delamination onset, the slope increase of the cumulative hits number for the four damage mechanisms of the FFRE composite was higher than that of the HFRE ones. This result is consistent with the higher value of G IC of the FFRE composite. In Region III, the acoustic activity of all damage mechanisms increases in a quasi-linear way until the final failure of the FFRE and HFRE composites. In contrast to Region II, the slope of the cumulative hits number of the four damage mechanisms is more important for the HFRE composite compared to the FFRE composite, probably due to the deviation of the crack path of the HFRE composite, which led to several flax fibres being torn out from the fractured surface of the flax layer. This could generate a large amount of fibre bridging at the propagation state leading to important distribution of the AE energy as well as an increase of the interlaminar fracture toughness of the HFRE composite. In the case of the GFRE composite in Region III, the number of hits of the matrix cracking and the fibrematrix debonding increases progressively until the global failure of the specimen. This increase is accompanied by a quasi-stabilisation of the number of hits derived from the delamination mechanism, in agreement with the steady state observed on the R-curve (Figure 5) and confirms that the GFRE composite exhibits an easier path for crack propagation due to the regular structure and the smooth surface of glass fibres (Figure 8c). fibre-matrix debonding, fibre and bundle failure and matrix cracking (Figures 8a andb). The images of the GFRE (Figure 8c andd) confirm the results presented in previous sections, particularly a regular structure with smooth glass fibres that explains the absence of fibre bridging, a clean delaminated surface and an easier path of crack propagation during the DCB test. Regarding the HFRE composite, a lot of torn out fibres were observed, confirming the existence of a large amount of fibre bridging (Figure 8e) requiring more energy for the propagation of cracks. In addition, Figure 8f shows some single flax fibres stretched from the flax yarns, resulting in several fibre failures, that explains the maximum value of the interlaminar toughness, G IC , of the HFRE composite (Figure 5).

SEM observations

Conclusions

In this work, the mode-I interlaminar fracture toughness (G Ic ) of flax, glass and hybrid flax-glass fibre woven composites (FFRE, GFRE and HFRE) was studied using a DCB test and discussed. The failure mechanisms are identified using AE measurements and observed by SEM images. For the FFRE composites, the large amount of flax fibre bridging occurring during crack propagation allows an initial fracture toughness of 1.2 times more than the GFRE composite and higher delamination resistance, even though the flax fibre volume content (40%) is lower than that of glass fibres (50%).

The differences in the morphology of the fibres and regularity of the structure explain these results.

The hybridisation of flax fibres with glass fibres in the composites, allows obtaining better interlaminar fracture toughness, G IC , during crack propagation despite an initial value of G IC close to that of GFRE and lower than the FFRE one. Thus, in HFRE laminates, glass fibres govern the initial toughness, and, then, at higher load, flax fibres play a larger role. The combination of the two types of fibres leads to deviations of the crack path, which are not observed for the other composites, probably at the origin of a greater resistance. It was also found that the initiation G Ic values of the GFRE and HFRE composites were rather close despite the difference of the fibre volume content between both composites (40% for HFRE vs. 50% for GFRE). At the propagation state, the HFRE composite exhibited the largest value of G Ic compared to other composites, for crack lengths between 55-85 mm.

For the HFRE specimen, a large amount of fibre bridging occurred during crack propagation with a deviation in the path of the crack. However, the crack showed a clear and easy path along the fibre layer for the FFRE end GFRE composites.

The composites containing flax fibres, i.e FFRE and HFRE, display four failure mechanisms during DCB tests, whereas only three are seen for composites with glass fibres. So, the acoustic events related to glass fibres failure do not exist. Only for the hybrid composite HFRE, flax fibres are stretched due to the arrangement between flax and glass fibres, which is in agreement with the maximum value of the interlaminar toughness during the crack propagation.

In conclusion, the hybridization of glass fibres with flax fibres in a suitable combination would offer an interesting solution when toughness must be increased. In addition, reducing the weight of the composite structure since the density of flax fibres is significantly lower than that of glass is a great advantage.

In future investigations, it would also be interesting to study the mode-II fracture toughness properties of studied composites for the purposes of finalizing this study. After that, since natural fibres are moisture sensitive, investigating the effect of wet ageing on mode-I and mode-II delamination resistance would be of interest. Another study may be interesting: analysis of the effects of through-thickness stitching on the mode-I interlaminar fracture toughness of flax and hybrid flaxglass fibre composites. In fact, the stitching process is characterised by the insertion of a throughthickness tufting yarn into the 2D perform of flax and glass fabrics. This could improve the delamination resistance in these composite materials.

