L Rolland

B Courbiere
email: blandine.courbiere@imbe.fr

V Tassistro
email: virginie.tassistro@univ-amu.fr

A Sansoni

T Orsière

W Liu
email: liu@cerege.fr

C Di

J Perrin

Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos

Keywords:

Our objective was to optimize the CA technique on mammal embryos. Materials and methods: 1000 frozen 2-cell embryos from B6CBA mice were used. Based on a literature review, and after checking post-thaw embryo viability, the main outcome measures included: 1) comparison of the embryo recovery rate between 2 CA protocols (2 agarose layers and 3 agarose layers); 2) comparison of DNA damage by the CA on embryos with (ZP +) and without (ZP-) zona pellucida; and 3) comparison of DNA damage in embryos exposed to 2 genotoxic agents (H 2 O 2 and simulated sunlight irradiation (SSI)). DNA damage was quantified by the % tail DNA. Results: 1) The recovery rate was 3,3% (n = 5/150) with the 2 agarose layers protocol and 71,3% (n = 266/ 371) with the 3 agarose layers protocol. 2) DNA damage did not differ statistically significantly between ZPand ZP+ embryos (12.60 ± 2.53% Tail DNA vs 11.04 ± 1.50 (p = 0.583) for the control group and 49.23 ± 4.16 vs 41.13 ± 4.31 (p = 0.182) for the H 2 O 2 group); 3) H 2 O 2 and SSI induced a statistically significant increase in DNA damage compared with the control group (41.13 ± 4.31% Tail DNA, 36.33 ± 3.02 and 11.04 ± 1.50 (p < 0.0001)).

The CA on mammal embryos was optimized by using thawed embryos, by avoiding ZP removal and by the adjunction of a third agarose layer.

Introduction

The exposure of parents to environmental toxicants, such as polycyclic aromatic hydrocarbons [START_REF] Einaudi | In vivo exposure to benzo(a)pyrene induces significant DNA damage in mouse oocytes and cumulus cells[END_REF][START_REF] Perrin | Tobacco consumption and benzo(a)pyrene-diol-epoxide-DNA adducts in spermatozoa: in smokers, swim-up procedure selects spermatozoa with decreased DNA damage[END_REF], Bisphenol A [START_REF] Goldstone | Urinary bisphenol A and semen quality, the LIFE study[END_REF], solvents [START_REF] Kolstad | Change in semen quality and sperm chromatin structure following occupational styrene exposure[END_REF], metals [START_REF] Thompson | Cadmium: toxic effects on the reproductive system and the embryo[END_REF][START_REF] Zhou | Evaluation of urinary metal concentrations and sperm DNA damage in infertile men from an infertility clinic[END_REF] or various therapies [START_REF] Bujan | Impact of lymphoma treatments on spermatogenesis and sperm deoxyribonucleic acid: a multicenter prospective study from the CECOS network[END_REF][START_REF] Esquerré-Lamare | Sperm DNA fragmentation after radioiodine treatment for differentiated thyroid cancer[END_REF][START_REF] Pecou | Ribavirin and pegylated interferon treatment for hepatitis C was associated not only with semen alterations but also with sperm deoxyribonucleic acid fragmentation in humans[END_REF][START_REF] Roti Roti | Acute doxorubicin insult in the mouse ovary is cell-and follicle-type dependent[END_REF] may induce DNA damage in male and female germ cells. DNA damage in parental germ cells can lead to reproductive issues, such as reduced fertilization, impaired early embryonic development, a decreased pregnancy rate and increased miscarriage rate [START_REF] Simon | Paternal influence of sperm DNA integrity on early embryonic development[END_REF][START_REF] Zhao | Whether sperm deoxyribonucleic acid fragmentation has an effect on pregnancy and miscarriage after in vitro fertilization/ intracytoplasmic sperm injection: a systematic review and meta-analysis[END_REF]. The transmission of paternal germ cell DNA damage to preimplantation embryos has been demonstrated in humans [START_REF] Zenzes | Detection of benzo(a)pyrene diol epoxide-DNA adducts in sperm of men exposed to cigarette smoke[END_REF], although DNA repair occurs in the zygote [START_REF] Ménézo | DNA damage and repair in human oocytes and embryos: a review[END_REF]. This observation raises the question of evaluating DNA damage in preimplantation embryos because, currently, no genotoxicity test on the embryo has been validated.

The comet assay is a simple and rapid test for evaluating DNA damage in eukaryotic cells [START_REF] Speit | The comet assay as an indicator test for germ cell genotoxicity[END_REF]. It allows for the visualization of denatured DNA fragments after ex-nucleus migration by electrophoresis. After staining, the obtained shape mimics a "comet" with the head containing intact DNA and the remaining parts of the chromosome and the tail containing relaxed DNA loops or broken DNA fragments [START_REF] Horváthová | DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization[END_REF]. This test is validated by toxicological regulatory agencies for the assessment of DNA damage in somatic cells (OECD, 2014). The comet assay is also used by researchers for the assessment of DNA damage in sperm and male germ cells [START_REF] Baumgartner | The comet assay in male reproductive toxicology[END_REF][START_REF] Perrin | Evolution of DNA strand-breaks in cultured spermatocytes: the comet assay reveals differences in normal and γ-irradiated germ cells[END_REF][START_REF] Preaubert | Cerium dioxide nanoparticles affect in vitro fertilization in mice[END_REF] and in oocytes [START_REF] Berthelot-Ricou | Genotoxicity assessment of mouse oocytes by comet assay before vitrification and after warming with three vitrification protocols[END_REF], 2011a, 2011b;[START_REF] Courbiere | Ultrastructural interactions and genotoxicity assay of cerium dioxide nanoparticles on mouse oocytes[END_REF][START_REF] Einaudi | In vivo exposure to benzo(a)pyrene induces significant DNA damage in mouse oocytes and cumulus cells[END_REF]. Studies using the comet assay on animal embryos are scarce and use heterogeneous protocols and species [START_REF] Blerkom | A microscopic and biochemical study of fragmentation phenotypes in stage-appropriate human embryos[END_REF][START_REF] Fabian | Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay[END_REF][START_REF] Harrouk | Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo[END_REF][START_REF] Hwang | Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos[END_REF][START_REF] Ju | Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro[END_REF][START_REF] Kitagawa | Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos[END_REF][START_REF] Müller | Comet assay studies indicate that caffeine-mediated increase in radiation risk of embryos is due to inhibition of DNA repair[END_REF][START_REF] Natarajan | Effect of α-tocopherol supplementation on in vitro maturation of sheep oocytes and in vitro development of preimplantation sheep embryos to the blastocyst stage[END_REF][START_REF] Rajesh | Effect of vitamin A supplementation at different gaseous environments on in vitro development of pre-implantation sheep embryos to the blastocyst stage[END_REF][START_REF] Sturmey | DNA damage and metabolic activity in the preimplantation embryo[END_REF][START_REF] Takahashi | Assessment of DNA damage in individual hamster embryos by comet assay[END_REF][START_REF] Takahashi | Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay[END_REF]Thiyagarajan andValivittan, 2009a, 2009b;[START_REF] Tranguch | Nitric oxide synthase production and nitric oxide regulation of preimplantation embryo development[END_REF][START_REF] Tsuda | Detection of pyrimethamineinduced DNA damage in mouse embryo and maternal organs by the modified alkaline single cell gel electrophoresis assay[END_REF][START_REF] Webster | Measurement of DNA damage by the comet assay in rat embryos grown in media containing high concentrations of vitamin K1[END_REF]. As comet assay is a very sensitive test, the analysis of fresh embryos by comet assay requires the proximity between animal facilities and laboratory. Moreover, the analysis of fresh embryos does not allow control or additional analyses if required, notably after in vivo exposure or for an hypothetical use for regulatory issues.

The aim of our study was to optimize the comet assay for application on frozen mouse embryos, in order to simplify the handling and allow subsequent analysis.

Material and methods

All of the chemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) unless otherwise stated.

Institutional Review Board

Institutional Review Board approval (C2EA-14) was obtained after submission to the National Ethics Committee on Animal Experimentation. All of the experimental protocols and animal handling procedures were approved by The National Ethics Committee on Animal Experimentation.

Source of embryos

In total, 1000 frozen 2-cell embryos were purchased from the ImmunoPHEnomique Center (CIPHE, Luminy, Marseille, France). Briefly, the embryos were obtained by natural mating on superovulated B6CBA females aged 5 weeks. 48 h after mating, the oviduct was flushed and the two-cells embryo obtained were cryopreserved. We used a slow freezing protocol with a controlled rate freezing machine and 1.5 M propanediol as a cryoprotectant, according to the procedure described by the European Mouse Mutant Archive (EMMA) (EMMA-The European mouse mutant archive, 2013a; [START_REF] Hagn | EMMA-the European mouse mutant archive[END_REF].

Embryo thawing

The embryos were thawed according to the procedure described by the European Mouse Mutant Archive (EMMA) (EMMA-The European mouse mutant archive, 2013b; [START_REF] Hagn | EMMA-the European mouse mutant archive[END_REF]. Briefly, the straw was thawed at room temperature (RT), and then the embryos were rinsed in 4 successive M2 medium drops and placed in KSOM medium (Embry-oMax®, Merck Millipore, Darmstadt, Germany).

