Lise Picaut

Léa Trichet

Olivier Ronsin

Bernard Haye

Isabelle Génois

Tristan Baumberger

Gervaise Mosser
email: gervaise.mosser@upmc.fr

Pure dense collagen threads from extrusion to fibrillogenesis stability

Keywords: collagen, extrusion process, fibrillogenesis, biomaterials, threads, tendon

In tendons, collagens reach a concentration of 60%-85% of the dry weight of the tissue, collagen I being one of the major structuring agents. Collagen is found under its fibrillated state and organized in a highly anisotropic and hierarchical manner. In a first step to synthetize a mime of a tendon, we built a set up to extrude highly viscous acidic pure molecular collagen solutions (60 mg ml -1 ) into meters of regular threads of 300 μm to 600 μm in diameter. Eight different extruding and fibrillogenesis buffers were tested for their ability to generate stable anisotropic threads, with collagen fibrils characterized by the 67 nm-D period and optimally aligned parallel to the thread axis. Thanks to a follow up over 15 days, we pinned down one condition for which both diameter and anisotropy of the threads were stable throughout extrusion, fibrillogenesis and storage. The threads presented characteristic collagen fibrils and mechanical tests indicated a Young modulus around 1.7 MPa and an ultimate tensile strength of 0.3 MPa. Since neither chemical cross-linkers nor dehydrating processes were used, our synthesis process raises no biocompatibility or structure preservation concerns for tissue engineering applications, opening routes for further developments.

Introduction

Collagens are being used in biomaterials synthesis since they are the most abundant components of connective tissues. For instance in tendon, collagens reach a concentration of 60%-85% of the dry weight of the tissue [START_REF] Screen | Tendon functional extracellular matrix: TENDON FUNCTIONAL EXTRACELLULAR MATRIX[END_REF], collagen I being the major structuring bio-component. The extracellular matrix (ECM) of tendons and ligaments is composed of many other biochemical components such as non-collagen proteins and proteoglycans [START_REF] Zhang | Development of tendon structure and function: regulation of collagen fibrillogenesis[END_REF][START_REF] Screen | Tendon functional extracellular matrix: TENDON FUNCTIONAL EXTRACELLULAR MATRIX[END_REF].

The notion of fibrillogenesis is important since, in vivo, collagen is found under its fibrillar form and more over organized in a hierarchical manner from collagen I molecules to fascicles [START_REF] Benjamin | Structure-function relationships in tendons: a review[END_REF]. Fibrils are characterized by a '67 nm-D' period resulting from the longitudinal arrangement of the collagen molecules within the fibrils [START_REF] Kadler | Collagens at a glance[END_REF]. Molecules, fibrils and fascicles are aligned in a preferred orientation in relation to the function of the tissue. For instance, excised rat-tail tendons present an overall alignment of the bundles along the tendon axis. This is clearly seen by cross-polarized optical microscopy. They also reveal units, ranging from 10 to 100 μm, of alternating oblique orientations to the tendon axis. The structure resulting from the repetition of those units is referred to as 'crimp' [START_REF] Nicholls | Crimping in rat tail tendon collagen: morphology and transverse mechanical anisotropy[END_REF][START_REF] Dlugosz | Transmission electron microscope studies and their relation to polarizing optical microscopy in rat tail tendon[END_REF], is a highly compliant element and was suggested to be a natural shock-absorber [START_REF] Wang | Mechanobiology of tendon[END_REF]. Elaborations of tendon-like scaffolds are inspired by this hierarchical construct and this anisotropy [START_REF] Park | Biological responses of ligament fibroblasts and gene expression profiling on micropatterned silicone substrates subjected to mechanical stimuli[END_REF][START_REF] Wang | Cell orientation determines the alignment of cell-produced collagenous matrix[END_REF]. In this project, in order to explore new paths to produce scaffolds for tendon reconstruction, we focused on the self-assembly of collagen of dense solutions following an extrusion process.

Tendons can present chronic and/or acute injuries due to intrinsic and/or external factors [START_REF] Morais | Current approaches and future trends to promote tendon repair[END_REF]. The etiology of tendinopathies is indeed often recognized as being multifactorial [START_REF] Thomopoulos | Mechanisms of tendon injury and repair: TENDON INJURY AND REPAIR[END_REF][START_REF] Young | Etiology and epidemiology of Achilles tendon problems (Achilles Tendon)[END_REF][START_REF] Oliva | Calcific tendinopathy of the rotator cuff tendons Sports[END_REF] with causes including work and sports activities, drugs [START_REF] Lewis | Fluoroquinolones and tendinopathy: a guide for athletes and sports clinicians and a systematic review of the literature[END_REF], age and biomechanical imbalance [START_REF] Morais | Current approaches and future trends to promote tendon repair[END_REF]. Spontaneous regeneration is limited due to a poor vascularization and a low number of cells. Treatments can be conservative, surgical, or both, with or without rehabilitation programs [START_REF] Thomopoulos | Mechanisms of tendon injury and repair: TENDON INJURY AND REPAIR[END_REF][START_REF] Lomas | The past, present and future in scaffoldbased tendon treatments[END_REF][START_REF] Sun | Conservative interventions for treating Achilles tendon ruptures Cochrane Database of Systematic Reviews The Cochrane Collaboration[END_REF][START_REF] Seida | Comparative effectiveness of the nonoperative and operative treatments for rotator cuff tears Agency[END_REF]. Surgical treatments go from sutures to insertion of metallic implants or grafts, which can be auto, xeno or allografts. Synthetic grafts made of polyester (Dacron®), polytetrafluoroethylene (PTFE or Teflon®) [START_REF] Stapp | Implantable materials and grafts in tendon surgery Reconstructive Surgery of the Foot and Leg: Update 93[END_REF], silicone rubber [START_REF] Williams | Experimental evaluation of a teflon tendon prosthesis[END_REF], polypropylene (Marlex® mesh) [START_REF] Choksey | Repair of neglected Achilles tendon ruptures with Marlex mesh[END_REF], polyethylene teraphtalate (PET, devices Orthotape® and Orthocoupler®) [START_REF] Morrey | Synthetic mesh augmentation of acute and subacute quadriceps tendon repair[END_REF][START_REF] Abdullah | Usage of synthetic tendons in tendon reconstruction[END_REF] are also used. Depending on the system, more or less promising physiological results were obtained with, in some cases, no re-rupture occurrence observed at the level of the graft consolidation. Alongside with those synthetic grafts, grafts made out of biopolymers are also being developed to mimic as well as possible the biochemistry and three-dimensional organization of tendon in order to optimize the biocompatibility (i.e. absence of immune rejection or toxicity [START_REF] Mertz | What is biocompatibility?: a new definition based on the latest technology[END_REF], proper host-response [START_REF]and Drug Administration 2016 Use of International Standard ISO 10993-1[END_REF], promotion of cell adherence, proliferation and differentiation).

Another important characteristic of tendons is their mechanical properties characterized by the Young modulus and the ultimate tensile strength (UTS) as assessed under uniaxial loading along their longitudinal axis. Those vary from 0.5 to 1.9 GPa for the Young moduli and from 50 to 120 MPa for the UTS [START_REF] Ladd | Coelectrospun dual scaffolding system with potential for muscle? tendon junction tissue engineering[END_REF][START_REF] Jung | Role of biomechanics in the understanding of normal, injured, and healing ligaments and tendons[END_REF]. Such values have not yet been achieved in wet conditions with simple extruded collagen-based thread scaffolds. Drying before rehydration led to threads with Young Modulus of 46 MPa and UTS of 10 MPa [START_REF] Cornwell | Crosslinking of discrete self-assembled collagen threads: effects on mechanical strength and cell-matrix interactions[END_REF][START_REF] Pins | Effect of static axial strain on the tensile properties and failure mechanisms of self-assembled collagen fibers[END_REF][START_REF] Dunn | Optimization of extruded collagen fibers for ACL reconstruction[END_REF][START_REF] Kato | Formation of continuous collagen fibres: evaluation of biocompatibility and mechanical properties[END_REF] while the association with chemical cross-linking strategies made those values reach 766 MPa and 110 MPa for Young modulus and UTS respectively [START_REF] Cornwell | Crosslinking of discrete self-assembled collagen threads: effects on mechanical strength and cell-matrix interactions[END_REF][START_REF] Pins | Effect of static axial strain on the tensile properties and failure mechanisms of self-assembled collagen fibers[END_REF][START_REF] Dunn | Optimization of extruded collagen fibers for ACL reconstruction[END_REF][START_REF] Kato | Formation of continuous collagen fibres: evaluation of biocompatibility and mechanical properties[END_REF]. However, those treatments bring up concerns relative to biocompatibility and structure preservation. Thus new developments of scaffolds for tendon augmentation, and even more so for tendon replacement need to be undertaken to achieve scaffolds mimicking at best the tendon structural characteristics and mechanical properties to better resist solicitations and promote cell differentiation [START_REF] Tong | Functional replication of the tendon tissue microenvironment by a bioimprinted substrate and the support of tenocytic differentiation of mesenchymal stem cells[END_REF].

In order to address this subject, we made use of the lyotropic properties of acidic-collagen I solutions, which relate to the self-organization of collagen molecules into liquid crystal phases as soon as a critical concentration in collagen is reached [START_REF] Besseau | Liquid crystalline assemblies of collagen in bone and in vitro systems[END_REF]. These liquid crystals can be stabilized thanks to a pH increase to create organized fibrillar scaffolds. The pH increase triggers the fibrils formation while preserving the long-range liquid-crystalline organization [START_REF] Gobeaux | Fibrillogenesis in dense collagen solutions: a physicochemical study[END_REF]. We combined this approach with an extrusion process to generate high-density, anisotropic, collagen threads. Using a homemade extruding apparatus (figure 1), we focused on determining the best conditions to induce a stable sol-gel transition while preserving the alignment of the molecules and achieving optimal mechanical properties. For this, eight different conditions varying mainly in their ionic strengths were tested and further narrowed down to a total of three and then one condition.

Results

Since the goal of our work was to find the best conditions to synthesize dense collagen threads that would be stable over time, present anisotropic organization of collagen fibrils and have the highest mechanical properties, we have, all along our work, narrowed down conditions, varying mainly in their ionic strengths (table 1), to determine those providing threads of optimized characteristics.

