Hypodynamia Alters Bone Quality and Trabecular Microarchitecture

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ORIGINAL RESEARCH

Hypodynamia Alters Bone Quality and Trabecular Microarchitecture

Eric Aguado^1,2 · Guillaume Mabilleau² · Eric Goyenvalle^1,2 · Daniel Chappard²

Received: 6 September 2016 / Accepted: 7 January 2017

tissue level. Confined chicken represents a new model for the study of hypodynamia because bone changes are not created by a surgical lesion or a traumatic method. Animals have a reduced bone mass and present with an altered bone matrix quality which is less mineralized and whose collagen contains less crosslinks than in control chicken.

Keywords Hypodynamia · Sedentarity · FTIR · Bone quality · Animal model · Chicken

Introduction

Bone is capable of sensing and adapting to the mechanical loads although the mechanisms are not yet fully under- stood. At the present time, considerable interest has been developed in the study of osteocytes as mechanoreceptors and mechanotransducers, providing information to other bone cells (osteoclasts, lining cells, and osteoblasts) [1].

Muscular contraction and gravity are the main mechanical forces acting on bone. Bone loss has been reported in prolonged spaceflights in spationauts and animals [2, 3]

and countermeasures based on specific exercise programs have been proposed in Mir and ISS space stations for elimi- nating the deconditioning due to prolonged microgravity [4]. Ground-based studies on paraplegic patients or bed-rest subjects have confirmed that bone mass is reduced [5–7].

In all these circumstances, a decline in muscle mass is observed in parallel with bone loss [8, 9]. Sedentarity (or hypokinesia) has also been found to affect muscle and bone in parallel [10, 11]. Loss of muscle mass (sar- copenia) or poor muscle performance associated with reduced muscle function has raised a considerable interest and the effects of exercise training or various sports have been studied in a large number of reports [12–14].

Abstract Disuse induces a rapid bone loss in humans and animals; hypodynamia/sedentarity is now recognized as a risk factor for osteoporosis. Hypodynamia also decreases bone mass but its effects are largely unknown and only few animal models have been described. Hypodynamic chicken is recognized as a suitable model of bone loss but the effects on the quality have not been fully explored. We have used ten chickens bred in a large enclosure (FREE group); ten others were confined in small cages with little space to move around (HYPO group). They were sacrificed at 53 days and femurs were evaluated by microcomputed tomography (microCT) and nanoindentation. Sections (4 µm thick) were analyzed by Fourier Transform InfraRed Microspectroscopy (FTIR) to see the effects on mineralization and collagen and quantitative backscattered electron imaging (qBEI) to image the mineral of the bone matrix.

Trabecular bone volume and microarchitecture were significantly altered in the HYPO group. FTIR showed a significant reduction of the mineral-to-matrix ratio in the HYPO group associated with an increase in the carbonate content and an increase in crystallinity (calculated as the area ratio of subbands located at 1020 and 1030 cm⁻¹) indicating a poor quality of the mineral. Collagen maturity (calculated as the area ratio of subbands located at 1660 and 1690 cm⁻¹) was significantly reduced in the HYPO group.

Reduced biomechanical properties were observed at the

* Daniel Chappard

daniel.chappard@univ-angers.fr

1 ONIRIS, Ecole Nationale Vétérinaire, route de Gachet, 44307 Nantes Cedex 3, France

2 GEROM Groupe Etudes Remodelage Osseux et

bioMatériaux, IRIS-IBS Institut de Biologie en Santé, CHU d’Angers, Université d’Angers, 49933 ANGERS Cedex, France

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Reduced muscle activity affects not only bone mass but also bone quality. Bone quality is a “term that describes the set of characteristics that influences bone strength and explains the interrelationships of these characteristics” [15]. A large number of factors including bone micro- and macro-architecture, mineralization degree of the matrix, collagen integrity, and remodeling level have been identified to influence biomechanical strength of the bone matrix. However, in vivo studies are limited to a small number of methods (pQCT, microindentation) which have confirmed that increasing muscle activity improves bone quality [10, 16, 17]. A growing number of methods have been reported in the literature to accu- rately analyze and quantify bone quality in vitro, i.e., on bone samples [18].