The number of thawed embryos used for each experiment performed in this study is presented in Table 1.

Embryo viability and culture conditions

To validate the use of frozen embryos instead of fresh embryos for comet assay, we assessed viability and blastulation rates in thawed embryos. After 1 h of recovery in KSOM, the 2-cells embryos were examined under a microscope and were classified as lysed or intact. The survival rate was defined as the ratio of the intact embryos to the total thawed embryos.

The embryos were then cultured for 24 h in a humidified chamber at 37 °C (95% air/5% CO 2 ) to determine viability. The viability rate was defined by the number of 2-cell embryos moving to the 4-8 cell stage to the total intact embryos. The embryos were cultured for 24 h more to determine the evolution rate to the blastocyst stage, assessing the number of blastocysts compared to the total intact embryos. For each separate experiment, 20 embryos were used (5 experiments = 100 embryos) (Table 1).

Literature review of the comet assay protocols used for mammalian embryos.

A literature review using PubMed with the key words "comet assay" and "embryo" was conducted. We excluded studies using non-mammalian species, post-implantation embryos, studies with incomplete data about the comet assay technique and studies applying the comet assay on other cells than embryos. We collected the following information from the protocols: species, embryo production, zona pellucida (ZP), embryo mixing technique in agarose, number of layers, lysis solution/duration and temperature used, incubation/electrophoresis duration, number and stage of embryos deposited per slide and technique used for the quantification of DNA damage.

The main technical details of the published protocols are presented in Table 2. We selected 14 studies using the comet assay on animal embryos matching our criteria, out of 118 studies retrieved. In these studies, fresh embryos were all obtained by natural mating [START_REF] Fabian | Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay[END_REF][START_REF] Harrouk | Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo[END_REF][START_REF] Müller | Comet assay studies indicate that caffeine-mediated increase in radiation risk of embryos is due to inhibition of DNA repair[END_REF][START_REF] Takahashi | Assessment of DNA damage in individual hamster embryos by comet assay[END_REF][START_REF] Tranguch | Nitric oxide synthase production and nitric oxide regulation of preimplantation embryo development[END_REF][START_REF] Webster | Measurement of DNA damage by the comet assay in rat embryos grown in media containing high concentrations of vitamin K1[END_REF] or IVF [START_REF] Hwang | Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos[END_REF][START_REF] Ju | Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro[END_REF][START_REF] Kitagawa | Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos[END_REF][START_REF] Natarajan | Effect of α-tocopherol supplementation on in vitro maturation of sheep oocytes and in vitro development of preimplantation sheep embryos to the blastocyst stage[END_REF][START_REF] Rajesh | Effect of vitamin A supplementation at different gaseous environments on in vitro development of pre-implantation sheep embryos to the blastocyst stage[END_REF][START_REF] Sturmey | DNA damage and metabolic activity in the preimplantation embryo[END_REF][START_REF] Takahashi | Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay[END_REF]Thiyagarajan andValivittan, 2009a, 2009b). This review of the literature shows that there is no standardization of the protocol: Seven different animal species were used; The ZP was removed in 4 studies; The protocols for the transfer of the embryos in the LMP agarose layer were heterogeneous: a minimum of half a blastocyst and a maximum of 20 cleavage embryos were transferred, and the volume of LMP varied from 4 μL to 50 μL; The lysis and electrophoresis protocols were also heterogeneous. No publication described the recovery rate of the embryos in the agarose layer after lysis and electrophoresis.

The findings of this literature review were used in the following paragraphs and tests to optimize comet assay. 2.5. Impact of the number of agarose layers on the embryo recovery rate

All of the published comet assay protocols on mice embryos [START_REF] Fabian | Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay[END_REF][START_REF] Harrouk | Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo[END_REF][START_REF] Hwang | Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos[END_REF][START_REF] Kitagawa | Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos[END_REF][START_REF] Müller | Comet assay studies indicate that caffeine-mediated increase in radiation risk of embryos is due to inhibition of DNA repair[END_REF][START_REF] Natarajan | Effect of α-tocopherol supplementation on in vitro maturation of sheep oocytes and in vitro development of preimplantation sheep embryos to the blastocyst stage[END_REF][START_REF] Rajesh | Effect of vitamin A supplementation at different gaseous environments on in vitro development of pre-implantation sheep embryos to the blastocyst stage[END_REF][START_REF] Sturmey | DNA damage and metabolic activity in the preimplantation embryo[END_REF][START_REF] Takahashi | Assessment of DNA damage in individual hamster embryos by comet assay[END_REF][START_REF] Takahashi | Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay[END_REF]Thiyagarajan andValivittan, 2009a, 2009b;[START_REF] Tranguch | Nitric oxide synthase production and nitric oxide regulation of preimplantation embryo development[END_REF][START_REF] Webster | Measurement of DNA damage by the comet assay in rat embryos grown in media containing high concentrations of vitamin K1[END_REF] used 2 layers of agarose except one [START_REF] Ju | Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro[END_REF]. A glass microscope slide is initially coated with 1% high or normal-melting point (HMP, NMP) agarose (the first agarose layer), and then, the embryos are mixed with a drop of low-melting point (LMP) agarose and placed on the first layer (the second agarose layer). We compared the impact of two protocols on the rate of cell loss. In Protocol 1, as described by Berthelot-Ricou et al. (2011a) we used 2 layers. In protocol 2, we used 3 layers (as described in the "Alkaline comet assay" section). For each protocol, the recovery rate was defined by the number of embryos found and analyzed on a slide at the end of the protocol compared to the total embryos initially deposited on the slide. For each of the 5 separate experiments, 30 embryos were used with protocol 1 and 30 embryos with protocol 2 (300 embryos) (Table 1).

2.6. Zona pellucida impact on comet assay.

To determine the impact of the ZP on the comet assay, we tested normal embryos (ZP+) and zona-free embryos (ZP -). In the ZP-embryos, the ZP was digested by an acidic Tyrode's solution at RT and was washed three times in M2 medium before use as described by Nicolson et al. [START_REF] Nicolson | Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs[END_REF]. The comet assay (Protocol 2) was performed on both control ZP+ and ZP-embryos and on ZP+ and ZPembryos exposed to H 2 O 2 . For each of the 5 separate experiments, 30 embryos were used for control (15 ZP+, 15 ZP-) and 30 for exposed embryos (15 ZP+, 15 ZP-) (300 embryos) (Table 1).

Exposure conditions

The subsequent experiments were performed on embryos with an intact zona pellucida (ZP+). To validate the sensitivity of the assay, we used two positive controls of well-known chemical and physical genotoxic agents (Berthelot-Ricou et al., 2011a;[START_REF] Kumaravel | Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals[END_REF][START_REF] Miranda-Vilela | Gene polymorphisms against DNA damage induced by hydrogen peroxide in leukocytes of healthy humans through comet assay: a quasi-experimental study[END_REF]: H 2 O 2 and Sun Simulated Irradiation (SSI). For the H 2 O 2 group, the embryos were placed in a 220 μM H 2 O 2 solution for 6 min at 4 °C in the dark. SSI induces DNA damage by irradiation to light. For the SSI group, light irradiation was carried out with a Suntest CPS + solar simulator (Atlas Material Testing Technology BV, Moussy le Neuf, France) equipped with a xenon arc lamp (1100 W) and special glass filters restricting transmission of light below 290 nm and near IR-blocking filter. The addition of glass filters limited the spectral emission to the 320-800 nm band (UVA-Visible light). The irradiation dose was 120 kJ/m 2 . The temperature of the samples was kept at 4 °C by using a regulated cooling system during the irradiation experiments. For each set of experiments, the control group consisted of embryos incubated for 1 h in KSOM medium. For each of the 5 separate experiments, 20 embryos were analyzed for each condition (control, H 2 O 2 or SSI) (300 embryos) (Table 1).

Alkaline comet assay

Protocol 2 for the alkaline comet assay was based on the protocol described by Berthelot-Ricou et al. (2011a) for oocytes with some modifications to adapt the method for embryos. We first covered glass microscope slides with HMP agarose (1.6% in PBS) and then dried these slides overnight at 20 °C. A 50 μL drop of 1% LMP agarose in PBS at 37 °C was placed on the first agarose layer, and the embryos were immediately transferred from the culture medium into this drop (under a stereo microscope to visualize the embryos). The resulting suspension was then gently mixed with the 1% LMP agarose. After a few seconds of solidification, the agarose drop with the embryos was surrounded by an 80 μL ring of 1% LMP agarose, covered with a coverslip (24 × 32 mm) and then kept 20 min at 4 °C for solidification (the second layer). After removing the coverslip, a third layer of 80 μL of 1% LMP agarose was deposited and was covered with a coverslip (24 × 32 mm) before solidification at RT. After removing the coverslips, slides were lysed in a freshly prepared lysis solution (LS) (2.5 M NaCl, 100 mMol Na2EDTA, 10 mMol Tris-HCl, pH 10, 1% Triton X-100, and 10% DMSO in ultrapure water) for 90 min at 4 °C. DNA unwinding was performed with an alkaline solution (1 mMol Na2EDTA and 300 mMol NaOH) for 20 min at 20 °C in a 30.5 × 22 cm horizontal electrophoresis unit. Next, electrophoresis was conducted for 30 min (25 V, 300 mA) in the same buffer solution at RT. After the electrophoretic run, the slides were neutralized 3 times with 0.4 M Tris-HCl (pH 7.5), rinsed with ultrapure water, dipped into 100% methanol (HPLC grade purity solvent), and dried overnight at RT. Staining was performed with a propidium iodide solution (0.1 mg/mL), and the slides were examined with a fluorescence microscope at a 400 × final magnification (Carl ZEISS Axio Imager A2, Bayern, Germany). The digital pictures were analyzed, and the determination of the % tail DNA was performed with a CCD Nikon camera (DS-Fi2, Nikon, Champigny-sur Marne, France) and Komet software (version 7.0, Nottingham, UK).

