A Le Duigou

G Chabaud

R Matsuzaki

M Castro

Tailoring the mechanical properties of 3D-printed continuous flax/PLA biocomposites by controlling the slicing parameters

Keywords: Natural fiber, 3D printing, mechanical testing

3D printing represents a great opportunity for biocomposites to be developed for the first time on the same time scale as their synthetic counterparts. The purpose of this article is a global investigation of the relationship between slicing parameters (i.e. Layer Height (LH), Interfilament Distance (ID), Number of Trips (NT), Number of Layers (NL)), microstructure and induced tensile properties in order to propose 3D printed continuous flax/PLA biocomposites with tailored mechanical properties.

The Layer Height controls filament compaction during printing. The decrease in LH from 0.6 to 0.2 mm results in a reduction in layer thickness, a decrease in porosity, and an increase in tensile properties (+210% for stiffness and strength). The increase in NL from 1 to 10 results in higher compaction ratio due to the compaction effect of layer n on layer n-1 and superior tensile properties (+ 50 % in modulus and + 73% in strength).

NT and ID control the space available for the filament between the nozzle and adjacent filaments respectively. This significantly affects the average layer thickness and the overall geometry of the sample. Increasing NT from 1 to 18 reduces the space available for the polymer matrix flow for each filament due to the neighboring effect. Generally, the 3D printing process with continuous-flax/PLA composites provides a wide range of tensile properties that are still considerably higher than the current published values for printed short natural fiber composites and in the same range as printed continuous glass fiber/PA composites.

I. Introduction

Additive manufacturing which builds physical 3D geometries by successive addition of material [START_REF]Terminology, Additive manufacturing -General principles[END_REF] is an emerging technology to produce the next generation of composite materials. Within this category, Fused Filament Fabrication (FFF), more commonly referred to as 3D printing, is increasingly being examined for pure polymers, short fiber-reinforced composites, and recently for continuous fiberreinforced composites. 3D-printed polymers and short fiber composites exhibit moderate properties due to a large void amount, lack of orientation, and low fiber content [START_REF] Kabir | A critical review on 3D printed continuous fiber-reinforced composites: history, mechanism, materials and properties[END_REF]. Continuous fiber composites generally offer high mechanical performance to meet structural design requirements [START_REF] Blok | An investigation into 3D printing of fi bre reinforced thermoplastic composites[END_REF] and were first introduced in the scientific community by Van der Klifft [START_REF] Van Der Klift | 3D Printing of Continuous Carbon Fiber Reinforced Thermo-Plastic (CFRTP) Tensile Test Specimens[END_REF] with the FFF process.

Continuous fiber-reinforced composites are mainly reinforced with glass, carbon, or Kevlar fibers [START_REF] Blok | An investigation into 3D printing of fi bre reinforced thermoplastic composites[END_REF][2] [START_REF] Mei | Tailoring strength and modulus by 3D printing different continuous fibers and filled structures into composites[END_REF]. Due to environmental concerns, biobased and natural fiber composites are also increasingly being investigated. Although the development of biocomposites is still in its infancy in terms of technological maturity, 3D printing is a great opportunity for biocomposites to develop for the first time on the same time scale as their synthetic counterparts. Recently, Matsuzaki et al. [START_REF] Matsuzaki | Threedimensional printing of continuous-fiber composites by in-nozzle impregnation[END_REF] and Le Duigou et al. [START_REF] Duigou | 3D printing of continuous flax fiber reinforced biocomposites for structural applications[END_REF] have proposed natural fiber-reinforced polylactic acid (PLA) that outperforms the mechanical properties of discontinuous natural fiber 3D-printed composites and could compete with conventional manufacturing processes [START_REF] Duigou | A Review of 3D and 4D printing of natural fibers biocomposites[END_REF]. The non-planar printing of continuous fiber-reinforced biocomposites has also been evidenced [START_REF] Zhang | Three-Dimensional Printing of Continuous Flax Fiber-Reinforced Thermoplastic Composites by Five-Axis Machine[END_REF]. However, due to recent developments, there is a lack of knowledge on the relationship between the printing parameters, the slicing parameters, and the resulting architecture and mechanical properties, especially for continuous natural fiber-printed composites.

Compared to conventional manufacturing, 3D printing is a layer-by-layer technique that offers innovation in term of reduced design-to-manufacture time scale [START_REF] Dizon | Mechanical characterization of 3D-printed polymers[END_REF], overall costs [START_REF] Ngo | Additive manufacturing (3D printing): A review of materials, methods, applications and challenges[END_REF] and production waste [START_REF] Mitchell | Additive Manufacturing-A Review of 4D Printing and Future Applications[END_REF], Interestingly, with 3D printing, complex microstructured material (gradient, distribution…) and tailored properties can be envisaged [2][3].

Printing parameters generally include nozzle geometry, printing speed, printing trajectory, nozzle temperature, bed temperature, and calibration. Replacing the round nozzle with a square nozzle significantly increases the interlaminar shear strength of the polymer due to a large reduction in porosity [START_REF] Papon | Effects of fiber surface treatment and nozzle geometry in structural properties of additively manufactured two-phase composites[END_REF]. Choosing the right temperature based on the thermal properties of the material has a significant influence on productivity and print quality. High temperatures contribute to better interlaminar bonding and reduced void occupancy and lead to improved mechanical properties [START_REF] Kabir | A critical review on 3D printed continuous fiber-reinforced composites: history, mechanism, materials and properties[END_REF].

Guessasma et al. [START_REF] Guessasmaa | Understanding the microstructural role of bio-sourced 3D printed structures on the tensile performance[END_REF] have suggested that wood-based biocomposites have stable tensile properties over a range of 210-250°C. Yang et al. [START_REF] Yang | Effect of extrusion temperature on the physico-mechanical properties of unidirectional wood fiber reinforced polylactic acid composite (WFRPC) components using fused deposition modeling[END_REF] have shown a slight decrease in tensile and flexural properties at extrusion temperatures between 200 and 230°C, but an increase in compressive strength (+ 15.1 %) and internal bond strength (+24.3%). The higher extrusion temperature showed better compatibility at the fiber/PLA interfaces and good adhesion between the extruded filament segments.

Printing speed has a minor effect on the tensile strength of continuous fiber 3D-printed composites [START_REF] Tian | Interface and performance of 3D printed continuous carbon fiber reinforced PLA composites[END_REF] but has a significant influence on the production cost [START_REF] Ning | Additive manufacturing of carbon fiber-reinforced plastic composites using fused deposition modeling: Effects of process parameters on tensile properties[END_REF]. Based on their review work, Mazzanti et al. [START_REF] Mazzanti | FDM 3D Printing of Polymers Containing Natural Fillers: A Review of their Mechanical Properties[END_REF] state that the reduction in high temperature residence time implied by faster printing speed can prevent the degradation of natural fibers. In another study, they also claim that an increase in porosity from 3.15% to 8.7% was observed in the temperature range 210-240°C, due to the change in filament cross-section that is not compensated for by print height and filament accumulation at the nozzle [START_REF] Guessasma | Microstructure and Mechanical Performance of 3D Printed Wood-PLA/PHA Using Fused Deposition Modelling: Effect of Printing Temperature[END_REF].

Slicing parameters include raster angle (in-plane angle), filling pattern, printing height, interfilament distance or nozzle diameter, filling pattern (e.g. honeycomb, hexagonal, triangular), filament orientation, and build orientation (out-of-plane) [START_REF] Kabir | A critical review on 3D printed continuous fiber-reinforced composites: history, mechanism, materials and properties[END_REF].

Ning et al. [START_REF] Ning | Additive manufacturing of carbon fiber-reinforced plastic composites using fused deposition modeling: Effects of process parameters on tensile properties[END_REF] have evidenced that specimens with 0° fiber orientation have the highest mechanical properties compared to 90° and 45° angles. Chabaud et al. [START_REF] Chabaud | Hygromechanical properties of 3D printed continuous carbon and glass fiber reinforced polyamide composite for outdoor structural applications[END_REF] confirm that a very high anisotropic ratio (E0°/E90°) is observed in 3D-printed Carbon and Glass/PA composites, which is underlined by the low transverse properties induced by the low interfilament properties. Liu et al. [START_REF] Liu | Three-dimensional printing of poly(lactic acid) bio-based composites with sugarcane bagasse fiber: Effect of printing orientation on tensile performance[END_REF] have examined the effect of printing orientation (0°, 90°, ±45° and 0°/90°) on the tensile strength of sugarcane bagasse fiber/PLA biocomposites. Longitudinal printing at 0° was the best solution, with a 7% improvement compared to the cross-solution (± 45° and 0°/90°) and 20% improvement compared to transverse printing at 90°. Le Duigou et al. [START_REF] Duigou | 3D printing of wood fiber biocomposites: From mechanical to actuation functionality[END_REF] observe more drastic evolutions between 0° and 90° that also depend on the distance between the printed filaments. Thus, 0° printing could exhibit two (2x) or three times (3x) more resistance than printing at 90°.

