Franconi Florence

Olivier Lefranc

Amandine Radlovic

Laurent Lemaire
email: laurent.lemaire@univ-angers.fr

; Lemaire

Can magnetisation transfer Magnetic Resonance Imaging help for the follow-up of synthetic hernia composite meshes fate? A pilot study

Keywords:

Purpose:

This study aims at evaluating the non-invasive Magnetic Resonance Imaging (MRI) technic to visualize a synthetic composite hernia mesh using a rodent model and to document the integration of this device over 4 months.

Methods:

Uncoated polyethylene terephthalate mesh and synthetic composite mesh-faced on the visceral side with a chemically engineered layer of copolymer of glycolide, caprolactone, trimethylene carbonate, and lactide to minimize tissue attachment-were placed intraperitoneally in rats, facing the caecum previously scraped to promote petechial bleeding and subsequent adhesions. Meshes fate follow-up was performed 4, 10, and 16-weeks post-implantation using a rodent dedicated high field MRI. Magnetization transfer (MT) images were acquired, associated with pneumoperitonealMRI performed after intraperitoneal injection of 8 mL gas to induce mechanical stress on the abdominal wall.

Results:

Uncoated meshes were clearly visible using both T2-weighted and MT imaging during the whole study while composite meshes conspicuity was not so evident on T2-weighted MRI and could be improved using MT imaging. Adhesions and collagen infiltration were massive for the uncoated meshes as expected. On the contrary, composite meshes showed very limited adhesion, and, if any, occurring at the edge of the mesh, starting at the fixation points.

Conclusions:

Magnetization transfer imaging allows to detect mesh integration and, associated with pneumoperitoneum, was able to probe the effective minimizing effect of the synthetic polymeric barrier on visceral attachments. However, magnetization transfer imaging could not unambiguously allow the visualization of the mesh through the polymeric barrier.

Introduction

Hernia textile-based meshes are intended for the reinforcement of abdominal wall soft tissue where weakness exists. Intra-abdominal meshes are frequently used for ventral hernia repair (VHR) and are associated with a low hernia recurrence rate when compared to simple surgical suturing of the abdominal wall hernia defects [START_REF] Cevasco | Ventral Hernia Repair with Synthetic, Composite, and Biologic Mesh: Characteristics, Indications, and Infection Profile[END_REF]. Intra-abdominal hernia mesh key characteristics include excellent biocompatibility so it integrates well into the abdominal wall tissue, and low to lack of adhesiogenicity minimizes abdominal adhesion to the mesh [START_REF] Cevasco | Ventral Hernia Repair with Synthetic, Composite, and Biologic Mesh: Characteristics, Indications, and Infection Profile[END_REF]. One strategy to decrease the adhesiogenicity of textile-based intra-abdominal meshes is to coat one of the surfaces of the mesh with a minimizing tissue attachment barrier that decreases the risk of abdominal adhesion to form. This type of mesh is called composite mesh. Despite the usage of composite meshes with optimal characteristics post-operative complications may still occur due to both the direct contact between the mesh and the abdominal viscera and the inflammatory reaction that occur as a consequence of the implantation of the mesh in living tissue and of the resulting wound healing process. One of the leading complications associated with the use of intra-abdominal meshes is the development of abdominal adhesions [START_REF] Lefranc | Reinforcement Materials in Soft Tissue Repair: Key Parameters Controlling Tolerance and Performance -Current and Future Trends in Mesh Development[END_REF]. There is therefore a critical need for non-invasive medical imaging tools and techniques to assess and document postimplantation mesh evolution and to better diagnose complications such as intra-abdominal adhesion. Computed tomography and ultrasonography are the commonly used non-invasive medical imaging technique used to clinically postoperatively monitor VHR patients [START_REF] Patil | Mind the gap: imaging spectrum of abdominal ventral hernia repair complications[END_REF]. These methods remain with limited performances when it comes to detect thin polymeric materials and abdominal visceral adhesions. Mesh visualization using Magnetic Resonance Imaging (MRI) technique has been achieved after contrast agent incorporation into meshes to make them MRI-visible [START_REF] Ciritsis | Time-Dependent Changes of Magnetic Resonance Imaging-Visible Mesh Implants in Patients[END_REF][5][START_REF] Blanquer | New magnetic-resonance-imagingvisible poly(epsilon-caprolactone)-based polyester for biomedical applications[END_REF][START_REF] Krämer | A Concept for Magnetic Resonance Visualization of Surgical Textile Implants[END_REF][8][START_REF] Schulz | UVtriggered photoinsertion of contrast agent onto polymer surfaces for in vivo MRI-visible medical devices[END_REF]. A recent preclinical study showed that a collagen coated mesh can be imaged over time using Amide Proton transfer MRI [START_REF] Franconi | Early postsurgical visualization of composite mesh used in ventral hernia repair by amide proton transfer MRI[END_REF]. Regarding abdominal adhesions to the mesh, Cine-MRI in humans [START_REF] Langbach | Adhesions to Mesh after Ventral Hernia Mesh Repair Are Detected by MRI but Are Not a Cause of Long Term Chronic Abdominal Pain[END_REF] and in experimental rodents using pneumoperitoneal-MRI [START_REF] Franconi | Imaging visceral adhesion to polymeric mesh using pneumoperitoneal-MRI in an experimental rat model[END_REF] was proven useful to document this side effect. Despite the large range of offered contrasts, the high spatial resolution and the absence of radiation exposure, MRI usually does not allow to easily image the meshes and remains an emerging technique for the exploration of VHR complications.

The aim of this study was therefore to evaluate the integration and the minimization of visceral attachments of an intra-abdominal synthetic hernia mesh, using long-term non-invasive MRI longitudinal follow-up. The Behaviour of a macroporous polypropylene mesh, covered on one side with a barrier layer of copolymer of glycolide, caprolactone, trimethylene carbonate, and lactide, was explored in a rodent caecal abrasion model [START_REF] Franconi | Imaging visceral adhesion to polymeric mesh using pneumoperitoneal-MRI in an experimental rat model[END_REF][START_REF] Harris | Analysis of the kinetics of peritoneal adhesion formation in the rat and evaluation of potential antiadhesive agents[END_REF] and compared to the fate of a bare polyethylene terephthalate hernia mesh.