Statistical analysis

The % tail DNA was used to determine the DNA damage in each embryo. The experiments were replicated 5 times for each group. A ttest or Pearson's Chi-squared test as appropriate for statistical analysis was performed using StatView software. The % tail DNA is expressed as the means ± standard error of the mean (S.E.M.). A probability of p < 0.05 was considered significant.

Results

Embryo survival and culture

After thawing and recovery in KSOM, the overall survival rate of the 5 separate experiments was 85% (85/100: 85 intact embryos and 15 lysed embryos). After a 24 h culture, the viability rate was 99% (84 2cell embryos moving to the 4-8 cell stage out of 85 intact embryos). After a 48 h culture, the blastocyst rate was 82% (70 blastocysts obtained from in vitro culture of 85 intact 2-cell embryos).

Impact of the number of agarose layers on the embryos recovery rate

The overall recovery rate in the 5 different experiments using Protocol 1 (two agarose layers) was 3,3% (n = 5/150). With Protocol 2 (three agarose layers), the overall recovery rate was significantly increased to 71,3% (n = 107/150) (p < 0.0001).

Impact of the zona pellucida on the comet assay

The impact of the ZP on the Comet Assay is illustrated in Fig. 1. No statistically significant difference was observed in DNA damage for the ZP-and ZP + control groups (12.60 ± 2.53% tail DNA vs 11.04 ± 1.50, p = 0.5837, respectively). The ZP-and ZP + groups after H 2 O 2 exposure were also not statistically significant different (49.23 ± 4.16% Tail DNA vs 41.13 ± 4.31, p = 0.1820, respectively).

Alkaline comet assay

Fig. 2 shows the aspects of the comets observed in the control groups (A) and in the groups exposed to the genotoxic agents (B, C). The control embryos showed a low basal level of DNA damage. Fig. 3 shows that H 2 O 2 and SSI induced a statistically significant increase in DNA damage compared with the control group (41.13 ± 4.31% Tail DNA, 36.33 ± 3.02 and 11.04 ± 1.50, p < 0.0001, respectively).

Discussion

Thawed embryos can be used for comet assay

We describe, for the first time, the use of the alkaline comet assay on thawed mouse embryos by optimizing protocols that were previously published in the literature. Control mouse thawed embryos showed a low basal level of DNA damage, comparable to the level of DNA damage observed in control sperm [START_REF] Preaubert | Cerium dioxide nanoparticles affect in vitro fertilization in mice[END_REF] and control oocytes (Berthelot-Ricou et al., 2011a) in our hands. In the literature, we selected 14 studies using the comet assay on mammal embryos matching with our criteria, and no study was performed on frozen embryos. Among the 3 studies in mice [START_REF] Fabian | Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay[END_REF][START_REF] Müller | Comet assay studies indicate that caffeine-mediated increase in radiation risk of embryos is due to inhibition of DNA repair[END_REF][START_REF] Tranguch | Nitric oxide synthase production and nitric oxide regulation of preimplantation embryo development[END_REF], only Fabian et al. evaluated the evolution rate to the blastocyst stage in embryos obtained by natural fertilization, and it was estimated to be 88%. This result suggests that the freezing/thawing step in our protocol did not affect the embryo quality. The use of frozen embryos simplifies the handling by allowing the researcher to dissociate the location and the timing of the embryo collection from the embryo analysis, which facilitates collaboration between laboratories. The use of frozen embryos also allows subsequent analysis if required.

Deleting the removal of zona pellucida improves feasibility. Many authors remove the ZP before the comet assay, in order to study individual blastomeres and to allow DNA migration [START_REF] Harrouk | Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo[END_REF][START_REF] Hwang | Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos[END_REF][START_REF] Ju | Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro[END_REF][START_REF] Müller | Comet assay studies indicate that caffeine-mediated increase in radiation risk of embryos is due to inhibition of DNA repair[END_REF][START_REF] Sturmey | DNA damage and metabolic activity in the preimplantation embryo[END_REF][START_REF] Webster | Measurement of DNA damage by the comet assay in rat embryos grown in media containing high concentrations of vitamin K1[END_REF]; nevertheless, they did not study the impact the ZP on the downstream results. Moreover, this step requires the use of a chemical and physical stress that could unnecessarily damage the embryo and induce DNA damage. In a previous study of our group (Berthelot-Ricou et al., 2011a), we performed the comet assay on mouse oocytes with and without ZP, and no significant difference was found in the DNA damage in both of the groups. In the present study, we did not observe significant differences in DNA damage in the ZP-and ZP + embryos, indicating that the presence of the ZP did not affect the embryo lysis and/or DNA migration. Deleting the ZP removal step simplifies the protocol, saving time and improving feasibility.

Adding a third layer of agarose decreases the embryo loss

One of the main issues of our study was to reduce embryo loss during lysis and electrophoresis. As the main difficulty of embryos studies is the low number of available samples, this issue was very important. The significant embryo loss we experienced with Protocol 1 (two agarose layers) was surprising. Indeed, we never observed such a significant loss during the comet assay with Protocol 1 on mouse oocytes (Berthelot-Ricou et al., 2011b, 2011a, 2013;[START_REF] Courbiere | Ultrastructural interactions and genotoxicity assay of cerium dioxide nanoparticles on mouse oocytes[END_REF][START_REF] Einaudi | In vivo exposure to benzo(a)pyrene induces significant DNA damage in mouse oocytes and cumulus cells[END_REF][START_REF] Greco | A new approach for the oocyte genotoxicity assay: adaptation of comet assay on mouse cumulus-oocyte complexes[END_REF], which sizes are comparable to the embryo size. In the studies using the comet assay on embryos, selected by our literature review, the authors used protocols with 2 agarose layers and deposited between 1 and 20 embryos per glass slide (Table 2), which is comparable with Protocol 1 (20 embryos, 2 agarose layers). None of these studies described the ratio between the number of embryos deposited and the number of recovered embryos after lysis and electrophoresis. Nevertheless, we can assume that embryo loss was not as high as in the present study. Our hypothesis is that the important embryo loss we observed with Protocol 1 could be related to the alteration of ZP due to the freezing-thawing protocol: dehydratation due to cryprotectant could induce hardening and thinning of the ZP [START_REF] Cavusoglu | Ultra-structural alterations in in vitro produced four-cell bovine embryos following controlled slow freezing or vitrification[END_REF][START_REF] Trounson | Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo[END_REF]. These alterations could modify the interaction between the ZP and the agarose, and increase the embryo loss during the steps of coverslip removal and/ or lysis. The use of a third agarose layer dramatically decreased the rate of embryo loss.

Limitations

In our study, we measured DNA damage by the % Tail DNA, as recently recommended by the OECD (OECD, 2014). The use of the % Tail DNA gives a clear indication of the appearance of the comets, and it is also linearly related to the DNA break frequency over a wide range of levels of damage [START_REF] Ersson | The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments[END_REF][START_REF] Hartmann | Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop[END_REF]OECD, 2014;[START_REF] Uno | JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: I. Summary of pre-validation study results[END_REF]. Therefore, the amount of DNA damage we observed could not be easily compared with the DNA damage in the other studies we selected. Indeed, the tail moment and/or the tail length measurements are also reported herein (Table 2), as these studies were published before the OECD recommendations. To better evaluate the sensitivity of our optimized protocol, despite the inability to compare these factors, we used two well-known genotoxic agents as positive controls. Nevertheless, % Tail DNA should be used for DNA damage quantification. Another limitation is that we performed the Comet Assay experiments on thawed embryos after an in vitro exposure to two well-known genotoxic agents; our protocol should also be tested to detect embryo DNA damage induced after in vivo mice exposure associated to DNA damage in gametes [START_REF] Einaudi | In vivo exposure to benzo(a)pyrene induces significant DNA damage in mouse oocytes and cumulus cells[END_REF][START_REF] Singh | The antimalarial agent artesunate causes sperm DNA damage and hepatic antioxidant defense in mice[END_REF].

Conclusion

Our optimized protocol of the comet assay on thawed embryos allows the rapid detection of primary DNA damage in the mouse embryo. We simplified the protocol by showing that the lack of ZP removal does not alter DNA lysis and migration. In addition, we demonstrated that the adjunction of a third agarose layer decreases embryo loss and allows for the study of a small amount of embryos. This new optimized tool could be used to analyze DNA damage in mice embryos after in vivo exposure of the mothers to environmental agents.

