Rémi Bourgoin
email: bourgoin.remi@gmail.com

Françoise Cornelis

Julien Masliah-Planchon

Catherine Genestie

Marick Laé

SCCOHT/ tumeur rhabdoïde ovarienne : A propos d'un cas Title SCCOHT/ ovarian rhabdoid tumor: A case report Nom des auteurs

Déclarations d'intérêts

Les auteurs déclarent n'avoir aucun conflit d'intérêt.

Résumé

Nous rapportons l'observation d'une patiente de 22 ans consultant pour des douleurs abdominopelviennes aiguës pour laquelle le diagnostic de « carcinome » à petites cellules de type hypercalcémiant (en anglais, small cell carcinoma of hypercalcemic type : SCCOHT) / "tumeur rhabdoïde ovarienne" a été posé.

Le « carcinome » à petites cellules de type hypercalcémiant est une tumeur rare et agressive survenant chez la femme jeune.

Le diagnostic de cette tumeur et la prise en charge doivent être rapides en raison de son agressivité. A travers cette observation, nous préciserons les aspects épidémiologiques, diagnostiques, moléculaires et les discussions à propos de son appellation.

Summary

We report the case of a 22-year-old patient with acute abdominopelvic pain. The diagnosis of hypercalcemic small cell carcinoma (SCCOHT) / ovarian rhabdoid tumor has been made. Small cell carcinoma of hypercalcemic type is a rare and aggressive tumor that occurs in young women.

The diagnosis of this tumor and the management must be rapid in view of its aggressiveness.

Through this observation, we specify the epidemiological, diagnostic, molecular aspects and discussions about its name.

Introduction

Le « carcinome » à petites cellules de type hypercalcémiant (en anglais, small cell carcinoma of hypercalcemic type : SCCOHT) / "tumeur rhabdoïde ovarienne" est une tumeur rare survenant chez la jeune femme avant la ménopause. Il s'agit d'une tumeur ovarienne de grande taille, unilatérale et agressive avec 50% de localisations péritonéales et une faible survie au diagnostic.

Malgré son appellation, l'hypercalcémie est inconstante. Cette tumeur est caractérisée par une perte de l'expression de la protéine BRG1 en immunohistochimie. Cette protéine, codée par le gène SMARCA4, fait partie du complexe de remodelage de la chromatine SWI/SNF qui un régulateur épigénétique majeur.

Nous rapportons l'observation d'un « carcinome » à petites cellules de type hypercalcémiant chez une femme de 22 ans. A travers cette dernière, nous précisons les aspects épidémiologiques, diagnostiques, moléculaires et les discussions à propos de son appellation.

Cas clinique

La patiente, âgée de 22 ans, nullipare, sans antécédents, consulte pour des douleurs pelviennes et une masse pelvienne. Au scanner, il existe une tumeur pelvienne suspecte de 20 cm de diamètre, hétérogène, à double contingent, tissulaire et liquidien, semblant à point de départ annexiel gauche. Il s'associe, une ascite et des nodules péritonéaux. La calcémie et les marqueurs sériques Beta-HCG, alpha-foetoprotéine, CA125 sont normaux. La laparotomie exploratrice met en évidence une tumeur ovarienne gauche, des nodules péritonéaux au niveau de la fosse iliaque droite, de l'iléon, du cul de sac de Douglas et de l'épiploon, une ascite. L'annexe controlatérale est normale. Un diagnostic de tumeur maligne peu différencié est posé lors de l'examen extemporané.

Une omentectomie et l'exérèse des nodules péritonéaux sont effectuées.

Macroscopiquement, la tumeur de 22 cm est solide, de couleur beige avec des remaniements hémorragiques, kystiques et nécrotiques.

Discussion

Le « carcinome » à petites cellules de type hypercalcémiant, appelé « small cell carcinoma of hypercalcemic type » (SCCOHT), a été décrit par Scully en 1979(1) et Dickersin en 1982 (2). Il s'agit d'une tumeur rare touchant les femmes jeunes de 24 ans d'âge moyen (4 mois -44 ans) (3, 4). Elle se manifeste le plus souvent par une masse pelvienne comme dans notre observation. La tumeur est de grande taille (15 cm en moyenne), unilatérale dans 99% des cas et agressive avec 50% de localisations secondaires au diagnostic. La mortalité est de plus de 65% à 2 ans, avec une médiane de survie de 18 mois. L'hypercalcémie paranéoplasique est inconstante (2/3 des cas), et rarement symptomatique (3,4).

La morphologie

Macroscopiquement, cette tumeur est en grande partie solide, de couleur beige, avec des remaniements kystiques, hémorragiques et nécrotiques. Concernant la sensibilité, il a été rapporté d'exceptionnelles SCCOHT montrant une conservation de l'expression de BRG1 et une altération moléculaire de SMARCA4 (10). Inversement, d'exceptionnelles SCCOHT sans mutation de SMARCA4 présentent une perte d'expression de BRG1, suggérant des altérations post-transcriptionnelles (6). Sur des prélèvements biopsiques, il convient d'être prudent pour poser le diagnostic de SCCOHT devant une tumeur présentant une perte d'expression de BRG1. En effet, les tumeurs de la granulosa adulte ou juvénile), ainsi que les tumeurs desmoplastiques à petites cellules rondes, les sarcomes d'Ewing et les tumeurs du stroma gonadique, peuvent avoir une expression de BRG1 hétérogène mais sans perte complète de l'expression de BRG1 (5, 10). Ces tumeurs n'ont pas de mutation de SMARCA4.

L

  'analyse morphologique montre une tumeur développée aux dépens de l'ovaire, d'architecture diffuse et pseudo-folliculaire (Figures1 et 2). Les cellules tumorales sont le plus souvent de taille petite à moyenne, monotones, au noyau hyperchromatique et finement nucléolé, au cytoplasme peu abondant plus ou moins clarifié (Figure3). Dans certains secteurs, on note des cellules très atypiques parfois de morphologie rhabdoïde (Figure4). L'activité mitotique est élevée. Le liquide d'ascite contient de nombreuses cellules tumorales. Les nodules péritonéaux sont tumoraux. L'annexe controlatérale est indemne.L'étude immunohistochimique montre une positivité des cellules tumorales pour les anticorps anti : AE1/AE3, EMA, CD99, CD10, CD56, inhibine (focalement), calrétinine (focalement), synaptophysine (focalement). Les cellules tumorales sont négatives pour les anticorps anti-: CK7, CK20, CD45-RB, WT1-N, chromogranine-A, PS100, SALL-4, TTF1, RO, RP. Le Ki67 est à 60%. Le phénotype fait discuter les diagnostics de sarcome d'Ewing et de tumeur à cellules de la granulosa de type adulte. L'absence de remaniement du gène EWSR1 en FISH va à l'encontre du diagnostic de tumeur d'Ewing. L'absence d'expression de la protéine FOXL2 et de mutation du gène FOXL2 n'est pas en faveur du diagnostic de tumeur de la granulosa. L'analyse immunohistochimique complémentaire a montré une perte d'expression de BRG1 (protéine encodée par le gène SMARCA4) en présence de témoins internes positifs ce qui est en faveur du diagnostic de « carcinome » ovarien à petites cellules de type hypercalcémiant (SCCOHT) / « tumeur rhabdoïde ovarienne ». (Figure 5). L'analyse d'autres protéines du complexe SWI/SNF montre une perte d'expression de BRM (protéine encodée par SMARCA2) sans perte d'expression d'INI1 (protéine encodée par SMARCB1). Afin de confirmer le diagnostic de « carcinome » à petites cellules de type hypercalcémiant, une analyse moléculaire a été réalisée. Le séquençage de l'ADN tumoral par NGS sur bloc FFPE a mis en évidence une mutation somatique du gène SMARCA4 à l'état homozygote (c.3229C>T ; p.Arg1077*). Le séquençage de l'ADN constitutionnel par technique Sanger a identifié la même mutation du gène SMARCA4 à l'état hétérozygote. L'évolution clinique a été caractérisée par l'apparition d'une lésion intra-hépatique suspecte d'allure secondaire. La patiente a bénéficié d'une première chimiothérapie s'inspirant des protocoles pédiatriques des tumeurs rhabdoïdes avec lesquelles les SCCOHT partagent quelques similitudes biologiques (VDCy-IE : Vincristine, Doxorubicine, Cyclophosphamide, Ifosfamide, Etoposide) et une deuxième ligne de chimiothérapie (« EpSSG-NRSTS 2005 », un protocole pédiatrique pour les sarcomes localisés hors rhabdomyosarcomes). Une chimiothérapie intrapéritonéale (CHIP) a été proposée devant la progression péritonéale et hépatique, mais la mauvaise tolérance hématologique des cures avec notamment l'apparition d'une aplasie fébrile ont limité les possibilités thérapeutiques. La patiente est décédée d'une évolution polymétastatique un an après le diagnostic.