Materials and methods:

In vivo mesh implantation

The study was performed following the regulations of the French Ministry of Agriculture and the protocol was approved by the Committee for the Ethics of Animal Experiments of the 'Pays de la Loire' (Permit no. APAFIS#1733-2015083114252179) in accordance with the EU Directive 2010/63/EU. Two types of meshes were used in this study. Each type of mesh was implanted in Sprague-Dawley rats (240-320g, Angers University/Hospital animal Facility, Angers, France) (n=6 per type of mesh). The first type of mesh was a synthetic composite mesh composed of a non-resorbable macroporous polypropylene textile coated on one side with an absorbable synthetic resorbable barrier (copolymer glycolic acid/lactic acid/trimethylene carbonate/caprolactone) for minimizing tissue attachment to the mesh [START_REF] Medtronic | Parietene DS Composite (TM) mesh[END_REF]. The second type of mesh was a polyethylene terephthalate (PET) multifilament textile without barrier. The PET was used as a positive control for the development of soft tissue visceral attachment [START_REF] Franconi | Imaging visceral adhesion to polymeric mesh using pneumoperitoneal-MRI in an experimental rat model[END_REF]15] to the mesh. Tissue attachments was induced , using a caecal abrasion and a peritoneal defect model previously described [START_REF] Franconi | Imaging visceral adhesion to polymeric mesh using pneumoperitoneal-MRI in an experimental rat model[END_REF][START_REF] Harris | Analysis of the kinetics of peritoneal adhesion formation in the rat and evaluation of potential antiadhesive agents[END_REF]. Briefly, fifteen minutes before the surgery, rats were injected intramuscularly with 10µg/kg Buprenorphine (Vetergesic, Sogeval, France) for perioperative pain relief. Under isoflurane anaesthesia (Isoflurane Belamont, France, 5-2%, O2: 0.8L/min) a midline incision of the skin and abdominal wall, the caecum was externalized and abraded using gauzes and a scalpel. The caecum was then allowed to air dry and during this period, a surgical defect was created by removing the peritoneum and some muscle fibers of the abdominal wall facing the cecum. The tested meshes (25mm X 15mm) were sutured to the abdominal wall over the peritoneal defect with a non-absorbable 6/0 suture with one stitch at each corner of the article. The composite mesh was placed with the minimizing tissue attachment barrier facing the abraded cecum. The linea alba was sutured with a non-absorbable suture and the cutaneous layer was closed using a running stitch of an absorbable suture. A dressing was applied to the abdomen. The rats were allowed to recover in individual cages for 24h, before being housed in pair to respect their social behaviour.

In vivo Magnetic Resonance Imaging.

MRI examination of the abdominal cavity was performed with a 7T scanner (Biospec 70/20 Avance III, Bruker, Wissembourg, France) equipped with a BGA12S gradient system (675mT/m) and a 72-mm proton volume resonator for radiofrequency excitation and signal reception. All imaging acquisitions were performed using ParaVision ® 6.0.1, under isoflurane anesthesia (5-2%, O2: 0.8L/min). Animal body temperature was maintained throughout the experiment by hot water circulation in the animal bed. Respiration was monitored using a small animal monitoring and gating system, model 1025, SA instrument, NY, USA. MRI visualization of the mesh was performed at 4, 10 and 16 weeks after implantation. The latest time point was chosen according to preliminary studies that have shown that the polymeric synthetic barrier resorbs within 16 weeks postimplantation [START_REF] Medtronic | Parietene DS Composite (TM) mesh[END_REF]. The 10-week time point was supposed to offer an intermediate situation whereas at 4-weeks, the polymeric film should still be in place and the remodelling/inflammation associated the mesh integration should not jeopardize the image quality. For the follow-up, all animals were scanned at weeks 4 and 10. At week-10, three animals of each group (composite mesh or PET implanted groups) were also scanned by pneumoperitoneal-MRI to visualize possible tissue adhesions before being euthanized for autopsy. The remaining 6 animals (composite mesh n=3 and PET n=3) were imaged 16 weeks after implantation to explore mesh visualization and possible visceral attachments before being euthanized for autopsy.

Mesh visualization and pneumoperitoneal-MRI by T2 weighted (T2-W)

After a set of scout images, respiration gated T2 weighted fast spin-echo images (TE/TR = 23/920ms, echo train length= 8, slice thickness = 1 mm, field of view of 55mm, matrix 256², in plane-resolution = 0.215 mm, 8 averages; with an active fat saturation using a frequency selective 90° pulse, Gaussian, 2.61ms, BW 1050 Hz, applied to a frequency offset of 3.5ppm relative to water, with gradient spoiler to suppress transverse magnetization produced by the pulse) with up to 12 slices were acquired for mesh visualization in the axial plane. This same sequence was also used to visualize tissue adhesions by pneumoperitoneal-MRI. In this case, an abdominal wall displacement was induced before images acquisition. A 5-mm skin incision was performed, under isoflurane anaesthesia, on the right side of the rat abdomen. A 2-mm outer diameter trocar was used to pass through the muscular layer, allowing the introduction of a 1-mm diameter polypropylene catheter, which was then used for the injection of 8 mL of 0.22μm filtered air within the rat abdomen to induce abdominal distension [START_REF] Franconi | Imaging visceral adhesion to polymeric mesh using pneumoperitoneal-MRI in an experimental rat model[END_REF].

Mesh visualization by Magnetization Transfer (MT) MRI

Magnetization transfer images were obtained with a gradient echo FLASH sequence (TE/TR = 2.4/33ms, flip angle 10°, one slice of 1mm thickness, field of view of 55mm, matrix 139x172 reconstructed to 256², in plane-resolution = 0.215 mm, 40 averages and fat saturation active) with or without saturation. The saturation was obtained using a Gaussian radiofrequency pulse of 20ms with a peak B1 amplitude of 25µT at an offset frequency of 2000Hz. Magnetization transfer ratio (MTR) was measured on MTR calculated images according to the following formula 100*(MToff-MTon)/MToff. MTR at the mesh level was measured from regions of interest drawn on the T2 anatomical images, pasted on the MTR images after thresholding. Thresholding was performed by eliminating the populations of low or high MTR pixels whose occurrence was less than 20% of the population of the most represented pixels. This thresholding allowed to remove obviously inaccurately calculated MTR leading to a reduction of the surface area of about 20%.

