NANOCI—Nanotechnology Based Cochlear Implant With Gapless Interface to Auditory Neurons

Copy igh © 2017 O ology & Neu o ology, Inc. Unau ho ized ep oduc ion o his a icle is p ohibi ed. NANOCI—Nano echnology Based Cochlea Implan Wi h Gapless In e ace o Audi o y Neu ons y§§Pascal Senn, yMa a Roccio, yS e an Hahnewald, zClaudia F ick, zMonika Kwia kowska, zMasaaki Ishikawa, zPe e Bako, §Hao Li, §F ed ik Edin, §Wei Liu, §Helge Rask-Ande sen, jjIlma i Pyykko¨, jjJing Zou, jjMa ika Manne s o¨m, {He be Keppne , {Alexand a Homsy, {Edi h Laux, {Miguel Lle a, #Jean-Paul Lellouche, #S ella Os o sky, #Ehud Banin, #Aha on Gedanken, #Nina Pe kas, U e Wank, Ka l-Heinz Wiesmu ¨lle , yyPa el Mis ı´k, yyHe al Bena , yyCa olyn Ga nham, yyClaude Jolly, zzFilippo Gande , zzPe e Ul ich, zMa cus Mu ¨lle , and zHube Lo¨wenheim Uni e si y Depa men o ORL, Head & Neck Su ge y, Inselspi al; y Depa men o Clinical Resea ch, Uni e si y o Be n, Be n, Swi ze land; z Depa men o O o hinola yngology–Head & Neck Su ge y, Uni e si y o Tu ¨bingen, Tu ¨bingen, Ge many; §Depa men o Su gical Sciences, Sec ion o ORL, Uppsala Uni e si y, Uppsala, Sweden; jj Hea ing and Balance Resea ch Uni , Depa men o O o hinola yngology and The Finnish Cen e o Al e na i e Me hods, Uni e si y o Tampe e, Tampe e, Finland; { Hau e Ecole A c Inge ´nie ie, HES-SO - Uni e si y o Applied Sciences Wes e n Swi ze land, La Chaux-de-Fonds; #Depa men o Chemis y, The Cen e o Ad anced Ma e ials and Nano echnology and The Mina and E e a d Goodman Facul y o Li e Sciences, Ba Ilan Uni e si y, Rama Gan, Is ael; EMC Mic ocollec ions GmbH, Tu ¨bingen, Ge many; yy MED-EL GmbH, Wo ldwide Headqua e s, Innsb uck, Aus ia; zz SCIPROM Sa ` l, Rue du Cen e 70, S -Sulpice; and §§Depa men o Clinical Neu osciences, Se ice o ORL and HNS, HUG, Uni e si y Hospi al o Gene a, Gene a, Swi ze land Cochlea implan s (CI) es o e unc ional hea ing in he majo i y o dea pa ien s. Despi e he emendous success o hese de ices, some limi a ions emain. The bo leneck o op imal elec ical s imula ion wi h CI is caused by he ana omical gap be ween he elec ode a ay and he audi o y neu ons in he inne ea . As a consequence, cu en de ices a e limi ed h ough 1) low equency esolu ion, hence sub- op imal sound quali y and 2), la ge s imula ion cu en s, hence high ene gy consump ion ( esponsible o signi ican ba e y cos s and o impeding he de elopmen o ully implan able sys ems). A ecen ly comple ed, mul ina ional and in e disciplina y p ojec called NANOCI aimed a o e - coming cu en limi a ions by c ea ing a gapless in e ace be ween audi o y ne e ibe s and he cochlea implan elec ode a ay. This ambi ious goal was achie ed in i o by neu o ophin-induced a ac ion o neu i es h ough an in a- cochlea gel-nanoma ix on o a modi ied nanoCI elec ode a ay loca ed in he scala ympani o dea ened guinea pigs. Func ionally, he gapless in e ace led o lowe s imula ion h esholds and a la ge dynamic ange in i o, and o educed s imula ion ene gy equi emen (up o i e old) in an in i o model using audi o y neu ons cul u ed on mul i-elec ode a ays. In conclusion, he NANOCI p ojec yielded p oo o concep ha a gapless in e ace be ween audi o y neu ons and cochlea implan elec ode a ays is easible. These indings may be o ele ance o he de elopmen o u u e CI sys ems wi h be e sound quali y and pe o mance and lowe ene gy consump ion. The p esen o e iew/ e iew pape summa izes he NANOCI p ojec his o y and highligh s