  Histologiquement, l'architecture est diffuse en nappes avec de rares nids, cordons ou travées et un aspect pseudo-folliculaire dans 80% des cas. Les cellules sont petites, rondes, monotones. Elles ont des noyaux hyperchromatiques et finement nucléolés. Leur cytoplasme est peu abondant plus ou moins clarifié. L'activité mitotique est augmentée. La nécrose tumorale est fréquente. Dans 50% des cas, il s'associe une composante à grandes cellules qui peut être focale, prédominante ou exceptionnellement exclusive (variante dite à grandes cellules d'aspect rhabdoïde). Les cellules tumorales ont des noyaux fortement atypiques avec un nucléole proéminent et un cytoplasme éosinophile en verre dépoli. Rarement, les cellules sont fusiformes ou clarifiées. Il existe, dans 10% des cas, des kystes bordés par un épithélium mucineux. Le stroma est peu abondant.Morphologiquement, de par l'architecture diffuse ou pseudo-folliculaire, les diagnostics différentiels sont principalement le carcinome à petites cellules de type pulmonaire primitifs de l'ovaire ou métastatique et les tumeurs à cellules de la granulosa de type adulte ou juvénile. D'autres diagnostics différentiels peuvent être évoqués : tumeur germinale, carcinome indifférencié, sarcome d'Ewing, tumeur desmoplastique à petites cellules rondes, sarcome CIC-DUX4, mélanome, lymphome, sarcome du stroma endométrial ou la localisation secondaire d'un carcinome. L'immunohistochimie L'immunohistochimie, montrant une perte d'expression de la protéine BRG1, permet d'évoquer le diagnostic de SCCOHT et d'éliminer les diagnostics différentiels. L'immunohistochimie BRG1 doit être interprétée en présence de contrôles internes positifs tels que : les cellules endothéliales, les cellules inflammatoires, les fibroblastes, les cellules de la granulosa. Les cellules du stroma ovarien et les cellules thécales sont en revanche négatives ou faiblement positives. (5, 6, 7). L'immunophénotypage de ces tumeurs montre une expression des marqueurs épithéliaux (CKAE1/AE3, EMA, CK7), WT1-N, CD10, calrétinine, une expression possible des marqueurs neuroendocrines (chromogranine-A, synaptophysine, CD56), de SALL-4 (50%). L'inhibine, la PS100, la desmine, le TTF1, FOXL2 sont négatifs. Dans tous les cas, une relecture dans le réseau d'expertise des tumeurs malignes rares de l'ovaire (TMRO) ou dans le réseau RRePS (réseau de référence en pathologie des sarcomes) est souhaitable. La biologie moléculaire La perte d'expression de BRG1 est le reflet d'une mutation inactivatrice du gène SMARCA4 qui appartient au complexe SWI/SNF de remodelage de la chromatine (complexe BAF (BRG1/Brahma Associated Factors). Ce complexe est requis pour la réplication, la réparation et l'activation transcriptionnelle de gènes réprimés dont la chromatine passe d'un état d'hétérochromatine à un état ouvert par modulation des nucléosomes. Il joue un rôle épigénétique majeur et a une fonction de suppresseur de tumeurs. Il est constitué de 10-15 sous-unités codées par 28 gènes dont SMARCB1 (ou BAF47, INI1), SMARCA4, SMARCA2. Des mutations de ce complexe sont retrouvées dans environ 20% de tous les cancers. Les SCCOHT sont caractérisées par un profil génétique simple avec des déséquilibres génomiques restreints au locus de SMARCA4 en 19p12. Il s'agit typiquement de mutations non-sens, des délétions-insertions avec décalage du cadre de lecture, des mutations splice-site et plus rarement des mutations faux sens (6,8, 9). Ces mutations sont souvent accompagnées d'une perte hétérozygotie (LOH) de l'allèle sauvage. Ces mutations sont présentes dans toutes les cellules tumorales indiquant leur rôle « driver » dans l'oncogenèse des SCCOHT. Une mutation constitutionnelle est retrouvée dans 40 à 50% des cas (9,11). Les cas familiaux surviennent dans le syndrome de prédisposition aux tumeurs rhabdoïdes (RTPS2, OMIM 613325) (SCCOHT et rarement tumeur rhabdoïde du système nerveux ou des tissus mous) Sensibilité / Spécificité de l'IHC et de la biologie moléculaire. L'interprétation de l'immunohistochimie anti-BRG1 doit être précise et nécessite une absence complète de marquage des cellules tumorales avec un contrôle interne positif intact. Clarke et al., (10) décrit dans une étude et revue de la littérature (129 SCCOHT et 2600 diagnostics différentiels de tous types de tumeurs gynécologiques de sarcomes et de mélanomes) (11, 12, 13) une sensibilité de la perte de BRG1 en immunohistochimie de 94% et une spécificité proche de 100% (10). Concernant la spécificité, de rares adénocarcinomes ovariens à cellules claires (3,8%) et d'exceptionnels carcinomes indifférenciés de l'ovaire (4,5%) sont les seules autres tumeurs ovariennes à perdre l'expression de BRG1. Parmi les autres tumeurs gynécologiques, 1% des adénocarcinomes endométrioïdes de l'endomètre, 7.5% des sarcomes stromaux de l'endomètre, et 27% des carcinomes indifférenciés de l'endomètre présentent une perte d'expression de BRG1 (10).

18)IconographieFigure 1 .Figure 2 .Figure 3 .Figure 5 .

 1235 Figure 1. Architecture diffuse en nappe sans stroma (coloration HES, x10). Diffuse pattern (hematein-eosin-safran staining, x10) Figure 2. Architecture pseudo-folliculaire (coloration HES, x10). Pseudofollicular pattern (hematein-eosin-safran staining, x10) Figure 3. Cytologie plutôt monotone. Noyaux finement nucléolés. Cytoplasme peu abondant (coloration HES, x40). Cytology rather monotonous. Nuclei finely nucleated. Scarce cytoplasm (hematein-eosin-safran staining, 40) Figure 4. Variante à grandes cellules avec un aspect rhabdoïde : noyaux fortement atypique, nucléole proéminent, cytoplasme éosinophile en verre dépoli au sein d'un stroma myxoïde ou oedémateux (coloration HES, x40). Large-cell variant with a rhabdoid features: strongly atypical nuclei, prominent nucleolus, abundant and eosinophilic cytoplasm within a myxoid or edematous stroma (hematein-eosin-safran staining, x40) Figure 5. Immunomarquage par l'anticorps anti-BRG1 : perte d'expression dans les noyaux tumoraux. Notez le marquage conservé dans les cellules normales (coloration HES, x20). Immunohistochemistry with anti-BRG1 : loss of expression by tumor nuclei. Note the positive internal controls. (hematein-eosin-safran staining, x20).

  La catégorisation des SCCOHT dans les « miscellaneous tumors » dans la classification OMS 2014 des tumeurs gynécologiques (12) n'est pas satisfaisante mais reflète le fait que l'histogénèse des SCCOHT ne soit pas encore établie. Certains auteurs plaident pour une tumeur épithéliale, une tumeur germinale (13), une tumeur des cordons sexuels ou la rapproche des tumeurs rhabdoïdesConclusionLe « carcinome » à petites cellules de type hypercalcémiant (small cell carcinoma of hypercalcemic type : SCCOHT) est une tumeur rare et agressive survenant chez la femme jeune. Chan-Penebre E, Armstrong K, Drew A, Grassian AR, Feldman I, Knutson SK et al. Selective Killing of SMARCA2-and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models. Mol Cancer Ther. 2017 May;16(5):850-860. 17) Kim KH1, Kim W, Howard TP, Vazquez F, Tsherniak A, Wu JN et al. SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2. Nat Med. 2015 Dec;21(12):1491-6.