Histological analysis

At 16-week time point, macroscopical observations of the meshes were performed before careful harvesting and fixation in formalin solution for further histological preparation and analysis.

For slide preparation, the samples were dried, dehydrated, and embedded in paraffin. Transversal microtome cutting (4µm slide thickness), on the medial and distal areas of the samples was performed. For each slide, 2 samples were prepared and stained by Haematoxylin-Eosin (HE) and Masson Trichrome (TM).

The slides were then scanned using an Aperio Scanscope CS2, Leica. Collagen content surrounding the meshes were evaluated using ImageJ. Cellular identification was performed according to cell morphology.

Statistics

This study is mainly descriptive. No statistics are carried out on the MTR data points as they are numerously too limited. The statistical power associated with the different tests is too low in this context to consider implementing them in a relevant and adequate way.

Results:

After 4 weeks, bare PET meshes were clearly visible during MRI examination on all rats, appearing as a heterogeneous line on T2 weighted images, up to 1.5mm thick, (figures 1 a and 2 a). For all the animals, the mesh was located right over the cecum. The PET mesh contrast on the T2 weighted images stayed stable for the 16 weeks study period in all the animals (figure 2 a, e, i). PET meshes were clearly visible on MTR images throughout the entire experimental procedure (figure 2 b, f & j). The mean MTR for the PET mesh (n=6) is 757 at 4 weeks, 754 at 10 weeks & 741 at 16 weeks (figure 3). The synthetic composite meshes detection was not

d, f, h, j, l) and corresponding T2-weighted images (a, c, e, g, I, k) of PET (pointed by a red arrow) and composite mesh (pointed by a green arrow) meshes at 4 (a-d), 10 (e-h) and 16 (i-l) weeks after implantation. The caecum is pointed by the yellow mark.

For better contrast, magnetization transfer images were acquired. Four weeks after implantation, the composite meshes appear as a thin line with an MTR ratio of 753 whereas the abdominal wall was characterized by an MTR ratio of 834 (figure 3).

Figure 3: Evaluation of MTR evolution with mesh integration. Red circles ( ) correspond to MTR values measured for the bare 3D PET mesh and blue squares ( ) to values measured for the composite mesh. The dashed box corrresponds to the the normal values in abdominal wall.

At later experimental points, the MTR ratio at the mesh level was equivalent to the MTR ratio within the abdominal wall (figure 3) suggesting an efficient integration of the mesh without massive remodeling (figure 2 h,l). This observation has to be considered in the light of the histological analysis showing the thin layer of collagen surrounding the composite meshes as opposed to the substantial collagen deposit onto the thicker PET mesh (figure 4A). A detailed analysis of the histological slices 16 weeks post-implantation reveals that in the composite mesh vicinity (figure 4B, collagen production is limited and represents, in surface, 25-30% of the implant. Giant cells, illustrating the reaction to foreign body are numerously limited (figure 4D). Regarding the bare PET mesh, the collagen deposit is more important as it represents 40-50% of the implant surface. Giant cells are also limited in numbers (figure 4C). Alongside with mesh detection, visceral attachments to the meshes at 10 weeks and 16 weeks post-implantation, using pneumoperitoneal-MRI before necropsy was assessed. This technique consists in the injection of gas within the abdomen of the rats in order to displace the abdominal wall at distance from the visceral tissue. As the meshes are attached to the abdominal wall when implanted, if subsequent adherences occur, any displacement of the wall will pull the visceral tissue and reveal the attachment. Ten weeks (figure 5a) or 16 weeks after implantation (figure 5c), massive adhesions are observed between the bare PET meshes and the caecum, the latest remaining in contact with the mesh despite the gas injection. This close contact was confirmed at necropsy (figure 6), as expected for a positive control. On the contrary, none of rats implanted with the synthetic composite meshes presented massive caecal adhesion or visceral attachment, the injected air being freely distributed between the guts and the mesh. Only focal adhesions at fixation point (suture) or to conjunctive tissue (figures 5b and 6b) were observed.

Discussion:

MRI is not the common method in clinical practice to monitor mesh outcome even though this method is reported in some preclinical or clinical researches [START_REF] Franconi | Early postsurgical visualization of composite mesh used in ventral hernia repair by amide proton transfer MRI[END_REF][START_REF] Franconi | Imaging visceral adhesion to polymeric mesh using pneumoperitoneal-MRI in an experimental rat model[END_REF][START_REF] Lambertz | MRI Evaluation of an Elastic TPU Mesh under Pneumoperitoneum in IPOM Position in a Porcine Model[END_REF]17] and present the major advantage to provide the ability for long term follow up of the same individual in a non-invasive manner.