The print pattern on the edge (turn) of the specimen influences the transverse tensile behavior and properties. The filling pattern (e.g. rectangular, hexagonal, triangular) influences the strength and stiffness of the Nylon composite reinforced with CF, FG, and Kevlar [START_REF] Mei | Tailoring strength and modulus by 3D printing different continuous fibers and filled structures into composites[END_REF]. Rectangular filling with a large concentric fiber ring shows the highest tensile strength, followed by hexagonal and triangular filling. Changing the filling percentage from 30% to 90% multiplies the tensile strength and stiffness by factor 2 and 1.5 respectively for wood/PLA biocomposites [START_REF] Vigneshwaran | Statistical analysis of mechanical properties of wood-PLA composites prepared via additive manufacturing[END_REF]. The printing pattern, whether rectangular, triangular, or hexagonal, does not seem to influence the final properties of biocomposites [START_REF] Vigneshwaran | Statistical analysis of mechanical properties of wood-PLA composites prepared via additive manufacturing[END_REF]. Torrado-Perez et al. [START_REF] Perez | Fracture surface anlysis of 3D printed tensiles specimens of novel ABS based materials[END_REF] have shown that depending on the printing direction, i.e. build orientation (XYZ or ZXY plane), the effect of the natural fiber is different with a larger decrease in the out-of-plane direction compared to the in-plane one. Due to the high porosity of the Jute/ABS filament, a rupture of the trans-filament is observed.

Lower layer thickness results in higher packing density and high inter-bonding strength, which leads to high interfacial properties on continuous fiber composites [16][17]. The printing height of 0.3 to 0.05 mm improved tensile strength from 20.5 to 35.5 MPa and stiffness from 2567 to 3542 MPa [START_REF] Ayrilmis | Effect of printing layer thickness on water absorption and mechanical properties of 3D-printed wood/PLA composite materials[END_REF],

confirming the work of Le Duigou et al. [START_REF] Duigou | 3D printing of wood fiber biocomposites: From mechanical to actuation functionality[END_REF] and Dong et al. [START_REF] Dong | Additive manufacturing of mechanical testing samples based on virgin poly (lactic acid) (PLA) and PLA/wood fiber composites[END_REF] on similar wood/PLA biocomposites. The thickness of the layer also influences the microstructure and porosity level of the biocomposites. Le Duigou et al. [START_REF] Duigou | 3D printing of wood fiber biocomposites: From mechanical to actuation functionality[END_REF] and Ayrilmis et al. [START_REF] Ayrilmis | Effect of printing layer thickness on water absorption and mechanical properties of 3D-printed wood/PLA composite materials[END_REF] have evidenced the effect of the height of the layer on the porosity of wood/PLA. An increase in layer height from 0.05 to 3 mm induces greater porosity (Fig. 12), increased water absorption, and reduced tensile properties (30% modulus and 40%

strength) [START_REF] Ayrilmis | Effect of printing layer thickness on water absorption and mechanical properties of 3D-printed wood/PLA composite materials[END_REF].

The Interfilament Distance (ID) or hatch space describes the extent of the overlap between adjacent filaments, i.e. the distance to the center of the filaments. Tian et al. [START_REF] Tian | Interface and performance of 3D printed continuous carbon fiber reinforced PLA composites[END_REF] have shown that increasing the interfilament distance from 0.4 to 1.8 mm results in a significant reduction in the flexural properties of continuous carbon fiber/PLA composites. A minimum Interfilament Distance value of 0.4 was found, from which excessive overlap is observed and the printing process is disrupted. To the authors' knowledge, no work has been done on natural fiber composites.

The development of biocomposites is still in its infancy in terms of technological maturity, so 3D

printing is an incredible opportunity for them to develop for the first time on the same time scale as their synthetic counterparts. The objective of the present work is to describe for the first time the relationship between the slicing parameters, the resulting microstructure and geometry as well as the mechanical properties of new PLA composites reinforced with continuous flax fibers. In doing so, it will be possible to obtain a new class of high-performance biocomposites printed in 3D with tailorable properties. The effect on Layer Height (LH), Number of Trips (NT), Number of Layers (NL) and

Interfilament Distance (ID) on the porosity content and tensile properties are presented and discussed.

Materials and methods

Materials

Continuous flax yarns were provided by SAFILIN. They have a low twist (320 Turn/meter) and a low linear density of 68 Tex (or g/km) to guarantee high mechanical properties [START_REF] Baets | Determination of the optimal flax fiber preparation for use in unidirectional flax-epoxy composites[END_REF].

PolyLactic Acid Ingeo TM 3260HP (PLA) were supplied by NatureWorks ® .

Filament production

The filaments were produced by a co-extrusion process combining polymer coating of the flax yarn and impregnation. More information can be found in a previous article [START_REF] Duigou | 3D printing of continuous flax fiber reinforced biocomposites for structural applications[END_REF]. The filament diameter is 503 ± 47 µm, which is slightly larger than for continuous glass or carbon /PA equivalents [START_REF] Chabaud | Hygromechanical properties of 3D printed continuous carbon and glass fiber reinforced polyamide composite for outdoor structural applications[END_REF]. Table 1 shows the extrusion parameters that were used for the continuous flax/PLA biocomposite filaments. Biocomposites were printed with a customized Prusa MK3s printer. An extrusion motor was settled to control the amount of printed filament and feed the nozzle with continuous filament. Next, a flat head nozzle was used to compress the filament and entrain it during printing. The temperature of the nozzle and the hot plate was set at 195°C and 60°C respectively. The printing speed was 6 mm/s.

All samples were printed with a rectilinear filling pattern, oriented either at 0° (longitudinal) along the

x-axis without any contour. The readers can see picture in previous work [START_REF] Duigou | 3D printing of continuous flax fiber reinforced biocomposites for structural applications[END_REF]. The printing trajectory was controlled by a manually written Gcode and then transferred to the Repetier Host.

Characterization of the mechanical properties

The geometry of the tensile test specimens was set to L: 100; W: 17, and t: 1 mm. Tensile tests were performed along the fiber direction (longitudinally) on printed samples according to ISO 527-4 standards [START_REF]propriétés en traction -Partie 4: Conditions d'essai pour les composites plastiques renforcés de fibers isotropes et orthotropes[END_REF] for sample geometry. 3 mm thick Glass/epoxy tabs were glued (Loctite 406) to printed laminates to reduce stress concentration when clamping in the jaws. Prior to mechanical characterization, the samples were stored at 23°C and 50% RH until a constant mass was observed.

Tensile tests were conducted on an MTS 5kN device with a cross-head displacement speed of 1 mm/min and a controlled environment (23°C and 50% RH). The tensile strain was measured with a clip-gauge (L0 = 10 mm) and the longitudinal tensile modulus EL1 was determined over a range of strains between 0.05 % and 0.1 %. This original procedure was suggested in many articles [START_REF] Shah | Damage in biocomposites: Stiffness evolution of aligned plant fiber composites during monotonic and cyclic fatigue loading[END_REF][30] [START_REF] Monti | Mechanical behaviour and damage mechanisms analysis of a flax-fiber reinforced composite by acoustic emission[END_REF] to fulfil the specificity of natural fiber composites with regard to the characterization of stiffness. Five samples were systematically tested for each characterization.

Porosity and microstructure analysis

Porosity content Vp were measured using ASTM D2734-16 [START_REF] Astm D | Standard Test Methods for Void Content of Reinforced Plastics[END_REF]. The equation for this standard is as follows:

= 1 - 1 - + (Eq. 1)
In this equation, Vp is the porosity content, dC is the density of the composite, wf is the fiber weight fraction, dm is the density of the matrix (1.2 from the datasheet of PLA), and df is the density of the fiber (1.4 g/cm 3 [START_REF] Gall | Recommended flax fiber density values for composite property predictions[END_REF]). Five density measurements were made by volumetric and gravimetric tests and then were arithmetically averaged.

The microstructures of the reinforced filament and printed parts were observed in optical microscopy (Olympus BX51) at different magnifications (x5, x10, x20). The image analysis was performed using ImageJ software to evaluate porosity. The porosities were manually color-coded, then the images were binarized, and the surface of black areas was finally calculated using the software. Ten images were used for each system. SEM observations were performed on glass and flax preforms. Samples were sputter-coated with a thin layer of gold in an Edwards Sputter Coater and analysed with a Jeol JSM 6460LV SEM at 20 kV.

3 Results and discussions An average diameter of 503 ± 47 µm was measured, which is similar to those previously published [START_REF] Duigou | 3D printing of continuous flax fiber reinforced biocomposites for structural applications[END_REF] and slightly larger than commercial continuous glass or carbon/polyamide filaments [START_REF] Goh | Characterization of mechanical properties and fracture mode of additively manufactured carbon fiber and glass fiber reinforced thermoplastics[END_REF].

The low dispersion of flax yarns in the PLA matrix is shown by the deviation of the matrix and the fiber-rich areas (Fig. 1) from the center of the filament. This seems to be attributable to the coextrusion die.