	1) Dickersin GR, Kline IW, Scully RE. Small cell carcinoma of the ovary with hypercalcemia: a 9) Clarke BA, Witkowski L, Ton Nu TN, Shaw PA, Gilks CB, Huntsman D et al. Loss of SMARCA4 16)
	report of eleven cases. Cancer. 1982;49:188-197 (BRG1) protein expression as determined by immunohistochemistry in small-cell
	carcinoma of the ovary, hypercalcaemic type distinguishes these tumours from their
	2) Young RH, Oliva E, Scully RE. Small cell carcinoma of the ovary, hypercalcemic type. A mimics. Histopathology. 2016 Nov;69(5):727-738.
	(9,11,15). Cette entité également dénommée « tumeur rhabdoïde ovarienne » demeure classée comme clinicopathological analysis of 150 cases. Am J Surg Pathol. 1994;18:1102-1116
	Witkowski et al (9) ont postulé qu'il pourrait y avoir un lien de développement entre les tumeurs « miscellaneous ovarian tumors » dans la Classification OMS 2014 des tumeurs gynécologiques et 10) Karnezis AN, Wang Y, Ramos P, Hendricks WP, Oliva E, D'Angelo EJ et al. Dual loss of the
	tératoïdes / rhabdoïdes atypiques extracrâniennes (AT / RT) et les SCCOHT par la morphologie non dans les carcinomes. Son origine n'est pas déterminée (épithéliale, mésenchymateuse, 3) Estel R, Hackethal A, Kalder M, Münstedt K. Small cell carcinoma of the ovary of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and
	focale rhabdoïde dans les contingents à grandes cellules et par le fait qu'un syndrome de germinale, neuroendocrine ?). hypercalcaemic type: an analysis of clinical and prognostic aspects of a rare disease on the specific for small cell carcinoma of the ovary, hypercalcaemic type. Pathol. 2016
	Voici un tableau récapitulatif des caractéristiques immunohistochimiques et moléculaires des prédisposition de la tumeur rhabdoïde familiale associée à la mutation SMARCA4 a été décrit au Le diagnostic est à évoquer devant une perte d'expression de BRG1 en immunohistochimie basis of cases published in the literature. Arch Gynecol Obstet. 2011 Feb;238(3):389-400.
	SCCOHT et ses diagnostics différentiels : cours des dernières années (14). La majorité des tumeurs rhabdoïdes étant associées néanmoins à (sensible et spécifique) au niveau d'une tumeur gynécologique non classique surtout chez jeune
	Ewing/ Tumeur patiente. Dans ce contexte, la perte d'expression concomitante de BRG1 et BRM est très spécifique 4) Karanian-Philippe M, Velasco V, Longy M, Floquet A, Arnould L, Coindre JM, et al.
	SCCOHT SMARCA4 (BRG1) loss of expression is a useful marker for the diagnosis of ovarian small Granulosa desmoplastique des SCCOHT. 11) Kurman RJ, Carcangiu ML, Herrington CS, Young R (Eds.): Who Classification of tumors of
	Immunohistochimie cell carcinoma of the hypercalcemic type (ovarian rhabdoid tumor): a comprehensive the Female Reproductive Organs, Vol4. International Agency for Research on Cancer, Lyon
	Perte d'expression de analysis of 116 rare gynecologic tumors, 9 soft tissue tumors, and 9 melanomas. Am J Surg Expression conservée de BRG1 (ou perte Expression conservée de BRG1 (ou perte 2014.
	BRG1 Pathol. 2015;39:1197-1205	d'expression	d'expression
	hétérogène) Expression conservée 12) McCluggage WG, Witkowski L, Clarke BA, Foulkes WD. Clinical, morphological and hétérogène) Expression conservée Expression conservée
	d'INI1 Absence d'expression de FOXL2 immunohistochemical evidence that small-cell carcinoma of the ovary of hypercalcaemic d'INI1 d'INI1 5) Ramos P, Karnezis AN, Craig DW, Sekulic A, Russell ML, Hendricks WP et al. Small cell Absence d'expression Expression de FOXL2 de FOXL2 carcinoma of the ovary, hypercalcemic type, displays frequent inactivating germline and type (SCCOHT) may be a primitive germ-cell neoplasm. Histopathology. 2017
	somatic mutations in SMARCA4. Nat Genet. 2014 May;46(5):427-9. Jun;70(7):1147-1154.
	Biologie moléculaire
	Mutation SMARCA4	Mutation FOXL2	Remaniement EWSR1
	6) Reisman DN, Sciarrotta J, Bouldin TW, Weissman BE, Funkhouser WK. The expression of 13) Schneppenheim R, Frühwald MC, Gesk S, Hasselblatt M, Jeibmann A, Kordes U et al.
	Karnesis a montré que les analyses immunohistochimiques couplées de BRG1 et BRM (protéine the SWI/SNF ATPase subunits BRG1 and BRM in normal human tissues. Appl Germline nonsense mutation and somatic inactivation of SMARCA4/BRG1 in a family with
	Actuellement, des essais thérapeutiques avec notamment des inhibiteurs deEZH2/PRC2 SMARCA2) montrent une spécificité de 100% pour le diagnostic de SCCOHT (11). De très Immunohistochem Mol Morphol. 2005 Mar;13(1):66-74. rhabdoid tumor predisposition syndrome. Am J Hum Genet. 2010 Feb 12;86(2):279-84.
	exceptionnelles SCCOHT présentent une perte d'expression d'INI1 et une mutation de SMARCB1 (6). Déclaration d'intérêts. (répresseur transcriptionnel agoniste à SWI/SNF) sont en cours. (17,18). Les auteurs déclarent ne pas avoir de conflits d'intérêt. 7) Jelinic P, Mueller JJ, Olvera N, Dao F, Scott SN, Shah R et al. Recurrent SMARCA4 mutations 14) Foulkes WD, Clarke BA, Hasselblatt M, Majewski J, Albrecht S, McCluggage WG et al. No
	Le stade TNM est le facteur prédictif le plus important et on identifie, dans les stades Ia, les in small cell carcinoma of the ovary. Nat Genet. 2014 May;46(5):424-6. small surprise -small cell carcinoma of the ovary, hypercalcaemic type, is a malignant
	L'identification d'une mutation de SMARCA4 n'est pas spécifique de SCCOHT. Elles ont été décrites facteurs de bon pronostic suivants : un âge > 30 ans, une calcémie pré-opératoire normale, une rhabdoid tumour. Pathol. 2014 Jul;233(3):209-14.
	dans divers carcinomes tels que ceux du poumon, du pancréas, du sein, de l'endomètre et de la taille tumorale < 10 cm et une absence de grande cellule (3). L'analyse de plus grande série de 293 8) Witkowski L, Carrot-Zhang J, Albrecht S, Fahiminiya S, Hamel N, Tomiak E et al. Germline Références prostate et récemment dans le sarcome thoracique SMARCA4 déficient (16). SCCOHT a montré que l'âge> 40 ans, le stage FIGO et le type de traitement (chimiothérapie à haut and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, 15) Le Loarer F, Watson S, Pierron G, de Montpreville VT, Ballet S, Firmin N et al. SMARCA4
	dose) sont les facteurs pronostiques les plus importants (19). Scully Tumors of the ovary and maldeveloped gonads, AFIP, 1979 hypercalcemic type. Nat Genet. 2014 May;46(5):438-43. inactivation defines a group of undifferentiated thoracic malignancies transcriptionally
	related to BAF-deficient sarcomas. Nat Genet. 2015 Oct;47(10):1200-5.
	Questions de nomenclatures

la présence d'une mutation germinale de SMARCB1, un autre élément clé du complexe SWI / SNF. et al., (15) suggèrent que les SCCOHT soient renommées « tumeur rhabdoïde maligne de l'ovaire » arguant notamment que certaines nomenclatures pouvaient être déroutantes comme : « variante à grande cellule du carcinome à petites cellules de type hypercalcémiant sans hypercalcémie ». Karnesis relie également les SCCOHT aux tumeurs rhabdoïdes en raison de leur morphologie (cellules petites/rhabdoïdes), cytogénétique diploïde, altérations du complexe SWI/SNF et mauvais pronostic (11).

Traitement et pronostic

Les traitements sont proposés par les cliniciens membres du réseau de tumeurs malignes gynécologiques rares. En raison de la présence d'une mutation récurrente « driver » de SMARCA4, des protocoles de chimiothérapies sont inspirés des traitements des tumeurs rhabdoïdes.

Il convient d'éliminer les principaux diagnostics différentiels que sont le carcinome à petites cellules de type pulmonaire, les tumeurs de la granulosa de type adulte et juvénile ainsi que les mélanomes, sarcomes, carcinomes.

L'identification une mutation « driver » récurrente (mutation biallélique de SMARCA4) dans les SCCOHT permet d'envisager de nouvelles approches thérapeutiques.

Une confirmation du diagnostic de SCCOHT est souhaitable par analyse du statut mutationnel somatique de SMARCA4. En présence d'une mutation somatique, il est primordial de proposer une consultation d'oncogénétique pour réalisation d'une analyse constitutionnelle, 40 à 50% des SCCOHT présentant une mutation germinale de SMARCA4 (syndrome de prédisposition aux tumeurs rhabdoïdes).


Diego Alfonso Godoy Pérez

Alberto Moreira

Jorge Junior

Virginie Roche

Jean-Claude Lepretre

Conrado Ramos

Moreira Afonso

Nagle Dilermando

Gabriel Hitoshi Travessa

Claudemiro Asato

Walter Jose Bolfarini

Botta

Jean- Claude Lepretre

Dilermando Nagle Travessa

Gabriel Hitoshi Asato

Claudemiro Bolfarini

Walter Jose Botta

Severe plastic deformation and different surface treatments on the biocompatible Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys: Microstructural and phase evolutions, mechanical properties, and bioactivity analysis

Keywords: Ti13Nb-13Zr and Ti35Nb7Zr5Ta alloys, Severe Plastic Deformation, Surface Anodization, Surface Chemical Treatment, Oxide Surface nanostructures, Surface modification

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

INTRODUCTION

For many decades, materials science and implantology scientists have been seeking advances in device designs that may be valuable for clinical employment in substitution of body parts. In this field, one of the main target-tissues is the bone due to its particular properties and the opportunity to undertake its functional rehabilitation utilizing bulk biocompatible implants together with proper surgical procedures.

Within the above context, Ti and its alloys are nowadays more relevant and sought materials for several designed implants for applications in orthopedics and dentistry. In the last decades, favorable biocompatibility and adequate corrosion resistance of Ti and Ti-based alloys, two of the necessary characteristics for a metallic implant, have been confirmed in numerous experimental and clinical studies. Titanium-based implants show outstanding mechanical performance regarding load capacity (maximum load, bending, and fatigue strength) [START_REF] Leinenbach | Fatigue and cyclic deformation behaviour of surfacemodified titanium alloys in simulated physiological media[END_REF][START_REF] Bosco | Instructive coatings for biological guidance of bone implants[END_REF][START_REF] Bosco | Surface engineering for bone implants: a trend from passive to active surfaces[END_REF] and stiffness; this last property acts favorably to control bone cell phenotypic requirement [START_REF] Engler | Matrix elasticity directs stem cell lineage specification[END_REF][START_REF] Reilly | Intrinsic extracellular matrix properties regulate stem cell differentiation[END_REF].

The mechanical properties of Ti and Ti-alloys are close to the properties of bone tissues meeting one essential prerequisite for the success of orthopedic implants. With values of Young's modulus and hardness similar to the ones of the bone, the stress shielding effect is diminished and the possibility of bone resorption, which may lead to loosening of the implant from the bone, is reduced [START_REF] Sabirov | Bulk Nanostructured Materials with Multifunctional Properties[END_REF].