The major limitation of MRI to image intraabdominal meshes is linked to the thinness of the polymeric mesh filaments and their short signal relaxation times T2 that make them appear usually as dark fine lines or spots on the T2-weighted MR images. This characteristic was even reinforced with the association of iron oxide nanoparticles within the filament [17][START_REF] Awada | Controlled Anchoring of Iron Oxide Nanoparticles on Polymeric Nanofibers: Easy Access to Core@Shell Organic-Inorganic Nanocomposites for Magneto-Scaffolds[END_REF][START_REF] Sindhwani | Short term post-operative morphing of sacrocolpopexy mesh measured by magnetic resonance imaging[END_REF]. However, this type of contrast enhancement can also be jeopardized by the normal post-surgical evolutions or complications such as seroma, local inflammation, or foreign body reaction [START_REF] Patil | Mind the gap: imaging spectrum of abdominal ventral hernia repair complications[END_REF]. Besides, the abdominal environment is complex, with peristaltic or respiratory displacement that can impair the image quality when long acquisition time is required, even though the latter can be easily compensated with a respiration-triggered acquisition. So, depending on the adjacent structures signal (moderate intensity for abdominal wall or fat, no signal in the caecum), the mesh can be difficult to distinguish from the background. The emergence of barrier coated meshes, covered on one face with biological material such as hyaluronic acid/carboxymethyl cellulose (Sepramesh TM ) or collagen (Parietex TM ) to minimize visceral attachments once implanted into the peritoneal cavity could also be of interest from an MRI detectability point of view. Indeed for collagen coated, the presence of amide function can be used to create a contrast using amide proton transfer [START_REF] Franconi | Early postsurgical visualization of composite mesh used in ventral hernia repair by amide proton transfer MRI[END_REF]. Nowadays, attention is also given to fully synthetic meshes with chemically engineered coating to minimize attachment. In the present study, we are exploring the MRI ability to document the fate of a synthetic composite mesh after implantation in a Rodeheaver type hernia model [START_REF] Franconi | Imaging visceral adhesion to polymeric mesh using pneumoperitoneal-MRI in an experimental rat model[END_REF][START_REF] Harris | Analysis of the kinetics of peritoneal adhesion formation in the rat and evaluation of potential antiadhesive agents[END_REF] as compared to a non-coated nonresorbable polyethylene terephatlate (PET) mesh used as a positive control for tissue attachment [START_REF] Franconi | Imaging visceral adhesion to polymeric mesh using pneumoperitoneal-MRI in an experimental rat model[END_REF]. The studied composite mesh composed was made of non-absorbable polypropylene monofilament knitted textile, to favor tissue integration, and faced on one side with the absorbable synthetic copolymer of glycolide, caprolactone, trimethylene carbonate and lactide to minimize tissue attachment was proposed. As the polymeric synthetic barrier is to be resorbed within 105 days [START_REF] Medtronic | Parietene DS Composite (TM) mesh[END_REF], the follow up was planned for 4 months. In all animals enrolled in the present study and at each time points, whatever the mesh type, the prostheses were localised using standard T2weighted MRI (figure 2). The wider appearance of the thicker PET mesh compared to the composite mesh was mainly due to a higher deposit of collagen (figure 4). As expected, PET meshes bearing rats' present massive attachment of the caecum membrane in contact with the mesh, as suggested by the 'wavy shape' and confirm on pneumoperitoneum MRI (Figure5) or at necropsy (figure6). This phenomenon was observed in all six rats. It should be noted that the localisation by the massive rat caecum, appearing as a hyposignal, greatly facilitate the mesh delineation. On the contrary, for the composite mesh the depiction can only be gained due to the high image resolution that can be achieved with the dedicated rodents MRI (figure 2). Moreover, the presence of the adherence limiting coating led to minimal adhesions between the caecum and the mesh as seen on the pneumoperitoneum MRI (figure 5) and at necropsy (figure 6), which does not allow to benefit from intrinsic contrast brought by the ceacum. The originality of this work stands on the evaluation of magnetization transfer MRI (MT-MRI) to depict mesh through the adherence limiting polymeric face and characterize the integration and remodelling associated with the implantation. MT-MRI is based on the magnetization exchange between two proton pools, the macromolecular bound water protons and the unbound water ones [START_REF] Chen | Threedimensional ultrashort echo time cones (3D UTE-Cones) magnetic resonance imaging of entheses and tendons[END_REF][START_REF] Henkelman | Magnetization transfer in MRI: a review[END_REF]. We have hypothesized that the presence of the polymeric resorbable barrier should be hydrated and therefore could constitute the bound water protons pool and could therefore allow the visualization of this barrier, at least at the early time point. However, and as shown in figure 3 The MTR measured at the coated or uncoated mesh level 4 and 10 weeks post-implantation are similar and reduced compared to normal muscle, suggesting that the contrast is more driven by the inflammation induced by the implantation rather than to the polymeric film. The persistence of the composite mesh contrast observed on the MT images at 16 weeks is not consistent with mesh lifespan as it has been established that the barrier is resorbed within 105 days [START_REF] Medtronic | Parietene DS Composite (TM) mesh[END_REF] and the change may reflect the natural evolution from an inflammatory situation to an integrative situation with deposit of a thin layer of neo-collagen [START_REF] Laurent | Quantitative and qualitative assessment of articular cartilage in the goat knee with magnetization transfer imaging[END_REF] as observed on the trichrome staining. Consequently, depiction of the adhesion limiting polymeric mesh layer could not be achieved using MT imaging. Nevertheless, and according to the MTR evolution with time, MT imaging might be a useful tool to evaluate the integration of the mesh and collagen deposit associated with the implant. The normalization of the MTR at the composite mesh level reflecting the efficient integration whereas the remaining low MTR value at the control bare-PET mesh reflecting the thick collagen deposit within the multifilament's structure.

CONCLUSION:

A durable ventral hernia repair success is based on a good abdominal wall integration linked to fibrin deposition on mesh knitted pattern induced by a moderate inflammatory response and fibroblast influx. However, this inflammatory response has the potential to induce large abdominal adhesions and be source of pain. The barrier layer of the composite mesh is there to minimize the tissue attachment to the meshes. Non-invasive follow up of meshes to document mesh tissue integration remains a challenge, especially for meshes without contrast agent incorporation.

Magnetization transfer magnetic resonance imaging, a technique based on the dynamic exchange of water molecules between hydrated macromolecules and the pool of unbounded water molecules appears as a promising technique as shown in the present pre-clinical study and, associated with pneumoperitoneal-MRI, allows the unambiguous evaluation of the barrier effect on tissue adhesion, allows the evaluation of the integration, but does not unambiguously allow the depiction of the mesh through the thin synthetic layer.

Figure 1 :

 1 Figure 1 : Meshes implantation: meshes were fixated by four stitches to the abdominal wall (blue mark) facing the caecum (yellow mark). Axial T2-weighted images show the meshes localisation 4 weeks after implantation of PET (a) pointed by a red arrow and of composite mesh (b) by a green one. evident on T2-weighted images. The meshes appeared as a thin line of up to 0.5mm of thickness (figures 1 b and 2 c). Ten and sixteen weeks after implantation, the visualization remained not obvious (figure 2 g, k).

Figure 2 :

 2 Figure 2: Representative axial magnetization transfer (b, d, f, h, j, l) and corresponding T2-weighted images (a, c, e, g, I, k) of PET (pointed by a red arrow) and composite mesh (pointed by a green arrow) meshes at 4 (a-d), 10 (e-h) and 16 (i-l) weeks after implantation. The caecum is pointed by the yellow mark.

Figure 4 :

 4 Figure 4 : Representative histological slice of bare 3D PET (frames A-C) and composite (frames B-D) meshes 16 weeks post implantation. Frames A & B are Masson trichrome staining and frames C & D are enlargement of H&E staining. Yellow arrows point at giant cells and stars at yarns.