Animal models have been extensively used to develop analogous conditions to model human diseases. Reduc- ing muscle function has been done by a number of surgical and non-surgical techniques in order to develop disuse osteopenia in laboratory animals (see review in [19, 20]). Among the most popular, the hindlimb unloading by tail suspension has been extensively studied by NASA and other space agencies [2] and appears to be more suitable than neurectomy, cordectomy, or any other surgical methods. Other techniques have been proposed to avoid the nociceptive changes in tail-suspended animals and the endocrine modifications observed [21]. We have proposed the BTX model in which a single injection of botulinum toxin in a muscle causes a severe unilateral amyotrophy associated with a unilateral bone loss [22, 23]. However, osteopenia is caused by a neurologic alteration at the muscle synapse. Recently, we have proposed a new model without nerve or muscle lesion in the broiler chicken [24]. When chicken are grown in a reduced surface environment, they develop normally (without growth disorder) but have a reduced bone mass associated with histological changes of the bone matrix.

The confined chicken represent a suitable model to explore the impact of hypokinesia on bone quality.

The aims of the present study were to characterize further the alteration of bone matrix by a panel of laboratory techniques covering several aspects of bone quality. Microcomputed tomography was used to evaluate microarchitecture of trabecular bone; Fourier transform Infrared spectroscopy and imaging was used to characterize the mineral and organic phase of the calcified bone matrix and nanoindentation allowed the measure- ments of biomechanical properties. A group of hypodynamic chicken was compared with a group of free-walking chicken of the same age and fed similarly.

Material and Methods Animals

The experimental protocol was approved by the Regional ethical committee at Oniris, National Veterinary School.

The protocol has been described recently [24]. Briefly ten chicken of the rapidly growing strain 857 K were grown in a large enclosure where they could walk freely (group FREE); ten others were kept in a small cage with little space to move around (group HYPO) (Fig. 1). Both groups were housed in the same conditions of temperature (near 20 °C) and light cycle (12-h/12-h light/dark cycle).

All chickens were given an equal amount of normal poultry food with normal calcium, phosphorus, and vitamin D contents. Chickens were killed at 53 days of age by elec- tronarcosis (representing the elapsed time to reach maturation and poultry marketing). Bones were excised, fixed for 10 days in 10% buffered formalin, and kept until analysis by microCT and histological embedding.

Fig. 1 Experimental conditions used in the present study: a chicken grown in a large enclosure, group FREE, and b hypodynamic chicken kept in small cages, group HYPO

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Microcomputed X‑ray Tomography (microCT)

MicroCT was performed on the distal right femur with a SkyScan 1172 X-ray computed microtomograph (Bruker MicroCT, Kontich, Belgium). Bones, still in the fixative to prevent desiccation, were scanned according to a routine protocol using 80 kV, 100 µA, and a 0.5 mm aluminum fil- ter. The pixel size was fixed at 19.28 µm and a 0.25° rota- tion angle was applied at each step. For each bone, a stack of 2D sections was obtained. The CTAn software (SkyS- can, release 1.13.11.0) was used to measure the trabecular bone volume and microarchitecture at the secondary spongiosa of the tibia after global thresholding [25]. The first image selected for analysis was just under the growth plate (excluding the primary spongiosa), and then 131 sections were selected (corresponding to 2.5 mm). The volume of interest (VOI) was designed by interactively drawing a polygon on each 2D section. Only a few number of poly- gons need to be drawn (e.g., on the first section, several at the middle, and on the final section) since a routine facility calculated all the intermediary masks by interpolation. The VOI comprised only trabecular bone and marrow cavity.

The following parameters were measured in the VOI (TV, in mm³) according to the recommendations of the Ameri- can Society for Bone and Mineral Research [26]:

• Trabecular bone volume (BV/TV, in %) represents the percentage of the cancellous space occupied by trabecular bone in the VOI.

• Microarchitectural parameters include trabecular thickness (Tb.Th, expressed in µm), trabecular number (Tb.N, expressed in /mm), and trabecular separation (Tb.Sp, expressed in µm). The other indexes determined were as follows: (i) structure model index (SMI) indicating the composition of trabecular bone in the form of rods or plates, the values of which range between zero (ideal plate structure model) and three (ideal rod structure), and (ii) trabecular pattern factor (Tb.Pf) which quantifies the connectivity of the trabecular scaffold and is negative in highly connected networks.