The porosities are mainly located in the flax yarn that gives a porosity vp of 2.2 ± 0.8 %, which is low compared to filaments that are produced including short natural fiber (8.4< vp <47 %) [22][35], or in the range of polyamide filaments reinforced with continuous glass (vp =1.34 ± 0.13%) or carbon (vp =1.85 ± 0.30%) [START_REF] Chabaud | Hygromechanical properties of 3D printed continuous carbon and glass fiber reinforced polyamide composite for outdoor structural applications[END_REF].

Different volume contents of the fibers have been tested (not shown here), but it is not easy to obtain high quality printed samples and a good production rate. Thus, in the present work, the developed cFF/PLA filaments contain a volume fraction of 26.4 ± 1.1 %, which is slightly lower than the previously published batch and the commercial high performance carbon/polyamide filaments [START_REF] Chabaud | Hygromechanical properties of 3D printed continuous carbon and glass fiber reinforced polyamide composite for outdoor structural applications[END_REF][34].

Influence of Layer Height (LH) on the mechanical properties of cFF/PLA printed composites

The Layer Height (LH) is a slicing parameter that defines the distance between the nozzle and the surface to be printed. The resulting thickness of a printed ply due to the compression of the nozzle on the heated bed is noted Layer Thickness (LT) (Fig. 2). Here, a nozzle with plate geometry was used to promote compaction during printing. A wide range of Layer Height (LH) measurements was investigated and the thickness of the corresponding layer was measured on a single printed layer (Fig. 3a, black solid symbols). The relationship between these quantities is not linear, and there is a difference between the programmed Layer Height and the actual layer thickness. Two regimes may be observed : Below 0.3 mm programmed height, the actual layer thickness is always greater than the programmed one. This leads to an overcompaction of the material as evidenced by the value of the compression ratio around 2. The latter is defined as the ratio of the initial filament diameter (503 ± 47µm) to the measured ply thickness (Fig. 3a, white symbols). This can be explained by the twisted structure of the continuous flax yarn, which prevents complete compaction of the filament by the nozzle during printing. It can be argued that printing many layers with each actual layer thickness greater than LH can result in the printing nozzle being crushed when the actual thickness of layer n exceeds the LH of layer n+1.

Above the threshold of 0.25-0.3 mm, there is a monotonic increase in the actual thickness with LH until the filament can no longer be printed because LH is greater than the filament diameter. An LH of 0.7 mm is the maximum value that was successfully printed. Within this range, the actual thickness is always slightly less than the preset values, which means that undercompaction occurs.

Layer Height (mm) programmed on Gcode The Layer Height controls the microstructure of continuous Flax/PLA printed biocomposites: Figure 3b shows that for a low LH (e.g. 0.2 mm) where there is overcompaction, a misalignment of the flax fibers is observed (Fig. 3b). The increase in LH allows the yarn to be less compacted, which is clearly observed on the surface of the samples, and the fiber reinforcement appears to be more rectilinear. The porosity content has been evaluated on samples with three different LHs, 0.2, 0.3 and 0.6 mm (Fig. 4a). The porosity level follows an exponential increase with Layer Height from vp = 2 ± 0.6% for LH of 0.2 mm to 17.6 ± 3.9% for LH=0.6 mm.

Layer Height (mm) Thus, a smaller LH creates a smaller space between the layers where the bonding and interdiffusion mechanisms have apparently been triggered (Fig. 4b) due to a higher compression ratio (Fig. 3a . This change in the microstructure is expected to accelerate the moisture sorption mechanism, as has been evidenced elsewhere on printed short-wood-fiber/PLA biocomposites [START_REF] Duigou | 3D printing of wood fiber biocomposites: From mechanical to actuation functionality[END_REF][25], while maintaining constant hygroexpansion [START_REF] Gager | Understanding the effect of moisture variation on the hygromechanical properties of porosity-controlled nonwoven biocomposites?[END_REF]. The modification of LH also induces a linear reduction in both the EL1 stiffness and strength of continuous flax fiber/PLA composites (Fig. 5a). This confirms that the compression ratio, which represents the pressure applied to the filament during printing, can modulate the microstructure and mechanical properties by influencing the bonding mechanism between the printed layers. The longitudinal tensile modulus (EL1), strength (σL) and strain at break (εL) are gathered in Table 2. The highest tensile properties found for LH = 0.2 mm are in the same range as those previously published, taking into account the slight difference of fiber volume fraction (26.4 ± 1.1 % against 30.5%) [START_REF] Duigou | 3D printing of continuous flax fiber reinforced biocomposite for structural applications[END_REF]. By comparison, similar materials (Flax (30wf%)/PLA) manufactured with thermocompression have exhibited a more brittle behavior with a sudden rupture around 0.8% of strain [START_REF] Duigou | A multiscale study of the interface between natural fibers and a biopolymer[END_REF]. The twisted yarn architecture of continuous flax/PLA may contribute to tougher tensile properties that may be advantageous for structural design. A Rule of Mixture (RoM) is applied to estimate the flax fiber reinforcement properties by back calculation (with Em =3.5 GPa [START_REF] Naturworks | Ingeo TM Biopolymer 3260HP Technical Data Sheet[END_REF] and Vf = 26.5%). A fiber stiffness of 60 GPa is calculated, which is relevant for the properties of flax fibers [START_REF] Baley | Average tensile properties of French elementary flax fibers[END_REF] and other work on the conventional manufacture of flax fiber composites [27][41]. As a result, the FFF of continuous flax fiber biocomposites retained the reinforcement potential of flax fibers, even though thermal cycles were applied (co-extrusion and printing processes).

Analysis of fracture analysis of cFF/PLA revealed changes with variation of Layer Height (Fig. 5b, c andd). For LH = 0.2 and 0.3 mm, there is an initial transverse crack followed by propagation along the tensile axis. Regardless of the Layer Height, there are numerous fiber pull-outs (Fig 5c andd).

However, when LH reaches 0.6 mm, the fracture mechanism is modified with delamination triggered by the high porosity content (white arrows in Fig 5d ). The Number of Trips, hence the effect of neighboring filaments, influences the thickness of the printed layers. For NT greater than 6, the sample thickness is almost constant and then depends on the LH values, as evidenced in Figure 3a. Below NT = 6, there is a drastic reduction in the thickness of the layer, the limit of which is given by the thickness and compressibility of the flax yarn (Fig. 1). This result highlights the constraining effect of the neighboring filaments during compression by the nozzle, which prevents the polymers from spreading. During printing, the nozzle compresses the filament on the bed or the previous composite layer, but the volume of the printed filament must be constant (considering the reduction in the volume of porosity due to the compression ratio). Thus, due to the constraining effect of the neighboring filament, the polymer matrix cannot flow in a lateral Distance ID controls the gap between the flax yarns, which should influence the internal cohesion of the sample, the fiber content for a constant sample width, but also the mechanical properties [START_REF] Gladman | Biomimetic 4D printing[END_REF]. The tested values have been selected because they enable the printing of high-quality material. Above ID = 0.8 mm, there is a lack of cohesion between the filaments, making it impossible to carry out mechanical tests.

The sigmoid trend in layer thickness with NT is broadly similar to Figure 6a with an increase in composite thickness with NT until stabilization occurs around NT = 10. This is observed regardless of the ID. However, the Interfilament Distance considerably influences the variation in layer thickness between low and high Number of Trips (Fig. 8a). A high ID (ID = 0.8) that is greater than the initial filament diameter (503 ± 47µm) flattens the curve with a reduction in the thickness of the final layer.

In contrast, a low ID of 0.4, which is smaller than the filament diameter, drastically increases the thickness of the composite and leads to a deviation from the preset thickness given by the Layer Height (LH = 0.3 mm) and also on the compression ratio (real thickness/initial filament diameter) due to filament overlapping. preset Interfilament Distance (0.4, 0.6, and 0.8 mm) due to the reduced flow of the polymer matrix.

Here, the space available for the printed filament is bound to the distance between neighboring filaments (ID) (Fig. 9). Reducing the lateral space between the filaments, and keeping the volume of the material implies a flow of the polymer in the direction of thickness. This also implies a slight modification of the microstructure with a higher porosity content for ID = 0.4 mm (Vp (ID =0.4 mm) = 5.4 ± 0.8% ; Vp (ID =0.6 mm) = 3.2 ± 0.7%; Vp (ID =0.8 mm) = 2.3 ± 0.9%) that may be due to overlapping filaments. The Number of Trips (NT) does not affect the stiffness of the printed samples as standard deviations overlap. However, the strength is altered and decreases as the NT increases. This may be due to a statistically uncertain number of defects due the larger sample size. The Number of Layers (NL) represents the number of printed layers. Figure 11a shows that for an LH = 0.2 or 0.3 mm, the printing of several layers induces a linear reduction of the average layer thickness from 0.3 to 0.2 mm. The average layer thickness is calculated by dividing the total thickness by the Number of Layers. The trend is radically different when the LH is set at 0.6 mm with a thickness that increases slightly with NL (Fig. 11a). In addition, for the latter case, beyond 6 printed layers, the increase in the distance between the nozzle and the surface is so great that compaction is considerably reduced, and the printing process with adhesion to the previous layer could not take place. The increase in the NL induces a continuous increase in the actual layer thickness through the laminate with a thinner layer for the first layer compared to the last printed layers (Fig. 11 c). Thus, printing an n layer affects the previous n-1 layer by providing additional compressive stresses on the molten composite. The more NL, the greater the compression on the previous layers. Therefore, to achieve a constant compression ratio throughout the printing process, a function of the LH values should be proposed rather than a constant value. Work should also be conducted on the effect of yarn on the compressibility of the filament.