Figure 5 :

 5 Figure 5 : Representative pneumoperitoneal-MRI of PET (a, c), pointed by a red arrow, and composite mesh (b, d), pointed by a green arrow, acquired 10 (a-b) and 16 (c-d) weeks after implantation are presented. The caecum is pointed by the yellow mark and the injected air by the pink one.

Figure 6 :

 6 Figure 6 : Representative necropsy of PET (a), pointed by a red arrow, and composite mesh (b) , pointed by a green arrow, meshes 16 weeks after implantation. The caecum is pointed by the yellow mark and the abdominal wall by the blue one.

Conflict of interest disclosure:

O. Lefranc

Authors' contribution:

The authors confirm contribution to the paper as follows: Study design and conception: O. Lefranc


Christophe Coquelet

Esther Neyrolles

Alain Valtz

Antonin Chapoy

On the Phase Behaviour of the CO2 + N2O4 system at low temperatures

Keywords: Bubble pressure, PVT, Thermodynamic Modelling, Carbon dioxide, Nitrogen Oxides

Carbon dioxide capture transportation and storage is one of the technologies that can be employed to reduce CO 2 emissions from power plants. Unfortunately, in the post combustion capture process, CO 2 is not pure and contains impurities like SO 2 , NO x , N 2 , O 2 and Ar for example. In this paper, Vapour-Liquid-Equilibrium (VLE) of a binary system composed of CO 2 and N 2 O 4 /NO 2 have been investigated. The equipment used is based on the "staticsynthetic" method with a variable cell to determine the bubble pressure or saturated pressure of the system. The setup was used to obtain bubble point data at four isotherms (253.43, 273.43, 293.43, 303.43) K and pressures up to 7.3 MPa. The accuracies of the measured temperature and pressure were estimated to be 0.03 K and 0.12 kPa, respectively. The Peng-Robinson equation of state (PR78 EoS) is used to represent the isothermal P, x data.

Introduction

To reduce greenhouse gas emissions in the atmosphere, carbon dioxide emissions must decrease as they are mainly responsible for global warming [START_REF] Pachauri | Changements climatiques 2014: rapport de synthèse : 1 contribution des Groupes de travail I, II et III au cinquième Rapport d'évaluation du 2 Groupe d'experts intergouvernemental sur l'évolution du climat[END_REF]. Most of the CO 2 emissions come from the sectors producing electricity and heat [2]. One of the solutions envisaged to cut emissions is Carbon Capture Utilization and/or Storage (CCUS). It consists of the capture of CO 2 fumes that come out of the combustion plant before their emissions to the atmosphere and to store them underground or reuse them. One of the issues with CCUS is the presence of impurities in the CO 2 fumes, like nitrogen, oxygen, water, sulphur oxides, nitrogen oxides (NO 2 , N 2 O 4 , NO), etc... To be able to treat the fumes correctly, the thermodynamical properties of the CO 2 and N 2 O 4 binary system must be known. Another application for this study is the rise of the use of CO 2 as a supercritical green solvent in the chemical industry [3].

For example, for the oxidation of a certain type of cellulose, nitrogen dioxide is used as an oxidant in supercritical CO 2 . In the context of the JIP called "Phase Behaviour and Thermophysical Properties of Acid Gases and Related Fluid Systems" started in 2012, Vapour Liquid Equilibrium of several binary systems were investigated (Coquelet et al. [4] and Chapoy et al. [5]). In the paper published by Coquelet et al. [4], it was shown that there is a lack of data concerning binary systems involving NO 2. The only available data on CO 2 and NO 2 binary system are published in the paper by Camy et al. [6]. One reason concerning the fact that there is no VLE data with NO 2 in the context of CCS is that its concentration is very low in fumes. The concentration of NO 2 in the fumes after a combustion plant are at most 400 ppm (mole) according to ISO Technical Report 27921 [7]. Another reason can be the high toxicity of nitrous dioxide. And finally, the chemical analysis of NO 2 is difficult, because nitrous dioxide is reactive. Nitrogen tetraoxide is formed by a monomer dimer association in the vapour phase from nitrogen dioxide, following the reaction . Taking into account temperature and pressure, we have examined which species is the predominant one at ambient and low temperature conditions. This reaction occurs in gas phaseand we have assumed ideal gas conditions.

Considering the expression of the chemical potential in the ideal gas condition, the equilibrium constant of this reaction can be expressed following Eq. 1 with P 0 the reference pressure (1 atm in general):

(

This equilibrium constant is also called K p relative to the partial pressures. The equilibrium constant of a reaction is related to its free reaction enthalpy following Eq. 2:

(2)

The Gibbs free energy of formation of NO 2 and N 2 O 4 and the Gibbs free energy of reaction are presented in Table 1. From the data in Table 1, the equilibrium constant K N2O4 can be correlated using Eq. 3:

(3)

The value calculated at 298 K is . Yoshino et al. [START_REF] Yoshino | High-resolution absorption cross 30 section measurements of NO2 in the UV and visible region[END_REF] One important piece of information required for our work is the dissociation fraction. In Table 2, the dissociation fraction of N 2 O 4 (x see Table 3), from Pascal and Baud [START_REF] Pascal | Traité de chimie minérale[END_REF] and Pascal [START_REF] Pascal | Azote, Phosphore, Arsenic, Industries de l'Azote[END_REF] are presented at different temperatures. The dissociation fraction of N 2 O 4 is expressed following the reaction presented in Table 3. From Table 3, we obtained Eq 4 (with P°=1 bar).

(4)

To obtain the dissociation fraction, Eq. 4 has to be solved. Considering a total pressure of 1 atm, the dissociation fraction of N 2 O 4 was calculated using our equilibrium constant and the one from Vosper. The results are presented in Table 2. An increase of pressure of the reaction

[17] works in favour of the production of N 2 O 4 , or a reduction in pressure shifts the reaction to the production of NO 2 . To quantify this affirmation, the dissociation fraction at 298 K is presented as a function of pressure in Figure 22, the data used are estimated from Eq. 4. As can be seen, the dissociation fraction of N 2 O 4 is low indicating that the majority compound remains N 2 O 4 . The experimental conditions of our study are high pressure (between 0.3 and 7 MPa) and low temperature (between 217 and 303 K). At these conditions, the ideal gas assumption is not valid and the fugacities have to be included. The dissociation constant can be calculated as:

with  the fugacity coefficient (lower than 1 when the pressure increase).