  In order to associate the obtained classes with their corresponding damage mechanisms, the characteristics of the recorded waveforms of each group of signals were analysed and compared with the literature results. Table2displays the AE characteristics of each signal clustering for the three studied composites. The signals of Class A are characterised by amplitude values ranged between 32-60 dB with duration and number of counts lower than 70 µs and 38, respectively. Moreover, the characteristics of these signals are substantially similar for the three composites and present very low energies (<100 aJ). Note that the epoxy matrix is the single component that is common to the three composites. The same characteristics were obtained in literature results for unidirectional flax/Elium composites under tensile[START_REF] Monti | Mechanical behaviour and damage mechanisms analysis of a flax-fibre reinforced composite by acoustic emission[END_REF][START_REF] Haggui | Static and fatigue characterization of flax fiber reinforced thermoplastic composites by acoustic emission[END_REF], cyclic and fatigue tests[START_REF] Haggui | Static and fatigue characterization of flax fiber reinforced thermoplastic composites by acoustic emission[END_REF] indicating that class A can be associated with the matrix cracking mechanism. The signals of Classes B and C also appear for the three composites with identical characteristics. More precisely, the signals of Class B are identified at an amplitude between 39 and 68 dB, a duration of 48 to 216 µs and a number of counts between 18 and 107. These characteristics are assigned to the fibre-matrix debonding in accordance with the literature[START_REF] Monti | Mechanical behaviour and damage mechanisms analysis of a flax-fibre reinforced composite by acoustic emission[END_REF][START_REF] Assarar | Acoustic emission characterization of damage in short hemp-fiber-reinforced polypropylene composites[END_REF][START_REF] Haggui | Static and fatigue characterization of flax fiber reinforced thermoplastic composites by acoustic emission[END_REF][START_REF] Bravo | Characterization of Tensile Damage for a Short Birch Fiber-reinforced Polyethylene Composite with Acoustic Emission[END_REF][START_REF] Bravo | Development of novel green and biocomposite materials: Tensile and flexural properties and damage analysis using acoustic emission[END_REF][START_REF] Habibi | Experimental characterization of short flax fiber mat composites: tensile and flexural properties and damage analysis using acoustic emission[END_REF][START_REF] Meraghni | Micromechanical modelling of matrix degradation in randomly oriented discontinuous-fibre composites[END_REF]. The class C presents signals characteristics (50-82 dB amplitude, 115-331 µs duration and 36-169 count range) slightly higher than those observed for the class B. According toMitchell et

Figure 7

 7 Figure7for the three composites. According to the shape of the cumulative hits number of the

Figure 8 shows

 8 Figure 8 shows SEM images of the composite cross sections after DCB tests recorded using a Jeol

FiguresFigure 1 Figure 2 .Figure 3 .Figure 4 .Figure 6 .Figure 7 .Figure 8 .

 1234678 Figures

Table 1 .

 1 Layer configuration, nominal thickness and fibre volume content of studied laminates

	Tables

Layer configuration Nominal thickness (mm) (flax/glass) Fibre volume content V f (flax/glass)

	FFRE	10/0	[F 5 ] s	4.98/0.00	0.40/0.00
	HFRE	6/6	[(G/F) 6 ]	3.21/1.35	0.26/0.14
	GFRE	0/20	[G 10 ] s	0.00/4.17	0.00/0.50
	F: Flax fabric, G: Glass fabric, s: symmetric stacking sequences.

Table 2 .

 2 Density, values of initiation fracture toughness and specific fracture toughness (the ratio of G Ic to the material density) for the studied laminates

	Material	Density of composite	Fracture toughness	Specific fracture toughness
		(kg/m 3 )	(J/m 2 )	(x 10 -3 J.m/kg)
	FFRE	1272.1 ± 8.1	1079.2 ± 66.4	848.40 ± 5.34
	HFRE	1411.5 ± 13.1	944.8 ± 76.7	669.40 ± 6.17
	GFRE	1888.4 ± 10.4	922.9 ± 30.2	488.70 ± 2.68

Table 3 .

 3 AE characteristics of each signal clustering for all composites

	Material	AE parameter	Class A	Class B	Class C	Class D

Acknowledgements

The authors would like to gratefully acknowledge Cyril Marsiquet from "Laboratoire de Physique et Mécanique Textiles" for his technical support and contribution to the use of the SEM facility.