Transparency document

The http://dx.doi.org./10.1016/j.tiv.2017.07.010 associated with this article can be found, in online version.

  vitro fertilization; LMP: low melting point; NMP: normal melting point; LS: lysis solution (2.5 M NaCl; 100 mM Na2EDTA; 10 mM Tris-HCl, pH 10; 1% Triton X-100); PK: proteinase K; DMSO: dimethyl sulfoxide; SLS: sodium lauroyl sarcosinate; SDS: sodium dodecyl sulfate; RT: room temperature; OTM: Olive Tail Moment; Z: zygote (one cell); C: cleavage stage embryo; M: morula stage embryo; B: blastocyst stage embryo.

Fig. 1 .

 1 Fig. 1. No impact of zona pellucida for 2-cell embryo comet assay. ZP-: zona-free embryo; ZP+: intact embryo; H 2 O 2 : exposure to hydrogen peroxide (220 μM H 2 O 2 ). No statistical difference was observed on DNA damage between ZP-et ZP+ embryo on control group and on H 2 O 2 exposure group. *: p < 0.01 between DNA damage in Control and in H 2 O 2 exposed embryos. t-Test.

Fig. 2 .

 2 Fig. 2. Examples of different aspects of DNA damage as detected by the comet assay on mouse 2-cell embryos with zona pellucida. A: Control group: intact 2-cell embryo after comet assay process. B: 2-cell embryo exposed in vitro for 5 min to 220 μMol H 2 O 2 . C: 2-Cell embryo exposed in vitro to Simulated Sun Irradiation. The direction of electrophoresis was from left to right, and the comet tail containing the DNA fragments was stained by iodide propidium 0.1 mg/mL. When 2-cell embryo DNA is intact, the two nuclei remain separated (A). When 2-cell embryo DNA is damaged, the two nuclei overlap and appear merged. (B, C). Arrows show the polar bodies. Scale bar, 100 μm. Magnification × 400.

Fig

  Fig. % Tail DNA of mouse 2-cells embryo with zona pellucida exposed to two genotoxic agents and measured by comet assay. Control; H 2 O 2 , exposure to hydrogen peroxide (220 μM); SSI, exposure to simulated sunlight irradiation (120 J/cm 2 ); ***statistical significance (p < 0.001). t-Test.

Table 1

 1 Experiments performed in the study and number of thawed embryos used for each one.Published protocols of comet assay on preimplantation embryos.

	Experiments performed	Embryo stage used Protocol used after embryo thawing	Nb of separate experiments Total nb of 2-cells embryos used
	Assessment of embryo viability and blastulation	2-Cell	2-Cells embryo in vitro culture to	5	20 × 5 = 100
	rates		blastocyst stage
	Impact of the nb of agarose layers on embryo	2-Cell	CA Protocol 1	5	30 × 5 = 150
	recovery rate	2-Cell	CA Protocol 2	5	30 × 5 = 150
	Impact of ZP on CA results	2-Cell	CA Protocol 2	5	60 × 5 = 300
	Impact of genotoxic agents exposure on DNA	2-Cell	CA Protocol 2	5	60 × 5 = 300
	damage
	Total				1000

CA: comet assay (Protocol 1 uses 2 agarose layers; Protocol 2 uses 3 agarose layers), ZP: zona pellucida.

L. Rolland et al.

Toxicology in Vitro 44 (

2017

) 266-272

Table

2

  ). No statistical difference was observed on DNA damage between ZP-et ZP+ embryo on control group and on H 2 O 2 exposure group. *: p < 0.01 between DNA damage in Control and in H 2 O 2 exposed embryos. t-Test.

Acknowledgments

The authors thank technicians from CECOS Laboratory (AP-HM La Conception) for technical help.

Funding

This work was conducted thanks to the support of the A*MIDEX project "CREER" (n ANR-11-IDEX-0001-02), which was funded by the "Investissements d'Avenir" French Government program and managed by the French National Research Agency (ANR). The study received additional funding from "Association Laurette Fugain" (no. 2014/01), the ARC Foundation (no. PJA 20141201596), and the Aix Marseille University Foundation (no. 16/07/01).

Conflicts of interest

None declared.


Mathieu Luglia

S Criquet

Max Sarrazin

Fabio Ziarelli

Daniel Guiral

Functional patterns of microbial communities of rhizospheric soils across the development

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


Yoshiki Nagatani
email: nagatani@ultrasonics.jp

Séraphin Guipieri

Vu-Hieu Nguyen

Christine Chappard

Didier Geiger

Salah Naili

Guillaume Haїat

Clara Ceron

Hung-Son Phan

Three-dimensional Simulation of Quantitative Ultrasound in Cancellous Bone using the Echographic Response of a Metallic Pin

Keywords: FDTD Simulation, Cancellous Bone, Quantitative ultrasound (QUS), 3D Model, Discopathy

Degenerative discopathy is a common pathology which may require spine surgery. A metallic cylindrical pin is inserted into the vertebral body to maintain soft tissues and may be used as a reflector of ultrasonic wave to estimate bone density. The first aim of this paper is to validate a 3D model to simulate the ultrasonic propagation in a trabecular bone sample in which a metallic pin has been inserted. We also aim at determining the effect of changes of bone volume fraction (BV/TV) and of positioning errors on the quantitative ultrasound (QUS) parameters in this specific configuration. The approach consists in coupling finite difference time domain simulation with X-ray microcomputed tomography. The correlation coefficient between experimental and simulated speed of sound (SOS) (respectively broadband ultrasonic attenuation (BUA)) was equal to 0.90 (respectively 0.55). The results show a significant correlation of SOS with BV/TV (R = 0.82), while BUA values exhibit a non-linear behavior versus BV/TV. The orientation of the pin should be controlled with an accuracy of around 1° in order to obtain accurate results. The results indicate that using the ultrasonic wave reflected by a pin has a potential to estimate the bone density. SOS is more reliable than BUA due its lower sensitivity to the tilt angle.

I. INTRODUCTION

Degenerative discopathy (DD) is one of the most common etiology of spinal degeneration and may require spine surgery [START_REF] Hughes | The pathogenesis of degeneration of the intervertebral disc and emerging therapies in the management of back pain[END_REF] . Spinal fusion is one of the possible options to treat intervertebral disc (ID) degeneration. In spinal fusion surgery, various implantable devices are used to connect two adjacent vertebrae such as cages, plates, screws and a variety of other fusion materials. In some cases, external fixations (such as pedicle screws) may be used in order to reinforce the biomechanical stability of the vertebral structure [START_REF] Lee | Accelerated degeneration of the segment adjacent to a lumbar fusion[END_REF] . However, the choice of the method used for pedicle screw insertion (use of cement, length and diameter of the screws) remains empirical and depends on bone biomechanical properties [START_REF] Shea | Designs and techniques that improve the pullout strength of pedicle screws in osteoporotic vertebrae: current status[END_REF] .

Another technique for the treatment of ID degeneration is total disc replacement (TDR), which has the advantage of preserving spinal motion [START_REF] Vital | Total disc replacement[END_REF] and maintaining a mobility between the two adjacent vertebrae [START_REF] Hughes | The pathogenesis of degeneration of the intervertebral disc and emerging therapies in the management of back pain[END_REF] . However, the long-term stability of implants used in TDR remains difficult to obtain due to problems of bony fixation and anchorage to the host spine, which depends on the vertebral bone properties. Moreover, a risk of impaction fracture of the vertebrae still remains, depending on bone properties. For all the aforementioned reasons, a reliable method to assess bone biomechanical properties would be of interest in spine surgery because the number of osteoporotic patients increases with age.

Dual-energy X-ray Absorptiometry (DEXA) is currently the only method available in clinical practice to estimate vertebral bone quantity and is sometimes used before spine surgery. It remains difficult to use DEXA to obtain a reliable information on vertebra L-5 (which is an important site of DD), because of the presence of soft tissue disrupting the measurements.

DEXA can only retrieve bone mineral density (BMD), which is not sufficient to determine bone biomechanical properties [START_REF] Nih | Osteoporosis prevention, diagnosis, and therapy[END_REF] since BMD does not provide information on bone microstructure nor material properties. Quantitative ultrasound (QUS) techniques represent a complementary approach to DXA [START_REF] Laugier | Bone Quantitative Ultrasound[END_REF][START_REF] Garra | Measurements of Ultrasonic Backscattered Spectral Centroid Shift from Spine in vivo: Methodology and Preliminary Results[END_REF] because ultrasound being mechanical wave, QUS can be used to retrieve bone biomechanical properties.