Consequently, in comparison to the case where ideal gas state is considered, the reaction will compensate and increase the dissociation fraction of N 2 O 4 , not so much than that pressure effect which decrease the dissociation fraction. Also, we have considered that only N 2 O 4 is present in the liquid and solid phases [18] as the density is very important and it favours the formation of dimer. The analysis previously done also indicates that it will be difficult to quantify the real composition of mixtures composed of CO 2 , N 2 O 4 and NO 2 due to the association/dissociation reaction, especially using gas chromatography.

P /MPa

Consequently, we have decided to measure the bubble pressure using a "static synthetic" method (i.e. a PVT cell /variable volume cell). The bubble pressure data are correlated with the Peng Robinson Equation of State (PR78 EoS) [START_REF] Robinson | The characterization of the heptanes and heavier 1 fractions for the GPA Peng-Robinson programs[END_REF]. For the data analysis of the vapour phase of our experimental data, we have made the approximation that the mixture NO 2 -N 2 O 4 is always under N 2 O 4 form since in our conditions the dissociation fraction seems to be low.

Also, we have estimated by variation of temperature (synthetic-isochoric method) the appearance and disappearance of the solid phase at fixed feed composition. The Solid-Liquid -Vapour (SLV) phase diagram of the binary system is estimated with the same model. Our experimental conditions are from 253 to 303 K for the bubble pressure measurements and at a minimum temperature of 217 K for the SLVE data. The pressure range goes from 0.3 to 7

MPa.

Materials and methods

Materials

The chemicals used in this study are listed in Table 4. No further purification of the chemical products was required.

Experimental set-up

The experiment equipment used was previously presented and described by Coquelet et al. [START_REF] Coquelet | Experimental Determination of 3 Thermophysical Properties of Working Fluids for ORC Applications[END_REF] and the schematic diagram of the apparatus is presented in Figure 3. At equilibrium conditions, each bubble pressure was determined between 2 and 3 times, average values are reported, along with relative standard deviation σ. Expanded uncertainties (k = 2) U(T) = 0.03 K 4 5

Results

Bubble

The experimental bubble points of the system were correlated using the Peng Robinson 6 equation of state (PR78 EoS) Eq. 5 [START_REF] Robinson | The characterization of the heptanes and heavier 1 fractions for the GPA Peng-Robinson programs[END_REF] with the Mathias-Copeman alpha function (Eq. 6) 7

[21] and the classical van der Waals mixing rules (Eq. 7). 8

    b v b b v v a b v RT P       (5) 9 With     , R C T a a  ; and i C i C i C P T R a 2 2 45724 . 0  , i C i C i P RT b 07780 . 0  10   2 3 3 2 2 1 1 1 1 1                                        C C C T T c T T c T T c T  (6) 11   i j ij j i a z z a , with   ij j i ij k a a a   1 and   i i i b z b (7) 12
The properties of each component are reported in Table 6. 13 [24] and Giauque and Kemp [START_REF] Giauque | The Entropies of Nitrogen Tetroxide and Nitrogen 16 Dioxide. The Heat Capacity from 15°K to the Boiling Point. The Heat of Vaporization 17 and Vapor Pressure. The Equilibria N 2 O 4 =2NO 2 =2NO+O 2[END_REF]. The adjusted parameters are found in Table 6 and the 3 deviations (Bias and Absolute Average Deviations AAD) are presented in Table 7. The 4 vapour pressure calculations obtained with the PR 78 EoS and the original alpha function are 5 also compared in Table 7. As seen in Figure 5, the PR78 EoS combined with the Copeman alpha function can reproduce the literature experimental vapour pressure from the 7 triple point to the critical point. 8 The experimental and modelling results for each different isothermal condition are plotted in 1 dew point calculated with the PR78 and k ij from Eq. 9. 8 9

The binary interaction parameters between carbon dioxide and Nitrogen tetraoxide were 10 adjusted directly to VLE data through a modified Simplex algorithm using an objective 11 function based on bubble pressure calculations given in Eq. 8. 12 (8)

The values of the adjusted binary interaction parameter, k ij , for each individual experimental 13 isotherm are plotted in Figure 7 and listed in Table 8. The binary interaction parameters, k ij , 14 can be correlated as a function of the temperature using the following Eq. 9. 15

Table 9 presents the AAD (Average Absolute Deviation) and Bias on bubble pressure 1 calculation, estimated from, respectively, Eqs. 10 and 11. In Table 9 using Eq. 9. 7 8

As seen from Figure 8, a fair agreement between our work and the literature data can be 9 observed. We can also note that the data of Camy et al. [6] are showing larger scattering. 10 11

Solid-Liquid-Vapour (SLV) study of the CO 2 + N 2 O 4 binary system 12

For each composition of the CO 2 + N 2 O 4 mixture studied, the temperature of appearance and 13 disappearance of a solid phase was experimentally determined. Considering the melting 14 temperatures (Table 5) of each component, considerate is assumed that solid phase is mainly 15 composed of N 2 O 4 . The temperature of the system was decreased until the solidification was 16 noticeable to the eye, as presented in Figure 9. This decrease in temperature was stepwise. 17 With this experimental apparatus, the lowest achievable temperature is around 213 K, so we 7 only determine the melting point above this temperature. The experiments were conducted for 8 three different compositions. The results are presented in Table 9. 9 Expanded uncertainties (k = 2) u(T) = 2 K, u(P) = 0.002 MPa 12 13

The chosen approach for modelling the Solid-Liquid (SL) phase diagram of the CO 2 + N 2 O 4 14 binary system, is the fugacity (f i ) equality, following Eq. 10. It is assumed that the solid 15 phases stay pure. 16 [START_REF] Yoshino | High-resolution absorption cross 30 section measurements of NO2 in the UV and visible region[END_REF] The fugacity of the solid phase can be estimated, with Eq. 11, according to Prausnitz et al. 17 6). 2