MD Marion Chambaz

Mikael Verdalle-Cazes

MD Charlotte Desprez

MD Lucie Thomassin

MD Cloé Charpentier

Sebastien Grigioni

MD Laura Armengol-Debeir

MD Valérie Bridoux

MD Guillaume Savoye

MD Céline Savoye-Collet
email: celine.savoye-collet@chu-rouen.fr

MD Sébastien Grigioni

Deep remission on magnetic resonance imaging impacts outcomes of perianal fistulizing Crohn's disease

Keywords: Crohn's disease, Magnetic resonance imaging, Anal fistula, anti-TNFα

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Crohn's disease (CD) is a chronic inflammatory bowel disease. Perianal disease can be a complication of CD with a major negative effect on patients' quality of life [START_REF] Schwartz | The natural history of fistulizing Crohn's disease in Olmsted County, Minnesota[END_REF]. Fistulas, anal fissures, abscesses and anorectal strictures occur in one-third of patients with CD, either before or after the diagnosis of CD [START_REF] Schwartz | The natural history of fistulizing Crohn's disease in Olmsted County, Minnesota[END_REF][START_REF] Cosnes | Epidemiology and natural history of inflammatory bowel diseases[END_REF]. The treatment of perianal CD requires combined surgical and medical therapies including antibiotics, anti-tumor necrosis factor (TNF)-α and immunosuppressants [START_REF] Schwartz | Guidelines for the multidisciplinary management of Crohn's perianal fistulas: summary statement[END_REF][START_REF] Jakobovits | Metronidazole therapy for Crohn's disease and associated fistulae[END_REF][START_REF] Dejaco | Antibiotics and azathioprine for the treatment of perianal fistulas in Crohn's disease[END_REF][START_REF] Present | Infliximab for the treatment of fistulas in patients with Crohn's disease[END_REF][START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF][START_REF] Colombel | Adalimumab for maintenance of clinical response and remission in patients with Crohn's disease: the CHARM trial[END_REF][START_REF] Sandborn | Adalimumab induction therapy for Crohn disease previously treated with infliximab: a randomized trial[END_REF][START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF][START_REF] Sciaudone | Treatment of complex perianal fistulas in Crohn disease: infliximab, surgery or combined approach[END_REF][START_REF] Gecse | Results of the Fifth Scientific Workshop of the ECCO [II]: Clinical Aspects of Perianal Fistulising Crohn's Disease-the Unmet Needs[END_REF]. The use of anti-TNF-α deeply improves the management of perianal CD. Anti-TNF-α have demonstrated their efficacy as induction therapy and also as maintenance therapy in disease remission [START_REF] Present | Infliximab for the treatment of fistulas in patients with Crohn's disease[END_REF][START_REF] Ford | Efficacy of biological therapies in inflammatory bowel disease: systematic review and meta-analysis[END_REF]. The first placebo-controlled trial with infliximab as induction therapy showed complete closure of fistulas in 50% of patients [START_REF] Present | Infliximab for the treatment of fistulas in patients with Crohn's disease[END_REF].

Immunosuppressors and duration of seton drainage are also associated with better outcomes [START_REF] Bouguen | Long-term outcome of perianal fistulizing Crohn's disease treated with infliximab[END_REF]. Maintenance therapy is necessary to increase sustained response and patients require dose adjustments [START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF]. Initial doses could be intensified or decreased, based on clinical response.

Magnetic resonance imaging (MRI) is a highly accurate non-invasive modality and allows morphological assessment and classification of perianal fistulas [START_REF] Gecse | Results of the Fifth Scientific Workshop of the ECCO [II]: Clinical Aspects of Perianal Fistulising Crohn's Disease-the Unmet Needs[END_REF][START_REF] Tougeron | Predicting factors of fistula healing and clinical remission after infliximab-based combined therapy for perianal fistulizing Crohn's disease[END_REF]. MRI is considered as the gold standard imaging technique. An MRI-based score (Van Assche score) is used to describe the anatomy, complexity and activity of fistulas [START_REF] Van Assche | Magnetic resonance imaging of the effects of infliximab on perianal fistulizing Crohn's disease[END_REF]. On MRI, fistula activity is represented by semiological items as contrast enhancement and T2 hyperintensity.

Several authors have reported that changes in MRI patterns can be followed [START_REF] Tougeron | Predicting factors of fistula healing and clinical remission after infliximab-based combined therapy for perianal fistulizing Crohn's disease[END_REF][START_REF] Van Assche | Magnetic resonance imaging of the effects of infliximab on perianal fistulizing Crohn's disease[END_REF][START_REF] Ng | Prospective evaluation of anti-tumor necrosis factor therapy guided by magnetic resonance imaging for Crohn's perineal fistulas[END_REF][START_REF] Bell | Response of fistulating Crohn's disease to infliximab treatment assessed by magnetic resonance imaging[END_REF][START_REF] Karmiris | Long-term monitoring of infliximab therapy for perianal fistulizing Crohn's disease by using magnetic resonance imaging[END_REF].