Bones were then embedded undecalcified in poly(methyl methacrylate) (pMMA) as previously described [27].

Fourier Transform InfraRed Microspectroscopy and Imaging (FTIR and FTIRI)

FTIR acquisition was done according to previous speci- fications [28, 29]. The short-term formaldehyde fixation used here is compatible with FTIR analysis of mineralized tissues [30–33]. Sections, 4 µm in thickness, were prepared with a Polycut S microtome (Leica, Rueil Mal- maison, France) and sandwiched between two barium

fluoride (BaF₂) optical windows (IR grade). The samples were then analyzed with a Bruker Hyperion 3000 microscope (Bruker optic, Ettlingen, Germany) equipped with a liquid nitrogen-cooled Mercury Cadmium Telluride (MCT) detector and purged with dried air, and interfaced with a vertex 70 spectrometer (Bruker optic). A 15X Casseg- rain objective and condenser with a numerical aperture of 0.4 was used. Infrared spectra were collected at a spectral resolution of 4 cm⁻¹ in transmission mode. 32 co-added scans were used for background and samples. FTIR data processing was performed with the Bruker’s proprietary Opus software (release 6.5). Twenty spectra (15 µm apart in a 45 × 60 µm square matrix) were obtained in three different regions of three independent trabecula per bone sample. For all animals, the area of analysis was always selected in the central part of the secondary spongiosa to ensure the homogeneity of the results. The contribution of the embedding pMMA was spectrally subtracted after nor- malization of the peak located at 1730 cm⁻¹. The spectra were cut between 800 and 1800 cm⁻¹, baseline corrected with an elastic correction method, vector normalized in the amide 1 region (1585–1710 cm⁻¹), and averaged. Spectra were also subjected to curve fitting in the υ1 υ3 phosphate band (900–1200 cm⁻¹) and the amide 1–amide 2 region (1485–1720 cm⁻¹) according to the second derivative and the Levenberg–Marquardt algorithm using the Grams/AI 8.0 software as reported previously [34].

The evaluated IR spectral parameters were (i) mineral- to-matrix ratio, calculated as the integrated area of the υ1υ3 phosphate band over the amide 1 region (1585–1720 cm⁻¹) and used as an index of mineral content [35]; (ii) mineral crystallinity, which reflects the apatite size and perfection and calculated as the area ratio of the 1030 cm⁻¹ over the 1020 cm⁻¹ subbands [36]. A lower ratio indicates a higher mineral crystallinity; (iii) collagen maturity, related to the relative proportion of the two major collagen crosslinks, namely pyridinium (Pyr, non-reducible) and deH-DHLNL (reducible), and calculated as the area ratio of the 1660 and 1690 cm⁻¹ subbands [37]; and (iiii) carbonate-to- phosphate ratio, which reflects the carbonate content in bone and calculated as the ratio of integrated areas of the υ2 CO₃²⁻ region (850–890cm⁻¹) and υ1υ3 phosphate band [36].

For FTIRI, sections were imaged using a 64 × 64 ele- ment Focal Plane Array (FPA) detector (coupled with the microscope) to provide 4096 spectra simultaneously from interest areas. The images were collected at a resolution of 8 cm⁻¹ with 32 co-added scans for background and samples, in the transmission mode in the spectral region 900–2000 cm⁻¹. Images of the trabeculae were obtained by a stitching of 3 × 3 elemental images provided by the FPA with the Opus software. Three spectral images were acquired on separated trabeculae from each sample. Each

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image was ratioed against the background imaging spectra collected under identical conditions. Color-coded images of mineral-to-matrix ratio, mineral crystallinty, collagen maturity, and carbonate content (using the intensity of the υ3 carbonate band at 1415 cm⁻¹ over the υ1υ3 phosphate band [38]) distributions were produced in each group for qualitative comparison using a Matlab routine (release 7.10; Math Works, Natick, MA). The calculated spectroscopic parameters were expressed as color-coded images, with the color of a pixel corresponding to a certain range of values.