The experimental SLV measurements were calculated using PR-EoS and the binary 3 interaction parameters tuned on VLE measurements (Eq 9). The predictions of the model are 4 in fair agreement with the measured melting points. As seen in Figure 10, the PR78 EoS 5 estimations are in relatively fair agreement at 250 K, but they deviate from the experimental 6 values at lower temperatures. This mixture is predicted to present an eutectic point with a 7 molar composition in CO 2 close of 0.8 (0.68 mas fraction) with a temperature close to 208 K. 8 with k ij from Eq. 8; dotted line: calculation based on fugacity estimated from CO 2 as 13 pure solid phase with k ij from Eq. 8 14 15 Additional measurements are required to have a better characterisation of the region close to 16 the eutectic point. 17 The pressuretemperature equilibrium behaviour of the CO 2 + N 2 O 4 binary system is 5 qualitatively illustrated in Figure 11. The thermodynamic model considered in this paper did 6 not allow for a complete description of the actual behaviour of the system, i.e., the 7 dissociation degree of N 2 O 4 was not measured. In 2008, Belkadi et al. [START_REF] Belkadi | Modeling the vapor-liquid 37 equilibrium and association of nitrogen dioxide/dinitrogen tetroxide and its mixtures with 38 carbon dioxide[END_REF] have applied the 8 crossover soft-Saft model to describe the CO 2 +NO 2 /N 2 O 4 system. The authors have 9 considered NO 2 has a self-associating molecule. The authors have shown that the NO 2 is a 10 strong associating fluid and a relatively good agreement was observed with the data from 11 Camy et al. but with values of deviation increasing with the pressure and the temperature. 12 With this model, they have shown that the liquid phase is mainly composed of N 2 O 4 at low 13 temperature (T<300K). An alternative method would be to use the approach developed by 14 Anderko [33]. The author has considered the chemical theory approach where the 15 compressibility factor is the sum of two contribution (physical and chemical) 16 . The Peng Robinson Eos can be considered to calculate the physical part of the 1 compressibility factor. For the chemical part, the Mecke-Kempter assumption (Eq. 12 with P 0 2 the standard pressure 0.101 MPa, v the molar volume and K the equilibrium constant) can be 3 considered with the equilibrium constant given by Eq. 3. 4 (12) 5 Using all the new data presented in this paper, such a model can be developed. 6

Conclusion 7

The bubble pressures of the CO 2 + N 2 O 4 binary system were experimentally measured 8 between 253 and 303 K using a static-synthetic apparatus. The new data were compared to 9 literature data and good agreement was found. Binary interaction parameters k ij of PR78 EoS 10 were adjusted assuming that only nitrogen tetraoxide was present in the liquid phase, and a 11 temperature dependency of the k ij was found. The same equipment was also used to measure 12 the melting temperature of three mixtures rich in N 2 O 4 . Based on our experimental data, it 13 appears that the binary system presents a eutectic point. However, further measurements are 14 required for a final validation. 15 16

Acknowledgments

Figure 1 :

 1 Figure 1: Variation of log 10 (K N2O4 ) as a function of inverse of temperature. Dotted line: Equation 1. Symbols (values from literature: ():Srivastava and Barua [11], (×): Reference 2 in Powell and Adams [12], (): Reference 1 in Powell and Adams [12], ():Powell and Adams [12], (): Calculated values from correlation from Hurtmans et al.[START_REF] Hurtmans | Integrated band intensities in N 2 O 4 in 41 the infrared range[END_REF]).

Figure 2 :

 2 Figure 2: The dissociation fraction of N 2 O 4 as a function of the pressure at 298 K estimated from Eq. 4; red curve: minimum pressure of the study

Figure 3 :

 3 Figure 3: Flow diagram of the apparatus. DAU: Data Acquisition Unit; DDD: Digital Displacement Display; DT: Displacement Transducer; GC: Gas Cylinder; LB: Liquid Bath; LVi: Loading Valve; P: Piston; PD: Piston Monitoring; PN: Pressurized Nitrogen; PP: Platinum Probe; PT: Pressure Transducer; PV (VP): Vacuum Pump; R: Gas Reservoir; SD: Stirring Device; SB: Stirring Bar; ST: Sapphire Tube; TR: Thermal Regulator; Vi: Valve; VVCM: Variable Volume Cell.

Figure 4 :

 4 Figure 4: Picture of the cell with the CO 2 + N 2 O 4 mixture at low temperature

Figure 6 :

 6 Figure 6. 2

8 9 10 1-Figure 8 :

 108 Figure 7: k ij parameter as a function of the temperature 2 3 The only available literature data for comparison are from Camy et al. [6]. They have studied 4 this binary system using a similar experimental technique. The data are presented in Figure 8 5 and are predicted with the PR78 EoS with k ji estimated from Eq. 9. 6 Bubble point of the CO 2 + N 2 O 4 binary system as a function of the CO 2 (1) 4 mole fraction between 253 and 303 K. Experimental data from Camy et al. [6]: : 298 5 K; : 313 K; : 323 K; Dashed line: calculated from PR78 EoS with k ij calculated 6

4

  Then the temperature was increased stepwise until the N 2 O 4 solid disappeared. Those steps 1 were repeated several times until the temperatures between freezing and melting were around 2 2 K apart. 3 Figure 9: Picture of the cell with SLV of the CO 2 + N 2 O 4 mixture. 5 6

  fugacity of the pure compound, ∆H S  L : the melting enthalpy, T m : temperature of 1 the melting point (see Table

9 10 Figure 10 :

 1010 Melting temperature as a function of the CO 2 mol fraction; : experimental 11 data; dashed line: calculation based on fugacity estimated from N 2 O 4 as pure solid phase 12

1 Figure 11 :

 111 Figure 11: Pressure-temperature equilibrium behaviour for the CO 2 -N 2 O 4 system (S 1 : 2 solid CO 2 ; S 2 : solid N 2 O 4 ; VP: vapour pressure; V=L: vapour-liquid critical locus). 3 4

17

  This work was a part of the JIP project "Impact of Common Impurities on Carbon Dioxide 18 Capture, Transport and Storage" which the phase-III was conducted jointly at Heriot-Watt 19 University in Edinburgh, UK and MINES ParisTech in France in 2017-2021. The authors 20 would like to gratefully acknowledge the sponsors of the project: GALP Energia, Linde AG 21 Engineering Division, Petronas, Petrobras, Equinor, TOTAL. 22 References 23

Table 1 : Gibbs free energy of formation of NO 2 and N 2 O 4 and Gibbs free energy of reaction of association. T /K Δ f G° /J.mol -1 ΔrG°a ssociation /J.mol -1 NO 2(g) a N 2 O 4(g)