Clinically, perianal activity is assessed according to fistula drainage [START_REF] Present | Infliximab for the treatment of fistulas in patients with Crohn's disease[END_REF]. Nevertheless, clinical response often contrasts with fistula healing on MRI [START_REF] Ng | Prospective evaluation of anti-tumor necrosis factor therapy guided by magnetic resonance imaging for Crohn's perineal fistulas[END_REF][START_REF] Bell | Response of fistulating Crohn's disease to infliximab treatment assessed by magnetic resonance imaging[END_REF][START_REF] Karmiris | Long-term monitoring of infliximab therapy for perianal fistulizing Crohn's disease by using magnetic resonance imaging[END_REF]. It has been demonstrated that an active fistula track might persist despite clinical healing (fistula classified as clinically closed) [START_REF] Tozer | Long-term MRI-guided combined anti-TNF-α and thiopurine therapy for Crohn's perianal fistulas[END_REF]. Cessation of treatment following clinical remission but before eradication of the track on MRI could be responsible for recurrences. In that regard, combined MRI and clinical evaluation should be preferred to clinical evaluation alone. In luminal CD, deep remission is defined as endoscopic mucosal remission associated with clinical remission. This concept has become a therapeutic goal in emerging treat-to-target approaches. Mucosal healing is associated with lower hospitalization rates, fewer surgical interventions and less relapse [START_REF] Baert | Mucosal healing predicts sustained clinical remission in patients with early-stage Crohn's disease[END_REF][START_REF] Colombel | Deep remission: a new concept?[END_REF]. Fistula healing on MRI could also be considered as a target. There are very limited data regarding the long-term outcomes and complications in CD patients with perianal fistulas in both clinical and MRI remission.

The aim of this study was to evaluate the long-term course and complications of CD patients with perianal fistulas treated with anti-TNF-α based on their clinical remission and healing on MRI.

Materials and Methods

Subjects

Between 2000 and 2017, all consecutive patients with fistulizing perianal CD treated with maintenance anti-TNF-α and evaluated by repeated MRI in our tertiary care center, were retrospectively reviewed. This retrospective study, without dedicated intervention, was approved by the Institutional Review Board and informed consent was waived. All patients had a confirmed diagnosis of CD. Patients were diagnosed by clinical, histological, radiological, and endoscopic examinations and had proven history of active perianal fistulas.

An initial perianal MRI examination was performed before the induction treatment.

Patients' characteristics (age, sex, smoking status, family history of inflammatory bowel diseases, localizations of disease according to Montreal criteria, mean duration of CD) were assessed [START_REF] Satsangi | The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications[END_REF]. Patients with an abscess had surgical drainage with setons associated with antibiotics, when appropriate. All patients had at least 12 months of anti-TNF-α therapy. They received induction therapy either with adalimumab (160, 80, 40 mg at week 0, 2 and 4) or with infliximab (5 mg/kg at weeks 0, 2 and 6). Immunosuppressant drugs could be associated (azathioprine, methotrexate or purinethol). This induction treatment was followed by maintenance therapy (5 mg/kg of infliximab every 8 weeks or adalimumab 40 mg every week). Each treatment could be optimized by decreasing the interval between two injections or by increasing doses or could be switched with other biologic drugs in case of failure (i.e. certolizumab, ustekinumab or vedolizumab). Seton removal was usually performed after induction treatment with anti-TNF-α between weeks 6 and 14. However it could be delayed at the appreciation of the clinician in charge in case of significant and persistent leakage.

MRI examination

An MRI examination was performed in all patients, before the start of anti-TNF-α therapy and then after at least 12 months of anti-TNF-α therapy. MRI examination was performed on a Philips Achieva 1.5 Tesla (Philips Medical Systems, Best, the Netherlands) using a torso phased-array coil. Patients were placed in supine position; the pelvis was centered on the coil. T2-weighted two-dimensional (2D) turbo spin-echo (TSE) sequences [START_REF] Van Assche | Magnetic resonance imaging of the effects of infliximab on perianal fistulizing Crohn's disease[END_REF]. Complexity of the fistula tracks (single unbranched: 1, single branched: 2, multiple: 3); location regarding the sphincter (inter/extrasphincteric: 1, transsphincteric: 2, suprasphincteric: 3); extension (infralevatoric: 1, supralevatoric: 2); hyperintense appearance in T2-weighted sequences (absent: 0, mild: 4, pronounced: 8); presence of abscesses (hyperintense fluid collections > 3 mm in T2-weighted sequences: 4), and rectal wall involvement (thickened rectal wall: 2) were recorded. In addition, fistula enhancement after gadolinium injection was analyzed as present or absent.

Clinical and MRI evaluations

The fistula drainage assessment has been utilized to standardize the clinical assessment according to Present's criteria [START_REF] Present | Infliximab for the treatment of fistulas in patients with Crohn's disease[END_REF]. Drainage of fistula openings was studied under gentle finger compression and identified as open and actively draining or closed. Clinical remission was defined as the absence of persisting draining fistulas self-reported by the patient or the absence of any draining fistulas at examination. Clinical examination was performed by a senior gastroenterologist specialized in inflammatory bowel disease (IBD).