The Number of Layers (NL) has a small effect only on the overall sample width and the average filament width (Fig. 12) with a slight linear increase with NL for LH of 0.2 and 0.3 mm. This is caused by the additional compression when printing many layers. Thus, the almost constant value of the filament width with increasing NL at a fixed value of LH = 0.6 mm confirms the relationship between additional layer, compression, and filaments width. The consequence of various NL on the tensile properties and fracture behavior of cFF/PLA composite is evidenced in Figure 13a andb. Table 4 shows the mechanical data. The increase in NL from 1 layer to 10 layers implies a linear increase in tensile modulus (+ 50 %), which is due to the greater compaction of the printed filament. Higher tensile strength (+ 73%) and similar strain at break are also measured when 10 layers are printed (Table 4). This confirms that the load transfer between the PLA matrix and the flax yarn reinforcement is more efficient with a higher Number of Layers, as indicated by observation of the fracture surface (Fig. 13b). In fact, a reduced number of dry yarns is observed for NL = 10 compared to biocomposites with NL = 1 due to the compacting effect on the impregnation of the flax yarns.

Number of Layers

In a similar way to the continuous synthetic fiber composite printed by the Makforged® (but for a different reason) [START_REF] Chabaud | Hygromechanical properties of 3D printed continuous carbon and glass fiber reinforced polyamide composite for outdoor structural applications[END_REF], the evolution of cFF/PLA printed biocomposite properties with their geometry has to be carefully taken into account in the design process. The dispersion of stiffness and strength is important and shows that for a similar Flax/PLA material, with a similar fiber volume fraction, the 3D printing process can give a wide range of tensile properties (from single to double for strength and from single to triple for stiffness) depending on the slicing parameters (LH, ID, NT and NL); this finding opens the design space for biocomposite parts. detail elsewhere [START_REF] Duigou | 3D printing of continuous flax fiber reinforced biocomposite for structural applications[END_REF] but can relate to the difference in fiber quality, linear yarn weight, and fiber content. Previous work carried out in the same laboratory on a similar continuous Flax/PLA composite [START_REF] Duigou | 3D printing of continuous flax fiber reinforced biocomposite for structural applications[END_REF] resulted in tensile properties superior to those presented here (≈ + 30%). This result is probably due to the difference in fiber content (Vf =26.5 instead of 30.5%), the change of flax fiber spool (similar 68 Tex from Safilin), the geometry of the sample (width = 17 mm vs. 10 mm), and the printer (Prusa MK3s vs. Prusa i3). This whole experiment demonstrates that this technology is still in its infancy, and further work is required to make it a consistent and reliable process. Compared to conventional processes (e.g. thermocompression, vacuum-assisted infusion) infusion (black dots in Figure 14b andc) applied to long flax fiber composites, continuous flax/PLA printed composites offer competitive tensile properties, especially if evaluated for a similar fiber volume content (Fig. 14b andc). Layer Height is a crucial parameter to control the compaction of filament during printing. Decreasing LH from 0.6 to 0.2 mm induced a reduced layer thickness, lower porosity content, and higher tensile properties (+210% for stiffness and strength).

3. 1

 1 Microstructure of the Continuous flax/PLA biocomposites filament before printing Continuous flax/PLA biocomposites filaments (cFF/PLA) exhibit irregularities on their surface that have led to imperfectly circular filaments (Fig.1), mainly due to the co-extrusion process and the twisted architecture of the fiber bundles.

Figure 1

 1 Figure 1 Cross-section of Continuous flax/PLA biocomposites filaments made by co-extrusion process

Figure 2

 2 Figure 2 Schematic view of the printing process for Continuous Flax/PLA biocomposites and the corresponding slicing parameters, i.e. Interfilament Distance (ID), Number of Layers (NL), Layer Thickness (LT) and Number of Trips (NT).

Figure 3 (

 3 Figure 3 (a) Evolution of the measurement of the actual layer thickness (LT) on a layer after the printing process as a function of the programmed Layer Height (LH) for cFF/PLA composites. (b) Photograph of the cFF/PLA composites surface with different LHs. The dotted lines represent the edges of each sample.

Figure 4 (

 4 Figure 4 (a) Evolution of the porosity level (Vp) as a function of the Layer Height (LH) of cFF/PLA composites, The dotted line represents an exponential fit of the experimental data. (b) Micrographic section of cFF/PLA composites with different LH (from 0.2 to 0.6 mm). The black areas correspond to the porosity. The Number of Trips (NT) is 18 and the Number of Layers (NL) is 5.

  , white symbols). An LH of 0.6 mm results in very high porosity content, mainly located in the interlaminar areas in Fig4b)

Figure 5 ( 3 . 3

 533 Figure 5 (a) Evolution of tensile modulus (EL1) and tensile strength as a function of Layer Height of cFF/PLA composites. (b) Fracture surface microscope observation of cFF/PLA composites with various LH (from 0.2 to 0.6 mm). (c) SEM micrography of cFF/PLA composites with LH = 0.2 mm. SEM micrography of cFF/PLA composites with LH = 0.6 mm. The white arrows indicate that porosity has caused failure. (NT = 10).

Figure 6 (

 6 Figure 6 (a) Evolution of the actual layer thickness (measured on one layer) as a function of the Number of Trips (NT) for different Layer Height (0.2, 0.3 and 0.6 mm). (b) Schematic view of the effect of Number of Trips on the thickness and width of printed cFF/PLA composites.

  greater thickness is achieved. LH is also changing this situation. A schematic view is proposed to reproduce the phenomenon in Figure6bwhere 1, 3 or 5 Number of Trips (NT) in a given layer are analyzed.Filament width follows an exponential decay with the Number of Trips (Fig.6band Fig.7a). Average filament width is calculated by dividing the total width of the sample by the Number of Trips. One should keep in mind that the width of the filament is fundamentally different from the Interfilament Distance (Fig.1). ID focuses on the distance between the flax yarns, while the width of the sample takes into account the overall geometry and the polymer flowing over the edge. Without an adjacent filament, the polymer matrix can migrate freely which leads to a wide sample width (Fig.7b). Printing a filament shows that a width of 1.764 mm is obtained while the increase of the NT drastically reduces the average width between the filaments to an almost constant value of 0.6 mm after NT=10 due to a constant spreading of the polymer on the edge. The choice of the Number of Trips affects the accuracy of the sample geometry. In fact, this value is finally close to the preset value of Interfilament Distance.A lower Layer Height of 0.2 or 0.3 mm logically increases the filament width at higher compression (Fig.6band Fig.7a) than LH=0.6 mm.

Figure 7

 7 Figure 7 (a) Evolution of filament width as a function of the Number of Trips (NT) for different Layer Heights (0.2, 0.3 and 0.6 mm). Filament width is the ratio between the total width of the sample and the Number of Trips. (b) Micrograph of a single printed filament (c) and several printed filaments showing the effect of the Number of Trips on the filament width.

Figure

  Figure 8a and b presents a similar experiment but with different Interfilament Distance (ID) of 0.4, 0.6

Figure 8 (

 8 Figure 8 (a) Evolution of the real thickness of the layer (on one layer) as a function of the Number of Trips (NT) for different Interfilament Distances (0.4, 0.6 and 0.8 mm). (b) Evolution of the filament width as a function of Number of Trips for different Interfilament Distances (0.4, 0.6 and 0.8 mm) for a cFF/PLA composite. The Layer Height is 0.3 mm.

Figure 8 b

 8 Figure 8 b confirms that the Number of Trips has a dramatic effect on the sample width and average

Figure 9 Figure 10

 910 Figure 9 Schematic view of the effect of Interfilament Distance (ID) during the variation of the Number of Trips as a function of the average thickness of the middle of the layer. The LH is kept constant at 0.3 mm.

Table 3 3 . 4

 334 Tensile modulus, tensile strength, and strain for various Interfilament distances (0.4, 0.6, 0.8) and Number of Trips (5, 10 and 18) for cFF/PLA printed composite. The tests were performed on one sample with a Number of Layer =1. The letter L in inedx refers to the longitudinal direction (along fiber direction) Influence of the Number of Layers (NL)

Figure 11 (

 11 Figure 11 (a) Evolution of the average layer thickness as a function of the Number of Layers (NL) for different Layer Heights (0.2, 0.3 and 0.6 mm) (NT = 10) (b) Evolution of the actual layer thickness as a function of layer digit (n°1 to 10), (c) Micrographic cross section of 10 layers of cFF/PLA showing the difference in architecture through the laminate (LH = 0.3 mm).