QUS techniques have been used clinically to estimate bone biomechanical properties at different sites such as the calcaneum [START_REF] Chin | Calcaneal quantitative ultrasound as a determinant of bone health status: what properties of bone does it reflect?[END_REF] , phalanx [START_REF] Olszynski | Multisite quantitative ultrasound for the prediction of fractures over 5 years of follow-up: the Canadian Multicentre Osteoporosis Study[END_REF] , tibia [START_REF] Maatta | Association between low-frequency ultrasound and hip fractures --comparison with DXA-based BMD[END_REF] , femur [START_REF] Barkmann | A device for in vivo measurements of quantitative ultrasound variables at the human proximal femur[END_REF][START_REF] Liu | Signal of Interest Selection Standard for Ultrasonic Backscatter in Cancellous Bone Evaluation[END_REF][START_REF] Jiang | Analysis of Apparent Integrated Backscatter Coefficient and Backscattered Spectral Centroid Shift in Calcaneus in vivo for the Ultrasonic Evaluation of Osteoporosis[END_REF] or radius [START_REF] Sasso | Analysis of the most energetic late arrival in axially transmitted signals in cortical bone[END_REF] using transverse [START_REF] Mathieu | Radial anatomic variation of ultrasonic velocity in human cortical bone[END_REF] and axial [START_REF] Tatarinov | Multi-frequency axial transmission bone ultrasonometer[END_REF] transmission devices. However, it remains difficult to assess vertebral bone density due to accessibility issues, the main difficulty consisting in positioning several transducers around the vertebral body during surgery.

Fortunately, when surgery is performed with an anterior approach, metallic cylindrical pins are commonly inserted into the vertebral body in order to maintain soft tissues. The pins are then removed from bone tissue at the end of the surgery. A recent experimental work by our group [START_REF] Guipieri | Ultrasound Speed of sound measurements in trabecular bone using the echographic response of a metallic pin[END_REF] has suggested to use this pin as a reflector in order to carry out Speed of Sound (SOS) measurements in trabecular bone. It remains difficult to achieve an accurate bone microstructure characterization using usual echographic techniques [START_REF] Garra | Measurements of Ultrasonic Backscattered Spectral Centroid Shift from Spine in vivo: Methodology and Preliminary Results[END_REF][START_REF] Chin | Calcaneal quantitative ultrasound as a determinant of bone health status: what properties of bone does it reflect?[END_REF][START_REF] Tatarinov | Multi-frequency axial transmission bone ultrasonometer[END_REF] because of the strong attenuation and of the complexity of trabecular bone. For instance, multiple scattering phenomena have been evidenced, which makes it difficult to apply classical speckle models to extract quantitative information from the analysis of the backscattered signal. Therefore, the advantage of using a pin as a reflector compared to classical echographic techniques to achieve accurate and reproducible measurements in the echographic configuration. However, the physical determinants of the interaction between a trabecular bone sample including a pin and an ultrasonic wave remain difficult to understand due to the complexity of the phenomena involved. Moreover, the possibility of using normalized broadband ultrasonic attenuation (BUA) to characterize bone quantity has not yet been described in this given configuration. The sensitivity of the QUS parameters to experimental errors related to the relative position of the pin and of the transducer should be determined because it might jeopardize future in vivo measurements.

Coupling numerical simulation with high resolution imaging techniques has been shown to constitute a powerful approach capable of bringing further insight on the interaction between bone tissue and ultrasound. In particular, coupling 3-D FDTD simulation tools with 3-D images obtained from microcomputed tomography (µCT) has been employed in the past to model the ultrasonic propagation in trabecular bone [START_REF] Nagatani | Applicability of finite-difference time-domain method to simulation of wave propagation in cancellous bone[END_REF][START_REF] Kaufman | Ultrasound simulation in bone[END_REF][START_REF] Hosokawa | Development of a Numerical Cancellous Bone Model for Finite-Difference Time-Domain Simulations of Ultrasound Propagation[END_REF] .

The aim of this paper is to develop a numerical model obtained using the coupling of threedimensional finite difference time domain simulation with X-ray µCT in order to simulate the ultrasonic wave propagation occurring in trabecular bone samples in which a metallic has been inserted. The numerical model aims at taking into account the ultrasonic propagation in trabecular bone as well as the reflection of the ultrasonic wave on the pin (whose axis is perpendicular to the direction of propagation). More specifically, we aim at i) validating the numerical model by comparing the QUS parameters (SOS and BUA) obtained experimentally and numerically, ii) assessing the effect of variation of bone volume fraction on the QUS parameters obtained in this given configuration and iii) assessing the effects of changes of the relative orientation of the pin and of the sensor axis on the QUS parameters.

II. MATERIALS AND METHOD

A. Experimental measurements

Twenty-one trabecular bone specimens were obtained from bovine femurs similarly as in reference 18. However, two specimens were excluded from the present study due to a problem which occurred when cutting specimens after the experimental measurements because it was not possible to obtain their 3-D image accurately. As a result, only nineteen specimens were employed in this study, which had already been employed in reference 18.

Soft tissues were removed manually using a hand bistoury and the samples were then cut in the proximal region in order to obtain cubic specimens (with a size of approximately 25*25*25mm) using an electric saw.

The ultrasound measurements were carried out similarly as in reference 18. Briefly, all samples were immersed in water at room temperature (which was monitored) and degassed before each measurement. A circular transducer (CMP 89, Sonaxis, Besançon, France) with a resonance frequency equal to 500 kHz and a diameter of the active surface equal to 16.0 mm was positioned in contact with one side of the cubic sample. The ultrasonic transducer was connected to a pulse-receiver amplifier (5052A, Panametrics, Waltham, MA, USA) and used in echographic mode. The received waveform was recorded by a PC via an A/D converter of 100 MHz sampling rate with 12-bit resolution (Spectrum, Grosshansdorf, Germany). The rf signal obtained with the transducer in contact with the bone sample was then recorded and is denoted Sb(t) in what follows.

The same stainless steel pin (Surgiway, Paris, France) as the one used during surgery was then inserted into the bone specimens in a similar manner what is done in the operating room. The pin was a 3.95 mm diameter 240 mm long cylinder made of stainless steel. A clamp was employed to hold the specimens tightly so that no relative movement of the transducer compared to the bone sample is allowed during the insertion of the pin. The distance between the surface of the transducer and the axis of the pin was equal to 11.0 mm. The distance between the pin and the transducer was kept constant thanks to the experimental device shown in Fig. 2 of reference 18, which allows a reproducible insertion of the pin thanks to the presence of a hole with a diameter matching precisely that of the pin. The pin was inserted in this hole, which is rigidly attached to the structure supporting the transducer, when performing the experiments in water as well as when hammering the pin into bone tissue. The rf signal obtained with the pin inserted in the bone sample was then determined and is given by Sp(t) in what follows. The echo of the pin immersed in water was also measured and the rf signal obtained was denoted by Sw(t). As shown in 18, the range of variation obtained for the experimental values BUA, SOS and BV/TV is equal to 18.1 to 43.3 dB/MHz/cm, 1520 to 2200 m/s, and 10.0 to 39.9 %, respectively.

After the ultrasound measurements were performed, the bone samples were cut in the plane perpendicular to the transducer axis and containing the axis of the pin in order to assess bone volume fraction. Only the region of the sample located between the transducer and the pin was considered. The size of the analyzed sample was equal to 10*10*10 mm. An X-ray µCT device (Skyscan1176® scanner, Skyscan, Kontich, Belgium) was used to obtain the 3-D image of the bone specimen with a resolution of 17.7 µm. The CT images were binarized in order to separate the image into bone tissue and liquid. The threshold was chosen so that the value was in the middle of the histogram of CT images. The value of BV/TV (bone volume fraction) was then determined for each sample.

B. Three-dimensional FDTD numerical simulation

In the framework of the linear elasticity, a 3-D elastic finite-difference time-domain (FDTD) method was used to simulate wave propagation inside the trabecular bone specimens. FDTD methods take into account wave propagation in solids as well as mode conversion. The software packages SimSonic3D (available at http://www.simsonic.fr/) was used in this study [START_REF] Bossy | Three-dimensional simulations of ultrasonic axial transmission velocity measurement on cortical bone models[END_REF] .

Table I shows the material properties used for all media, which were derived by the data of cortical bone and considered as elastic and isotropic [START_REF] Sasso | Analysis of the most energetic late arrival in axially transmitted signals in cortical bone[END_REF][START_REF] Yamato | Correlation between hydroxyapatite crystallite orientation and ultrasonic wave velocities in bovine cortical bone[END_REF] . A Gaussian pulse with a center frequency equal to 500 kHz and similar to the one used experimentally was employed in the simulation. The parameters shown in Table 1 correspond to material properties at room temperature. Figure 1 shows an example of the geometrical configuration used in the simulation. The specimen was immersed in water. The original spatial resolution obtained with the X-ray µCT device (17.7 µm) was reduced to 35.4 µm in the simulation model in order to obtain a compromise between a reasonable computation time and an acceptable precision, similarly as what was done in [START_REF] Nagatani | Applicability of finite-difference time-domain method to simulation of wave propagation in cancellous bone[END_REF][START_REF] Nagatani | Two-wave behavior under various conditions of transition area from cancellous bone to cortical bone[END_REF] . A circular transducer with a diameter equal to 10 mm acting in echographic mode was located in contact with one side of each sample.