Although pure titanium and Ti6Al4V alloys are widely used for commercial titanium-based implant materials [START_REF] Ratner | Biomaterials science: an introduction to materials in medicine[END_REF], there are important concerns regarding their elastic moduli, still much higher in comparison with the bones (10-30 GPa) [START_REF] Wintermantel | [END_REF].

Pure titanium continues to be the choice when corrosion resistance is the most serious concern, such as in dentistry.

The α+β Ti6Al4V alloy is the choice when mechanical properties are more relevant; its mechanical behavior, chemical stability, and microstructure can be modified by heat treatment or thermomechanical processing [START_REF] Long | Titanium alloys in total joint replacement-a materials science perspective[END_REF][START_REF] Ko | Effects of temperature and initial microstructure on the equal channel angular pressing of Ti-6Al-4V alloy[END_REF][START_REF] Semiatin | Flow behavior and globularization kinetics during hot working of Ti-6Al-4V with a colony alpha microstructure[END_REF][START_REF] Seshacharyulu | Hot working of commercial Ti-6Al-4V with an equiaxed α-β microstructure: materials modeling considerations[END_REF].

However, there are increasing concerns about the cytotoxicity effects of Al and V ions that can dissolve into the body as a consequence of corrosion of the Ti6Al4V alloy [START_REF] Long | Titanium alloys in total joint replacement-a materials science perspective[END_REF] resulting in hostile reactions with the bone tissue [START_REF] Laing | Tissue reaction in rabbit muscle exposed to metallic implants[END_REF].

Therefore, a more recent intent is the development of single-phase β-titanium alloys where toxic and allergic elements are not present, with superior mechanical properties, and excellent workability. Also, β-Ti alloys present intrinsically lower elastic moduli than the ones for α-Ti or α+β Ti6Al4V alloy [START_REF] Long | Titanium alloys in total joint replacement-a materials science perspective[END_REF][START_REF] Niinomi | Fatigue characteristics of metallic biomaterials[END_REF].

The newest generation of titanium alloys for orthopedic applications, which include Ti12Mo6Zr2Fe (TMZF) [START_REF] Wang | The Characterization of Ti-12Mo-6Zr-2Fe A New Biocompatible Titanium Alloy Developed for Surgical Implant[END_REF], Ti15Sn4Nb2Ta0.2Pd [START_REF] Okazaki | Effect of alloying elements on mechanical properties of titanium alloys for medical implants[END_REF], Ti13Nb13Zr (TNZ) [START_REF] Davidson | New surface-hardened, low-modulus, corrosion-resistant Ti-13Nb-13Zr alloy for total hip arthroplasty[END_REF], and Ti29Nb13Ta4.6Zr (TNZT) alloys [START_REF] Niinomi | Fatigue performance and cyto-toxicity of low rigidity titanium alloy, Ti-29Nb-13Ta-4.6Zr[END_REF], has attained lower modulus and improved biocompatibility. A further comprehensive and up-to-date synopsis on the mechanical behavior, impact of thermomechanical processing on the microstructure, and alloying of Ti-based alloys can be found elsewhere [START_REF] Long | Titanium alloys in total joint replacement-a materials science perspective[END_REF][START_REF] Geetha | Ti based biomaterials, the ultimate choice for orthopaedic implants-a review[END_REF].

The mechanical properties of Ti-based alloys may be further improved by microstructural refinement using severe plastic deformation (SPD) methods such as the equal channel angular pressing (ECAP), high-pressure torsion (HPT) and others, which are capable of leading grain sizes to the ultrafine or even to the nanosize range. In this way, SPD may improve wear resistance, and the bioactivity by inducing apatite precipitation and intensified biological response when compared to coarse-grained alloys, because of Hall-Petch strengthening, higher surface energy, augmented number of nano-grooves, and a considerable rise in the cell adhesion/proliferation [START_REF] Kim | In vitro biocompatibility of equal channel angular processed (ECAP) titanium[END_REF].

The spontaneous passive oxide layer on the surface of Ti-based materials assists the required chemical features mentioned above, making these alloys proper to be applied in orthopedic and dental implants [START_REF] Hernández-López | Correlation of the nanostructure of the anodic layers fabricated on Ti13Nb13Zr with the electrochemical impedance response[END_REF]. This layer, mainly composed by TiO2, yields chemical inertness in distinct media, thus protecting the metallic surface.

However, despite this feature, inert Ti is scarcely able to combat infections or even to effectively control any particular process related to bone cell interactions, which are helpful for bone healing implants [START_REF] Hazan | Bone growth to metal implants is regulated by their surface chemical properties[END_REF].

Hence, the enhancement of Ti surfaces to solve, if not both, at least one of these two problems became an important issue and several modifications and coating methods on the titanium surface are now available, which can lead to the improvement in bone formation or even in decreasing the chance of infections [START_REF] Bosco | Instructive coatings for biological guidance of bone implants[END_REF][START_REF] Bosco | Surface engineering for bone implants: a trend from passive to active surfaces[END_REF].

Osseointegration, which refers to the structural and functional relationship between the bone tissue and the surface of an implant [START_REF] Albrektsson | Oral implant surfaces: Part 1--review focusing on topographic and chemical properties of different surfaces and in vivo responses to them[END_REF], is strongly affected b y the properties of the implant surface. Such properties influence the bone reaction (rate, quantity, and quality) and for instance, protein adsorption and cell attachment [START_REF] Junker | Effects of implant surface coatings and composition on bone integration: a systematic review[END_REF] are critically and directly affected by the implant composition and charges. Surface chemical composition influences the hydrophilicity, which can assist the synergies between cells and biological fluids [START_REF] Zhao | High surface energy enhances cell response to titanium substrate microstructure[END_REF][START_REF] Buser | Enhanced bone apposition to a chemically modified SLA titanium surface[END_REF].

Several techniques of surface treatments have been investigated and implemented [START_REF] Wennerberg | On implant surfaces: a review of current knowledge and opinions[END_REF][START_REF] Wong | Effect of surface topology on the osseointegration of implant materials in trabecular bone[END_REF], aiming to favor the osseointegration mechanism and thus providing superior stability over the bone healing process and enabling faster implant loading procedures [START_REF] Beutner | Biological nanofunctionalization of titanium-based biomaterial surfaces: a flexible toolbox[END_REF][START_REF] Wennerberg | Effects of titanium surface topography on bone integration: a systematic review[END_REF].

Different modifications on Ti and Ti-alloys surfaces can be found reported in the literature [START_REF] Oliveira | Osteoblasts behavior on chemically treated commercially pure titanium surfaces[END_REF][START_REF] Oliveira | Surface chemical treatment of ultrafinegrained Ti-6Al-7Nb alloy processed by severe plastic deformation[END_REF][START_REF] Oliveira | Gene expression of human osteoblasts cells on chemically treated surfaces of Ti-6Al-4V-ELI[END_REF][START_REF] Zemtsova | Modern techniques of surface geometry modification for the implants based on titanium and its alloys used for improvement of the biomedical characteristics[END_REF]. Remarkably, one valuable and easy way to treat surfaces was the use of an acid medium to generate a uniform micron/submicron surface porosity followed by an alkali treatment for producing either a sponge or a coral-like nanotopographic substrate, which allowed modulation of cellular interactions [START_REF] Oliveira | Osteoblasts behavior on chemically treated commercially pure titanium surfaces[END_REF].

Nevertheless, one must regard with precaution the vast number of implant types present in the market because they differ in surface properties and other characteristics [START_REF] Binon | Implants and components: entering the new millennium[END_REF]. In this way, it appears to be logical considering new implant surfaces as new biomaterials [START_REF] Coelho | Basic research methods and current trends of dental implant surfaces[END_REF] because distinct and unique surface properties, which might affect the bone-to-implant reaction, may be achieved by different methods for surface modification.

The present work aimed to analyze the structural and microstructural evolutions and mechanical properties of the Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys in different conditions of heat treatment and severe plastic deformation by the method of highpressure torsion (HPT). It also aimed to evaluate the surfaces' morphologies and chemistry of the alloys, processed in different conditions, after surface modification for micro-nano topographic conversion by either anodization or chemical treatment. Some results regarding the Ti13Nb13Zr alloy were recently published [START_REF] Peréz | Surface anodization of the biphasic Ti13Nb13Zr biocompatible alloy: Influence of phases on the formation of TiO2 nanostructures[END_REF]; however, here, a more in-depth analysis is performed as a result of the comparative analysis between the two types of alloys. Finally, the resultant in-vitro bioactivities were analyzed to evaluate a possible synergy between processing and surface treatment

-Materials and Methods

Cylindrical bars of 10 mm in diameter of biomedical Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys (ERCATA GmbH, Development Co., Ltd., Germany) were used in this study. The bars were sliced out in disk-shaped samples of 1 mm thick using wire electro-discharge machining. Such disks were heat-treated from the β field at 750 °C for 1 hour (above the β transus), after which they were fast water quenched.

High-pressure torsion (HPT) was used to severely deform the samples at the room temperature, utilizing a pressure of 4.5 GPa, applying three turns, and employing a rotation speed of 3 rpm. X-ray diffraction (XRD) was used for the structural analysis, utilizing a Siemens D5000 diffractometer with CuKα radiation and a scanning speed of 2°/min from 30° to 90°. To perform the indexation of phases present in both alloys, the following CIF files were used: ICSD 253841 for the α' phase, ICSD 168830 for α phase, and ICSD 645545 for the β phase.