Figure 12

 12 Figure 12 Evolution of the average filament width as a function of the Number of Layers (NL) for different Layer Heights (0.2, 0.3 and 0.6 mm) (NT = 10) for a cFF/PLA composite.

Figure 13 (

 13 Figure 13 (a) Evolution of tensile modulus and tensile strength as a function of Number of layers (NL) (for NT = 10, ID = 0.6 mm and LH =0.3 mm), (b) Fracture surface of cFF/PLA composite with NL of 1 and 10.

Figure 14

 14 Figure 14 Graph of tensile modulus and tensile strength (a) [43][6][22][44][45][46][24][47][15]. Evolution of Tensile modulus (b) and tensile strength (c) as a function of fiber volume fraction for cFF/PLA in relation to the literature values for the conventional manufacture (injection molding, thermocompression or infusion) of natural long fiber composites [48][30][31][49][50][51][52][53][54][27].

  Compared to the work of Matsuzaki et al.[START_REF] Matsuzaki | Threedimensional printing of continuous-fiber composites by in-nozzle impregnation[END_REF] on continuous Jute/PLA (black dot in Fig14a), the current work yield higher the tensile modulus and the strength. The reasons are described in more Tensile strength (MPa)

  an emerging technology aimed at producing the next generation of composite materials that are currently limited by the absence of applied materials. In the context of current environmental concerns, it has become necessary to develop biobased materials with a lower environmental footprint. 3D printing represents a great opportunity for biocomposites to develop for the first time on the same time scale as their synthetic counterparts. Most natural fiber composites used for 3D printing include short natural fibers with moderate mechanical properties. One alternative is the development of continuous natural reinforced polymer to improve those mechanical properties. The purpose of this article was, for the first time, to bring deeper insights in the relationship between slicing parameters (Layer Height (LH), Interfilament Distance (ID), Number of Trips (NT) and Number of Layers (NL)) on the final sample geometry (printing accuracy), the microstructure and the tensile properties in order to envisage a new class of 3D printed continuous flax/PLA biocomposites with tailored performance.

Table 1 Extrusion parameters to produce continuous flax/PLA filaments

 1

		Continuous flax yarn filament
	Die size (mm)	0.6 (diameter) x 20 (length)
	Extruder temperature (°C)	180
	Die temperature (°C)	190
	Oven temperature (°C)	120
	Consolidating system (mm)	40 x 7
	Puller speed (m/min)	1
	Extrusion speed (rpm)

Table 2 The tensile properties of cFF/PLA composites for different Layer Heights (LH). The letter L in index refers to the longitudinal direction (in the direction of the fibers)

 2

	LH (mm)	EL1 (GPa)	σL (MPa)	εL (%)
	0.2	17.2 ± 0.7	183 ± 22	1.5 ± 0.2
	0.3	14.6 ± 0.6	166 ± 31	1.9 ± 0.1
	0.4	12.1 ± 0.7	163 ± 5	1.7 ± 0.1
	0.6	9.5 ± 0.4	87 ± 1	1.4 ± 0.1

  across all plies. Figure11b and cevidences the evolution of the actual thickness of each layer as a function of the layer digit.

	and compression ratio
	ID (mm)	EL1 (GPa)	σL (MPa)	εL (%)
	0.4	12.9 ± 0.9	135 ± 10	1.4 ± 0.1
	0.6	15.6 ± 1.1	186 ± 4	1.9 ± 0.2
	0.8	17.1 ± 1.5	147 ± 16	1.6 ± 0.3
	NT	EL1 (GPa)	σL (MPa)	εL (%)
	5	11.6 ± 0.9	130 ± 19	1.7 ± 0.2
	10	11.1 ± 1.3	132 ± 14	1.6 ± 0.3
	18 (full sample)	11.2 ± 1.2	112 ± 11	1.6 ± 0.2
				Increasing
	the Number of Layers from 1 to 10 does not generate a homogeneous distribution of layer thickness

Table 4 Tensile properties of cFF/PLA composite for different Number of Layers (NL). The letter L in index refers to the longitudinal direction (along fiber direction)

 4

	Figur
	es
	14a,
	b and

(Fig 14c)

. Overall, the mechanical properties are always considerably higher than for printed short natural fiber composites (respectively blue and red dots in Fig

14a

).

Layer thickness was limited by the compressibility of the twisted flax yarn while the relation between LH and the real layer thickness was linear. Both over-and undercompaction were observed for low to high LH. This result implies that when printing numerous layers, the compaction ratio (initial filament diameter/real layer thickness) varied. Increasing Number of Layers (NL) from 1 to 10 brought higher compaction ratio and higher tensile properties (+ 50% in modulus and + 73% in strength). The compaction varied along the cross section with the first plies being the more compressed. Number of Trips (NT) and Interfilament Distance (ID) played the role of "neighboring filaments" that influenced the polymer matrix flow during the printing process by controlling the available space for the filament between the nozzle and the adjacent filaments. This considerably affected the average layer thickness and the overall sample geometry. Increasing NT from 1 to 18 reduced the available space for the polymer matrix flow for each filament. Thus, the sample geometry is more accurate and the average filament width (total width/ NT) followed the preset Interfilament Distance. A decrease of ID from 0.8 to 0.4 mm emphasized this effect. These conclusions may be extended to other 3D-printed materials but the amplitude of phenomena should depend on the rheological properties of the matrix and the compressibility of yarns.

Overall, the 3D printing process presented here with continuous flax/PLA composites yielded to a wide range of tensile properties that were always drastically higher than the current published values on short natural fiber-printed composites. The tensile properties are in the same range as those continuous glass/PA-printed composites. This confirms the potential of continuous flax fiberreinforced printed composites to be high performance composite materials with a large range of reachable properties. This open the era of new design of biocomposites with the added-value of a versatile and disruptive manufacturing process.

5-Acknowledgments

The authors wish to acknowledge "Region Bretagne" and MITI "BIOMIMETISME" CNRS for financial support. We would also like to thank SAFILIN for flax yarns supplying. The article was post-edited by Nicole Geslin.

-Credit author statement


Amélie Battais

Victor Barrère

W Apoutou N'djin

Aurélien Dupré

Michel Rivoire

David Melodelima
email: david.melodelima@inserm.fr

Fast and Selective Ablation of Liver Tumors by High-Intensity Focused Ultrasound Using a Toroidal Transducer Guided by Ultrasound Imaging: The Results of Animal Experiments

Keywords: Ultrasound, HIFU, therapy, liver, VX2, in vivo, rabbit, animals

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

INTRODUCTION

Colorectal liver metastases (CLMs) represent a major public health issue [START_REF] Bray | Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians[END_REF]. Hepatic resection is the gold standard in the treatment of CLMs and is currently the best treatment option for long-term survival. Five-year survival rates of up to 51% have been reported after resection [START_REF] Nordlinger | Arbeitsgruppe Lebermetastasen und-tumoren in der Chirurgischen Arbeitsgemeinschaft O, Australasian Gastro-Intestinal Trials G, Federation Francophone de Cancerologie D. Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial[END_REF]. Liver resection and tumorectomy are widely accepted as a potentially curative treatment for liver metastases from the gastro-intestinal tract. However, the indications for liver resection and even more for tumorectomy in the treatment of liver metastases from non-colorectal primary tumors are still unclear [START_REF] Chen | Effect of Time to Surgery of Colorectal Liver Metastases on Survival[END_REF][START_REF] Heinrich | The current role of liver surgery in the treatment of colorectal liver metastases[END_REF][START_REF] Maeda | Hepatectomy for liver metastases in non-colorectal, non-neuroendocrine cancer patients. The survival benefit in primary unresectable cases[END_REF]. This discrepancy may be due to the fact that liver metastases from other cancers typically occur in the setting of widespread systemic failure, whereas tumors arising within the portal system possess a greater proclivity for establishing truly isolated liver metastases. Currently, surgical management of liver metastases primarily involves interventions that focus on achieving potential cure; therefore, patient selection is limited to those in whom the liver is the only site of failure or the dominant site of disease.

Despite progress in surgery [START_REF] Adam | Two-stage hepatectomy: A planned strategy to treat irresectable liver tumors[END_REF] and downsizing obtained with chemotherapy [START_REF] Folprecht | Neoadjuvant treatment of unresectable colorectal liver metastases: correlation between tumour response and resection rates[END_REF], only a minority of patients are eligible for surgery (less than 20%).