Extrusion and maturation

All threads look similar at the time of the extrusion in all buffers (figures 2 and SI-1 is available online at stacks.iop.org/BPEX/4/035008/mmedia). Their surfaces present a 'sharkskin' roughness, generally =

´( )

v v p D D out 2 p in
. In order to apply the same shear condition for different inner diameters, the output velocity must be the same. The collagen solutions are extruded at v out =50±/-2 mm s -1 . A steel structure holds the syringe with its needle as well as an optical glass cell (Hellma®) containing the extrusion bath. Holes are drilled to create optical windows. The whole set-up is put inside a temperature-controlled enclosure. The extrusion bath can be removed at any time during the process to collect the extruded thread, while keeping the syringe in place. A quasi-parallel beam is created with a light source (S) limited by a diaphragm placed at the focal length of a convergent lens. The light beam illuminates the glass cell and the extrusion flow is followed thanks to a video camera equipped with a macro-objective and placed on the other side. Acquisition speeds of 600 frames/s can be reached. This optical system allows us to obtain extrusion images of a high contrast level as shown in the figures 2(A), (B). 'T' corresponds to the thermal regulation of the extrusion chamber. observed when extruding viscoelastic fluids [START_REF] Larson | Instabilities in viscoelastic flows[END_REF][START_REF] Denn | Extrusion instabilities and wall slip[END_REF]. The roughness amplitude is of the order of 20 μm for a thread diameter of 685 μm as measured on figure 2(A). Noticeably, after averaging the sharkskin roughness, the swelling ratio, measured just downstream of the die exit (the so called die-swell, of mere viscoelastic origin), remains close to 1 within the experimental error, i.e. the die swelling -an emblematic viscoelastic effect [START_REF] Miller | Swelling behavior in extrusion[END_REF][START_REF] Tanner | A theory of die-swell[END_REF]-is absent under the experimental conditions of this study. Figure 2 illustrates the threads synthesized using buffers 1, 7 and 8 as visualized during extrusion thanks to the optical system (figures 2(A) and (B)), and after the two weeks of maturation under polarized light (C) and in between crossed polarizers (D). We see that the threads diameters evolve differently depending on the buffers. No changes are observed in condition 7, while swelling and shrinkage are respectively observed in conditions 1 and 8. In figure 2(D), threads are positioned at nearly 45°to both polarizers. They appear homogeneously bright along the thread axis. In the case of condition 1, the bright signal concerns only the central part of the thread. Figure 3(A) shows the evolution of the threads diameter over time during the maturation process for all buffers and figure 3(B) shows the plot of the swelling ratio after 10 days as a function of the ionic strength. The former shows that diameters evolve over 10 days and then reach a plateau. The latter reveals three groups of behaviors. For the first one, consisting in a cluster of conditions 1 to 6, we find a D/D in >1 indicative of a swelling of the threads. For the second one, consisting in buffers 7, we observe a ratio D/D in =1, indicating that the diameters remain constant over time. Finally for the third group, buffers 8, we observe D/D in <1, in agreement with the observed shrinkage of the collagen thread diameters. Data are summarized in table 1.

At this stage, we chose to focalize only on conditions 1, as representative of the cluster of conditions 1 to 6, as well as on conditions 7 and 8.

Structural characterization

Figure 4 shows the external morphology and ultrastructures of threads obtained with fibrillogenesis buffers 1, 7 and 8 as seen by SEM. At low magnifications (200 M, 1 kM, 5 kM, top three lines), we observe the overall characteristics of the threads. In condition 1 (A), threads present an outskirt of large 'hairs' while, in condition 8 (C), threads present a smooth shell with hills and valleys oblique to the thread axis with some localized cracks revealing the internal ultrastructure. In condition 7 (B), the threads are smooth or with ridges running longitudinally along the thread axis with a few 'hairs' sticking here and there. At higher magnifications (bottom line), in condition 1, we see large (≈560 nm) linear fibers or bunches of compact fibrils (≈150 nm) on top of more tortuous fibrils (≈70 nm). The fibers present a large striation of ≈180 nm and sub-striations of approximatively 67 nm. In condition 8, we observe a smooth top shell under which linear fibers and fibrils (approximatively 150 nm in diameter) can be seen. The fibers present large striations of varied lengths and a sub striation of ≈67 nm. Helical fibers are also clearly seen. In condition 7, densely packed wavy fibrils with diameters of ≈100 nm are seen with the 67 nm cross-striation. The fibrils are fused in a more or less wavy pattern. Some larger striations can also be seen.

Table 1. Fibrillogenesis buffers and collagen threads diameters ratios after 10 days of maturation. The table gives the reference number of each buffer, its composition and final ionic strength calculated as indicated in material and method section. D/D in gives an indication of the swelling of the threads in each buffer over the time of maturation. Din is the inner diameter of the extrusion needle and D the diameter of the thread. In this table the ratio D/Din is given at Day 10. Note that the pH was adjusted to 7.4 for all buffers. Figure 5 shows the distribution of the fibrils as well as their morphology as seen by transmission electron microscopy. Figures 5(A)-(C), are cross-sections of threads respectively made in buffers 1, 7 and 8, at low magnification on the top part and high magnification on the lower part. The pictures show views from the external (Ex) part to the internal part (In) of the threads as indicated by the side arrows. In 5 A, we see that the thread presents very large fibrillar entities (≈500 nm) on the outmost edge of the thread. Further in, we observe large (≈250 nm) individualized fibers that do not present any specific alignment. Finally, in the core part of the thread, we observed thin fibrils (≈40 nm) tightly packed, most of them being cut perpendicular to their axis thus giving the dotted features on the photo. Figure 5(B) shows ultra structure of a thread made in buffers condition 7. We see a cohabitation of thick (≈80 nm) fibrils together with packages or aggregates of thin fibrils (≈20 nm). The 67 nm D-period is clearly seen. In figure 5(C), we observe a section of a thread made in buffers condition 8. Here, we observe dense packages (≈200 nm) of small fibrils (≈25 nm) in majority aligned with the axis of the thread in the external part and more randomly orientated especially in the core.

No

Mechanical properties

Figure 6 shows the results of the mechanical tests and the threads after breakage. Figure 6(A) shows typical stress curves as a function of the strain obtained for the 3 selected conditions 1, 7 and 8. Figure 6(B) shows typical profile of thread extremities after breakage. figure 6(C) shows Young modulus and Ultimate Tensile Strength obtained for the three conditions as measured over more than 4 threads for each of them. We see that the highest mechanical properties are obtained for threads extruded and maturated in PBS5X that exhibit a Youngs modulus of Figure 2. 60 mg ml -1 collagen thread extrusion and maturation. Figures 2(A) and (B) shows the threads extrusions in extrusion buffers 1, 7 and 8. Figure 2(A) corresponds to the extrusion through a 650 μm inner diameter needle in condition 1, while figures 2(B)-(D) correspond of extrusion in the three buffers through a 390 μm inner diameter needle. A sharkskin pattern is observed on all threads, although is less pronounced in condition 8. Figure 2(C) shows the threads after the two weeks of maturation in the respective extrusion buffers. We observe the increase and shrinkage in diameters of threads extruded in condition 1 and 8 respectively, while the diameter of the threads in condition 7 does not evolve. The thread extruded and matured in buffers 1 shows a core that is denser than the shell. Figure 2(D) shows the matured threads as seen in between cross-polarizers (crossed arrows) and at around 45°f rom both polarizers. We see that in all the three conditions, the threads appear very bright indicating an overall alignment of the molecules. For the thread made in buffer 1, only the core shows birefringence. The shell, which limit is seen on the overlaid inset taken in bright field mode, appears dark and remains invisible. This suggests a loss of molecular orientation along the thread axis.

Discussion

The main objective of this work was to optimize the synthesis protocol in order to reproducibly obtain homogeneous, high-density, anisotropic collagen threads with the best mechanical properties, that is high Young modulus and high UTS. As criteria we have selected: (1) the stability of the thread upon storage in the fibrillogenesis buffers to avoid swelling or contraction, (2) the ultrastructure of the threads, (3) their mechanical properties.

Another figure of merit is the straightness of the thread. This one can be affected by several gross instabilities [START_REF] Larson | Instabilities in viscoelastic flows[END_REF][START_REF] Denn | Extrusion instabilities and wall slip[END_REF]. It is controlled both by the extrusion velocity and the needle diameter. In the present work, we chose the extrusion speed according to the analysis previously made on alginate samples [36] so as to limit instabilities. We performed most of the present study with a needle of 390 μm diameter to produce threads easier for post-production processes (such as weaving), as compared to collagen threads with similar properties produced in the other studies, which diameters range from 50 to 200 μm [START_REF] Cornwell | Crosslinking of discrete self-assembled collagen threads: effects on mechanical strength and cell-matrix interactions[END_REF][START_REF] Dunn | Optimization of extruded collagen fibers for ACL reconstruction[END_REF][START_REF] Kato | Formation of continuous collagen fibres: evaluation of biocompatibility and mechanical properties[END_REF][START_REF] Cavallaro | Collagen fabrics as biomaterials[END_REF]. Noticeably, threads extruded at D in =650 μm had similar structural and mechanical properties than those at 390 μm diameter with adapted extrusion speed. Remarkably, upon extrusion, the thread diameter remains equal to the inner diameter of the needle. It entails that the viscoelastic iconic effect known as 'die swelling', which may result in an up to 3-fold increase of the extrudate diameter with respect to the nominal (needle) one [START_REF] Miller | Swelling behavior in extrusion[END_REF], is inhibited with our collagen solutions. This unexpected and still unexplained behavior is important since it makes it possible to control directly the thread diameter. This observation is puzzling since, even for a Newtonian fluid, a die swelling ratio of 1.1 is observed [START_REF] Tanner | A theory of die-swell[END_REF]. Gross slip would suppress both wall shear and normal stresses responsible for die swelling. However, it unlikely takes place in our case, since the presence of the sharkskin is both indicative of a strong adhesion of the solution at the wall and of high shear rates required to develop significant normal stresses. At this stage, one can tentatively suggest that the resulting strong anisotropy of the collagen solution could be one factor precluding the die swelling. The small irregularity of the piston drive is smoothed out by the fluidic circuit itself, which has a low pass cutoff frequency (<1 Hz) much smaller than the periodicity of the steps (100-1000 Hz). Consequently, the otherwise non-periodic sharkskin roughness cannot be attributed to the stepped nature of the drive.