 1

	b

a Data from Chase et al. (1964a) [8] b Data from Chase et al. (1964b) [9]

Table 2 : The dissociation fraction of N 2 O 4 (x) at different temperatures T /K x x from our equilibrium constant (Eq. 4) x from Vosper equilibrium constant equation Pascal and Baud[15]

 2

	299.85	0.1995	0.2005	0.1927
	312.95	0.2923	0.3137	0.3008
	333.35	0.5284	0.543	0.5222
	353.75	0.7991	0.7605	0.7404
	373.25	0.8923	0.8893	0.8762
	394.65	0.9623	0.9545	0.9479
	413.15	1	0.9783	0.9748
			Pascal [16]
	313.15	0.2944	0.3157	0.3027
	333.15	0.5266	0.5406	0.5199
	353.15	0.7589	0.7551	0.7349
	363.15	0.9269	0.833	0.816
	373.15	0.892	0.8888	0.8757
	393.15	0.9588	0.9516	0.9447
	413.15	1	0.9783	0.9748

Table 3 : Reaction of dissociation of N 2 O 4 balance (x is the dissociation fraction) Reaction: N 2 O 4 2 NO 2

 3

	t = 0	1	0
	t	1 -x	2 x
	Partial pressure

Table 4 : Purities and suppliers of the chemicals used

 4

	Chemicals	CAS number	Purity GC a	Supplier
	CO 2	124-38-9	99.995 vol%	Messer
	N 2 O 4	10544-72-6	99.95 vol%	Air Liquide

a Gas Chromatography

pressure of the CO 2 + N 2 O 4 binary system

	Bubble point (BP) data of the CO 2 + N 2 O 4 system have been measured at 253.43, 273.43,
	293.43 and 303.43 K. The results are reported in Table 5 with the associated calculated
	uncertainties.

Table 5 : Experimental and calculated isothermal bubble pressure data for the CO 2 (1) + 1 N 2 O 4 (2) mixture system according to the mole fraction of CO 2 x 1 and their standard

 5

	2
	3				uncertainties.
		T /K	253.43	273.42		293.43		303.43
	x 1	u(x 1 )	P /MPa u(P)	P /MPa	u(P)	P /MPa	u(P)	P /MPa	u(P)
	0.254	0.004	0.726	0.005	1.095	0.005	1.608	0.003	1.916	0.006
	0.545	0.002	1.165	0.006	1.952	0.000	3.026	0.002	3.685	0.001
	0.716	0.004	1.469	0.002	2.490	0.003	3.891	0.004	4.741	0.004
	0.833	0.003	1.756	0.010	2.925	0.008	4.622	0.013	5.659	0.013
	0.942	0.005	1.872	0.004	3.274	0.002	5.274	0.006	6.518	0.007
	1	0.00002	1.997	0.001	3.519	0.009	5.751	0.007	7.301	0.012

Table 6 : Properties of CO 2 and N 2 O 4 pure components a 14

 6 The parameters of the Mathias-Copeman alpha function were adjusted to experimental vapour 1 pressure of N 2 O 4 using several literature data:Scheffer and Treub [22], Egerton [23], Smith 2

	Compound T c /K	P c / MPa	C 1	MC C 2	C 3	ω	T m /K	P t /MPa	∆H S  L /J.mol -1
	CO 2	304.13 7.373 0.7138 -0.4422 2.4364 0.224 216.58 0.518	8652.3
	N 2 O 4	431.4	10.1 2.0690 -3.7228 3.1108 0.834 261.85 0.019	14650

a Data from DIPPR 801 database 15

Table 7 :

 7 Deviations between experimental and calculated vapour pressures 9

		Bias/%		AAD/%
	Alpha Function	Mathias Copeman	Classical Mathias Copeman Classical
	All data	-0.02	-0.5	1.0	12
	Scheffer and Treub [22]	-0.6	-15	0.9	15
	Baume and Robert [26]	-0.8	5.2	1.1	9.7
	Mittasch et al. [27]	0.02	-2.4	0.4	9.9
	Tsymarnyi [28]	-0.8	-10	1.1	10
	Smith [24]	0.8	13	1.6	13
	Stoddart [29]	2.9	17	3.4	17
	Scheffer and Treub [30]	-1.0	5.0	1.0	12
	10
	Figure 5:

Vapour pressure of N 2 O 4 as a function of the temperature from 261.9 to 430 K, 11

		10				10
		8 9	Scheffer and Treub (1913) Stoddart (1945) Smith (1927)			Scheffer and Treub (1913) Stoddart (1945) Smith (1927) Tsymarnyi (1970)
		7	Tsymarnyi (1970) Mittasch et al. (1926)			1	Mittasch et al. (1926) Baume and Robert (1919)
	MPa	5 6	Baume and Robert (1919) Scheffer and Treub (1912) PR-EoS	p/ MPa		Scheffer and Treub (1912) PR78 PR + MC
	P/	4
		3				0.1
		2
		1
		0				0.01
		263.15	313.15	363.15 T / K	413.15	263.15	283.15	303.15	323.15	343.15 T / K	363.15	383.15	403.15	423.15
	14
	15

: data from [22]; : data from [26]; ▲: data from [27]; •: data from [28]; : data 12 from [24]; : data from [29]; : data from [30]; red line: calculated with PR78 EoS. 13 Green line: Calculated with PR78 EoS with the original alpha function.

Table 8 : k ij parameter and F function

 8 the deviations between 2 the model and the data from Camy et al. [6] are also presented. 3

	(10)
	(11)

Table 9 : Deviations in Pressure using the Peng Robinson Equation of State with the 6 Mathias Copeman Alpha function and the classical mixing rules. 7 T /K BIASP /% AADP /% Camy and al. [6]

 9

		298.15	7.90	7.90
		313	3.18	5.70
		328.45	-3.65	3.65
		All T	2.25	5.46
		253.43	-0.36	3.14
		273.42	0.14	2.04
	This work	293.43	0.23	1.86
		303.43	0.46	1.66
		All T	0.12	2.17

Table 10 : Experimental melting point data on the CO 2 (1) + N 2 O 4 (2) at three mole 10 fraction in CO 2 x 1 binary system 11

 10

	T m (K)	x 1	u(x 1 )	P /MPa
	249.83	0.254	0.004	0.56
	230.15	0.545	0.002	0.53
	217.05	0.716	0.004	0.44