Patients are eligible for hepatic resection if complete resection of colorectal metastases is possible with tumor-free margins. Additionally, the remaining functional hepatic volume must be sufficient to ensure adequate postoperative liver function [START_REF] Pawlik | Expanding criteria for resectability of colorectal liver metastases[END_REF]. Combining hepatic resection and intra-operative local destruction, such as radiofrequency ablation, cryosurgery ablation or microwaves is a widely accepted multidisciplinary treatment for unresectable colorectal cancer liver metastases to increase the number of patients treated with a curative intent [START_REF] Sasaki | Combined resection and RFA in colorectal liver metastases: stratification of long-term outcomes[END_REF]. However, all these techniques require intraparenchymal introduction of a probe, which can be a limit in some difficult regions [START_REF] Meijerink | Radiofrequency and Microwave Ablation Compared to Systemic Chemotherapy and to Partial Hepatectomy in the Treatment of Colorectal Liver Metastases: A Systematic Review and Meta-Analysis[END_REF].

Moreover, RFA is frequently limited by perfusion and CSA is limited by heat conduction [START_REF] Lin | Effect of heat sink on the recurrence of small malignant hepatic tumors after radiofrequency ablation[END_REF]). In addition, local recurrence has been reported especially for tumors with a diameter larger than 3 cm [START_REF] Van Amerongen | Radiofrequency ablation compared to surgical resection for curative treatment of patients with colorectal liver metastases -a meta-analysis[END_REF][START_REF] Wu | Radiofrequency ablation vs hepatic resection for solitary colorectal liver metastasis: a meta-analysis[END_REF].

High-intensity focused ultrasound (HIFU) has been proven effective in a wide range of clinical applications [START_REF] Copelan | High-Intensity Focused Ultrasound: Current Status for Image-Guided Therapy[END_REF][START_REF] Hsiao | Clinical Application of High-intensity Focused Ultrasound in Cancer Therapy[END_REF]. HIFU treatment does not require to puncture the parenchyma and monitoring is possible using B-Mode ultrasound imaging (Parmentier, et al. 2009). As current regional destruction techniques for treating CLMs make it possible to increase the number of patients treated with curative intend we developed a HIFU device that can be used during surgery. Such an approach is invasive but has the advantage to be less invasive than conventional intra-operative local destruction techniques since the HIFU treatment is performed without puncture of the liver and thus preserve the hepatic capsule. We have previously reported that the use of a HIFU transducer with a toroidal geometry can increase the ablation rate by increasing the focal volume [START_REF] Chenot | Intra-operative ultrasound hand-held strain imaging for the visualization of ablations produced in the liver with a toroidal HIFU transducer: first in vivo results[END_REF][START_REF] Melodelima | Ultrasound surgery with a toric transducer allows the treatment of large volumes over short periods of time[END_REF][START_REF] N'djin | Assisted hepatic resection using a toroidal HIFU device: an in vivo comparative study in pig[END_REF]. Several preclinical studies (in vitro and in vivo) have validated the concept of using toroidal HIFU focusing for inducing large ablations in a short amount of time without the need for mechanical scanning allowing to use the device by hand [START_REF] Caloone | Non-invasive high-intensity focused ultrasound treatment of the placenta: a preliminary in-vivo study using a simian model[END_REF], Melodelima, et al. 2009[START_REF] Vincenot | Electronic beam steering used with a toroidal HIFU transducer substantially increases the coagulated volume[END_REF]. Since this HIFU treatment is planned to be performed during an open procedure [START_REF] Dupre | First clinical experience of intra-operative high intensity focused ultrasound in patients with colorectal liver metastases: a phase I-IIa study[END_REF], Dupre, et al. 2017), the ultrasound propagation was not dependent on anatomical constraints, such as the position of the ribs or lungs. Therefore, the device was used and evaluated in an open procedure to provide a complementary tool to surgeons for hepatic resection. In normal liver tissues, the toroidal HIFU focusing strategy indeed confirmed promising results in terms of ablations rate, homogeneity and repeatability [START_REF] Dupre | First clinical experience of intra-operative high intensity focused ultrasound in patients with colorectal liver metastases: a phase I-IIa study[END_REF]. To date however, the impact of this focusing method on the ablation quality has not been clearly investigated in the case of heterogeneous target tissues, especially in presence of solid liver tumors. Here, we report an in-vivo evaluation of this unconventional treatment strategy in a rabbit liver tumor model. Particular attention was paid to the evaluation of the HIFU ablations obtained in this tumor model and the short-term efficacy of the treatment with regard to tumor growth.

MATERIALS AND METHODS

Therapeutic ultrasound device

The therapeutic transducer was a piezocomposite material (Imasonic, Voray-surl'Ognon, France) operating at a frequency of 3 MHz. The transducer has a toroidal shape 70 mm in diameter and was divided into eight radial ultrasound emitters. The radius of curvature was 70 mm, and each of the 8 emitters was divided into 32 individual transducers (Figure 1).

Then, the spatial location and intensity of the pressure field were controlled electronically by modulating the amplitude and the phase applied to each of the individual transducers. An ultrasound imaging probe working at a frequency of 7.5 MHz (Vermon, Tours, France) was placed in the center of the device and connected to a BK HAWK 2102 EXL scanner (B-K Medical, Herlev, Denmark) for guiding the treatment. The imaging plane was aligned with the HIFU acoustic axis. The device was in acoustic contact with the liver using an ultrasound coupling fluid (Ablasonic®, EDAP, Vaux-en-Velin, France). The Ablasonic® was contained in a sterile polyurethane envelope (CIV-Flex Transducer cover, CIVCO, Kalona, Iowa, USA) that covered the device, making it possible to use the HIFU system under sterile conditions.

Continuous flow (0.3 l/min) maintained the degassed coupling water at 20°C and allowed the cooling of the HIFU transducer during the treatment. A peristaltic Masterflex pump (L/S model 7518-60, Cole-Parmer Instruments Co., Chicago, IL, USA) drove the water around a closed cooling circuit.

The HIFU transducer-driving equipment in this experiment was similar to that reported previously [START_REF] Melodelima | Intraluminal high intensity ultrasound treatment in the esophagus under fast MR temperature mapping: in vivo studies[END_REF]; 32 channels of a 50 channel pattern generator (PG1050 Acute, Hsin Chuang City, Taiwan) generated 32 transistor-transistor logic (TTL) signals at the working frequency (3 MHz). Thirty-two power amplifiers (AHF 855, ADECE, Artannes, France) converted the TTL signals into amplified sine waves. The TTL-signals were AC-coupled into the amplifier which also performed bandwidth-limited signal amplification. The amplified signals were then band-pass filtered by their respective matching networks, resulting into amplified sine-waves that were required to drive the individual transducers. This system was developed to allow electronic beam steering while optimizing the number of power amplifiers. An individual element, i, of the first emitter (1 ≤ i ≤ 32), as well as the seven similar elements located at same position in the other seven radial emitters, were supplied in parallel by only one power amplifier. Inside each amplifier, a directional coupler generated 2 voltages, one that was directly proportional to the direct electrical power and one that was directly proportional to the reflected electrical power. The gain of each amplifier was adjusted using digital-analog output cards to control the acoustic power delivered in tissues during treatment. Finally, the user interface displayed the position of the HIFU focal region superimposed on the sonogram, making it possible to place the location of the ablation in specific tissues and visualize in real time the coagulated zone created during sonication.

Animals

Experiments were performed on 45 New Zealand rabbits from Charles River Laboratories (Ecully, France). While no model can truly represent CLM, VX2 tumors implanted in the liver of rabbits provide a good substitute with regard to the size of the animal used to test the HIFU device. Animals weighed between 2.5 and 3.2 kg on the day of arrival.

Implantation of VX2 liver tumors

VX2 cells were initially implanted in both hind limbs of a donor rabbit. Frozen VX2 cancer was extracted from frozen archives which had been used in previous research. Frozen VX2 tumor tissue kept in -80°C was thawed, minced, and isolated with a 100 µm cell strainer to obtain a uniform single-cell suspension from tissues. A venous catheter was placed in an auricular vein for the premedication, which was performed 15 min prior to anesthesia using a combination of ketamine (1.6 ml) and xylazine (0.1 ml). Anesthesia was performed by intravenous injection of 25% of the premedication dose and was maintained by intravenous injection of the same dose every 15 min. 1.0×10 7 of cells suspended in 500 µl of HBSS solution was injected into donor animals' right lateral quadriceps. After 4 weeks of tumor growth, the hind limb tumors were harvested, and small tumor fragments (approximately 25 mg) were dissected from the viable tumor tissue. The abdomen was shaved and cleaned with povidone iodine solution using the anesthesia protocol described above. These tumor portions were then implanted in the lateral right liver lobe of the 45 rabbits during a laparotomy procedure. The liver lobe and the abdomen were then sutured.