Nanoindentation

Nanoindentation tests were used to evaluate the mechanical properties at the tissue level. The pMMA blocks used for preparing the FTIR sections were polished with 1 µm diamond particles (Struers, France). Prior to nanoindentation testing, blocks were rehydrated for 24 h in saline at 4 °C and the tip area function and instrument frame compliance were calibrated using a fused silica sample. Twelve indentations, at a distance from osteocyte lacunae and/or micro- cracks, were randomly positioned in cortical bone with a NHT-TTX system (CSM, Peseux, Switzerland) equipped with a Berkowitch diamond probe as the indenter and an optical system to control where the indentations are positioned. Indentations were made up to a depth of 400 nm with a loading/unloading rate of 40 mN/min. At maximum load, a holding period of 15 s was applied to avoid creep- ing of the bone material. The following material properties at the tissue level were determined according to Oliver and Pharr [39]: hardness, indentation modulus, maximal force to obtain 400 nm depth, and dissipated energy.

Quantitative Backscattered Electron Imaging (qBEI) qBEI experiments were performed on the same blocks as nanoindentation and FTIRM. pMMA blocks were carbon- coated and observed with a scanning electron microscope (EVO LS10, Carl Zeiss Ltd, Nanterre, France) equipped with a five-quadrant semiconductor backscattered electron detector. The microscope was operated at 20 keV with a probe current of 250 pA and a working distance of 15 mm.

The backscattered signal was calibrated using pure carbon (Z = 6, mean gray level = 25), pure aluminum (Z = 13, mean gray level = 225), and pure silicon (Z = 14, mean gray level = 253) standards (Micro-analysis Consultants Ltd, St Ives, UK). The cortical and trabecular bone areas were imaged at a 150X nominal magnification, corresponding to a pixel size of 0.75 µm/pixel. The trabecular area was imaged 2 mm below the growth plate, whilst the cortical areas were imaged 10 mm below the growth plate. Four images per area were taken. Three variables were obtained

from the bone mineral density distribution: Ca_mean as the average calcium concentration, Ca_peak as the peak calcium concentration, and Ca_width as the width of the histogram at half maximum of the peak.

Statistical Analysis

Statistical analysis was performed with SYSTAT statistical software, release 13 (SYSTAT Software, Inc., San José, CA). Data are expressed as mean ± SD. Differences between groups were analyzed by the non-parametric Mann–Whitney U test. Differences were considered significant when p < 0.05.

Results

MicroCT Analysis

Results are shown in Table 1. A significant reduction in the trabecular bone volume could be evidenced both in 2D and 3D. Microarchitecture of the trabecular network appeared to be constituted of thin trabeculae (Fig. 2) in the secondary spongiosa with a large bundle of trabeculae in the central part of the metaphysis extending into the diaphysis. Quan- titative analysis evidenced a net reduction in the number of trabeculae with a concomitant increase in Tb.Sp in the HYPO group. However, no significant modification in trabecular thickness, SMI, or Tb.Pf was observed.

FTIR Analysis

FTIR evidenced a conjoined alteration of the mineral and organic phases of the bone matrix (Fig. 3). A significant reduction of the phosphate/amide ratio was noted in the HYPO group (p = 0.01), which was associated with an increase in the carbonate content (p = 0.05) and an increase in crystallinity (p = 0.02). Collagen maturity (evidenced by the crosslinks ratio) was significantly reduced in the HYPO group (p = 0.008).

Table 1 MicroCT analysis of the distal femoral epiphysis

Parameter Unit HYPO FREE p

BV/TV % 9.26 ± 0.53 10.82 ± 0.5 0.04

Tb.Th µm 62 ± 2 62 ± 1 NS

Tb.N /mm 1.49 ± 0.08 1.75 ± 0.06 0.02

Tb.Sp µm 626 ± 42 515 ± 23 0.02

SMI 1.80 ± 0.06 1.77 ± 0.04 NS

Tb.Pf 7.21 ± 0.61 6.63 ± 0.33 NS

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FTIRI

The spatial distribution of mineral–to-matrix, mineral crystallinity, carbonate-to-phosphate, and collagen maturity ratios is presented in Fig. 4 for typical distribution in the FREE and HYPO groups. As may be seen, the highest values (represented by “hot colors”) and low values (represented by “cold colors”) appear somewhat different for the two groups of animals with the lowest amount of mineral-to-matrix and collagen maturity ratios in the HYPO group. On the contrary, more “hot spots” are observed in the HYPO group regarding the mineral crystallinity and carbonate-to-phosphate ratios suggesting a less mature mineral in the HYPO group.