Confocal Laser Scanning Microscopy (CLSM) was performed to characterize the microstructure of the samples with high focus depth and surface resolution. The CLSM Olympus LEXT OLS 4000 was the equipment used for such analysis.

The surfaces of the samples were also inspected by scanning electron microscopy (SEM) to obtain higher resolution images. The equipment was the Zeiss Ultra-55 FEG microscope, equipped with an Energy Dispersive X-ray (EDX) microanalysis system with an SDD detector (BRUKER AXS-30mm²). Different beam energies were applied in the analyses: 6 keV for acquiring high spatial resolution secondary electron (SE) images, and 10 keV for EDX analysis.

Transmission electron microscopy (TEM), using orientation phase mapping, was performed in the FEI TECNAI G2, operated at 200 kV. Such microscope is equipped with an orientation and phase mapping precession unit NanoMEGAS (model ASTAR) and with a Digistar P1000 unit, which enables Automated Crystal Orientation Mapping (ACOM) in nanoprobe mode allowing collecting orientation/phase maps with a spatial resolution of about 1 nm. Such kinds of maps are comparable to utilizing electron backscatter diffraction (EBSD) in scanning electron microscopy (SEM), but with higher resolution, and it gives the ability for performing analyses in heavily deformed samples as after processing by HPT. As it is in EBSD systems, the identification of grains based on ASTAR results needs a certain critical misorientation angle in such a way all segments of boundaries with a misorientation angle above of the critical one will be recognized as grain boundaries. In this way, the software will measure misorientations among all pixel pairs, hence identifying the boundaries containing individual grains.

When different phases are present, using the same procedure, it is feasible to ascertain grain size distributions for distinct phases present in the sample. For such an analysis, the TSL OIM Analysis 7 software was used to determine the different phases present in the samples and to measure their grain size using ASTAR data. Grains were defined using boundaries with critical misorientations (θ) greater than 15°, and subgrains were determined by boundaries with misorientations between 2° and 15°. The indexation of phases present in both alloys using ASTAR data was performed utilizing the same CIF files for the indexation of XRD patterns were used: ICSD 253841 for the α' phase, ICSD 168830 for α phase, and ICSD 645545 for the β phase.

Vickers microhardness measurements were accomplished using a Stiefelmayer microdurometer, by applying a load of 300 g during 15 s. Microhardness mappings were constructed from hardness measurements along over several samples' radii. These measures were taken in the direction from the center to the edge of the specimen.

Measurements of elastic moduli of the samples were performed utilizing the free decay elastometer equipment SONELASTIC  from ATCP Physical Engineering.

The electrochemical anodization setup to generate oxide nanostructures on surfaces of undeformed and HPT-deformed samples comprised a standard two electrodes system with a platinum grid as the counter electrode, a Ti alloy disk as the working electrode, and a power supply (TCR Electronic measurements inc.). Counter and working electrodes were regularly held at a distance of almost 3 cm. For the anodization procedure, the potential of 20 V was applied for one hour. Glycerol was the working electrolyte and was composed of 25 vol% of water and 0.25 M of NH4F. The choices of anodization conditions and electrolyte were based in the previous work of our group [START_REF] Hilario | Influence of morphology and crystalline structure of TiO2 nanotubes on their electrochemical properties and apatite-forming ability[END_REF]. The temperature of the electrolyte was controlled to be 25 °C during the anodization process. As-anodized oxide nanostructures are amorphous, with little mechanical strength. Hence, as-anodized samples were annealed in air at 550 °C for two hours, thus crystallizing oxide nanostructures and mechanically reinforcing them.

Before anodization experiments, disk samples were successively ground with P320, P600, P1200, and P2400 grade SiC emery paper, and then cleaned with deionized water (Milli-Q®), ethanol and acetone.

Surface chemical treatment was accomplished by, firstly, wet-abrading the disks using a 1500 grit SiC abrasive paper, sonicating them in deionized water, washing in acetone, and drying in the air. After, the surface was acid etched by immersing the disks in a concentrated phosphoric acid at 80 o C during 30 min, after which they were promptly sonicated in deionized water and washed with acetone. Finally, the resulting surfaces were alkaline treated in a 10 mol L -1 NaOH solution at 60 o C during 24 h.

The analysis of the samples' surfaces after chemical treatment was performed by

Raman Spectroscopy employing the RENISHAW InVia Raman Microscope with an Argon Ion laser of 20 mW, whose wavelength was of 514 nm.

Wettability was evaluated through the static water contact angle with an optical tensiometer at room temperature. Measurements were accomplished just after dripping deionized water, thus preventing evaporation and other disturbances. The static contact angle was defined by fitting the Young-Laplace equation around the droplet.

-Results and Discussion

-Structural and Microstructural Characterizations

Figure 1 presents XRD patterns comparing both alloys in the undeformed and HPT deformed conditions. The indexation of XRD patterns of the Ti13Nb13Zr13 alloy presented in Figure 1a indicates the presence of the hcp α' and the bcc β phases before and after HPT. The figure also suggests an increase of the β phase after processing by HPT, probably induced by solute redistribution, which may lead to a prompt phase transformation of the coarse non-equilibrium metastable martensite (hcp α') during severe plastic deformation. Conversely, the indexation of the XRD patterns of Figure 1b reveals that, before deformation, the Ti35Nb7Zr5Ta alloy is mostly composed by the bcc β phase with the presence some residual hcp α phase, which practically vanishes after mechanical processing, also probably due to solute redistribution during HPT.

Peak broadening observed after deformation points to a very high grain refinement for both alloys. presented an average length of ~20 μm with thicknesses ranging from 1 to 2 μm. Figure 2b shows a single β phase with equiaxed grains forming the microstructure of the Ti35Nb7Zr5Ta alloy after heating and quenching. This result is coherent with the one presented in Fig. 1b because the amount of the residual hcp α phase appears to be very small when comparing peak intensities in the XRD pattern. After image analysis, the average grain size was evaluated to be around 240 μm.

The refinement of the microstructure and distribution of phases after deformation by HPT was assessed by ACOM-TEM analysis. Crystallographic mappings obtained for the two alloys are presented in Figure 3.

For the sake of comparison, Figures 3a and3b present equivalent bright-field (virtual bright field -VBF) images from the samples observed by conventional TEM where (3a) is for the Ti13Nb13Zr alloy and (3b) is for the Ti35Nb7Zr5Ta alloy. Both figures confirm a very high grain refinement after processing by HPT, as also observed elsewhere [START_REF] Afonso | From Porous to Dense Nanostructured β-Ti alloys through High-Pressure Torsion[END_REF]. Furthermore, it is possible to observe a non-homogeneous distribution of grains/subgrains sizes in the alloys and, comparing with Figure 2a, a redistribution of the α' phase after mechanical processing (Fig. 1a). The grain size distributions for the samples after processing by HPT were determined, and the measured average grain sizes were ~203 nm and ~112 nm for the Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys, respectively.

Figures 3c and3d present phase mappings, respectively for the Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys after processing by HPT. High-angle grain boundaries superimpose such maps for an easy and fast view of phases' positioning in the samples.

As it may be observed, Figure 3c shows that the β phase (blue) is being formed in between the non-equilibrium metastable acicular martensite (hcp α' -yellow) of the alloy Ti13Nb13Zr during severe plastic deformation. On the other hand, Figure 3d shows that the residual α phase (white) in the Ti35Nb7Zr5Ta alloy is being consumed and transformed into the β phase (blue) and is kept in β grain boundaries. Measurements obtained from colored regions in Figs. 3c and3d, indicate that the area fractions were ~77% of hcp α' and 23% of β in the Ti13Nb13Zr alloy, and ~93% of β and 7% of α in the Ti35Nb7Zr5Ta. The mean second phase particle sizes were ~91 nm and ~50 nm, correspondingly for the β phase present in the Ti13Nb13Zr alloy and α phase in the Ti35Nb7Zr5Ta alloy.

-Mechanical Properties

Figure 4 compares elastic modulus measurements for both materials and conditions (deformed and undeformed). All the samples acquired excellent resilience with a slight reduction after HPT, which is probably due to the increase of β phase in the Ti13Nb13Zr alloy and a reduction of α in the Ti35Nb7Zr5Ta alloy. In the case of the alloy Ti13Nb13Zr, average values were ~63 GPa for the as-quenched sample and ~60 GPa for the one processed by HPT. It is important to emphasize this astonishing result since such a modulus for water-quenched/aged samples of the Ti13Nb13Zr alloy was reported to be around 79 GPa [START_REF] Davidson | New surface-hardened, low-modulus, corrosion-resistant Ti-13Nb-13Zr alloy for total hip arthroplasty[END_REF]. In the same sense, the mean Young's modulus for the Ti35Nb7Zr5Ta alloy was reduced from ~45 GPa to ~44 GPa, which are very close to the values for human bones (10-30 GPa [START_REF] Wintermantel | [END_REF]).

Figure 5 presents hardness mappings after severe plastic deformation comparing results for the Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys. For comparison purposes, the hardness scale was set to be the same, which reduces mapping resolutions. Hence, the insets of the Figures are showing the equivalent mapping with different relative scales to provide more information regarding hardness variations along the samples' radius, which is typical behavior after HPT processing.