Pins et al (1995) [START_REF] Pins | Effect of static axial strain on the tensile properties and failure mechanisms of self-assembled collagen fibers[END_REF] have shown the importance of fibril alignment on the mechanical properties obtained, starting from a 10 mg ml -1 collagen thread, and using a multistep procedure with two air-dryingrehydration stages, interspersed by a stretching step of different spans to align and condense the collagen fibrils. The authors showed that on the final products, synthetized in absence of cross-linking, the linear Young modulus increased from 1, 8 MPa at 0% stretch to 46 MPa at 50% stretch, and UTS from 0, 9 MPa to 7 MPa. They made no ultrastructural study but showed the overall increased orientation by the increase of birefringence values. They concluded that collagen fibrils alignment within the thread increased its mechanical properties. Therefore, since after extrusion our threads presented good anisotropy along their axis, it seemed important to us that the Figure 3. Evolution of thread diameters upon maturation for the different buffers. After extrusion, the threads are left in the buffers for two weeks for a maturation process. Figure 3(A) presents the evolution of the diameters of the thread, which have been measured regularly along the two weeks. We see that depending on the buffer some threads see their diameter increasing and some decreasing. In condition 7 the diameter remains relatively stable throughout the two weeks period. Figure 3(B) shows the ratios D/D in , measured at Day 10, as a function of the ionic strength of the corresponding buffers. The ratios are negatively correlated to the ionic strength. maturation and storage stages maintained this anisotropy to ensure better mechanical properties. In this context, finding conditions for which the thread diameter remained stable appeared to us mandatory as any change could potentially reflect a reorganization of the collagen molecules or fibrils, and a loss of this alignment. In our experimental conditions, after fifteen days of maturation, we see by polarized light microscopy that the overall alignment of the threads was still present for the three conditions 1, 7 and 8, but only in the central part of the thread in condition 1. Since on the other hand our observations and measurements indicate that threads in condition 1 had swelled during the maturation process, we believe that the swelling only affected the external part, disturbing the alignment of collagen molecules and fibrils at the surface of the thread. This result points out the direct impact of the rate of fibrillogenesis upon the homogeneity of the thread. To further sustain this, we see that in the case of threads in buffers 8, we observe shrinkage correlation with sharkskin structure. Although this one was observed in all different buffers right after extrusion, it was only preserved in buffers 8. In this case, the fibrillation occurred more rapidly than the fluid relaxation and faster than the osmotic swelling. It has already been shown at low collagen concentrations, that many parameters such as collagen concentration, pH, ion types, temperature, are at play in the fibrillogenesis processes [START_REF] Mertz | Interactions of inorganic phosphate and sulfate anions with collagen[END_REF], and this is further confirmed at higher concentrations used in our systems. The effectiveness of phosphate anion in changing the rate of fibrillogenesis has been reported for long on diluted systems [START_REF] Hayashi | Factors affecting the interactions of collagen molecules as observed by in vitro fibril formation[END_REF][START_REF] Williams B R, Gelman R A, Poppke | Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results[END_REF]. Using pepsin-treated collagen (4 mg ml -1 ) Hayashi has shown that, at pH 6.8, with equal concentrations of monovalent and divalent ions, the time length to reach opacity was at a 4(A)-(C) correspond respectively to threads synthesized in condition 1, 7 and 8. The magnification increases from top to bottom (200 M, 1 kM, 5 kM, 30 kM). The cross-striated appearances of fibrils and fibers are clearly seen at 30 kM in all three conditions. In condition 1, a 67 nm striation is further integrated in a wider one of 180 nm. In condition 7, the 67 nm striations dominate although curly or wavy undulated fibrils, fused or not, and which wavelength or pitch give other repeated distances, are seen locally. In condition 8, the 67 nm striation is seen both on straight and on helical fibrils. peak for a concentration around 150 mM phosphate. At pH 6, the precipitation was first retarded from 130 to 230 mM then accelerated again at 310 mM, indicating also an effect of pH. In our experiments, at high collagen concentration, we observed roughly that the higher the ionic strength, the faster the opacification. Our study highlights phenomena at play in dense collagen solutions, moreover constrained in cylindrical geometries, and the possible role of kinetics formation in the final structures obtained.

The ultra-structural characterization of the different threads confirmed the core-shell nature of the thread synthesized in buffers 1. It revealed very large aggregated fibers with wide interspaces at its outmost edge, while in the internal part, smaller fibrils were found. We discarded this condition due to this pronounced difference from external to internal part. Threads made in conditions 7 and 8 both presented ultra-structures more homogeneous than in condition 1. However, in condition 8, the threads showed a diameter contraction together with the presence of a crusty and smooth surface. This crusty and smooth surface may result from an artifact of SEM preparation although TEM images also revealed a rather continuous limit of very small fibrils. We discarded this condition since we believed the small fibril diameters, Figure 5. Ultrastructure of threads after maturation in buffers 1, 7 and 8 as seen by TEM (transverse sections). All threads were extruded through a 390 μm inner diameter needle. Figures 5(A)-(C) correspond respectively to threads synthesized in condition 1, 7 and 8. Top: view at low magnification from the outskirt (when present) to the inner part of the thread. Bottom: view at higher magnification from the outskirt to the inner part of the thread. Collagen fibrils appear cut in all directions from longitudinal to transverse, in which case they appear as dots. In A, the thread presents three different structural characteristics when going from the outskirt of the thread to the inner part. In B and C the threads present more homogeneous appearances. In B two types of fibrils are observed, some with greater diameters than others, while in C most of the fibrils appear with a reduced diameter. smaller than those found in living tissues, exception made of cornea, would be unfavorable for cell attachment and colonization. Thus, although we observed two types of fibrils by TEM and SEM in the case of condition 7, the otherwise overall homogeneity of ultra-structure seemed to us more favorable for future developments. Concerning the 67 nm D-bands characteristic of fibrillar collagen, although the thread diameters evolved over four days of maturation before reaching stabilization, the D-period was still observed after two weeks in our three selected conditions (1, 7 and 8) indicating good and stable fibrillogenesis. This differs from the result of Uquillas et al (2011) who, on different collagen constructs and using SAXS, showed that the D-bands were present after 12 h incubation in PBS 1X but not in PBS 10X, and that this pattern also disappeared after 96 h incubation in PBS 1X [START_REF] Uquillas | Effects of phosphate-buffered saline concentration and incubation time on the mechanical and structural properties of electrochemically aligned collagen threads[END_REF].

Finally, concerning the mechanical properties of our threads, we see that threads made in condition 7 present the best properties. Indeed, they present the highest values of Youngs modulus (1.66±0.45 MPa), for the highest strain at break (48%±5%) and the largest area under the curve that is the highest energy dissipated during the rupture process. The difference in the breaking mode of the three types of threads is also seen on their profiles adopted after rupture: in case of buffers 1, the thread breaking is sharp and localized, whereas in case of buffers 8 the breaking propagates through a sliding mode resulting in fiber-like tips at both ends. In the case of condition 7, the tip profile presents small sharp breaks observed at different positions along the longitudinal axis. From those observations, it stands out that the mechanical properties obtained in condition 7 are the best. The values obtained in this condition may seem rather low compared to other reported scaffolds [27-29, 37, 45]. However, the use of concentrated collagen solutions enables reaching moduli in the MPa range in absence of any cross-linking, airdrying and dehydration, while respecting the requirements of biocompatibility and structural preservation. Moreover, the already good mechanical properties enable further bottom-up post-processing like braiding and weaving while preserving the qualities of the original threads.

In conclusion, the homemade set up built to extrude dense and highly viscous collagen solutions enabled us to extrude regular threads over meters for collagen at 60 mg ml -1 . Through the analysis of different buffers, we have selected a condition (PBS 5X). In this condition, the diameter of the extruded solution was preserved during fibrillogenesis and storage. Collagen fibrils, presenting the '67 nm-D period', with two diameters (80 and 20 nm) were generated, the majority of which were aligned parallel to the thread axis as revealed under cross-polarizers. However, electron microscopy reveals fibrils misaligned to this axis up to a 90°deviation. This local disorganization, compared to the overall alignment of the fibrils within the thread, most likely interferes with the preferential sliding along the thread axis upon traction. Thus it may contribute to the mechanical strength of the threads. The threads synthesized in this condition present a Young modulus of 1.66±0.45 MPa and an ultimate tensile strength of 0.33±0.08 MPa without the use of any cross-linkers or any drying step. We suggest those conditions to be good candidates for future development of tendon-like scaffolds in an upgraded synthesis process enabling the generation of a crimp-like structure.

Materials and methods

Collagen type I preparation

Type I collagen was purified from young Wistar or Sprague-Dawley rat tail tendons as previously described [START_REF] Tidu | Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration[END_REF]. Extracted tendons were washed several times with phosphate-buffered saline (PBS) to remove blood and fat traces. They were subsequently soaked and washed in 4 M NaCl solution to remove cells. Finally, tendons were extensively rinsed again with PBS and solubilized in 0.5 M acetic acid. After successive and selective precipitations, respectively at 0.3 M and 0.6 M NaCl, the final collagen I pellets were solubilized in 500 mM acetic acid and thoroughly dialyzed against the same solvent to completely remove salt content. The solutions were kept at 4 °C and centrifuged at 41 000×g (Beckman Coulter J26-XP) for 4 h before use. Sample purity was assessed by SDS-PAGE electrophoresis. Collagen concentration was determined from the acidic solution by titrating the amount of hydroxyproline.

Collagen solutions were concentrated using centrifugal filtration units (Vivaspin®, Sartorius, with a 100 kD cutoff) spun at 3000xg, 10 °C, until reaching the desired final concentrations (60 mg ml -1 ). Solutions were then placed in 1 ml syringes and degassed by centrifugation at 3000xg for 30 min at 10 °C.