Nanoindentation

Nanoindentation analysis revealed a significantly altered reduction in mechanical properties of the bone matrix at the tissue level. The indentation modulus, hardness, and maximal force were significantly reduced in the HYPO group as compared with FREE control animals (Fig. 5). On the contrary, dissipated energy was not significantly reduced in the HYPO group.

qBEI

qBEI analysis confirmed the findings observed in FTIR with significant reductions observed for Ca_mean and Ca_peak in both trabecular and cortical compartments, suggesting a lower calcium and thus mineral content in the bone matrix (Table 2). On the other hand, although slightly reduced in the HYPO group, Ca_width in both compartments, reflecting mineralization heterogeneity, was not significantly different as compared with FREE animals.

Fig. 2 MicroCT analysis of the lower femoral epiphysis of a chicken of the FREE group and b chicken of the HYPO group. Note the marked reduction in the amount of trabecular bone and the changes in micro-architecture

3.0 3.5 4.0

Phosphate/amide ratio

0.003 0.004 0.005 0.006 0.007

Carbonate/phosphate ratio

p = 0.01

FREE HYPO FREE HYPO

p = 0.05

0 1 2 3 4

Crosslinks ratio Crystallinity

FREE HYPO FREE HYPO

p = 0.008

p = 0.02

0 0.20 0.40 0.60 0.80 1.00

Fig. 3 FTIR analysis of the bone matrix of the femoral trabecular in the control (FREE) and hypodynamic (HYPO) groups of chicken

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Discussion

In the present study, we have used different established methods to examine bone quality at the tissue level in two groups of chicken [18]. The hypodynamic animals presented a net reduction in bone quality due to an altered bone microarchitecture at the femoral lower extremity. A reduction in the trabecular bone volume, due to a net reduction in the number of trabeculae (and an increased separation as a consequence), was evidenced by microCT. This finding confirms our previous results found in this model and noted both at the femur and tibia [24]. A reduced bone mass and altered trabecular microarchitecture have also been reported in humans with disuse osteoporosis [40, 41] and in animal models of disuse osteoporosis [20]. We have previously observed similar microarchitectural changes when disuse is induced by a single injection of Botulinum toxin (BTX) in the rodents [22, 42–44]. These findings were confirmed by other groups [45–47]. Hypodynamia, created by the walking limitation in the chicken, was previously found to have no effect on the growth of long bones but it induces alterations in the bone matrix evidenced by microCT and texture analysis. The absence of the effect on the growth of long bones evidences the absence of implication of stress

Fig. 4 FTIRI analysis of the bone matrix of the femoral trabeculae. Upper line: in a control animal of the FREE group.

Lower line: in a hypodynamic animal of the HYPO group.

First raw: imaging of mineral to matrix ratio, mineral crystallinity, carbonate to phosphate ratio, and collagen maturity.

For each image, the same look-up table used is figured.

Images (192 × 192 pixels) were obtained by stitching

0 100 200 300Hardness (MPa)

0 2 4 6 8 )aPG(suludomnoitatnednI 10

0 0.5 1.0 1.5 )Nm(ecroflamixaM2.0

0 100 200 300 400Dissipated energy (pJ)

p = 0.009

FREE HYPO FREE HYPO

p = 0.02

p = 0.01

Fig. 5 Nanoindentation study performed on the trabecular bone matrix of the femur in the chicken of the control (FREE) and hypodynamic (HYPO) groups

Table 2 Bone mineral density distribution analysis

*p < 0.05 versus FREE Compart-

ment Groups Ca_mean (%

Ca) Ca_peak (%

Ca) Ca_width (% Ca)

Trabecular FREE 14.2 ± 0.2 14.3 ± 0.3 3.4 ± 0.3 HYPO 13.3 ± 0.2* 13.3 ± 0.2* 2.8 ± 0.1 Cortical FREE 15.2 ± 0.2 16.0 ± 0.2 3.8 ± 0.5 HYPO 13.8 ± 0.3* 14.4 ± 0.4* 3.4 ± 0.2