The orientation of the specimen and of the direction of propagation was the same as the ones considered experimentally in [START_REF] Guipieri | Ultrasound Speed of sound measurements in trabecular bone using the echographic response of a metallic pin[END_REF] . In order to avoid wave reflection between the specimen and water at the opposite end of the transducer, the specimen model was duplicated using a planar symmetry relatively to the plane containing the pin axis and perpendicular to the transducer axis, as shown in Fig. 1. A cylindrical stainless steel bar with a diameter equal to 4.0 mm was inserted in the bone sample at a similar distance compared to the experimental case.

C. Determination of the QUS parameters

The QUS parameters were determined following a substitution method described in more details in reference 18. The same method described below was used for all experimental and simulated rf signals. Examples of wave propagation representation and simulated rf signals are shown in Fig. 2 and3, respectively.

For each bone sample, SOS and normalized BUA were determined 7 by comparing the rf signal obtained from the echo of the pin immersed in water (Sw(t)) and the echo of the pin in bone tissue (Sp(t)). However, as shown in Fig. 3b-1, it remains difficult to precisely distinguish the wave reflected by the inserted pin from the echoes generated by the microstructure of trabecular bone, which is due to the complex echographic response of the trabecular structure. Therefore, the signal corresponding to the ultrasonic response of bone tissue located between the pin and the transducer is removed by considering the echo of the pin only given by: D(t) = Sp (t) -Sb (t) , (1) where Sb (t) is the echo of the bone tissue without inserting a pin. The determination of D(t) corresponds to a simple way of distinguishing the echographic response of the pin. In what follows, SOS and BUA are determined by comparing D(t) (which corresponds to the echo of the pin immersed in bone tissue) and Sw(t) (which corresponds to the echo of the pin immersed in water, reference signal). SOS was determined following:

SOS: c = 2𝐿 2𝐿 𝑣 𝑤𝑎𝑡𝑒𝑟 + (𝑡 𝐷 -𝑡 𝑤 ) , ( 2
)
where vwater is the sound speed in water and L is the distance between the surface of the transducer and the pin and tD and tw are the time of the first maximum of D(t) and Sw(t), respectively. We chose to consider a time marker in the early part of the signal because such method has been shown to lead to a better correlation between SOS and BV/TV than when using time markers considering the entire signal (such as for example group or phase velocity) [START_REF] Haïat | Optimal prediction of bone mineral density with ultrasonic measurements in excised femur[END_REF] .

Moreover, normalized BUA was determined by the slope of the curve of the attenuation coefficient α(f) as a function of frequency in the 300-600 kHz range, with

α(𝑓) = 1 2𝐿 20log 10 |𝐴 𝑤 (𝑓)| |𝐴 𝐷 (𝑓)| , (3)
where AD(f) and Aw(f) are the amplitude spectrum of D(t) and Sw(t), respectively (see Fig. 4).

D. Effect of a variation of the orientation of the pin.

A potential source of error on the determination of the QUS parameters lies in a possible default of alignment between the transducer axis and the normal of the pin axis. Such effect remains difficult to estimate experimentally because a given bone sample cannot be reused after the pin insertion. Therefore, the effect of a variation of the orientation of the pin relatively to the orientation of the transducer on the determination of the QUS parameters was estimated by modifying the simulation domain. The axis of the pin inserted in trabecular bone was rotated around an axis perpendicular to the ultrasonic propagation for three representative specimens, as shown in Fig. 5. Here, the pin was rotated around an axis in the y direction crossing the pin at its center (shown by dots in Fig. 5). The effect of a variation of this tilt angle on the QUS parameters was investigated using virtual models for angles comprised between -16.7 degrees to +16.7 degrees for all samples, which leads to 171 different simulations.

E. Dilation algorithm

Image processing algorithms were used in order to modify bone volume fraction by using same CT images of a sample. First, the binarizing threshold [START_REF] Nagatani | Multichannel instantaneous frequency analysis of ultrasound propagating in cancellous bone[END_REF] applied to the original µCT images was varied. In addition to the technique consisting in varying the binarizing threshold, a 3-D dilation algorithm was applied for deriving high BV/TV models (between 50% and 65%) [START_REF] Haïat | Variation of Ultrasonic Parameters with Microstructure and Material Properties of Trabecular Bone: A 3D Model Simulation[END_REF][START_REF] Padilla | Estimation of trabecular thickness using ultrasonic backscatter[END_REF][START_REF] Jenson | Prediction of frequency-dependent ultrasonic backscatter in cancellous bone using statistical weak scattering model[END_REF][START_REF] Chaffai | Ultrasonic characterization of human cancellous bone using transmission and backscatter measurements: relationships to density and microstructure[END_REF][START_REF] Wear | The dependence of ultrasonic backscatter on trabecular thickness in human calcaneus: theoretical and experimental results[END_REF][START_REF] Luo | Computational methods for ultrasonic bone assessment[END_REF] : The algorithm replaces the liquid portion, which is surrounded by bone, with bone material so that the bone volume fraction of the model is increased. The use of these image processing algorithms constitutes a simple way of investigating the effect of BV/TV variation, allowing to obtain many different BV/TV values for the same sample. SOS and BUA values were then determined for each modified image.

III. RESULTS

Figure 6 shows the comparison between experimental and simulated results obtained for SOS and BUA. The correlation coefficient obtained between simulated and experimental SOS was equal to R = 0.90 (p < 0.001). The correlation coefficient obtained between simulated and experimental BUA is lower but the correlation is significant (R = 0.55, p = 0.015).

Figure 7 shows the variation of the simulated values of SOS and BUA as a function of BV/TV.

A significant linear correlation was obtained between SOS and BV/TV (correlation coefficient R = 0.82, p < 0.001). However, the linear correlation between BUA and BV/TV was not significant (p = 0.65), which will be discussed in section IV.

Figure 8 shows the variation of the experimentally measured values of SOS (R = 0.77, p < 0.001) and BUA (p = 0.40) as a function of BV/TV. Similarly to the simulated results, no significant linear correlation was found between experimental BUA and BV/TV.

Figure 9 shows the variation of the QUS parameters as a function of the incident angle for three representative samples. Figure 9(a) (respectively 9(b)) shows that an error of 6 degrees of the angle causes a variation of around 25~50 m/s for SOS (respectively around 5~10 dB/MHz/cm for BUA).

IV. DISCUSSION

An originality of this study is to consider the application of a method coupling highresolution imaging technique and FDTD numerical simulation (which has already been employed in the past with through transmission devices [START_REF] Hosokawa | Development of a Numerical Cancellous Bone Model for Finite-Difference Time-Domain Simulations of Ultrasound Propagation[END_REF][START_REF] Bossy | Three dimensional simulation of ultrasound propagation through trabecular bone structures measured by synchrotron microtomography[END_REF] ) to a configuration corresponding to the reflection of an ultrasonic wave of a metallic pin inserted in trabecular bone. The 3-D FDTD numerical model is validated by comparing experimental and numerical values of SOS. Moreover, we now show the behavior of BUA (both experimental and numerical) as a function of BV/TV and provide physical explanations for the results.

Another originality is to assess the sensitivity of the measurement method to variations of the tilt angle, which is important because it provides information on the requirement for precision of the orientation of the pin compared to the transducer axis for future developments of the device. Note that this sensitivity study would not have been possible by considering an experimental approach because the pin insertion can only be done once. More specifically, as shown in Fig. 9, an error of 6 degrees on the tilt angle causes a variation of around 25~50 m/s for SOS (respectively around 5~10 dB/MHz/cm for BUA). Moreover, the experimental errors of SOS (respectively BUA) measurements when realized in a transverse transmission configuration is equal to around 8 m/s (respectively 0.6 dB/cm/MHz) [START_REF] Laugier | Bone Quantitative Ultrasound[END_REF] . The comparison of the sensitivity of QUS parameters to the tilt angle and of the reproducibility typically obtained with transverse transmission devices can be used in order to determine the maximum acceptable error on the tilt angle, which is equal to 0.36 -0.72° for BUA and to 1.0 -1.8 ° for SOS. The acceptable error on the tilt angle for BUA measurements is significantly lower than SOS measurements, which indicates that the precision requirement is stronger in order to obtain a reliable BUA measurement compared to SOS. This result indicates that SOS may be a more robust indicator than BUA since it may be difficult to practically control the error on the tilt angle with a precision lower than 1°.

A. Comparison between experiments and simulation

We checked that the range of variation of BUA obtained in the present study (10-50 dB/MHz/cm) is in agreement with previous findings (see for example 37,38). Different limitations may explain the differences between the experimental and numerical results

shown in Fig. 6 concerning both BUA and SOS values. First, bone material properties are not known and generic values are used instead. Second, bone material was assumed to be homogeneous and isotropic. Third, we considered a slightly different geometrical configuration in the simulation compared to the experiments because the diameter of the transducer was smaller (10 mm) in the simulation than in the experiments (16 mm). This choice was made i) in order to take into account the fact that piston mode is not obtained in real transducer and ii) due to a limitation in terms of size of the µCT device. Fourth, the bovine samples used herein have a higher density compared to human vertebral samples (in the range 7%-17%) [START_REF] Hulme | Regional variation in vertebral bone morphology and its contribution to vertebral fracture strength[END_REF][START_REF] Peyrin | Characterization of bone samples from synchrotron radiation µCT : evaluation of the segmentation of µMRI images, in Noninvasive assessment of trabecular bone architecture and the competence of bone[END_REF][START_REF] Mizuno | Propagation of two longitudinal waves in human cancellous bone: an in vitro study[END_REF] . However, this difference in terms of range of variation in BV/TV does not affect the validity of the results concerning the correlation between BV/TV and the QUS parameters, except that the nonlinear variation of BUA as a function of BV/TV is not likely to be encountered for vertebral bone samples. Following our previous study realized with the same samples [START_REF] Guipieri | Ultrasound Speed of sound measurements in trabecular bone using the echographic response of a metallic pin[END_REF] , we choose not to consider human vertebral samples due to ethical reasons since our aim was to validate the measurement protocol before its application to configurations closer to the clinical situation of interest.