Extrusion of dense collagen solutions

The homemade extrusion set up, schematized in figure 1, was built to allow: (i) in-sight visualization, (ii) application of large forces compatible with highly viscous solutions, (iii) precise monitoring of the extrusion speed and the buffers exchange during extrusion. Briefly, a 1 ml syringe, filled with a 60 mg ml -1 collagen solution in 500 mM acetic acid, was mounted with a 20 or a 23-gauge (inner diameter D in of 650 and 390 μm respectively) blunt stainlesssteel needle, and loaded vertically. A motorized stage was used to bring down a rod in contact with the syringe piston and to impose its speed. The extruded fluid velocity (about 50 mm s -1 ) was adjusted so as to ensure that the system was stable against the helical instability evidenced with alginate solutions [START_REF] Picaut | Experimental evidence of a helical, supercritical instability in pipe flow of shear thinning fluids[END_REF]. The collecting cuvette was filled with the fibrillogenesis buffer (pH 7.4). At the blunt needle exit, extruded threads, plunging into the fibrillogenesis buffer, were observed and followed by an optical through-hole mounting. Prior to their use, the threads were kept and left for maturation in fibrillogenesis buffer at room temperature for 2 weeks under gentle agitation to prevent them to stick together.

Several fibrillogenesis buffers were tested with ionic strengths ranging from 0.15 M to 1.65 M (table 1). The buffers' pH were adjusted prior use to pH 7.4 with HCl or NaOH solutions depending on the initial pH measured. Same buffers were used for extrusion and maturation. Ionic strength was calculated by taking into account all the ions present in the solution according to the equation = å

= I cz, i n i i 1 2 1
2 where c i is the molar concentration of each ion and z i the charge of that ion, assuming ideal solution.

Maturation

Collagen threads (60 mg ml -1 in collagen), were kept over two weeks at room temperature in the different buffers listed in table 1. Their diameters D were measured along the maturation process on at least 4 threads and on 3 different areas for each (n>12). For each buffer condition and each time, standard deviation of thread diameter was determined classically as the dispersion of those threads measurements by calculating the square root of the variance.

We define the swelling ratio as D/D in . D is measured and plotted over time. Standard Deviation of this ratio was calculated by:

D = ´D + ´D ( ) ( ) ( ) D D , D D D in D 1 2 1 2
in in where DD and DD in are the respective standard deviations of the thread diameter and the needle inner diameter measurements.

Microscopy analysis

For optical analysis, threads were put in a homemade thin glass-chamber filled with fibrillogenesis buffers to prevent dehydration during observation. Polarized light microscopy was performed using a transmission Nikon Eclipse E600 Pol, equipped with crossed polarizers and a Nikon DXM 1200 CCD camera.

For electron microscopy analysis, all samples were fixed using 3.63% glutaraldehyde in a cacodylate/saccharose buffer (0.05 M/0.3 M, pH 7.4) for 1 h at 4 °C. For scanning electron microscopy (SEM), the samples were then washed three times in a cacodylate/saccharose buffer (0.05 M/0.3 M, pH 7.4) and dehydrated through successive ethanol baths with increasing concentrations from 70% to 100% alcohol. Thereafter, samples were dried at the carbon dioxide critical point and gold sputtered (20 nm) for analysis. Samples were observed at 200 M, 1 kM, 5 kM and 30 kM with a Hitachi S-3400N SEM operating at 8 kV with a probe current around 95 μA. For transmission electron microscopy (TEM), samples were postfixed with 2% osmium tetroxide in cacodylate/saccharose buffer solution, washed in cacodylate/saccharose solution, dehydrated in successive ethanol baths (from 50% to 100%), and finally embedded in araldite. Embedded samples were sectioned on an Ultracut Reichert Jung as already described [START_REF] Tidu | Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration[END_REF]. 500 μm thin sections were stained with toluidine blue and observed on a Nikon Eclipse E600 Pol microscope equipped with a 40× /0.65 NA objective and a DXM 1200 CCD camera. 70 nm ultrathin sections were contrasted with uranyl acetate and observed on a transmission electron microscope FEI Tecnai spirit G2 operating at 120 kV. Images were recorded on a CCD Camera (Orius Gatan 832 digital) at 4.4 kM and 15 kM.

Mechanical tests

Mechanical tests were performed using a BOSE® ElectroForce® 3200 Series II Test Instrument. Specimens were attached to the stainless steel crossheads by gluing each extremity with a cyanoacrylate adhesive drop. Samples and crossheads were immersed in a glass Petri dish filled with fibrillogenesis buffers at room temperature. The thread diameter D was determined by optical microscopy assuming full cylindrical symmetry. Mechanical loading of the thread was achieved by driving one crosshead at a constant velocity of 0.1 mm per second with a stepping motor (0.1-micron steps). The loading force F was measured with a 220 g load cell. We defined the nominal tensile stress as σ=4 F/πD 2 . The nominal strain is defined as ε=(L -L 0 )/L 0 , where L is the distance between the glue points and L 0 the initial length. All measurements were performed up to the specimens rupture. The resulting stress-strain curves were plotted and analyzed. At least 4 different threads were tested for each condition. The initial « toe » of the σ(ε) curve (ε £ 2%) corresponds to the initial straightening of the thread. The Young modulus is determined as the slope dσ/dε in the following linear regime (typically, 2% < ε < 5%). The UTS is determined as the maximum stress of the σ(ε) curve.

Contributors

Lise Picaut, did the experiments, analyzed the data and participated in writing the article.

Léa Trichet, daily supervised the work and wrote the article.

Olivier Ronsin, programmed the extrusion set up software and set the tensile testing parameters.

Bernard Haye, prepared the sample for electron microscopy observations. Isabelle Génois, made the observations of the samples by scanning electron microscopy.

Tristan Baumberger, supervised the physical part of the work and wrote the article.

Gervaise Mosser, supervised the physico-chemical part of the work and wrote the article.

Figure 1 .

 1 Figure 1. Extruding homemade experimental set up. A syringe (Terumo®) of 1 ml contains the collagen solution to be extruded. It has an inner diameter D p equal to the piston diameter of 4.45 mm. Stainless steel blunt needles (Terumo®) of 390 or 650 μm inner diameters D in , respectively 23 G and 20 G, are set at the syringe end. A steel rod is clamped to a stepping motorized stage, which imposes a velocity v p ranging from 0 to 2000 μm s -1 . The average output velocity v out is calculated as follows: =

  1.66±0.[START_REF] Kato | Mechanical properties of collagen fibres: a comparison of reconstituted and rat tail tendon fibres[END_REF] MPa and an ultimate tensile strength of 0.33±0.08 MPa, while threads synthetized in the two other conditions yield 0.30±0.15 MPa and 0.13± 0.01 MPa respectively for Young modulus and UTS in condition 1, and of 0.88±0.14 MPa and 0.16± 0.02 MPa respectively in condition 8.

Figure 4 .

 4 Figure 4. Ultrastructure of threads after maturation in buffers 1, 7 and 8 as seen by SEM. All threads were extruded through a 390 μm inner diameter needle. Figures4(A)-(C) correspond respectively to threads synthesized in condition 1, 7 and 8. The magnification increases from top to bottom (200 M, 1 kM, 5 kM, 30 kM). The cross-striated appearances of fibrils and fibers are clearly seen at 30 kM in all three conditions. In condition 1, a 67 nm striation is further integrated in a wider one of 180 nm. In condition 7, the 67 nm striations dominate although curly or wavy undulated fibrils, fused or not, and which wavelength or pitch give other repeated distances, are seen locally. In condition 8, the 67 nm striation is seen both on straight and on helical fibrils.

Figure 6 .

 6 Figure 6. Mechanical characterization. All threads were extruded through a 390 μm inner diameter needle.Figure 6(A) shows the typical profiles obtained for the stress-strain curves in the three conditions 1, 7, 8.Figure 6(B) shows the three threads at the point of breakage. Note that the breakage end is rather sharp in condition 1, intermediate in condition 7 and elongated in condition 8.Figure 6(C) gives Young Modulus and Ultimate Tensile Strength for the three conditions 1, 7 and 8 as measured over 4 threads. See Young modulus and ultimate tensile strength definitions in materials and methods (4.5).

Figure 6 (

 6 Figure 6. Mechanical characterization. All threads were extruded through a 390 μm inner diameter needle.Figure 6(A) shows the typical profiles obtained for the stress-strain curves in the three conditions 1, 7, 8.Figure 6(B) shows the three threads at the point of breakage. Note that the breakage end is rather sharp in condition 1, intermediate in condition 7 and elongated in condition 8.Figure 6(C) gives Young Modulus and Ultimate Tensile Strength for the three conditions 1, 7 and 8 as measured over 4 threads. See Young modulus and ultimate tensile strength definitions in materials and methods (4.5).

Figure 6 (

 6 Figure 6. Mechanical characterization. All threads were extruded through a 390 μm inner diameter needle.Figure 6(A) shows the typical profiles obtained for the stress-strain curves in the three conditions 1, 7, 8.Figure 6(B) shows the three threads at the point of breakage. Note that the breakage end is rather sharp in condition 1, intermediate in condition 7 and elongated in condition 8.Figure 6(C) gives Young Modulus and Ultimate Tensile Strength for the three conditions 1, 7 and 8 as measured over 4 threads. See Young modulus and ultimate tensile strength definitions in materials and methods (4.5).

Figure 6 (

 6 Figure 6. Mechanical characterization. All threads were extruded through a 390 μm inner diameter needle.Figure 6(A) shows the typical profiles obtained for the stress-strain curves in the three conditions 1, 7, 8.Figure 6(B) shows the three threads at the point of breakage. Note that the breakage end is rather sharp in condition 1, intermediate in condition 7 and elongated in condition 8.Figure 6(C) gives Young Modulus and Ultimate Tensile Strength for the three conditions 1, 7 and 8 as measured over 4 threads. See Young modulus and ultimate tensile strength definitions in materials and methods (4.5).

Acknowledgments

LP was supported by a PhD grant of the Ecole doctorale 397 (Physique et Chimie des Matériaux) from MESR.

Conflicts of interest

There are no conflicts of interest to declare.