All clinical examinations were performed by the same physician dedicated to one patient. MRI interpretation was performed by a senior gastrointestinal radiologist (18 years of experience). Healing on MRI was defined as the total disappearance of T2 hyperintensity and contrast enhancement after gadolinium injection, along the entire fistula track; otherwise, it was considered as pathological on MRI.

Clinical and MRI evaluations were assessed and patients were classified either in the deep remission group or not. Deep remission was defined as the association of both clinical remission according to Present criteria and healing on MRI.

Outcomes

All reports mentioning the flare of luminal and perianal CD were reviewed. Flare was defined as the appearance or exacerbation of symptoms for over 2 consecutive weeks leading to an unscheduled visit to the out-patient clinic with or without therapeutic intervention or changes. All surgeries, as well as the number and duration of hospital stays were collected.

Discontinuation, optimization (increase in dosage) or changes (switch of drugs or addition of drugs) in CD-related treatment were also collected. An event was defined as flare, hospitalization, surgery, optimization or switch of therapy. We identified the cause of the event as perineal or luminal according to symptoms.

Statistical analysis

Qualitative data are described as percentage and quantitative data as median and maximal range values. The events were analyzed using survival analysis (Kaplan Meier test) and compared using Log Rank test (Mantel Cox). Comparison of patients according to their deep remission status was made by Chi 2 test for qualitative and Fisher test for quantitative variables. Results were considered significant with a p value of < 0.05.

Results

Patients' characteristics

Forty-eight patients were included. Their characteristics are listed in Table 1. Of these 48 patients, 30 were female. The median age was 32 years (14-70) and the mean duration of CD was 72 months (3-300 months). According to the Montreal classification, 6 patients (12%) were diagnosed with a penetrating complication (B3) [START_REF] Satsangi | The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications[END_REF]. Forty-five percent of the patients had ileo-colitis involvement (L3) at the time of diagnosis. Pure colonic disease was observed in 16 patients (33%) and isolated ileal location in 4 (8%). All patients had received anti-TNF-α therapy for at least 12 months before MRI and clinical evaluation, with a median treatment duration of 26 months (12-85) (Table 1).

Clinical and MRI evaluation

Thirty-two patients had pathological MRI. Among them, the median Van Assche score was 12 [START_REF] Cosnes | Epidemiology and natural history of inflammatory bowel diseases[END_REF][START_REF] Schwartz | Guidelines for the multidisciplinary management of Crohn's perianal fistulas: summary statement[END_REF][START_REF] Jakobovits | Metronidazole therapy for Crohn's disease and associated fistulae[END_REF][START_REF] Dejaco | Antibiotics and azathioprine for the treatment of perianal fistulas in Crohn's disease[END_REF][START_REF] Present | Infliximab for the treatment of fistulas in patients with Crohn's disease[END_REF][START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF][START_REF] Colombel | Adalimumab for maintenance of clinical response and remission in patients with Crohn's disease: the CHARM trial[END_REF][START_REF] Sandborn | Adalimumab induction therapy for Crohn disease previously treated with infliximab: a randomized trial[END_REF][START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF][START_REF] Sciaudone | Treatment of complex perianal fistulas in Crohn disease: infliximab, surgery or combined approach[END_REF][START_REF] Gecse | Results of the Fifth Scientific Workshop of the ECCO [II]: Clinical Aspects of Perianal Fistulising Crohn's Disease-the Unmet Needs[END_REF][START_REF] Ford | Efficacy of biological therapies in inflammatory bowel disease: systematic review and meta-analysis[END_REF][START_REF] Bouguen | Long-term outcome of perianal fistulizing Crohn's disease treated with infliximab[END_REF][START_REF] Tougeron | Predicting factors of fistula healing and clinical remission after infliximab-based combined therapy for perianal fistulizing Crohn's disease[END_REF][START_REF] Van Assche | Magnetic resonance imaging of the effects of infliximab on perianal fistulizing Crohn's disease[END_REF][START_REF] Ng | Prospective evaluation of anti-tumor necrosis factor therapy guided by magnetic resonance imaging for Crohn's perineal fistulas[END_REF][START_REF] Bell | Response of fistulating Crohn's disease to infliximab treatment assessed by magnetic resonance imaging[END_REF][START_REF] Karmiris | Long-term monitoring of infliximab therapy for perianal fistulizing Crohn's disease by using magnetic resonance imaging[END_REF]. The MRI characteristics of these patients are listed in Table 2. The most common fistula location was extra or inter-sphincteric (75%) and thirteen patients had an abscess. Sixteen patients had healing on MRI (Figure 1) and were considered to be in clinical remission. Ten patients were in clinical remission despite pathological MRI. There was no difference in patients' characteristics regarding the presence or absence of deep remission (Table 1).