The initial average hardness values in the as-quenched conditions were ~265 HV and ~183 HV, respectively for samples of Ti13Nb13Zr and Ti35Nb7Zr5Ta alloy. As it can be observed in Figure 5, after deformation by HPT, those values raised correspondingly to ~390 HV and ~320 HV, representing an increase of about ~47% for the Ti13Nb13Zr alloy and ~75% for the Ti35Nb7Zr5Ta alloy. Such Hall-Petch strengthening difference is expected since, as discussed above in Figure 3, the final grain size for the sample Ti35Nb7Zr5Ta was about 81% smaller than for sample Ti13Nb13Zr. However, in the case of the Ti13Nb13Zr alloy, one may suppose that the hardness behavior is not only due to the Hall-Petch effect because the initial and final hardness values were higher than for the Ti35Nb7Zr5Ta alloy. The presence of the martensitic phase may explain such a difference. The hcp α' martensite is a distorted hcp α lattice in parts of the alloy crystal. The hardness of martensite comes from the density of defects it contains, mostly from dislocations, which are formed to assist martensite formation.

-Surface treatments

-Anodized surfaces

Morphologies of heat-treated anodized surfaces were examined by a FEG-SEM.

Figures 6a-d show top views of Ti13Nb13Zr samples undeformed and deformed, and Ti35Nb7Zr5Ta samples undeformed and deformed, respectively. The insets in the Figures are higher magnification images of the respective region, showing details of the surface morphology. Cross-sectional views are presented in the supplementary material (Fig. S1), and Table 1 summarizes the morphologies and dimensions of the anodized nanostructures.

Figures 6a and6b present SEM images of top views' morphologies of the nanostructures after anodization and heat-treatment of the sample Ti13Nb13Zr, respectively for the undeformed and deformed conditions. As already reported earlier [START_REF] Peréz | Surface anodization of the biphasic Ti13Nb13Zr biocompatible alloy: Influence of phases on the formation of TiO2 nanostructures[END_REF], Figure 6a shows self-organized oxide nanotubes formed in the α' phase and oxide nanoporous in the β phase (please, see the inset in Figa.6a and 6c). If one observes the cross-sectional view in Figure S1a, it will be possible to note that both kinds of nanostructures are formed by tubes with dimensions described in Table 1. However, the difference between them is that nanotubes are noticeably separated from each other, while an oxide layer links the nanopores, whose diameters are smaller than the ones for nanotubes. Furthermore, there is a distinct contrast between the two nanostructures, with nanopores being bright, and nanotubes being dark. This distinct contrast was ascribed to the presence of the heavier β stabilizer (Nb) in the titanium dioxide. Thus, it can be hypothesized that there is a competition between the dissolution and production of oxides as a function of the different chemical composition in both phases [START_REF] Taveira | Initiation and growth of self-organized TiO2 nanotubes anodically formed in NH4F∕(NH4) 2SO4 electrolytes[END_REF].

The above-discussed aspects, regarding the presence of nanopores and nanotubes, were also observed in the deformed condition of the Ti13Nb13Zr alloy presented in Figure 6b, whose characteristics are resumed in Table 1. However, its surface is fuzzy with significant differences in the length of the nanostructures, producing depressions in the sample surface.

Concerning the quasi-single β phase Ti35Nb7Zr5Ta alloy, only nanopores were observed on the whole surface of both samples' conditions, undeformed (Fig. 6c) and deformed (Fig. 6d). In fact, this result confirms that the β phase produces only nanopores, whose characteristics (linking of pores) are better visualized in Figures 6c than in Figure 6a. Nevertheless, the surface of the deformed sample (Fig. 6d) is much fuzzier than the surface of the deformed Ti13Nb13Zr alloy (Fig. 6b), also presenting poor nanostructure formation leading to the presence of large voids on the surface and exposing the substrate. Furthermore, it is noteworthy that all the above nanostructures were amorphous after anodization (please, refer to Figure S2 in the Supplementary Material). Such amorphous structures, as observed earlier [START_REF] Hilario | Influence of morphology and crystalline structure of TiO2 nanotubes on their electrochemical properties and apatite-forming ability[END_REF], were crystallized in a mix of anatase and rutile after heat-treatment at 550 °C for two hours. However, even with the high amount of Nb and Zr in the Ti13Nb13Zr alloy and Nb in the Ti35Nb7Zr5Ta alloy, it is interesting noting the presence of only TiO2 oxides. Such presence of sole TiO2 was also found in many works in the literature, for example, for the Ti20Nb [START_REF] Jin | Fabrication and characterization of anodic oxide nanotubes on TiNb alloys[END_REF][START_REF] Jang | Electrochemical characteristics of nanotubes formed on Ti-Nb alloys[END_REF], Ti13Nb13Zr [START_REF] Stróż | Production, structure and biocompatible properties of oxide nanotubes on Ti13Nb13Zr alloy for medical applications[END_REF], and Ti15Mo [START_REF] Oliveira | Obtaining self-organized nanotubes on biomedical Ti-Mo alloys[END_REF] alloys. Jin et al. [START_REF] Liu | Surface modification of titanium, titanium alloys, and related materials for biomedical applications[END_REF] affirmed that the absence of Nb or Nb oxide reflections in the XRD pattern of as-anodized and annealed nanostructures might be consigned to the small quantity of Nb in such nanostructures due to the small diffusion coefficient of Nb in TiO2 compared to that of Ti. Definitely, by analyzing Table 2, which brings results of EDX analysis (a more sensitive technique) of nanostructures formed on samples surfaces after anodization and heat treatment one can confirm that the amounts of all other elements are small, and the presence of other oxides are below the detection limit of the XRD equipment. Nonetheless, a more precise analysis of the results in Table 2 can be observed in Table 3, which presents the stoichiometric analysis of the data shown in Table 2, that reveals almost the same quantity of TiO2 (anatase and rutile) and the presence of other oxides, other than TiO2, in a considerable total amount. Such other oxides are surely contaminating anatase and rutile in the nanostructures, being, as expected, more significant in the Ti35Nb7Zr5Ta alloy.

-Chemical Treatment by acid etching (H3PO4) and NaOH activation

SEM analysis was accomplished to assess the effect of chemical treatments on surface morphologies. Figures 7a-7d present SEM images for chemically modified surfaces respectively for undeformed and deformed Ti13Nb13Zr alloy, and undeformed and deformed Ti35Nb7Zr5Ta alloy. Table 4 presents the quantitative chemical EDX analysis of samples' surfaces for both compositions and processing conditions. The insets are higher magnification SEM images to give details of the surface morphology.

From these micrographs, one can observe that the acid plus alkali treatment induced the formation of a nano topographic sponge-like morphology on the samples' surface. However, it is noticeable that sponges' tentacles have quite different sizes, depending on the sample composition and mechanical processing condition; they decrease in the sequence of undeformed and deformed Ti13Nb13Zr alloy to undeformed and deformed Ti35Nb7Zr5Ta alloy. Observing such a trend of reducing tentacles' size, it appears that deformation influences such a decreasing in individual compositions. However, it is apparent that the composition and/or phases present in the samples' surface are also affecting the size of tentacles. Therefore, a more in-depth analysis is necessary to determine all possible factors influencing such behavior.

In this sense, Raman analysis was performed (please refer to Figure S3 in the supplementary material) and indicated that sodium titanate, anatase, and rutile are the main phases present in all the samples. Stoichiometric analysis of the data presented in Table 4, based on Raman analysis, is shown in Table 5, which indicates almost the same amount of TiO2 (anatase and rutile) and sodium titanate for all conditions, which are contaminated by a considerable amount of different oxides in the case of the Ti35Nb7Zr5Ta alloy, and smaller contamination in the Ti13Nb13Zr alloy. However, none of the total amounts of such other oxides present in the alloys follows the order of decreasing of tentacles' size, leading us to infer that a specific oxide may be contributing for such decrescent sequence. Analyzing Table 4, it appears that Zr, in the form of an oxide, follows the same sequence of tentacles' sizes. In other words, the higher the amount of Zr, the greater the size of tentacles. Therefore, one may infer that an extra amount of Zr induces to a length increase and thickening of corals' "tentacles", thus coarsening the surface layer in the same sense. Of course, as stated above, deformation also plays a role, but it is impossible to separate the effects of deformation from chemistry and to affirm which one would be most important.

-Surface Bioactivity

Osseointegration requires the development of a bone-like apatite layer on the implant surface, which must start after the implantation [START_REF] Liu | Surface modification of titanium, titanium alloys, and related materials for biomedical applications[END_REF]. The osseointegration is usually evaluated in association with its bioactivity, which is performed by soaking the implant material in SBF solution [START_REF] Liu | Surface modification of titanium, titanium alloys, and related materials for biomedical applications[END_REF], thus verifying its efficacy of apatite production.

Therefore, the bone-bonding ability of samples with treated surfaces correlated to polished surfaces was evaluated by immersing them for 1, 7, and 14 days in SBF.