ORCID iDs

Gervaise Mosser https:/ /orcid.org/0000-0002-9812-5340


Brice Ilharreborde

Sébastien Pesenti

Emmanuelle Ferrero

Franck Accadbled

Jean-Luc Jouve

Jérôme Sales De Gauzy

Keyvan Mazda

• Se ´bastien Pesenti

Je ´ro ˆme

Sales De Gauzy

Correction of hypokyphosis in thoracic adolescent idiopathic scoliosis using sublaminar bands: a 3D multicenter study

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

Introduction The comparison of implants and correction methods remain controversial in AIS. Excellent frontal and axial correction rates have been reported with all-screw constructs, but at the expense of sagittal alignment, which has a tendency to flatten postoperatively. Posteromedial translation using hybrid constructs seems to preserve and improve thoracic kyphosis (TK), but no series exist to date with a significant number of hypokyphotic patients. In addition, the measures of TK in 2D are often wrong in severe AIS due to axial rotation. The goals of this study were therefore to analyze the 3D radiological outcomes of a group of hypokyphotic AIS patients operated with sublaminar bands. Methods 35 consecutive AIS hypokyphotic patients (T4T12\15°) operated in three centers were included, with a minimum 2-year follow-up. The surgical technique was similar in all centers, associating lumbar pedicle screws and thoracic sublaminar bands. Posteromedial translation was the main correction technique, and no patient underwent prior anterior release. 3D spinal reconstructions were performed preoperatively, postoperatively and at the latest follow-up by an independent observer using SterEOS (EOS imaging, Paris, France), and 2D and 3D measurements were compared. In addition, a new 3D parameter [sagittal shift of the apical vertebra (SSAV)], reflecting the translation of the apical vertebra of the main curve in the patient sagittal plane, was described and reported. Results The age of the cohort was 16 years and the number of sublaminar bands used for correction averaged 6 (±1.5). T1T12 and T4T12 sagittal Cobb angles appeared to be overestimated on 2D postoperatively (3°, p = 0.002 and 4°, p \ 0.001, respectively). Hence, only 3D measurements were kept for the quantitative analysis of the postoperative correction. T4T12 TK significantly increased after surgery (average 8°± 7°, p \ 0.001), but 11 patients (31.4%) remained hypokyphotic. Seven out of the eight patients (87.5%) who presented a thoracic lordosis (i.e., T4T12 \0°) preoperatively were corrected after surgery (mean gain 16°± 4°). A posterior shift (positive SSAV) of the apical vertebra was reported in 24 patients (68.6%). In this subgroup, the mean SSAV was ?2 cm (±1). Good correlation was found between the SSAV and the postoperative change in 3D T4T12 kyphosis (r = 0.62). Conclusion Measures in 2D tend to overestimate sagittal alignment and are not sufficient to evaluate postoperative correction. SSAV is a new 3D parameter reflecting the TK change that needs to be further investigated and used in the future. This series confirms that sublaminar bands should be considered in hypokyphotic patients, since thoracic sagittal alignment was restored in 68.6% of the cases.

Keywords Adolescent idiopathic scoliosis Á Hypokyphosis Á Sagittal alignment Á Stereoradiography Á 3D analysis & Brice Ilharreborde i brice@hotmail.com

Introduction

The goal of adolescent idiopathic scoliosis (AIS) surgery is to achieve a 3D correction of the deformity and prevent curve progression of the unfused spine, while improving the overall cosmetic aspect of the trunk and preserving enough mobility for daily activities [1]. Correction has been reported with numerous systems, but the most popular technique currently relies on pedicle screws, with a recent emphasis on the axial correction of the deformity using direct vertebral derotation techniques [START_REF] Pankowski | Direct vertebral rotation versus single concave rod rotation: low dose intraoperative computed tomography evaluation of spine derotation in adolescent idiopathie scoliosis[END_REF]. However, all-screw constructs have been associated with higher hospital charges (27.6% annually in the USA) and with a tendency to flatten the thoracic spine, not observed with hybrid constructs in matched patients [3 6]. Maintaining or restoring thoracic sagittal balance is essential not only to avoid future junctional degeneration (proximal or distal), but also theoretically to improve pulmonary function, even if relevant clinical evidence is still lacking [7 9]. Analysis of the current existing literature regarding the consequences of AIS surgery on thoracic sagittal alignment is difficult due to multiple biases. First, most of the series include different Lenke-type curves, while the sagittal flattening of the spine mostly occur in Lenke 1 and Lenke 2 curves (single structural thoracic and double structural thoracic, respectively). Second, the analysis of postoperative results, even in this subgroup of thoracic structural curves (Lenke 1 and 2), is also biased by the distribution of the three Lenke's sagittal modifiers (-, N or ?). As a matter of fact, hyperkyphotic patients (sagittal modifier ?) tend to increase the mean postoperative thoracic kyphosis of the series, therefore leading to an overestimation of the surgical gain, while the number of the most challenging patients (sagittal modifier -, i.e., sagittal T4T12 Cobb \10°) is very often limited or not even reported. Last, radiological outcomes are usually assessed in 2D, and the value of such measurements in severe AIS is questionable due to apical rotation [START_REF] Newton | Defining the ''Three Dimensional Sagittal Plane'' in thoracic adolescent idiopathic scoliosis[END_REF].

While some authors advocate the use of larger diameter rods, higher screw density on the concave side or multiple facetectomies to optimize the correction of thoracic sagittal misalignment, several studies have recently emphasized the efficacy and safety of the posteromedial translation technique using sublaminar bands, even in stiff curves, with no need for previous thoracoscopic anterior release [11 14]. Since the EOS low-dose system is now accessible in routine clinical use, the goal of this multicenter study was to analyze the 3D radiological outcomes of a consecutive subgroup of hypokyphotic thoracic AIS patients, operated with sublaminar bands.

Materials and methods

Patients

Following institutional review board approval, a consecutive series of 43 hypokyphotic patients, operated for progressive Lenke 1 or 2 AIS in three university hospitals between June 2011 and May 2014 were retrospectively analyzed. Thoracic hypokyphosis was defined by a preoperative sagittal T4T12 Cobb angle \15°in both 2D and 3D measurements. A minimum 2-year follow-up was required. All patients were evaluated preoperatively in the early postoperative period (within 1 month) and at the latest follow-up. None of the patients had prior spinal surgery.

Surgical procedure

Fusion levels, implants number and localization at thoracic levels were selected according to the same criteria in all three centers. The only difference between departments regarding the operative strategy was the type of rod material used for correction (CoCr in center 1 and Ti in centers 2 and 3). All patients underwent posterior spinal fusion using hybrid constructs, combining lumbar pedicle screws [ISS (Implanet, Bordeaux, France) or Legacy (Medtronic, Minneapolis, USA)], concave thoracic sublaminar bands [Jazz (Implanet, Bordeaux, France)] and proximal hooks with 5.5 mm diameter rods (Fig. 1). No patient underwent prior anterior release before posterior fusion and no posterior osteotomy was performed during correction. The same perioperative blood saving strategy was used in all centers, associating intraoperative cell saver and tranexamic acid. Posteromedial translation was the main technique used for thoracic correction, using the progressive tension transmitted by the polyester bands to the laminae to bring the thoracic spine to the rods, manually precontoured in the sagittal plane. Spinal cord monitoring was systematically recorded.

Radiological measurements

All patients underwent low-dose stereoradiographs using the EOS system (EOS imaging, Paris, France) preoperatively, postoperatively (within 1 month), and at the latest follow-up, as previously described [START_REF] Ilharreborde | Angle measurement reproducibility using EOS three dimensional reconstructions in adolescent idiopathic scol iosis treated by posterior instrumentation[END_REF]. Spinal measurements were first performed in 2D (Kodak Carestream, Rochester, NY, USA) by an experienced independent spinal surgeon, and then 3D reconstructions were performed using SterEOS software (EOS imaging, Paris, France) by an independent Imaging reconstruction service (EOS 3DServices, Montreal, Canada). Inter-and intraobserver reliabilities of both Fig. 1 Preoperative and postoperative low dose radiographs of a Lenke 1 AIS patient treated by T3L3 posterior fusion using a hybrid construct with seven sublaminar bands methods have been previously reported [START_REF] Ilharreborde | Angle measurement reproducibility using EOS three dimensional reconstructions in adolescent idiopathic scol iosis treated by posterior instrumentation[END_REF][START_REF] Vidal | Reliability of cervical lordosis and global sagittal spinal balance measurements in adolescent idiopathic scoliosis[END_REF]. The following coronal radiographie parameters were recorded: Cobb angles of the main curve and contra-curves and Tl tilt (measured between the horizontal reference line and the upper endplate of Tl). Sagittal parameters included T1T12 and T4T12 thoracic kyphosis (TK), LlSl lumbar lordosis (LL), and pelvic parameters. 2D and 3D parameters were compared, and only hypokyphotic patients (i.e., sagittal T4T12 Cobb <15°) on preoperative 3D reconstructions were kept for analysis. ln addition, a new parameter [sagittal shift of the apical vertebra (SSA V)] was calculated from 3D reconstructions to illustrate the posterior translation of the thoracic spine. The SSA V corresponded to the projection in the axial plane (3D top view in patient' s reference) of the sagittal translation of the anatomie center of the apical vertebra of the main thoracic curve, in reference to the central hip vertical axis (CHV A) (Fig. 2). A positive value was given in case of posterior translation of the apex, while a negative value produced an anterior shift of the apical vertebra

Statistical analysis

Paired-samples Student' s t tests were used to analyze differences between 2D and 3D radiological measurements and to evaluate surgical outcomes. Pearson correlation test was used to compare the 3D postoperative gain in T4T12 kyphosis and the SSA V. All statistical tests were twotailed, and a p value <0.05 was considered significant. All statistical analyses were conducted using SPSS version 12.0 (SPSS Inc, Chicago, IL, USA).

Results

Demogr aphic and oper ative data Among the 43 consecutive Lenke 1 and 2 hypokyphotic AIS patients identified on 2D measurements, 8 were excluded because the personalized 3D reconstructions were not feasible. The reason was the existence of a lumbosacral transitional anomaly (lumbarized Sl or sacralized L5) altering vertebrae numbering on SterEOS software. The mean age of the 35 patients, therefore, kept for analysis was 16 years (±2). There were 11 boys and 24 girls, with a mean follow-up of 34 months (±8). Lenke 1 curves were the most frequent (30 cases, 85%), while Lenke 2 curves were identified in five patients (15%). The number of fused vertebrae averaged 12.5 (±1), and all fusions extended to L1 or below. The upper instrumented level (UIV) was Tl

P1 P2 f __ !~-~~~---------------------------------•-:

in 1 case (2.9%), T2 in 12 cases (34.2%), T3 in 20 patients (57.1%) and T4 in 2 cases (5.8%). The mean number of sublaminar bands used for correction at thoracic levels was six (±1.5).