Flare-free survival course

The median follow-up period was 62 months (5 -130). In patients with deep remission, the median time to a flare event was 96 months (69-116) versus 12 months [START_REF] Schwartz | Guidelines for the multidisciplinary management of Crohn's perianal fistulas: summary statement[END_REF][START_REF] Jakobovits | Metronidazole therapy for Crohn's disease and associated fistulae[END_REF][START_REF] Dejaco | Antibiotics and azathioprine for the treatment of perianal fistulas in Crohn's disease[END_REF][START_REF] Present | Infliximab for the treatment of fistulas in patients with Crohn's disease[END_REF][START_REF] Sands | Infliximab maintenance therapy for fistulizing Crohn's disease[END_REF][START_REF] Colombel | Adalimumab for maintenance of clinical response and remission in patients with Crohn's disease: the CHARM trial[END_REF][START_REF] Sandborn | Adalimumab induction therapy for Crohn disease previously treated with infliximab: a randomized trial[END_REF][START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF][START_REF] Sciaudone | Treatment of complex perianal fistulas in Crohn disease: infliximab, surgery or combined approach[END_REF][START_REF] Gecse | Results of the Fifth Scientific Workshop of the ECCO [II]: Clinical Aspects of Perianal Fistulising Crohn's Disease-the Unmet Needs[END_REF][START_REF] Ford | Efficacy of biological therapies in inflammatory bowel disease: systematic review and meta-analysis[END_REF][START_REF] Bouguen | Long-term outcome of perianal fistulizing Crohn's disease treated with infliximab[END_REF][START_REF] Tougeron | Predicting factors of fistula healing and clinical remission after infliximab-based combined therapy for perianal fistulizing Crohn's disease[END_REF][START_REF] Van Assche | Magnetic resonance imaging of the effects of infliximab on perianal fistulizing Crohn's disease[END_REF][START_REF] Ng | Prospective evaluation of anti-tumor necrosis factor therapy guided by magnetic resonance imaging for Crohn's perineal fistulas[END_REF][START_REF] Bell | Response of fistulating Crohn's disease to infliximab treatment assessed by magnetic resonance imaging[END_REF][START_REF] Karmiris | Long-term monitoring of infliximab therapy for perianal fistulizing Crohn's disease by using magnetic resonance imaging[END_REF][START_REF] Tozer | Long-term MRI-guided combined anti-TNF-α and thiopurine therapy for Crohn's perianal fistulas[END_REF] in patients with pathological MRI (p < 0.01), (Figure 2). In patients with deep remission, five events were observed; three patients had clinical perianal recurrence, one patient had an allergy to infliximab and one patient had luminal recurrence. Three patients in deep remission were lost to follow-up at 17, 61 and 130 months, respectively. In the pathological MRI group, 26 patients had an event; seventeen had perianal relapse and nine luminal recurrence. Two patients were lost to follow-up at 3 and 29 months, respectively.

Perineal flare-free survival course

In patients with deep remission, the median time to any perianal event was 116 months (95-130) versus 42 months (8-72) in patients with pathological MRI (p < 0.001), (Figure 3). Among patients in deep remission, three patients had clinical perianal recurrence at 26, 41 and 64 months, respectively. In the pathological MRI group, seventeen patients had perianal relapse.

Treatment during follow-up

All sixteen patients in deep remission were treated with infliximab. Three of them received adalimumab during their follow-up for perianal relapse, luminal recurrence and allergy to infliximab, respectively. Certolizumab was introduced in one patient due to anti-TNF-α intolerance. Five patients benefited from reduced dosage and three patients discontinued treatment. Two of the three patients had perianal relapse after discontinuation.

Among the group of patients with pathological MRI, 16 received infliximab and 16 were on adalimumab. Among the 16 patients on adalimumab, 12 were switched due to infliximab failure. Fourteen patients (44%) were optimized compared with 3 (13%) in the deep remission group (p < 0.05), (Table 3). Sixteen patients (50%) with pathological MRI had perianal surgery compared with 2 (12.5%) in the deep remission group (p < 0.05), (Table 3).

Hospitalizations

The mean number of hospital stays was 0.53±0.35 in the deep remission group versus 2.6± 0.5 in the pathological MRI group (p < 0.05). The mean duration of cumulative hospital stays was 0.75±0.52 days in the deep remission group versus 19.7±7.4 days in the pathological MRI group (p < 0.05), (Figure 4).

Discussion

In this study, 48 patients with perianal CD on anti-TNF-α treatment were followed for a median of 62 months. Flare-free and perianal event-free survival were statistically longer in patients with deep remission compared to patients with pathological MRI. Patients with pathological MRI needed four times more perianal surgeries than patients with deep remission. Deep remission also positively impacted the need for treatment changes and hospitalization.