The formation of an apatite layer was assessed by SEM-EDX chemical microanalysis (please, refer to Figure S4(1-6) in the supplementary material). The presence of hydroxyapatite was evaluated in terms of its constituent elements (Ca, P, and O) and the ratios between Ca/P, which should be around the theoretical values of 1.67 (Ca5(PO4)3(OH) Ca/P = 5/3 = 1.67). Furthermore, EDX spectra show the presence of small quantities of Mg and Na. Actually, Mg 2+ and Na + can substitute Ca ions in human bones' apatites, which are nonstoichiometric and contain vestiges of ions like Mg 2+ and Na + , and also anions like CO3 -2 , HPO4 -2 , F -and Cl - [START_REF] Hsu | Fabrication of nanotube arrays on commercially pure titanium and their apatite-forming ability in a simulated body fluid[END_REF]. SEM-EDX chemical microanalysis (Figure S4(1-6)) showed that even soaking for 14 days, none of the samples with polished surfaces did form apatite, i.e., they are not bioactive. This fact confirms earlier reports that although the initial oxide passivation layer can grant some corrosion resistance and biocompatibility for Ti and its alloys, the physicochemical characteristics of this layer do not stimulate bone formation [START_REF] Huo | Fabrication, modification, and biomedical applications of anodized TiO2 nanotube arrays[END_REF].

The above behavior was also observed for samples of the Ti35Nb7Zr5Ta alloy treated with acid etching using H3PO4 and activated with NaOH, indicating that, even with the presence of phosphate on the surface, the surface treatment does not result in bioactivity. Contrarily, with the same surface treatment, the Ti13Nb13Zr alloy showed bioactivity between one and seven days for deformed and undeformed conditions. Anodized surfaces of the undeformed Ti13Nb13Zr alloy started producing an apatite layer between one and seven days, while deformed samples showed to be bioactive since the first 24 hours. However, the same surface treatment proved to be not so effective for specimens of the Ti35Nb7Zr5Ta alloy. In this case, samples (deformed or not) only started producing apatite between the seventh and the fourth day.

The above results from SEM-EDX analyses may be considered qualitative because it gives information only regarding the presence or not of apatite. Therefore, apatite deposition was quantitatively estimated by weighing the samples before and after SBF soaking. Such an evaluation is summarized in Figure 8, which presents the average value of the three weighings. Error bars display the standard deviation.

As it can be observed, the quantitative analysis of Figure 8 endorses the above qualitative ones, regarding apatite deposition or not on the different samples and conditions.

It is noticeable that even having some bioactivity, the surfaces of anodized samples of deformed and mechanically processed Ti35Nb7Zr5Ta alloy produced an undermost amount of apatite after 14 days. In a whole, for the Ti35Nb7Zr5Ta alloy, it can be inferred that even with these two kinds of surface treatments, its bioactivity in the deformed and undeformed conditions are unsatisfactory.

Concerning the apatite formation for the Ti13Nb13Zr alloy after anodization, there was no precipitation of apatite on the surface of undeformed sample during the first day of soaking, while, on deformed samples, the apatite mass gain raised with the immersion time from the early 24 hours. This behavior leads us to infer that the smaller the grain size, the more significant the mass gain, which also proves the consequence of the kind of the surface for bioactivity, and, in this case, the redistribution and refinement of α' and β phases on the sample surface caused by HPT. However, if one compares these results with the ones for a cp α -Ti earlier published by our group [START_REF] Hilario | Influence of morphology and crystalline structure of TiO2 nanotubes on their electrochemical properties and apatite-forming ability[END_REF], it will be possible to observe that from the first day immersed in SBF, cp α-Ti samples showed more significant bioactivity than specimens of the Ti13Nb13Zr alloy. This behavior indicates the result of the distinct type of surfaces (only nanotubes for cp α-Ti and the mix of nanotubes and nanopores for the Ti13Nb13Zr alloy). Nonetheless, even considering the positive effect of grain size reduction in the Ti13Nb13Zr alloy, its bioactivity is much smaller than the one observed for α-Ti.

The chemical surface treatment of the undeformed Ti13Nb13Zr alloy was not as useful for activating the surface; however, better bioactivation was observed for the deformed samples. Finally, comparing anodization and chemical treatment, one may presume that anodization is much better for activating deformed and undeformed surfaces of both alloys than the chemical treatment.

Reasons for the above behavior are worthy of being understood given other good qualities of these implant materials, mainly Young's modulus. As stated in the introduction, implant surface properties will impact the bone response. The implant composition and charges will markedly and directly affect protein adsorption and cell fixing [START_REF] Junker | Effects of implant surface coatings and composition on bone integration: a systematic review[END_REF], which are also influenced by the hydrophilicity that is controlled by the surface chemical composition. Hydrophobic surfaces seem not to help symbiosis between cells and biological fluids when compared with hydrophilic ones [START_REF] Zhao | High surface energy enhances cell response to titanium substrate microstructure[END_REF][START_REF] Buser | Enhanced bone apposition to a chemically modified SLA titanium surface[END_REF].

From this reasoning, one would expect that hydrophilicity could be the first good reason for the behavior of apatite-forming ability on the different surfaces analyzed in this work. Figure 9 presents wettability results as measured by the contact angle of a droplet. Although all the samples surfaces are presenting a predominantly hydrophilic behavior (contact angles lower than 90°), anodized and chemically treated surfaces presented highly hydrophilic responses in comparison with those of polished surfaces.

If one observes Figure 9, despite presenting hydrophilic behavior, it is clear that polished surfaces of the Ti35Nb7Zr5Ta alloy and Ti35Nb7Zr5Ta alloy have shown statistically similar very low hydrophilicity. Even so, such surfaces could influence the adhesion of biological fluids. However, observing Figure 8, in none of the polished surfaces of both alloys for deformed and undeformed conditions, there was apatite formation. Therefore, specifically for untreated surfaces, their lower hydrophilicity may be the main reason influencing the non-formation of apatite.

Despite the excellent reasoning in elucidating the behavior of the apatite formingability on the surfaces of polished samples, hydrophilicity cannot explain alone the apatite-forming ability of anodized and chemically treated surfaces of the Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys, since similar highly hydrophilic responses were produced by their surfaces, when compared to the untreated ones. Therefore a more in-depth analysis is necessary for understanding the apatite forming-ability on surfaces of treated samples' of both alloys.

Therefore, concerning treated surfaces, besides hydrophilicity that is statistically similar for all treated surfaces, it appears that apatite formation on treated surfaces may be associated with the contamination of the surfaces by other oxides. The presence of such excess of contaminant oxides can be observed in Tables 3 and5, respectively for anodized and chemically treated surfaces. If one carefully observes the results in Tables 3 and5 and analyzes Figure 8 in the light of such findings, it will be possible to infer the effect of such contamination and its relationship with apatite-forming ability.

In other words, considering anodized samples, the less contaminated anodized surfaces of the Ti13Nb13Zr alloy led to an excellent apatite-forming ability. Such ability was much better for the deformed sample, which presented the smallest contamination among all surfaces, starting apatite formation in the early stage of the first day. Conversely, highly contaminated anodized surfaces of the Ti35Nb7Zr5Ta alloy resulted in poor apatite formation. However, considering chemically treated surfaces, comparing Tables 5 and3, one can observe that the levels of contaminant oxides in chemically treated and anodized surfaces are similar. Thus, also considering hydrophilicity, it should be expected that chemically treated surfaces would present the same level of apatite formation as it was for anodized ones. In other words, one could expect a similar apatite precipitation trend between the two alloy's compositions after surface chemical treatment. Nevertheless, it was observed after surface chemical treatment the poor apatite formation in the Ti13Nb13Zr alloy and no formation at all in the Ti35Nb7Zr5Ta alloy, suggesting that an additional factor may be influencing apatite formation on such kind of surface.

When analyzing all variables that could influence apatite formation in chemically treated surfaces, one should take a look in Figure 7. In such Figure, it is possible to observe an evolution of tentacles' sizes according to chemistry and deformation conditions. Deeply analyzing the evolution of such tentacles' sizes, it appears that there is a critical dimension to induce apatite formation better. Tentacles sizes decrease in the sequence of undeformed Ti13Nb13Zr, deformed Ti13Nb13Zr, undeformed Ti35Nb7Zr5Ta, and deformed Ti35Nb7Zr5Ta. Here, it is worth remembering the influence of Zr on the thickening of such tentacles, reinforcing the effect of contamination of other oxides in the apatite forming ability. Tentacles with sizes observed in the deformed Ti13Nb13Zr appear to be the critical one for the best apatite formation. Larger or much smaller sizes prejudice the apatite precipitation.

In a nutshell, an accurate summary of effects on apatite formation is as follows.

Surely, hydrophilicity is an essential factor, because low hydrophilicity led to nonformation of apatite in polished surfaces. Not only chemistry will affect apatite precipitation but also, besides hydrophilicity, different charges arising on the surfaces due to the presence of other oxides will worsen such precipitation in anodized and chemically treated surfaces. In the case of chemically treated surfaces, besides the two previous factors, tentacles' sizes appear to have a critical dimension to allow adhesion of biological fluids and thus favor apatite formation.

-Conclusions

The mechanical properties and bioactivity of the Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys were investigated as a function of the microstructural refinement, phases present, and the kind of surface modification (anodization and chemical treatment).

Commercial Ti alloys were heat treated and rapidly quenched. The Ti13Nb13Zr alloy presented a biphasic microstructure containing the α' and β phases. The Ti35Nb7Zr5Ta alloy also presented a biphasic microstructure composed mainly by the β phase and a small amount of the α phase.

HPT processing significantly reduced grain sizes, resulting in ultra-fine microstructures with grain sizes of ~203 nm and ~112 nm for the Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys, respectively. High hardness values were observed in both alloys, in the range of ~390 HV and ~320 HV, with outstanding (low) values of the elastic modulus 60 GPa and 44 GPa before and after deformation, respectively for the Ti13Nb13Zr and Ti35Nb7Zr5Ta alloys.