Treatment procedures

Treatments were performed 10 days after implantation of VX2 tumors. All rabbits were anesthetized using the same protocol as described previously. Fifteen rabbits were treated with toroidal HIFU ablation (Group 1). Treatments were performed under sterile conditions during laparotomy. Adjacent organs were carefully isolated from the liver using pads which also allowed to avoid liver movements during HIFU treatments. The HIFU probe was held by hand at the surface of the liver. The tumor to be treated was located using the integrated ultrasound imaging probe by measuring the dimensions of the tumor into several contiguous planes. The acoustic power was 70 W, and the sonication time was 40 seconds to cover the whole tumor with safety margins (i.e., margins ≥ 5 mm) without resorting to mechanical scanning. If incomplete treatment was identified on ultrasound imaging guidance, up to 3 single HIFU were allowed. Fifteen rabbits were included in group 2 for surgical resection. The liver resection technique was performed after laparotomy using a Kelly clamp claw, a suction device, and metallic clips (SLS-CLIP B2180-A, Vitalitec International) when necessary. The judgement of the HIFU treated area and surgical resected area in Group 1 and 2 made by the same surgeon.

Fifteen rabbits formed a control group (Group 3). For these animals, only the laparotomy was performed. At the end of the treatment, the laparotomy was closed in all animals with a running suture (2 planes). Groups 1 and 2 were compared to evaluate whether toroidal HIFU treatment increases the risk of tumor dissemination. Groups 1 and 3 were compared to evaluate treatment efficacy.

Pathologic examination

All animals were euthanized 11 days after treatment by a single intravenous injection of 5 ml of Dolethal® (Vetoquinol, Paris, France). A total hepatectomy was then performed to visually inspect the tumor and ablation. Dimensions of the tumor and ablation were measured along thin contiguous planes. The length (L, millimeters), width (W, millimeters), and depth (D, millimeters) of ablated area and of the tumor were measured in each animal, and the volume (V, cubic millimeters) was calculated as V = π/6 L × W × D (Di [START_REF] Matteo | USguided application of Nd:YAG laser in porcine pancreatic tissue: an ex vivo study and numerical simulation[END_REF].

Tumor metastasis was also evaluated. The lungs, kidney, abdominal cavity and pelvic cavity were examined by ultrasonography, especially lymph node metastasis. Metastasis was confirmed by anatomic examination. Once metastasis was identified, the metastasis was visually graded as follows: 0, the organ has normal structure and there is no newly generated mass; 1, there are 1-2 newly generated masses, and metastatic VX2 tumor is confirmed in the pelvic lymph nodes by pathological examination (0-1 cm in diameter); 2, there are 3-10 newly generated masses, and metastatic VX2 tumor is confirmed in the pelvic lymph nodes by pathological examination (1-2 cm in diameter); 3, there are more than 10 newly generated masses, and metastatic VX2 tumor is confirmed in the pelvic lymph nodes by pathological examination (>2 cm in diameter).

All collected samples were also evaluated microscopically. Representative portions of tissues were immersed in 10% formalin for fixation and set in paraffin. The samples were then stained with hematoxylin and eosin. The samples were treated and analyzed by the Anipath Laboratory (Lyon, France).

Statistical analysis

All images obtained from sonograms and gross pathology were interpreted and evaluated independently by three examiners. All examiners were blinded to the results of each other. Lin's Concordance Correlation Coefficient (CCC) was used to evaluate the inter-reader variability [START_REF] Lin | A concordance correlation coefficient to evaluate reproducibility[END_REF]). The CCC was interpreted using the classification of Partik et al. that ranges the CCC from inacceptable (<0.50) to excellent (>0.95) [START_REF] Partik | 3D versus 2D ultrasound: accuracy of volume measurement in human cadaver kidneys[END_REF].

Data were given as the mean values ± standard deviations (minimum value-maximum value). The studied criteria were the dimensions of the tumors measured via gross pathology.

The study was designed to show a difference of at least 25% in the tumor volumes for statistical significance at the 5% level between groups with a power of 95%. The statistical analyses were performed using R® software (The R Foundation for Statistical Computing, Vienna, Austria). Data at different time points were compared with one-way analysis of variance (ANOVA), followed by paired comparison with Student' t test The significance level for all tests was fixed at p = 0.05.

RESULTS

Figure 2 shows a VX2 tumor visualized with the integrated ultrasound imaging probe just before (Figure 2a) and just after (Figure 2b) one HIFU ablation. As previously described, HIFU lesions created in the liver with a toroidal transducer were seen as a hypoechoic (Melodelima, et al. 2009), whereas VX2 tumors appear as hyperechoic. Therefore, it was possible to clearly identify whether the treatment was performed with safety margins at the time of ablation and to apply another HIFU sonication when necessary. In this study, 12 VX2 tumors were treated with only one HIFU sonication, and 3 VX2 tumors were treated with two HIFU sonications. The total procedure time, including targeting, HIFU ablations and the time to perform ultrasound imaging averaged 14 ± 4 min (9 -24). The time for resection in group 2 was significantly longer and averaged 34 ± 8 min (22 -47). In all cases, the VX2 tumors were completely coagulated and surrounded by ablated liver tissue without secondary thermal lesions in surrounding organs. The coagulated tissues are clearly distinguishable from untreated tissue; the treated region has an off-white color, which is sometimes dark at the most heated points (Figure 3). The average treatment margins around the VX2 tumor were negative in all directions, with a minimum value of 3 mm. Based on ultrasound imaging, on the day of the treatment, the tumor volumes averaged 162 ± 20 mm 3 , 150 ± 19 mm 3 and 145 ± 21 mm 3 for the HIFU, resected and control groups, respectively. There were no significant differences between the tumor volumes on the day of the treatment of the groups. The correlation between the dimensions of the HIFU treated region measured using ultrasound imaging and the dimensions measured via gross pathology after autopsies was r = 0.88, p < 0.05 (Figure 4). The agreement between measurements made by the three observers ranges from satisfactory (0.765) to excellent (0.989). According to the classification of Partik et al. [START_REF] Partik | 3D versus 2D ultrasound: accuracy of volume measurement in human cadaver kidneys[END_REF], the CCC for dimensions measured via gross pathology were 0.765 (satisfactory), 0.815 (fairly good) and 0.901 (very good) for inter-readers 1 and 2, 1 and 3 and 2 and 3 respectively. The CCC for dimensions measured using ultrasound imaging were 0.989 (excellent), 0.879 (fairly good) and 0.869 (fairly good) for inter-readers 1 and 2, 1 and 3 and 2 and 3 respectively. Figure 5 shows that tumor volume was significantly lower at the time of autopsy (39 ± 9 mm 3 ) in the HIFU group than in the control group (2610 ± 700 mm 3 , p<0.0001). The tumor and the surrounding tissues treated by HIFU are still visible on gross pathology at the day of autopsy (Figure 3) but complete coagulation necrosis was confirmed in both tissues (normal and tumoral) by histological analysis (Figure 6). The tumor volume measured macroscopically in group 2 (resection) at the day of the resection was on average 157 ± 23 mm 3 . Secondary tumors were observed in 4, 10 and 5 animals in the HIFU group, control group and resected group, respectively. Tumor dissemination was significantly lower in the HIFU group (27%) than in the control group (67%) and all metastases were graded 0 (Table 1). There was no significant difference between tumor dissemination in the HIFU group and the resected group (33%). The dissemination sites were the stomach, the lungs and the peritoneum in all groups.

Histological findings supported these outcomes. Edges of the coagulated area were clearly delimited. The demarcation between treated and untreated liver was very sharp (approximately 200 µm). There was a halo of congestive tissues around all HIFU ablations.

The microscopic examination confirmed complete homogeneous coagulation necrosis in the tumors and in the safety margins for all HIFU ablations (Figure 6).

The animals tolerated the treatment well over the experimental period. The rabbits remained hemodynamically stable during the procedure. They recovered from the procedure within 1 h of termination of anesthesia and quickly resumed eating and normal behavior. The mean weight gain was 200 g (minimum 50 g -maximum 450 g), which is normal according to the age of the animals and the procedure that was conducted. There were no secondary HIFU lesions in adjacent organs.

DISCUSSION

The data from the present study demonstrated that successful rabbit liver tumor ablation can be achieved using an intraoperative toroidal HIFU transducer under ultrasound imaging guidance and therefore could be an effective treatment for localized liver metastases.

All tumors treated by HIFU were completely coagulated and therefore irreversibly destroyed, as confirmed by histological analyses. There was no complication related to the HIFU procedure.

A potential risk of focused ultrasound therapy was the possible enhancement of metastatic development if viable tumoral cells penetrate damaged blood vessels and then into the circulation. In our study on VX2 liver tumors, the metastatic rate observed in control rabbits (67%) was significantly higher than the metastatic rate observed in HIFU treated animals (27%). There was no significant difference between tumor dissemination in the HIFU group and the resected group (33%). These findings suggested that HIFU therapy with a toroidal transducer and applied to liver tumors should not create a risk of tumor dissemination. The recurrence rate observed in the HIFU treated animals provides convincing evidence that this approach is competitive with invasive surgical resection.

In previous work, we have shown that a conventional ultrasound imaging probe integrated in the center of the toroidal HIFU transducer can accurately visualize the coagulated zone created in the liver (Melodelima, et al. 2009). This result was confirmed in the present study. VX2 tumors were clearly visible as a hyperechoic region, and HIFU ablations appeared hypoechoic, leading to an accurate evaluation of the lesion size as well as the safety margins. This allowed to decide whether additional ablations were required in cases of incomplete ablation around tumors. Due to this reliable evaluation of the treatment, quantitative thermometry was not needed to achieve and validate the therapeutic endpoint.