Radiological measurements

Preoperative radiological values are reported in Table 1. The Cobb angle of the proximal thoracic curve appeared to be overestimated in 2D (average 5°, p = 0.003). The only significant difference between 2D and 3D measurements regarding preoperative sagittal alignment was the L1S1 lordosis, which was underestimated in 2D (mean 6°, p \ 0.001). The same finding was reported after surgical correction (Table 2). However, both T1T12 and T4T12 sagittal Cobb angles appeared to be overestimated in 2D postoperatively (3°, p = 0.002 and 4°, p \ 0.001, respectively). Hence, only 3D measurements were kept for the quantitative analysis of the postoperative correction (Table 3). T4T12 thoracic kyphosis significantly increased after the procedure (average 8°± 7°, p \ 0.001), but 11 patients (31.4%) still remained hypokyphotic according to Lenke's classification (i.e., T4T12 \10°) (Fig. 3). However, seven out of the eight patients (87.5%) who presented a preoperative thoracic lordosis (i.e., T4T12 \0°) were corrected after surgery (mean gain 16°± 4°). No significant difference was found between CoCr and Ti rods. A posterior shift (positive SSAV) of the apical vertebra was reported in 24 patients (68.6%). In this subgroup, the mean SSAV was ?2 cm (±1). Good correlation was found between the SSAV and the postoperative change in 3D T4T12 kyphosis (r = 0.62, Fig. 4). Significant spontaneous increases in T4T12 and T1T12 sagittal Cobb angles were observed during the follow-up (p = 0.002 and p \ 0.001, respectively), with a subsequent increase in the lumbar lordosis (p = 0.006) (Table 3). Similarly, the apex of the main thoracic curve significantly shifted posteriorly, while a significant loss of correction was also observed in the frontal plane (average 3°for the main curve and 4°for the proximal thoracic curve).

Discussion

While many surgeons currently advocate the use of thoracic pedicle screws for optimal care in AIS, sometimes associated with multiple Ponte osteotomies, sublaminar bands should be considered in hypokyphotic patients to reduce complication rates and in particular the risk of intraoperative concave screw failure due to pull-out forces [START_REF] Kleuver | Optimal surgical care for adolescent idiopathic scoliosis: an international consensus[END_REF][START_REF] Quan | Correction of main thoracic ado lescent idiopathic scoliosis using pedicle screw instrumentation: does higher implant density improve correction?[END_REF].

Thoracic sagittal alignment correction

Since modern implants have proved their efficacy to restore the frontal alignment in AIS, the residual challenges are axial and sagittal corrections. The axial component mostly has a consequence on the cosmetic aspect, reducing the rib hump, while correcting the thoracic hypokyphosis reduces the risk of future proximal junctional kyphosis (PJK), improves cervical balance and increases the overall thoracic volume [19 21].

Watanabe et al. have recently showed on 3D models that derotation maneuvers alter sagittal alignment. As a matter of fact, the anterior and convex overgrown vertebral walls become anterior, increasing the length of the anterior column at the expense of kyphosis [START_REF] Watanabe | Vertebral derotation in adolescent idiopathic scoliosis causes hypokyphosis of the thoracic spine[END_REF]. This might explain why many studies have reported a lordosing effect of pedicle screws at thoracic levels, which was not found when screws were used with posteromedial translation techniques [START_REF] Fletcher | Residual thoracic hypokyphosis after posterior spinal fusion and instrumentation in adolescent idiopathic scoliosis: risk factors and clinical ramifications[END_REF][START_REF] Clement | Relationship between thoracic hypokyphosis, lumbar lordosis and sagittal pelvic parameters in adolescent idiopathic scoliosis[END_REF]. More than the implants, the correction technique therefore appears to be the most important parameter to restore sagittal alignment. The results of this study focused on severe hypokyphotic AIS and confirmed that sublaminar bands used with posteromedial translation were able to correct the thoracic hypokyphosis in 68.6% of the cases, with a mean gain of 8°. In the 11 patients who remained hypokyphotic, the lack of correction can be explained by a lower tension applied on the bands, an insufficient sagittal contouring of the rods, or a greater stiffness of the spinal segments in the sagittal plane. Another hypothesis could be a greater axial correction, shifting the taller anterior and convex walls in the ventral direction of the spine, thus increasing the length of the anterior column; but this parameter was not evaluated in this study.

3D measurements

The main advantage of the current study is that only 3D measurements were kept for analysis. As a matter of fact, Newton et al. have demonstrated that most of the 2D sagittal Cobb measurements were biased and clearly underestimated the real thoracic spine flattening [START_REF] Newton | Defining the ''Three Dimensional Sagittal Plane'' in thoracic adolescent idiopathic scoliosis[END_REF]. The comparison of postoperative 2D and 3D values also confirmed these findings in our cohort, with four extra degrees on average in 2D. Standing position 3D measurements should therefore become the gold standard for sagittal balance assessment, while respecting the ''as low as reasonably achievable'' (ALARA) concept to reduce radiation exposure [START_REF] Icrp | ICRP publication 121: radio logical protection in paediatric diagnostic and interventional radiology[END_REF]. They are more reliable and accurate than the • proj ection in a random sagittal plane of the 3D deformity. The reference used in the cU1Tent study was the so-called "patient' s plane", based on the position of the hips and therefore consistent because of not being influenced by the patient's position in the cabin. Newton et al. have proposed preferring an addition of segmental planes, in which the local kyphosis is measured between each vertebra, but this method seems to be less relevant for clinical decisions [l 0). A new 3D parameter (SSA V) was also reported here for the first time. The 3D reconstructions allowed localizing the position of the apical vertebra in the patient's sagittal reference plane, and the shift of this vertebra was calculated postoperatively. The SSAV helped clearly understand the posterior translation of the thoracic spine, and therefore reflected the clinical impact of the procedure on sagittal balance. As a matter of fact, an increase of T1T 12 sagittal Cobb without positive SSAV rnight only represent a biased measure or even a PJK in some cases, since the surgical procedure would not have successfully pulled the spine posteriorly.

Limitations

This study presents some limitations. lndeed, patients were operated in three distinct centers by four different senior surgeons, using different tools for clinical assessment of the rib hump. Ti rods were used in two institutions, while the third one chose to perform the posteromedial translation technique with CoCr rods. However, no significant difference was found between the two types of rods in this specific population, and this cohort represents one of the largest series of hypokyphotic patients, who are very often underrepresented in AlS literature. ln addition, all measurements were performed in 3D using a reproducible software, and the reference plane was given by the position of the patient's hips for optimal accuracy and reliability. The spontaneous improvement of the thoracic sagittal alignment in an instrumented zone also needs to be further explored. Whether it is primitive or secondary to a physiological adaptation of the uninstrumented lumbar spine remains unclear. Longer follow-up rernains necessary to make sure that fusion will occur without further loss of correction, but no rod breakage or implant loosening was reported in this series. Finally, the correlation between the new 3D parameter, apical axial corrections, the functional outcomes, and in particular pulmonary fonction, as investigated by Yaszay et al., needs to be assessed in the future [START_REF] Yaszay | The effects of the three dimensional deformity of adolescent idiopathic scoliosis on pulmonary function[END_REF].

Compliance with ethical standard~

Conflict of interest The authors declare that they have no competing interests.

Funding Brice Ilharreborde, Keyvan Mazda and Jerôme Sales de Gauzy have received consultancy fees (unrelated to this study) from lmplanet (lmplanet, Bordeaux, France).

Fig. 2

 2 Fig. 2 Preoperative and postoperative 3D reconstructions of a Lenke 1 AIS curve, showing the posterior shift of the apical vertebra [positive SSAV (P2 P1) of T10]

Fig. 3

 3 Fig. 3 Preoperative and postoperative lateral views of 3D recon structions of a Lenke 1 AIS patient treated by T3L3 hybrid posterior fusion, showing the restoration of the thoracic sagittal alignment and the subsequent effect on the cervical spine

Table 1

 1

	Preoperative comparison between 2D and 3D	2D measurements	3D measurements	p
	measurements (N 35)	Mean	SD	Mean	SD
	Main curve (°)	56	11	53	10	0.090
	Proximal contra curve (°)	30	8	25	9	0.003
	Distal contra curve (°)	33	12	33	9	0.469
	T1 tilt (°)	3	5	2	7	0.288
	L1S1 lordosis (°)	4 0	1 4	4 6	1 1	0.001
	T4T12 kyphosis (°)	5	8	6	9	0.432
	T1T12 kyphosis (°)	11	10	13	9	0.154
	T1 slope (°)	8	6	8	8	0.077
	Pelvic incidence (°)	51	14	50	10	0.610
	Sacral slope (°)	41	9	41	8	0.961
	Pelvic tilt (°)	11	7	10	7	0.051
	Bold indicates significant p values

M 1 12 11 10 9 1 65 .. 3 2 1 01 0 1 1 1

Table 2

 2

	Postoperative comparison between 2D and 3D	2D measurements	3D measurements	p
	measurements (N 35)	Mean	SD	Mean	SD
	Main curve (°)	17	6	16	7	0.021
	Proximal contra curve (°)	18	6	15	7	0.195
	Distal contra curve (°)	8	6	9	6	0.806
	T1 tilt (°)	4	5	5	5	0.396
	L1S1 lordosis (°)	4 1	1 3	4 4	1 1	0.003
	T4T12 kyphosis (°)	17	7	13	8	0.001
	T1T12 kyphosis (°)	24	7	21	8	0.002
	T1 slope (°)	14	8	15	7	0.850
	Pelvic incidence (°)	50	12	50	11	0.827
	Sacral slope (°)	38	9	39	7	0.159
	Pelvic tilt (°)	1 5	1 2	1 2	8	0.021
	Bold indicates significant p values

Table 3

 3 Comparison between preoperative, postoperative and final follow up 3D parameters(N 35)

		Preoperative measurements	Postoperative measurements	Follow up measurements
		Mean	SD	Mean	SD	Mean	SD
	Main curve (°)	53	10	16*	7	19**	7
	Proximal contra curve (°)	25	9	15*	7	19**	8
	Distal contra curve (°)	33	9	9*	6	9	7
	T1 tilt (°)	2	7	5 *	5	5	6
	L1S1 lordosis (°)	46	11	44	11	51**	11
	T4T12 kyphosis (°)	6	9	13*	8	18**	8
	T1T12 kyphosis (°)	13	9	21*	8	27**	9
	T1 slope (°)	8	8	15*	7	18**	7
	CHVA apex sag (mm)	1.4	2	1.8	3	3**	2
	CHVA apex coronal	5	2	0.8*	1	1.4**	1
	Pelvic incidence (°)	5 0	1 0	5 0	1 1	5 0	1 1
	Sacral slope (°)	41	8	39*	7	41	8
	Pelvic tilt (°)	10	7	12*	8	9	8

* Significant difference between the pre and postoperative measurements ** Significant difference between the postoperative and final follow up measurements


Ecolab Nicolas Guillerault
email: nicolas.guillerault@univtlse3.fr

Eb A S T I E N D E L M O T T E ‡

S T Ep H A N I E B O U L Êt R E A U

Christine Lauzeral

Nicolas Poulet

E R I C S A N T O U L

Does the non-native European catfish Silurus glanis threaten French river fish populations?