Despite these limitations, a significant correlation was found between numerical and experimental SOS. However, the simulated values of SOS were slightly overestimated compared to the experimental results. The Bland and Altman method [START_REF] Bland | Statistical Methods for Assessing Agreement Between Two Methods of Clinical Measurement[END_REF] revealed a systematic bias (-115.6±50.2 m/s (95% CI)) of the numerically calculated SOS compared to the experimentally measured SOS. The same sources of discrepancies listed above may also hold to explain the difference between numerical and experimental SOS.

The weak correlation obtained between experimental and simulated BUA (which is comparable to the results obtained in reference 43) can be explained by the limitations described above as well as by the following limitations. In a previous report by Bossy et al. [START_REF] Bossy | Attenuation in trabecular bone: A comparison between numerical simulation and experimental results in human femur[END_REF] , a strong linear correlation was evidenced between measured and simulated BUA values with an R 2 -value of 0.83. One possible explanation for the good correlation found in Bossy et al. [START_REF] Bossy | Attenuation in trabecular bone: A comparison between numerical simulation and experimental results in human femur[END_REF] between experimental and numerical BUA in the case of a classical transverse transmission configuration and the relatively poor correlation found in the present study may be the effect of errors of the tilt angle on the pin compared to the axis of the transducer (see beginning of subsection IV for more details). Errors due to the tilt angle are not present in classical transverse transmission devices and are shown to be more important for BUA than on SOS. Therefore, a possible explanation for the relatively low correlation between experimental and numerical BUA may be the influence of errors on the tilt angle. Another possible explanation for the relatively low correlation between experimental and simulated BUA may be related to phase cancellation effects [START_REF] Cheng | Effects of Phase Cancellation and Receiver Aperture Size on Broadband Ultrasonic Attenuation for Trabecular Bone In Vitro[END_REF] , especially since simulation and experimental transducers had different sizes and therefore different amounts of phase cancellation. Since SOS is computed using a time marker considering the beginning of the signal, SOS may be less sensitive to phase cancellation effects than BUA.

Similarly as in reference 43, our results show that experimental values of BUA were higher than numerical values. Note that the model ignores absorption as a loss mechanism, which may be present in marrow-filled bone specimens [START_REF] Haïat | Velocity dispersion in trabecular bone: influence of multiple scattering and of absorption[END_REF] . However, as shown in reference 47, the fact that absorption was neglected cannot be used to explain the fact that experimental BUA is higher than theoretical BUA. Note that the effect of absorption on BUA is not understood because contradictory results have been obtained in the literature [START_REF] Pakula | Influence of the filling fluid on frequency-dependent velocity and attenuation in cancellous bones between 0.35 and 2.5 MHz[END_REF] .

B. Correlation between the QUS parameters and BV/TV

The results shown in Fig. 7(a) indicate a significant correlation between the simulated SOS values and BV/TV, in good agreement with the experimental results obtained in Fig. 4 of the reference 18. However, a slightly nonlinear relationship is obtained between BV/TV and SOS, with a peak around 30 or 35%, as shown in Fig. 7(a). This result must be interpreted with caution due to the low number of samples considered. Although most studies have found a linear relationship between SOS and BV/TV e.g. [START_REF] Laugier | Bone Quantitative Ultrasound[END_REF] , some studies have also found nonlinear variations [START_REF] Haïat | Fast wave propagation in trabecular bone: numerical study of the influence of porosity and structural anisotropy[END_REF] , which were explained by the presence of fast and slow waves propagating in the sample. The presence of such phenomena might explain the nonlinear variation of SOS as a function of BV/TV obtained herein. Note that such variations were not observed experimentally nor with the dilation/erosion model because of the particular conditions (in particular in relation with the degree of anisotropy) necessary to distinguish the fast and slow wave modes. Moreover, when fast and slow wave modes can be distinguished, phase cancellation effects [START_REF] Cheng | Effects of Phase Cancellation and Receiver Aperture Size on Broadband Ultrasonic Attenuation for Trabecular Bone In Vitro[END_REF] may occur, which may be due to a nonlinear variation of the attenuation coefficient as a function of frequency [START_REF] Haïat | Fast wave propagation in trabecular bone: numerical study of the influence of porosity and structural anisotropy[END_REF] and therefore a modification of the BUA value. Note that in this case, BUA cannot be defined.

Moreover, the variation of BUA as a function of BV/TV obtained experimentally (see Fig. 8b) and numerically (see Fig. 7(b)) is shown to be nonlinear, with a maximum value of BUA for a value of BV/TV of around 25 %. The second order polynomial regression curves are indicated in Fig. 7b and8b. The maximum value of the regression polynomial fit is comparable for the simulated and the experimental data (31.9 dB/MHz/cm in Fig. 7b and 33.5 dB/MHz/cm in Fig. 8b). Moreover, the maximum value of the regression polynomial fit is obtained for comparable values of BV/TV (25.3% in Fig. 7b and 24.2% in Fig. 8b). Although qualitatively comparable, these results are slightly different than those obtained experimentally by reference 49 who found a maximum value of 60 dB/cm/MHz for a value of BV/TV equal to 18 %. When considering samples with relatively low BV/TV values (between 5 and 10%), increasing BV/TV induces an increase of the size of the trabeculae, which act as scatterers [START_REF] Haïat | Independent scattering model and velocity dispersion in trabecular bone[END_REF]51 . An increase of the size of the trabeculae induces an increase of their scattering cross-section, and thus of the frequency dependent attenuation, which may lead to an increase of the BUA, as obtained in reference 30. However, a competing effect occurs when BV/TV values are higher (typically around 30 to 40%) since the pores may then act as scatterers instead of the trabeculae for samples with low porosity 52 , leading to the opposite behavior of attenuation as a function of porosity. However, more work is needed to understand these phenomena. Note that the modified Biot-Attenborough model and the scattering theory have also been employed in reference 53 to explain the nonlinear variation of BUA as a function of BV/TV.

In order to verify the aforementioned phenomenological explanation, investigations of the effect of BV/TV were performed by modifying in silico BV/TV values for three given samples using the erosion/dilation method. To do so, three representative specimens with different BV/TV values were selected (10.0%, 19.4%, and 35.7%, respectively). The results shown in BV/TV values lower than 17%, a significant correlation coefficient is obtained between BUA and BV/TV for simulated (R = 0.66) and experimental results (R = 0.72). These results are consistent with previous numerical results obtained with human samples [START_REF] Haïat | Variation of Ultrasonic Parameters with Microstructure and Material Properties of Trabecular Bone: A 3D Model Simulation[END_REF] .

C. Effect of the tilt angle.

The results shown in Fig. 9 indicate a significant effect of the tilt angle on the QUS parameters for the three representative specimens. Interestingly, the minimum value of SOS and of BUA are obtained for a relatively similar value of the tilt angle for each sample, which is not equal to 0°. These two values of tilt angles, for which minimum values of BUA and SOS are obtained, are noted TABUA and TASOS respectively in what follows. Figure 11 shows the relationship between the values of the tilt angles TABUA and TASOS for all samples.

The values shown in Fig. 11 were obtained using a parabolic regression of the QUS parameters as a function of the tilt angle in order to account for possible numerical errors.

One specimen which did not show a concave curve was rejected. A significant correlation (R = 0.82) was found between TABUA and TASOS. Moreover, the linear regression curve is given by the relation y = 1.16x -0.41 and is close to the identity line.

The aforementioned results can be explained as follows. If the ultrasonic propagation occurred in an isotropic medium, the minimum value of SOS would be obtained for a tilt angle equal to zero because it corresponds to a geometrical situation where the propagation distance between the sensor and the pin reaches its minimum value. However, in anisotropic media, the direction of propagation of the ultrasonic energy is not necessarily in the direction perpendicular to the emitter 54 . Let α denote the angle between the normal of the transducer and the direction of propagation of the energy. If α ≠ 0, the propagation distance will be equal to 𝐷/ cos(𝛼𝐷) when the tilt angle is equal to 0 and will be minimum and equal to D when the tilt angle is equal to α. Therefore, the anisotropic behavior of bone tissue may explain that the minimum value of the velocity is not obtained for tilt angles equal to 0, as shown in Figs. 9 and 11.