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hormones [48]. Glucocorticoids are well known to affect the growth cartilage and to induce a severe growth retar- dation in children under steroid therapy [49]. In addition, the weights of the animals were similar between the HYPO and FREE groups as previously reported (data not shown) [24]. Minor changes in osteoid parameters were observed but osteoid thickness remained normal in the HYPO group, indicating that mineralization is not impaired by hypodynamia and that glucocorticoids are not implied. In the present study, the quality of the bone matrix was studied by FTIR, a robust method to investigate both the organic (i.e. collagen) and mineral (i.e., hydroxyapatite) phases of the bone matrix [28, 50–52]. Bone collagen properties are important determinants of bone strength: the crosslinks content is altered in post-menopausal osteoporotic patients [52, 53] or idi- opathic osteoporosis [54]. Altered collagen properties have also been demonstrated by FTIR in animal models of bone diseases such as osteoporosis [55], osteogenesis imperfecta [56], and type I diabetes [34]. A reduced crosslinks ratio (subbands 1660/1690 cm⁻¹) was evidenced in the HYPO group. The crosslinks ratio in the type I fibrillary collagen of the bone matrix is directly linked with biomechanical properties such as viscoelasticity and tensile strength.

FTIR also evidenced changes in the mineral-to-matrix ratio in this group indicating a lower degree of mineralization of the bone matrix that was confirmed by qBEI analysis of the bone matrix. A limitation of the study could be represented by the use of formalin as a fixative. In a systematic assay of different fixatives and embedding media for bone, it was found that aqueous fixatives such as formalin could cause alteration in the crystallinity of the bone matrix and hence alcohol should be preferred [57]. However, alcohol alone is responsible for a considerable shrinkage of tissues and is no longer recommended for histological analysis [58] and formalin is preferred [27]. In the present study, the bones from both groups of animals were fixed for a short period of time to minimize the effects of formalin as reported by other authors [30–33].

The ratio of the 1030/1020 cm⁻¹ bands has also been shown to provide a measure of mineral crystallinity and maturity [36]. High values of this ratio indicate a reduced size of hydroxyapatite crystals. Chicken of the HYPO group had significantly higher values than those in the FREE group, confirming that the quality of the mineral was altered in parallel with alterations in the collagen- ous content. FTIR and FTIRI also evidenced an increase in the carbonate content of the bone matrix. The consequences of an alteration in both the organic and mineral phases of the bone matrix have consequences at the biomechanical level. Nanoindentation is a powerful technique to explore the biomechanical properties of the bone matrix [59, 60]. Biomechanical parameters determined by nanoindentation evidenced a decreased hardness of

the bone matrix in the HYPO group. A reduction in bone hardness has also been shown to be a risk factor for fracture in humans [54]. In a hypodynamia model developed in 2-year-old laying hens, prolonged exercise restriction also resulted in major bone loss together with reduced biomechanical properties of long bones [61].

In conclusion, we have confirmed that hypodynamia in the chicken induces changes in bone quality associated with reduced biomechanical properties of the bone matrix at the tissue level. Hypodynamia affected both the collagen maturation and the mineral phase of the matrix at the material level (the “nature level” according to [18, 62]). In addition, hypodynamia also affected the “microarchitectural level” by reducing the number of trabeculae and their spatial organization. The confined chicken represents a new animal model of hypodynamia/sedentarity to evaluate the effects on bone changes. The model presents the advantage of avoiding surgical or complex handling of animals and do not alter muscles or nerves.

The development of countermeasures, such as the return to free walking, may be beneficial in evaluating its effects on bone volume and bone quality.

Acknowledgements This work was made possible by grants from the Ministry of Research. Many thanks to Mrs. Lechat for secretarial assistance, N. Retailleau for microCT, and A. Mieczkowska for FTIR analysis.

Author contributions This study was conceived by EA and EG, and initiated by DC. Experimental analyses were designed by DC and performed by GM and the acknowledged technicians. Analysis of the data was performed by DC. The paper was written by DC and EA, the manuscript was revised by GM, and the final manuscript was approved by all the authors. There is no potential conflict of interest for all of the authors.

Compliance with Ethical Standards

Conflict of interest Eric Aguado, Guillaume Mabilleau, Eric Goy- envalle, and Daniel Chappard have no conflict of interest.

Human and Animal Rights and Informed Consent This article does not contain any studies with human participants performed by any of the authors. All procedures performed in the study were reviewed and approved by the ONIRIS Council on Animal Care (National Vet- erinary School of Nantes) and the Regional ethical Committee and conformed to the guidelines of the French Animal Care Committee.

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