Keywords: alien predator, before after analysis, biological invasion, control impact analysis, impact assess ment

1. The European catfish, Silurus glanis, was widely introduced in western Europe, and it has now established self-sustaining populations in numerous large rivers of western France. Using data collected from surveys conducted by the French National Agency for Water and Aquatic Environment (Onema) from 1989 to 2010 in more than 500 sites throughout the country (10636 electrofishing surveys), we investigated the potential impact of the European catfish on fish communities in French rivers. 2. In the first part of the analysis, we compared trends observed before and after the European catfish was established at given sites (before after analysis). Species richness, evenness and diversity decreased significantly after the European catfish was established at 1.4%, 1.4% and 5.8% of the sites, respectively. Total fish biomass and density decreased significantly at 6.6% and 2.9% of the sites, respectively. In the second part of the analysis, we compared sites with European catfish against sites lacking European catfish during the same period (with without analysis). Fish species richness was significantly higher in sites with European catfish. No significant differences in fish diversity, evenness, total fish biomass or density were found between sites with or without European catfish. 3. While our results indicate that the European catfish may in a few cases impact fish communities or populations, it does not appear to be responsible for a countrywide collapse in fish assemblages.

Introduction

Several threats to inland water ecosystems have been identified, among which invasions are recognised as serious menaces [START_REF] Dudgeon | Freshwater bio diversity: importance, threats, status and conservation challenges[END_REF]. Often resulting from accidental or intentional translocations of species by humans, invasions affect a wide range of plant and animal taxa in many lakes and rivers worldwide (Garc ıa-Berthou, 2007;Ricciardi & MacIssac, 2011). Freshwater fish are among the most introduced animals worldwide [START_REF] Copp | To be, or not to be, a non native freshwater fish[END_REF][START_REF] Almod | Freshwater fish introduc tions in Spain: facts and figures at the beginning of the 21 st century[END_REF], mainly because of escapes from aquaculture or intentional releases for recreational fisheries [START_REF] Copp | Propagule pressure and the invasion risks of non native freshwater fishes: a case study in England[END_REF][START_REF] Welcomme | Inland cap ture fisheries[END_REF]Cucher-ousset & Olden, 2011). Non-native game fish are often significantly larger than native species and occupy significantly higher trophic levels [START_REF] Donaldson | Con trasting global game fish and non game fish species[END_REF]. In freshwater ecosystems subject to strong top-down control [START_REF] Shurin | A cross ecosys tern comparison of the strength of trophic cascades[END_REF], fish introductions may have important effects [START_REF] Vander Zanden | Stable isotope evidence for the food web conse quences of species invasions in lakes[END_REF] such as changes to prey-size refugia and thus to predator prey equilibria [START_REF] Chase | Food web effects of prey size refugia: var iable interactions and alternative stable equilibria[END_REF][START_REF] Clavero | Invasive species are a leading cause of animal extinctions[END_REF] or energy fluxes [START_REF] Macavoy | Marine nutrient contributions to freshwater apex predators[END_REF].

One of the most popular and successful fish introductions in Europe has involved the European catfish, Silurus glanis. Originating in eastern Europe and western Asia, the species was introduced to France at the end of Analyses were conducted using R software (R Development Core Team, 2014). The impact of S. glanis was defined from the standpoint of a quantifiable negative effect on the recipient environment (sensu [START_REF] Gozlan | Introduction of non native freshwater fish: is it all bad?[END_REF].

Before after analysis

We considered impact as a significant decrease in total fish richness, native fish richness, diversity, evenness, biomass or density after S. glanis establishment at a site. Fish assemblages were so different among sites that it was impossible to perform a global analysis.

Silurus glanis was considered established at a given site as soon as one individual was caught (t 0 ). Sites that were sampled at least once before t 0 and after t 0 were selected. Among these, sites with at least four years of S. glanis presence were used for the analysis (n 112).

Differences between the descriptive variables in each site (total species richness, native species richness, diversity, evenness, biomass and density) recorded before t 0 and after t 0 were tested using the Mann Whitney test. When a significant negative difference was identified, the Spearman correlation and autocorrelation tests (rank 1) with time before t 0 were used to detect a general negative temporal trend experienced prior to t 0 and not attributable to the presence of S. glanis.

The before after analysis does not allow the assignment of negative trends observed in the field to S. glanis (i.e. correlation does not imply causation). We performed the before after analysis with a comparison of sites with and without S. glanis (with without analysis) to parallel the intervention analysis (IA) and impact versus references sites (IVRS) analyses of Stewart- [START_REF] Oaten | Temporal and Spatial variation in environmental impact assessment[END_REF].

With without analysis

The impact of S. glanis was judged according to whether sites with S. glanis (W-sites) exhibited significantly lower total species richness, native species richness, diversity, evenness, biomass or density than sites where environmental conditions would allow the presence of S. glanis but where it has never been recorded (while electrofishing or angling; Wt-sites).

The W-sites were selected using species distribution models (set from 236 occurrence and 874 absence sites throughout France), taking into account elevation, distance from source, slope, catchment surface, mean temperature in January and mean temperature in July. Because of the non-native status of S. glanis, many of these absences were not environmentally caused. We the 19th century and has now established self-sustaining populations in many French rivers [START_REF] Copp | Voracious invader or benign feline? A review of the environmental biology of European catfish Silurus glanis in its native and intro duced ranges[END_REF][START_REF] Poulet | Time trends in fish populations in metropolitan France: insights from national monitoring d ata[END_REF]. This long-lived predator may reach a total length of over 2.5 m, becoming twice as large as the native piscivore, northern pike (Esox Lucius).

The large number of videos broadcast and articles published in newspapers and angling magazines, feeding myths about the size or behaviour of specimens caught, and even some scientific publications (e.g. [START_REF] Cucherousset | Freshwater killer whales': beaching behavior of an alien fish to hunt land birds[END_REF], have resulted in strong views for or against S. glanis and deep concerns about its foraging behaviour and impact on freshwater biota. Although S. glanis has been hypothesised to affect waterbird populations in Spanish reservoirs [START_REF] Carol | Growth and diet of European catfish (Silurus glanis) in early and late invasion stages[END_REF], little is known about the ecology of the introduced S. glanis and there is no evidence that it as a voracious predator [START_REF] Copp | Voracious invader or benign feline? A review of the environmental biology of European catfish Silurus glanis in its native and intro duced ranges[END_REF]Syv€ aranta et al., 2010).

Using data collected from 10636 electrofishing surveys conducted within the last two decades in more than 500 sites throughout France, we aimed to evaluate the potential impact of S. glanis on riverine fish populations in the metropolitan area of France. Fish species richness, evenness, diversity, density and biomass were compared in different rivers (i) prior to and after S. glanis establishment (before after analysis) and (ii) between sites with and without S. glanis (with without analysis).

Methods

Fish survey

The French National Agency for Water and Aquatic Environments (Onema) has been monitoring freshwater fish populations in the whole river network of France for several decades. Sites were sampled during the lowflow period (from May to October) using standardised electrofishing procedures appropriate to prevailing river widths and depths. Streams were sampled by wading (mostly two-pass removal), while fractional sampling strategies were used in larger rivers (see [START_REF] Poulet | Time trends in fish populations in metropolitan France: insights from national monitoring d ata[END_REF]. Fish were identified to species, counted, weighed and then released.

Data analysis

Only surveys performed with the same sampling protocol at a given site were selected. Density and biomass were expressed as fish number and biomass per 100 m 2 . Species diversity was described using Shannon's diversity index and evenness using Pielou's evenness index.

thus chose an iterative ensemble modelling approach [START_REF] Lauzeral | Dealing with noisy absences to optimize species distribution models: an iterative ensemble modelling approach[END_REF] that combined six different statistical methods. Two-thirds of the sites were chosen at random and used to calibrate the models, and the other third was kept for independent evaluation. The calibrated model was then projected over France, giving rise to a potential species distribution. The selection of the calibration data set was repeated ten times. The ten resulting distributions were then summed, giving rise to a map of the level of habitat suitability. Each modelselected site (n 521) was submitted to the expertise of field technicians from Onema. First, the experts were asked to judge the probability of S. glanis presence in or near the site. In fact, especially in large rivers, European catfish is not always detected using electrofishing and the field technicians were sometimes aware of the presence of the species (from anecdotal evidence of anglers). Then, the experts were asked to judge the suitability of habitat at each site, to refine results from the model (10' 9 10' resolution). Some model-selected sites had local habitat features (not previously used in the model) that were not suitable for S. glanis (e.g. water velocity). Finally, 47 stations were confidently selected.

The previously selected Wt-sites were compared with W-sites showing homogenous environmental conditions. To identify these W-sites, a hierarchical classification (HC, Ward's method) was performed on the environmental data set containing both W-sites and Wt-sites. This hierarchical classification generated four groups. A redundancy analysis (RDA) showed significant differences between the groups (99% randomisation test, ADE4 package for R), although two groups of stations overlapped, revealing very close environmental conditions. These two groups, containing 46 of the 47 sites without S. glanis, were retained for further analysis.