The choice of developing a HIFU device dedicated to intraoperative use was supported by oncological necessities and technological challenges that remain unmet in HIFU technology. The thermal power of this device allows treatments that were shown to be not very dependent on perfusion. This is mainly due to the relatively high frequency (3 MHz) and to the small penetration depth when using an intra-operative approach (Melodelima, et al. 2009). Using HIFU during an open procedure facilitates the protection of adjacent viscera with protective gauzes that can be placed all around the liver. Gauzes inhibit ultrasound propagation and unintended HIFU damage to adjacent organs. An open procedure also facilitates the intraabdominal staging of malignancies. Despite improvements in imaging, liver metastases not detected via preoperative imaging are found in approximately 15% of patients during surgery [START_REF] Arita | Routine Preoperative Liver-specific Magnetic Resonance Imaging Does Not Exclude the Necessity of Contrast-enhanced Intraoperative Ultrasound in Hepatic Resection for Colorectal Liver Metastasis[END_REF]. Additionally, the use of ablation in conjunction with resection appears to be the best current strategy in curative treatment of liver metastases from colorectal cancer [START_REF] Tsitskari | The role of interventional oncology in the treatment of colorectal cancer liver metastases[END_REF]. Our clinical objective is not to use this HIFU device as a replacement for resection but as a complementary tool to surgery. This approach is currently used for RFA and CSA [START_REF] Pawlik | Combined resection and radiofrequency ablation for advanced hepatic malignancies: results in 172 patients[END_REF]. Like RFA, CSA and microwaves, combining hepatic resection with intra-operative HIFU ablation could also expand the number of patients who may be candidates for liver-directed surgical therapy but with a less invasive technique since the HIFU treatment is performed without puncture of the liver. Intraoperative ablations are widely employed in the management of CLM, regardless of whether they are associated with surgery.

The main limitation to the widespread clinical use of HIFU has been the length of time required to treat tumors several cubic centimeters in volume [START_REF] Knuttel | Early health technology assessment of magnetic resonance-guided high intensity focused ultrasound ablation for the treatment of earlystage breast cancer[END_REF][START_REF] Leslie | High-intensity focused ultrasound treatment of liver tumours: post-treatment MRI correlates well with intra-operative estimates of treatment volume[END_REF]. This fact compromises the technique for the most prominent applications, i.e., cancers where tumors may be very large and where therapeutic options are limited. Previous studies have been conducted to enlarge the ablated region using, for example, cavitation (Melodelima andCathignol 2004, Sokka, et al. 2003), phased array systems [START_REF] Held | Annular phased-array high-intensity focused ultrasound device for image-guided therapy of uterine fibroids[END_REF]), a split focus [START_REF] Sasaki | Effect of split-focus approach on producing larger coagulation in swine liver[END_REF] or an optimization algorithm for the focus pattern [START_REF] Lu | Focused beam control for ultrasound surgery with spherical-section phased array: sound field calculation and genetic optimization algorithm[END_REF]. The use of a toroidal HIFU transducer may fill this therapeutic gap, since it can rapidly induce significantly larger lesions. Because of its toroidal geometry, the transducer focal zone and the lesion induced were shown to be cone-shaped [START_REF] Caloone | High-intensity focused ultrasound using a toroidal transducer as an adjuvant treatment for placenta accreta: A preliminary ex vivo study[END_REF], Dupre, et al. 2017[START_REF] Vincenot | Electronic beam steering used with a toroidal HIFU transducer substantially increases the coagulated volume[END_REF]. Under these settings, the focal zone observed in the focal plane is a ring of 10 mm in diameter. Each of the eight emitters can focus on a distinct arc of circle (1/8 of the ring) and can contribute to the formation of approximately 1/8 of a conical single HIFU lesion.

In this study, all the transducers were working in phase in order to use the natural focusing of the torus. HIFU lesions were generated using a total acoustical power of 70 W by activating the eight emitters at the same time. Due to the geometrical characteristics of a torus, the ultrasound beams coming from each of the eight emitters intersect beyond the principal focal ring and form a secondary focal zone, which contributes to reinforce the homogeneity of the lesion at its center [START_REF] N'djin | Assisted hepatic resection using a toroidal HIFU device: an in vivo comparative study in pig[END_REF] to create the HIFU lesion of 2 cm in diameter by 2.5 cm on its major axis. Larger lesions can even be produced with this device by using an electronic beam steering up to 45 cm 3 in 130 seconds [START_REF] Vincenot | Electronic beam steering used with a toroidal HIFU transducer substantially increases the coagulated volume[END_REF]. Therefore, the treatment duration is significantly shortened using this new transducer geometry, and importantly, without resorting to mechanical scanning.

The coagulated zone is large enough for simply placement of the transducer at the right location to target the tumor and produce an ablation with safety margins in 40 to 370 seconds [START_REF] Dupre | Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases[END_REF]. Compared with surgery, this method of local treatment could allow the treatment of inaccessible juxtavascular metastases and therefore may have a role in treating unresectable colorectal liver metastases and may also be used in conjunction with resection to extend its limits and thus significantly increase the number of patients eligible for curative surgery.

There are some limitations to this study. This work was performed on rabbits to find a compromise between the size of the animal and the use of a tumor model. However, the volume of rabbit livers is far away from the size of human livers but physiology is similar.

Safety outcomes following surgery in a small animal model might vary from what would be expected with surgical technique in a person with liver metastases and many additional comorbidities. This study helps to provide convincing evidence that this approach is competitive with invasive surgical resection but not to evaluate the creation of ablations in depth. This point was previously validated in a healthy pig model (Melodelima, et al. 2009).

Moreover, VX2 tumors provide an excellent model for hepatic tumors with the ability to evaluate the metastatic rate under different treatment conditions. It is the most used tumor model for large animals but VX2 is a rabbit squamous cell cancer line and not a human liver metastatic model limiting the biological evaluation that can be made. However, this model is the closest possible and the most commonly used to evaluate image-guided interventions, particularly in cancer treatment [START_REF] Aravalli | Relevance of Rabbit VX2 Tumor Model for Studies on Human Hepatocellular Carcinoma: A MicroRNA-Based Study[END_REF]. In this study there was no group combining HIFU and resection. As mentioned previously this animal model allows to demonstrate successful liver tumor ablation using toroidal focusing intra-operatively but not to evaluate a complete multimodal management protocol as it would be performed in Humans including chemotherapy, surgical resection, radiation therapy… However, demonstrating the efficacy of this device to ablate hepatic tumors under sterile conditions with adequate ultrasonic guidance for targeting and determining the interventional end-point without promoting the metastatic rate makes it possible to consider clinical application. The main advantage of this treatment during an open procedure is to make it possible to consider associating the HIFU destruction technique with hepatic resection, which seems to be a relevant strategy in curative treatment methods for resecting accessible metastases and clearing residual disease with HIFU.

In conclusion, use of a toroidal HIFU transducer allows successful liver tumor ablation and therefore could be an effective treatment for localized liver metastases. The ultrasonic guidance was found to be adequate for targeting and determining the interventional end-point.

Quantitative thermometry was not needed to achieve and validate the therapeutic endpoint.

The recurrence rate provides convincing evidence that this approach is competitive with invasive surgical resection. The metastatic rate for this invasive (but less invasive than surgery and intra-operative local destruction techniques) approach was more favorable when compared with surgical resection. Based on these results and other preclinical work, a clinical study is ongoing using this device for the treatment of liver metastases. Table 1. Tumor metastasis and grade in group 1 (HIFU), 2 (Resection) and 3 (Control).

  The animals were kept on-site 7 days before the start of the experiments. The experiments were conducted at the Institute of Experimental Surgery of the Léon Bérard Cancer Center after local institutional review board approval. These animal experiments conformed to the requirements of the INSERM Office of Animal Experimentation and were in accordance with the legal conditions of the National Commission on Animal Experimentation.

  FIGURES CAPTIONS

Figure 2 .

 2 Figure 2. VX2 tumor visualized with the integrated ultrasound imaging probe (a) just before

Figure 3 .

 3 Figure 3. Examples of treated and untreated VX2 tumors. (a) Example of a HIFU lesion

Figure 4 .

 4 Figure 4. Correlation between dimensions measured on sonograms using the integrated

Figure 5 .

 5 Figure 5. Differences in tumor volume between groups in log scale. Statistical differences

Figure 6 .

 6 Figure 6. Microscopic examination confirming homogeneous necrosis with all HIFU ablations

ACKNOWLEDGEMENTS

The authors wish to thank the staff of the laboratory for experimental surgery for their aid in the animal study. This work was performed within the framework of the SIRIC LyriCAN grant INCa_INSERM_DGOS_12563 and was partly funded by the French National Research Agency (ANR-19-CE19-0027-01).