One third of patients were in deep remission after at least twelve months of anti-TNFα therapy. This rate is comparable to other studies [START_REF] Tougeron | Predicting factors of fistula healing and clinical remission after infliximab-based combined therapy for perianal fistulizing Crohn's disease[END_REF][START_REF] Ng | Prospective evaluation of anti-tumor necrosis factor therapy guided by magnetic resonance imaging for Crohn's perineal fistulas[END_REF][START_REF] Bell | Response of fistulating Crohn's disease to infliximab treatment assessed by magnetic resonance imaging[END_REF]. Ten among the 32 patients with pathological MRI were in clinical remission. This clinical-radiography discrepancy is consistent with previous studies. Nevertheless, other studies defined MRI healing without considering contrast enhancement and reported only the disappearance of T2 hyperintensity on treatment [START_REF] Van Assche | Magnetic resonance imaging of the effects of infliximab on perianal fistulizing Crohn's disease[END_REF][START_REF] Ng | Prospective evaluation of anti-tumor necrosis factor therapy guided by magnetic resonance imaging for Crohn's perineal fistulas[END_REF][START_REF] Bell | Response of fistulating Crohn's disease to infliximab treatment assessed by magnetic resonance imaging[END_REF][START_REF] Karmiris | Long-term monitoring of infliximab therapy for perianal fistulizing Crohn's disease by using magnetic resonance imaging[END_REF]. In previous studies from our center, we reported deep remission in nearly one third of patients and one third of patients in clinical remission had persisting active fistula tract on MRI [START_REF] Savoye-Collet | Fistulizing perianal Crohn's disease : contrast-enhanced magnetic resonance imaging assessment at 1 year on maintenance anti-TNF alpha therapy[END_REF][START_REF] Thomassin | MRI may predict deep remission in patients with perianal fistulizing Crohn's disease[END_REF]. We described MRI healing as disappearance of T2 hyperintensity and absence of contrast enhancement [START_REF] Savoye-Collet | Fistulizing perianal Crohn's disease : contrast-enhanced magnetic resonance imaging assessment at 1 year on maintenance anti-TNF alpha therapy[END_REF][START_REF] Thomassin | MRI may predict deep remission in patients with perianal fistulizing Crohn's disease[END_REF]. Indeed, contrast enhancement allows a distinction between inflammatory or fibrotic lesions and disappearance of contrast enhancement is related to clinical activity [START_REF] Savoye-Collet | Fistulizing perianal Crohn's disease : contrast-enhanced magnetic resonance imaging assessment at 1 year on maintenance anti-TNF alpha therapy[END_REF][START_REF] Thomassin | MRI may predict deep remission in patients with perianal fistulizing Crohn's disease[END_REF][START_REF] Rimola | Characterization of inflammation and fibrosis in Crohn's disease lesions by magnetic resonance imaging[END_REF][START_REF] Savoye | How deep is remission in perianal Crohn's disease and do imaging modalities matter?[END_REF][START_REF] Horsthuis | Perianal Crohn disease: evaluation of dynamic contrast enhanced MR imaging as an indicator of disease activity[END_REF]. Van Assche score appeared insufficient in the setting of anti-TNF-α evaluation; most of the items are linked to anatomical description and to complications of fistula tracks. As a consequence, we decided not to include the Van Assche score as part of our definition of MRI-based deep remission. In this study we did not use magnetization transfer or diffusion to monitor sub-clinical levels of perianal fistulas that have been recently reported as able to help lesion monitoring [START_REF] Dohan | Diffusion-weighted MRI in Crohn's disease: Current status and recommendations[END_REF][START_REF] Pinson | Magnetization transfer ratio for the assessment of perianal fistula activity in Crohn's disease[END_REF].

In this study, patients having achieved deep remission had better prognosis in terms of relapse and hospitalizations. Nearly half of the patients with pathological MRI had treatment optimization versus only 13% of patients in deep remission. This is one of the first studies to evaluate the long-term outcomes of patients with perianal CD according to their MRI and clinical evaluation after at least one year of treatment for perianal CD.

In luminal CD, deep remission has proved its interest [START_REF] De Chambrun | Current evidence supporting mucosal healing and deep remission as important treatment goals for inflammatory bowel disease[END_REF]. There are studies that have specifically investigated the long-term outcomes of patients who achieved deep remission in luminal CD. In the EXTENT study, a double-blind placebo controlled trial of adalimumab for the induction and maintenance of mucosal healing in ileocolonic CD, deep remission was assessed at week 12 in eleven patients [START_REF] Colombel | Adalimumab induces deep remission in patients with Crohn's disease[END_REF]. No patient in deep remission underwent surgery or hospitalization. Nine patients in the non-deep remission group were hospitalized and three patients required surgeries. A subsequent analysis of the ACCENT-1 study, suggested that mucosal healing and deep remission increased the duration of clinical remission and the time to clinical relapse [START_REF] Haens | Endoscopic healing after infliximab treatment for Crohn's disease provides a longer time to relapse[END_REF]. The STORI trial, a prospective multicenter study investigated infliximab withdrawal with continuation of azathioprine in patients with luminal CD in clinical and biological remission. Deep remission was associated with a reduced risk of relapse when infliximab was stopped [START_REF] Louis | Maintenance of remission among patients with Crohn's disease on antimetabolite therapy after infliximab therapy is stopped[END_REF].