To detect a potential time lag in S. glanis impact, Wtsites were compared with W-sites colonised for 0 2 years, 3 5 years, 6 10 years or more than 10 years during the same period of time (here, the last data recorded in the database, that is from 2006 to 2010). Significant difference in fish richness, native fish richness, diversity, evenness, biomass or density between Wt-sites and Wsites was identified using the Mann Whitney test.

Results

Before after analysis

Total fish species richness, native fish richness, evenness and diversity were potentially affected by S. glanis in 1.4% (1/69), 1.4% (1/69), 1.4% (1/69) and 5.8% (4/69) of S. glanis was affected at 5.8% (5/69) of sites.

The biomass of the three fish species most strongly represented (in terms of biomass) in the data set was potentially impacted by S. glanis in 3.3% (chub, Squalius cephalus) and 4.1% (barbel, Barbus barbus) of sites but with no impact identified for the European eel, Anguilla anguilla (Table 1).

The densities of the three fish species most strongly represented (in terms of density) during electrofishing (roach, Rutilus rutilus; chub and bleak, Alburnus alburnus) were significantly lower at 13%, 1.4% and 2.9% of sites, respectively, in relation to S. glanis introduction.

For the top predators, a significant decrease in biomass was detected in 8.3% (northern pike, Esox lucius) and 1.8% (Eurasian perch, Perca fluviatilis) of sites and a significant change in densities was found in 5.5% and 6.2% of sites, respectively. No significant change, however, was observed in either biomass or density of pikeperch, Sander lucioperca.

With without analysis

Irrespective of time since S. glanis introduction, no difference was found between W-sites and Wt-sites with respect to total evenness, diversity, biomass or density. A significant difference in total species richness was found between W-sites and Wt-sites (K W test; P 0.0036); most Wt-sites held fewer fish species than W-sites (mean values: Wt 14; W [0-2] 17; W [2-5] 17; W [5][6][7][8][9][10] 18; W >10 19). This difference originates from significant differences in both native and non-native fish species richness (K-W test; P 0.04 and P < 0.001, respectively).

The with without analysis showed no differences in biomass of the three most represented species (carp, European eel and chub), or densities of the three most numerically represented species (roach, chub and bleak) or biomass and density of the top-predators (northern pike, pikeperch and Eurasian perch) (Table 2).

Discussion

Comparisons between sites with and without S. glanis did not highlight any drastic changes in species richness during the study period, and no fish species extinction sites, respectively. Total fish biomass was potentially affected at 6.6% (4/61) of sites, while total biomass of fish other than S. glanis was potentially affected at 13.1% (9/61) of sites. The total density of sampled fish was potentially affected after S. glanis establishment at 2.9% (3/69) of sites, while total density of fish other than was attributed to S. glanis. On the contrary, sites with S. glanis had higher mean total, native and non-native species richness. Sites without catfish are probably less prone to colonisation because of low local habitat suitability, physical barriers such as dams or waterfalls, or lower anthropogenic propagule pressure [START_REF] Lockwood | The role of propagule pressure in explaining species invasions[END_REF], which limit the spread of S. glanis as well as other species of fish and thus do not permit a rise in species richness [START_REF] Daufresne | Climate change impacts on structure and diversity of fish communities in rivers[END_REF].

The with without analysis also revealed no significant differences in fish evenness, biomasses or fish densities, regardless of time since the invasion began. Nor was there a time lag between S. glanis establishment and any change over a period ranging from 0 to more than 10 years since S. glanis was first caught (i.e. when old and large individuals were probably not well represented). Some river basins are considered as non-equilibrium islands in which species extinctions (related to historical events) are not fully balanced by colonisations from neighbouring river basins (Oberdorff, Hugueny & Gu egan, 1997;[START_REF] Reyjol | Patterns in species richness and ende mism of European freshwater fish[END_REF]. Species-unsaturated rivers are more susceptible to invasion by non-native species because ecological space is less densely packed and interspecific competition is less intense [START_REF] Hutchinson | Homage to Santa Rosalia or why are there so many kinds of animals[END_REF][START_REF] Kennedy | Biodiversity as a barrier to eco logical invasion[END_REF]. In addition, S. glanis appears to be a scavenging predator, able to exploit a wide variety of prey sources [START_REF] Copp | Voracious invader or benign feline? A review of the environmental biology of European catfish Silurus glanis in its native and intro duced ranges[END_REF] so that, despite its size, the species does not occupy the highest trophic position in fish communities of large rivers in France (Syv€ aranta et al., 2009). Feeding on a wide spectrum of available prey alleviates predation pressure on any particular category or species of prey (except for some specialised S. glanis individuals; see Syv€ aranta et al., 2009;[START_REF] Cucherousset | Freshwater killer whales': beaching behavior of an alien fish to hunt land birds[END_REF].

Few sites have experienced a significant impact of S. glanis introduction, and this lessens the effect of S. glanis as one among the numerous variables driving river ecosystems. Some systems, such as ponds or streams, are known to be more sensitive to invasion than large systems [START_REF] Elton | The Ecology of Invasions by Animals and Plants[END_REF][START_REF] Gozlan | Introduction of non native freshwater fish: is it all bad?[END_REF], and anthropogenic alterations may have an additive or synergistic impact with S. glanis on the host system [START_REF] Light | Distinguishing between invasions and habitat changes as drivers of diversity loss among California's freshwater fishes[END_REF].

Possible impacts on a given species were detected in a small percentage (usually <5%) of sites in our analysis. Roach decreased to the greatest extent after S. glanis establishment. [START_REF] Wysujack | Can feeding of European catfish prevent cyprinids from reaching a size refuge?[END_REF] reported that S. glanis (TL ranging from 40 to 100 cm) feeds mostly on crayfish and roach. Predation by S. glanis on mostly smaller roach individuals, in comparison with predation by either northern pike or pikeperch, could explain why 15% of the sites experienced a significant decrease in roach density. Nevertheless, sites without S. glanis did not hold significantly more roach than sites with the invader, and neither roach biomass nor density decreased with time since S. glanis establishment. Climate change may affect roach reproduction [START_REF] Geraudie | Roach (Rutilus rutilus) reproductive cycle: a study of biochemical and histological parameters in a low contaminated site[END_REF] [START_REF] Poulet | Time trends in fish populations in metropolitan France: insights from national monitoring d ata[END_REF]. B>A: number of sites with a significant decrease in before after analysis, using the Mann Whitney test. rs: number of sites where the significant decrease in fish population was not associated with a temporal trend already established before S. glanis settlement, using the Spearman correlation test. The value of rs in % corresponds to the proportion of sites where S. glanis potentially affected fish species biomass or density. Significant increases in biomass or density (Mann Whitney test) are displayed in the column B<A.

Table 1 Results of the before after selection procedure of the sites exhibiting the Silurus glanis impact on biomass or density of the main freshwater fish species during the study period led to a decrease in pollution by phosphorus and organic substances [START_REF] Crouzet | L'eutrophisation des rivi eres en France : o u en est la pollution verte ? Les Donn ees de l[END_REF], while river incision led to an increase in water velocity (Wy zga, 2001;Kondolf, Pi egay & Landon, 2002). These changes could be responsible for declines in limnophilic or eurytopic populations such as those of roach or perch [START_REF] Jeppesen | Trophic structure, species rich ness and biodiversity in Danish lakes: changes along a phosphorus gradient[END_REF][START_REF] Olin | Fish community structure in meso trophic and eutrophic lakes of southern Finland: the rela live abundances of percids and cyprinids along a trophic gradient[END_REF][START_REF] Poulet | Time trends in fish populations in metropolitan France: insights from national monitoring d ata[END_REF].

Silurus glanis might potentially affect predatory species such as northern pike or pikeperch via predation or competition for food or habitat. However, densities of predators are often low (<1 individual 100 m À2 ), and the number of sites holding these species is often small; this may limit the robustness of our analysis for such species. Predatory fish are also very popular with anglers, potentially creating a significant pressure on their populations through harvesting. The population dynamics of such species are also blurred by numerous stocking episodes, for which statistics are often absent and where stocking efficiency may vary widely from year to year [START_REF] Arlinghaus | The characteristics of invasive fishes : what has been learned so far?[END_REF][START_REF] Arlinghaus | The conservation and fishery benefits of protecting large pike (Esox lucius L.) by harvest regulations in recreational fishing[END_REF] Our study showed no generalised impact of S. glanis on fish biomass, density or community structure in French rivers. Nevertheless, S. glanis may have local effects on fish populations since significant changes in fish biomass and density after S. glanis establishment were identified at several sites. Future studies should determine in greater detail whether S. glanis might change the structure of the food web in the decades after its establishment. Additionally, research should focus on potential effects on migratory species (Mac-Avoy et al., 2000) whose populations are not monitored by Onema's electrofishing survey.

  , and efforts to enhance water quality made

		Biomass				Density
	Species	Sampled sites	B>A	rs	B<A	Sampled sites	B<A	rs	B<A
	Abramis brama (N)	38	1	1 (2.6%)	0	44	1	1 (2.3%)	2
	Alburnus alburnus (N)	59	2	2 (3.4%)	1	69	2	2 (2.9%)	5
	Ameiurus melas (NN)	21	0		2	22	0		1
	Anguilla anguilla (N)	54	0		3	58	1		3
	Barbus barbus (N)	49	2	2 (4.1%)	0	51	1	1 (2.0%)	2
	Cyprinus carpio (NN)	23	0		0	24	0		0
	Esox lucius (N)	48	4	4 (8.3%)	0	55	3	3 (5.5%)	0
	Perca fluviatilis (N)	57	1	1 (1.8%)	0	65	4	4 (6.2%)	0
	Rutilus rutilus (N)	60	7	5 (8.3%)	0	69	10	9 (13.0%)	0
	Sander lucioperca (NN)	25	0		2	29	0		4
	Squalius cephalus (N)	60	2	2 (3.3%)	1	69	1	1 (1.4%)	0
	Tinca tinca (N)	42	0		0	53	0		0
	(N), native species; (NN), non native species

Acknowledgments

The authors wish to thank the numerous engineers and technicians who perform electrofishing and maintain the database. The authors also thank anonymous reviewers and the editor for useful comments on the manuscript.