A similar explanation can be used to explain the minimum value of BUA for nonzero tilt angles. If α ≠ 0, the incident angle of the energy will be equal to α when the tilt angle is equal to 0 and equal to 0 when the tilt angle is equal to α, which explains the higher BUA when the tilt angle is equal to 0.

Therefore, these results may be explained by the structural anisotropy of the samples, which is known to significantly affect the ultrasonic propagation [START_REF] Haïat | Optimal prediction of bone mineral density with ultrasonic measurements in excised femur[END_REF] . However, it was not possible to quantify the structural anisotropy of the sample and more work is needed to understand the effect of structural anisotropy on these results. Moreover, the structural anisotropy of trabecular bone affects the received waveforms, which has been evidenced for example in 48 in a through transmission configuration. In some cases, the wave may separate into two waves (fast wave and slow wave, see above), which makes it almost impossible to calculate BUA. In this paper, we selected the geometries where these two wave modes are confounded, which simplifies the analysis of BUA. Moreover, it is impossible to control the direction of wave propagation when performing such measurement in a clinical configuration. For these reasons, we chose not to assess the effect of the structural anisotropy on SOS and BUA.

D. Future potential in vivo measurements.

The present approach may be used to develop model based inversion approaches to be used in future in vivo measurements, in order to eventually lead to possible clinical procedures.

A dedicated device adapted to future clinical measurements was developed and is shown in Fig. 12. The device will be inserted around the vertebral body. It is composed of two main bodies (denoted A and B in Fig. 12), which are related by 2 hooks. In clinical practice, the entire device (parts A and B linked by the two closed hooks) will first be inserted between two vertebral bodies after disk resection. Then, the pin will be inserted in the hole located between A and B and will be guided by the device. Then, the ultrasonic measurement will be performed and the hooks will be opened by the surgeon, in order to remove the device, leaving the pin in bone tissue, so that the pin can be used to hold the surrounding soft tissue.

Therefore, the pin will not be specifically inserted for the measurement because it will also be used to hold soft tissue, similarly as what is done currently clinical practice. Using the device shown in Fig. 12 allows to insert a pin perpendicular to the transducer axis and positioned at a given distance of the transducer.

The choice of the frequency of 500 kHz was made to obtain a compromise between a "sufficiently high" value of the frequency to obtain a good spatial resolution and a "sufficiently low" value of the frequency in order to obtain acceptable SNR. In particular, when considering the sample with the highest attenuation (50 dB/MHz/cm), assuming a constant frequency dependence of the attenuation coefficient, leads to an attenuation of 110 dB at 1 MHz, which may be too strong compared to usual values of the signal to noise ratio.

Therefore, using a value of 1 MHz or higher for the frequency of the transducer could impact the robustness of future in vivo measurements, which justifies the choice of 500 kHz for the transducer frequency.

In this study, we used a planar contact transducer, but many studies in the literature employed focused transducers for bone characterization. We chose to consider a planar transducer for the following reasons. First, a planar transducer is adapted to the strong geometrical constraints due to the small space available between the vertebral bodies.

Second, using a focused transducer would imply an increased sensitivity on the angle of incidence due to a stronger loss of amplitude when the beam does not intercept the pin axis at its center. Third, we used a contact transducer in order to avoid the reduction of SNR for this preliminary study and to obtain an easy positioning of the transducer relatively to the bone sample, which is important in the measurement configuration.

The presence of cortical bone (which is around 1 mm thick in this region) could disrupt the measurements, due to possible reverberations at the interface between cortical and trabecular bone. This point should be checked in future works. However, the influence of the cortical bone layer could be relatively limited, as shown in 56, which evidenced that the behavior of the received two waves was not modified significantly by the presence of the cortical layer 56 . This paper suggests that the wave propagating in cancellous bone can survive even when it propagates through cortical layer. Moreover, the vertebral body is grinded during surgery. Moreover, the echo of the interface between trabecular and cortical bone arrives much earlier than the echo of the pin. Eventually, the echo of this interface is subtracted, which further decreases the negative impact of this interface.

V. CONCLUSION

The behavior of the ultrasonic wave reflected by a metallic pin inside cancellous bone was investigated using CT images of cancellous bone specimens. Moreover, the effect of an error on the tilt angle of the pin during the insertion procedure was investigated. The results showed that the angle of the pin strongly affects the value of BUA while it weakly affects the value of SOS. These results indicate that the proposed method using the ultrasonic wave reflected by a pin has a potential to estimate the bone density although the data is sensitive to the geometrical properties of the configuration obtained during the insertion protocol. Moreover, SOS seems to constitute a more reliable parameter compared to BUA due to its lower sensitivity of errors on the tilt angle and to its linear variation with BV/TV.

Fig. 10

 10 Fig.10 indicate i) a linear increase of SOS as a function of BV/TV for all samples and ii) a strongly nonlinear variation of BUA as a function of BV/TV. The nonlinear variation of BUA as a function of BV/TV (with a maximum value of BV/TV around 20 to 35 %) obtained for all samples constitutes a validation of the phenomenological explanation given above. However, the physiological values of BV/TV obtained for human vertebral trabecular bone is lower than around 17 % 30 . When considering the data shown in Fig.10, Fig.7(b), and Fig.8 for

Figures

  Figures

Figure 1 :Figure 2 :Figure 3 :Figure 5 :Figure 6 :

 12356 Figure 1: An example of the geometrical configuration of three-dimensional simulation. Figures (a)-(c) show cross-section diagrams and (d) shows the three-dimensional view. (Color online) Figure 2: Examples of the screenshots of the simulated wave distribution. Figure (a-1) to (a-3) show the results with pin and (b-1) to (b-3) shows without pin. (Color online) Figure 3: Examples of the simulated rf signals (a) without bone specimen and (b) with bone specimen. (b-1): original rf signals and (b-2): difference of the signals shown in (b-1) and namely the signals obtained with the pin and without the pin. (Color online)Figure 4: (a): normalized spectrum of the simulated rf signals and (b): frequency dependence of the attenuation coefficient derived using the two spectra shown in (a). (Color online) Figure 5: Simulation setup used for the evaluation of the effect of a variation of the pin orientation. The pin axis was rotated around an axis perpendicular to the ultrasonic

Figure 7 :

 7 Figure 7: Variation of the simulated values of SOS and BUA as a function of BV/TV.

Figure 8 :

 8 Figure 8: Variation of the experimentally measured values of SOS and BUA as a function of BV/TV.

Figure 9 :Figure 10 :Figure 11 :

 91011 Figure 9: Variation of the QUS parameters as a function of the incident angle for three different samples. (Color online) Figure 10: Variation of the QUS parameters as a function of in silico BV/TV values when a dilation/erosion process was applied to three specimens in order to modify bone volume fraction by adding/suppressing bone voxel at the surface of trabecular bone. (Color online) Figure 11: Relationship between the values of the tilt angle for which the values of BUA and SOS are minimum.

Figure 12 :

 12 Figure 12: A dedicated device adapted to future clinical measurements. (Color online)

  Fig.2

  Fig.4

  Fig.5

  Fig.7

  Fig.9

  Fig.11

  of SOS [degrees] R = 0.82

  Fig.12

  The 3-D FDTD numerical model has been validated by comparing experimental and numerical SOS. Interestingly, the behavior of BUA exhibits non-linear characteristics, with a maximum value for BV/TV values around 20 to 35 %, which corresponds to values higher than what is obtained for human vertebral trabecular bone.

Table I :

 I 51. Hasegawa S, Nagatani Y, Mizuno K, Matsukawa M. Wavelet transform analysis of ultrasonic wave propagation in cancellous bone. Jpn. J. Appl. Phys. 2010; 49:07HF28. 52. Luppé F, Conoir JM, Franklin H. Multiple scattering in a trabecular bone: Influence of the marrow viscosity on the effective properties. J. Acoust. Soc. Am. 2003; 113:2889-2892. Material properties used for bone tissue (generic values taken from [e.g. 7]) and for stainless steel (from [e.g. 55]) in the simulations.

	53. Lee KI, Yoon SW. Comparison of acoustic characteristics predicted by Biot's theory and
	the modified Biot-Attenborough model in cancellous bone. J. Biomech. 2006; 39:364-368.
	54. Royer D, Dieulesaint E. Elastic Waves in Solids I. Berlin: Springer; 2000. pp 1-374.
	55. Gray DE. ed. American Institute of Physics handbook, 3rd ed. New York: McGraw-Hill;
	1982. pp 3-78.
	56. Nagatani Y, Mizuno K, and Matsukawa M. Two-wave behavior under various conditions
	of transition area from cancellous bone to cortical bone. Ultrasonics. 2014; 54:1245-1250.
	Table

  ]

	Bone	Longitudinal Shear	4,000 2,000	2,000
	Water	1,483	1,000
	Stainless steel	Longitudinal Shear	5,882 3,144	7,900

Table I

 I

technicians of MSME laboratory, providing perfect computing environment. This study was supported in part by KAKENHI (Grant Number 25871038 and 16K01431) from the Japan Society for the Promotion of Science (JSPS) and the PRTS program (project OsseoWave n°ANR-13-PRTS-0015-02) of French National Research Agency (ANR). This paper has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No 682001, project ERC Consolidator Grant 2015 BoneImplant).