In perianal CD, Ng et al described deep remission in 30% of 26 patients by 6 months.

No relapse was observed at 12 months and 18 months, and 2 patients stopped their treatment at 6 months without any relapse (17). Tozer et al followed 41 patients for 3 years. Fifteen of these 41 patients assessed were in deep remission by 12 months [START_REF] Tozer | Long-term MRI-guided combined anti-TNF-α and thiopurine therapy for Crohn's perianal fistulas[END_REF]. Seven of the deep remission group relapsed. Relapses were observed in cases of treatment reduction or discontinuation. In our study, treatment reduction (dose reduction or immunosuppressant withdrawal) was performed in 29% of patients with deep remission. No relapse was observed.

Nevertheless, among three patients in deep remission who benefited from a therapeutic break, two patients relapsed. Therefore, drug de-escalation could be discussed in deep remission patients, with regular MRI to detect sub-clinical relapses. However, therapy discontinuation can not be recommended at this present time [START_REF] Legué | Outcomes of Perianal Fistulising Crohn's Disease Following Anti-TNFα Treatment Discontinuation[END_REF]. Moreover, recent data suggest that monitoring drugs through levels could be useful to optimize anti-TNF-α therapy. Yarur et al showed a significant association between serum infliximab levels and rates of fistula healing [START_REF] Yarur | Higher infliximab trough levels are associated with perianal fistula healing in patients with Crohn's disease[END_REF].

Despite its strengths, our study has some limitations related to its retrospective design and small cohort size. Furthermore, we did not evaluate perianal activity using standardized scores as the perianal disease activity index (PDAI). Trough levels were not available at the time of MRI in patients in deep remission and could not be analyzed in our cohort. Moreover, due to the retrospective design of the study cohort the impact of perianal fistulas on quality of life were not evaluated. But others have suggested that disease control has a strong impact on quality of life and patient-related outcomes [START_REF] Leong | Prospective validation study of the International Classification of Functioning, Disability and Health score in Crohn's disease and ulcerative colitis[END_REF]. The recently developed Disease Disability Index (IBD-DI) may also help to evaluate the disease burden in these challenging clinical situations [START_REF] Gower-Rousseau | Validation of the Inflammatory Bowel Disease Disability Index in a population-based cohort[END_REF].

In conclusion, our findings show that perianal deep remission based on MRI, is associated with better long-term outcomes of disease. Achieving deep remission seems to be a relevant target in the management of patients with fistulizing perianal CD. The timing of MR evaluation needs to be addressed and the next step will be to implement MR monitoring of fistulizing CD in our daily practice. We believe that MRI-based decisions could be helpful to adjust therapy in a treat-to-target approach. However, it seems dangerous to recommend deescalation therapy based on MRI findings without prospective evidence to support this practice.

Figure legends

(

  TR = 6000 ms, TE = 500 ms, scan time = 5 min, matrix of 312 × 512, field of view 250 mm) and T2-weighted 2D TSE sequences with spectral presaturation inversion recovery (SPIR) (TR = 2000 ms, TE = 50 ms, scan time = 5 min, matrix of 312 × 512, field of view 250 mm) were obtained. T1-weighted 2D TSE sequences with and without fat suppression with SPIR (TR = 500 ms, TE = 10 ms, scan time = 5 min, matrix of 285 × 384, field of view = 250 mm) sequences were performed after gadolinium enhancement after checking for normal renal function. Coronal and axial planes were oriented using the long axis of the anal canal. MRI evaluations were assessed by experienced gastrointestinal radiologists by retrieving images from the Institutional Picture Archiving and Communicating System. Each item was evaluated according to Van Assche score

Fig. 1 -Fig. 2 :Fig. 3 -Fig. 4 -

 1234 Fig.1-Comparison of initial (a) and follow-up (b) MRI of a patient with Crohn's disease on

Table 1 : Patients's characteristics -n (%)

 1

	Pathological MRI	Deep Remission	Patients
	(n = 32)	(n= 16)	(n = 48)

Acknowledgments

The authors are grateful to Nikki Sabourin-Gibbs, Rouen University Hospital, for her help in editing the manuscript.