Another feature produced by HPT was the increase of the β phase in the Ti13Nb13Zr alloy and a decrease of the α phase in the Ti35Nb7Zr5Ta alloy, making it almost single phase.

Anodization of the Ti13Nb13Zr alloy resulted in two different nanostructures, with nanotubes forming in the α'-phase and nanopores in the β-phase, irrespective to the spatial distribution of either phases or their refinement. In the case of the Ti35Nb7Zr5Ta alloy, only nanopores were observed.

Surface modification using acid etching followed by alkaline treatment resulted in a sodium titanate coral-like layer. Coral tentacles' sizes depended on the microstructure size, surface chemistry, and structure, being thicker for undeformed samples and much thinner for samples processed by HPT.

Bioactivity tests indicated that oxide nanostructures could activate the surfaces of both samples, but the bioactivity of the Ti13Nb13Zr alloy is much higher than for the Ti35Nb7Zr5Ta alloy, which did not present any bioactivity when chemical treatment was used. Contrarily, Ti13Nb13Zr alloy was active after both treatments, but much better for oxide nanostructures and samples deformed by HPT.

Reasons for apatite forming-ability behavior were related to hydrophilicity, different charges arising on the surfaces due to the presence of other oxides in both kinds of surface treatment and tentacle sizes in the case of chemically treated surfaces.

Figure and Table Captions

Figure 2

 2 Figure 2 compares the microstructures of both alloys in the undeformed condition obtained by confocal laser microscopy. Figure 2a, whose inset is a higher-resolution SEM picture, presents microstructural aspects of the Ti13Nb13Zr alloy and reveals the existence of retained bcc β phase and the coarse non-equilibrium metastable martensite (hcp α') phase precipitated after heating and quenching. XRD results presented in Fig 1a confirm such an observation. Measurements of grain size gave a mean initial size of about 250 μm for the β phase, and α' laths

Figure 1 -

 1 Figure1-XRD patterns comparing both alloys in the undeformed and HPT deformed conditions. (a) Ti13Nb13Zr alloy[START_REF] Peréz | Surface anodization of the biphasic Ti13Nb13Zr biocompatible alloy: Influence of phases on the formation of TiO2 nanostructures[END_REF], and (b) Ti35Nb7Zr5Ta.

Figure 2 -

 2 Figure 2 -Microstructures of both alloys in the undeformed condition obtained by confocal laser microscopy. (a) Ti13Nb13Zr alloy and (b) Ti35Nb7Zr5Ta alloy. The inset in Figure 1a is a higher-resolution SEM picture showing microstructure details.

Figure 3 -

 3 Figure 3 -ACOM-TEM analysis. Equivalent bright-field images obtained by TEM for (a) Ti13Nb13Zr and (b) Ti35Nb7ZR5Ta alloys. Phase mappings superimposed by maps of high-angle grain boundaries for (c) Ti13Nb13Zr alloy (β-Ti (bcc) in blue, and α' martensite (hcp) in yellow) and (d) Ti35Nb7ZR5Ta alloy (β-Ti (bcc) in blue, and α-Ti (hcp) in white).

Figure 4 -

 4 Figure 4 -Graph comparing elastic modulus values for the alloys Ti13Nb13Zr and Ti35Nb7Zr5Ta in both processing conditions, deformed, and undeformed.

Figure 5 -

 5 Figure 5 -Hardness mappings after severe plastic deformation comparing results for the Ti13Nb13Zr alloy in (a) and Ti35Nb7Zr5Ta alloy in (b). The insets of the Figures are showing the equivalent mapping with different relative scales to provide more information regarding hardness variations along the samples' radius.

Figure 6 -

 6 Figure 6 -FEG-SEM images showing the morphologies of heat-treated anodized surfaces. (a) undeformed Ti13Nb13Zr alloy. (b) deformed Ti13Nb13Zr alloy. (c) undeformed Ti35Nb7Zr5Ta alloy. (d) mechanically processed Ti35Nb7Zr5Ta alloy. The insets in the Figures are higher magnification images, showing details of the surface morphology.

Figure 7 -

 7 Figure 7 -SEM images for chemically modified surfaces, respectively, for (a) undeformed Ti13Nb13Zr alloy. (b) deformed Ti13Nb13Zr alloy. (c) undeformed Ti35Nb7Zr5Ta alloy. (d) mechanically processed Ti35Nb7Zr5Ta alloy. The insets are higher magnification SEM images to give details of the surface morphology.

Figure 8 -

 8 Figure 8 -Apatite mass gain after soaking specimens in SBF as a function of the immersion time, comparing polished surfaces and treated surfaces of deformed and undeformed samples.

Figure 9 -

 9 Figure 9 -Graph showing the results of contact angles measurements for all surface conditions.

Table captions Table 1 -

 captions1 Summary of observed morphologies and dimensions of the formed anodized nanostructures observed in Figures6 and S1.

Table 2 -

 2 Quantitative chemical EDX analysis of nanostructures on samples' surfaces for both compositions and processing conditions, after anodization and heat treatment.

Table 3 -

 3 Stoichiometric analysis of the data presented in Table2.

Table 4 -

 4 Quantitative chemical EDX analysis of samples' surfaces for both compositions and processing conditions.

Table 5 -

 5 Stoichiometric analysis of the data presented in Table2, considering the Raman analysis presented in FigureS3of the supplementary material.

Table 1 -

 1 Summary of observed morphologies and dimensions of the formed anodized nanostructures observed in Figures6 and S1.

		Ti13Nb13Zr alloy
	Condition	Morphology	Diameter (nm) Internal External	Length (nm)
	Nondeformed Nanotubes and nanopores	58±7	96±20	1839±72
	Deformed	Nanotubes and nanopores 47±10	94±11	1492±149
		Ti35Nb7Zr5Ta alloy
	Nondeformed	Nanopores	47±7	82±14	2494±23
	Deformed	Nanopores	36±3	69±7	1742±189

Table 2 -

 2 Quantitative chemical EDX analysis of nanostructures on samples' surfaces for both compositions and processing conditions, after anodization and heat treatment.

			Alloy
		Ti13Nb13Zr (at %)	Ti35Nb7Zr5Ta (at %)
		undeformed deformed undeformed deformed
	Ti	26.3±0.3 27.9±0.4	22.0±0.2 20.76±0.3
	Zr	2.6±0.1	2.7±0.1	1.5±0.1	1.5±0.1
	Nb	3.1±0.1	2.8±0.3	8.1±0.6	8.1±0.4
	Ta	0	0	0.56	0.59
	O	68.1±0.4 66.6±0.7	67.8±0.6 69.0±0.7

Table 3 -

 3 Stoichiometric analysis of the data presented in Table2.

				Alloy
			Ti13Nb13Zr (at %)	Ti35Nb7Zr5Ta (at %)
	Phase	Element undeformed deformed undeformed deformed
	TiO2	Ti	26.3±0.3 27.9±0.4	22.0±0.2 20.76±0.3
	Anatase or Rutile	O	52.6±0.4	55.9±0.7	44.0±0.6	41.7±0.4
	Other oxides	O	15.5±0.4	10.8±0.7	23.8±0.6	27.3±0.4

Table 4 -

 4 Quantitative chemical EDX analysis of samples' surfaces for both compositions and processing conditions.

				Alloy
		Ti13Nb13Zr (at %)	Ti35Nb7Zr5Ta (at %)
		undeformed deformed undeformed deformed
	O	66.4±0.7	66.5±0.6	70.2±0.5	69.9±0.7
	Na	1.4±0.1	1.1±0.1	1.2±0.1	1.1±0.1
	Zr	1.9±0.1	1.1±0.1	1.0±0.1	0.5±0.1
	Nb	2.5±0.4	2.0±0.3	3.6±0.1	3.8±0.2
	Ti	27.9±0.2	29.3±0.4	23.5±0.3	24.2±0.3
	Ta			0.5±0.1	0.5±0.1

Table 5 -

 5 Stoichiometric analysis of the data presented in Table2, considering Raman the analysis presented in FigureS3of the supplementary material.

				Alloy
			Ti13Nb13Zr (at %)	Ti35Nb7Zr5Ta (at %)
	Phase	Element undeformed deformed undeformed deformed
		Na	1.4±0.1	1.1±0.1	1.2±0.1	1.1±0.1
	Na4O4Ti	O	1.4±0.1	1.1±0.1	1.2±0.1	1.1±0.1
		Ti	0.34±0.1	0.3±0.1	0.3±0.1	0.3±0.1
	TiO2	Ti	27.6±0.2	29.1±0.4	23.2±0.3	23.9±0.3
	Anatase or Rutile	O	55.1±0.7	58.1±0.6	46.4±0.5	47.8±0.7
	Other oxides	O	9.9±0.7	7.3±0.6	22.7±0.5	21.0±0.7

Acknowledgments

The authors are grateful to the French Committee for the Evaluation of Academic and Scientific Cooperation with Brazil (COFECUB) and the Brazilian CAPES (Process number #88887.137755/2017-00) for funding the bilateral cooperation and also FAPESP under the grant number for the partial funding FAPESP Thematic # 2013/05987-8. The authors thank the Laboratory of Structural Characterization (LCE/DEMa/UFSCar) for using their facilities. AMJJ would like to acknowledge the CNPq (Brazil) under grant #301429/2017-0 and 409847/2016-0.