achie emen s o he indi idual wo k packages. Key Wo ds: Audi o y ne e egene a ion—BDNF—Cochlea implan —Gapless in e ace—Guinea pig—Hea ing loss—Hyd ogel—Mul i- elec ode a ay—Neu on–elec ode in e ace. O ol Neu o ol 38:e224–e231, 2017. Cochlea implan s (CI) ha e become he gold s anda d ea men o acqui ed and congeni al dea ness, allowing he es o a ion o unc ional hea ing in he majo i y o ecipien s. As o oday o e 300,000 de ices a e in use. Despi e he emendous success o cu en sys ems, he e is a subs an ial a iabili y o pe o mance ac oss CI-use s. In some cases, sound quali y is poo o music and onal languages, and use s may s uggle in noisy en i onmen s. Add ess co espondence and ep in eques s o Pascal Senn, M.D., Depa men o Clinical Neu osciences, Se ice ORL & CCF, Hoˆpi aux Uni e si ai es de Gene` e (HUG), Rue Gab ielle Pe e -Gen il 4, 1211 Gene a, Swi ze land; E-mail: [email p o ec ed] M.M. and H.L. ha e equal con ibu ion o he a icle. Disclosu e: The NANOCI-p ojec (www.nanoci.o g) was unded by he Eu opean Union unde he g an ag eemen no. 281056 in he 7 h ame wo k p og amme. The au ho s disclose no con lic s o in e es . This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-Non Comme cial-No De i a i es License 4.0 (CCBY-NC-ND), whe e i is pe missible o download and sha e he wo k p o ided i is p ope ly ci ed. The wo k canno be changed in any way o used comme cially wi hou pe mission om he jou nal. DOI: 10.1097/MAO.0000000000001439 e224 O ology & Neu o ology 38:e224–e231 Copy igh ß2017 The Au ho (s). Published by Wol e s Kluwe Heal h, Inc. on behal o O ology & Neu o ology, Inc. Copy igh © 2017 O ology & Neu o ology, Inc. Unau ho ized ep oduc ion o his a icle is p ohibi ed. The majo bo leneck o op imized s imula ion is he ana omical gap be ween he implan ed elec ode a ay and he s imula ed egions o he audi o y ne e. As a consequence o he gap and he associa ed cu en sp ead du ing a s imulus pulse, housands o audi o y neu ons a e s imula ed om de ices ea u ing only be ween 12 and 22 elec odes (depending on he manu ac u e ). Whe eas mo e han eigh independen channels a e needed in challenging lis ening si ua ions (1), CI use s ypically pe o m as i hey ha e only abou eigh channels o in o ma ion (2). The main eason o his is sp ead o exci a ion om he elec odes and in e e ence ac oss channels. Any a emp o inc ease channel numbe s is limi ed by his signi ican ‘‘c oss alk’’ o elec odes o o e lapping elec ical ields. Hence, he esolu ion and speci ici y o neu onal s imula ion is low, which is he main eason o subop imal sound quali y and a iabili y o speech and music pe cep ion. Addi ionally, he high ampli udes con ibu e o a high ene gy consump ion associa ed wi h signi ican main enance cos s o ba e ies. Mo eo e , high ene gy consump ion challenges he de el- opmen o ully implan able sys ems, which would ha e se e al ad an ages such as inc eased usabili y o small child en, in isibili y, hea ing while sleeping and be e accep ance by pa ien s. I is gene ally acknowledged ha elimina ing he gap be ween neu ons and he elec ode a ay, and inc easing he numbe o ac i e channels, should g ea ly imp o e he e ec i eness o audi o y ne e s imula ion (3). The NANOCI p ojec , he e o e, was concei ed wi h he aim o c ea e a gapless man:machine in e ace in he cochlea by a ac ing he pe iphe al p ocesses o he audi o y ne e owa ds he elec ode a ay (Fig. 1). Such a gapless in e ace could open he doo o a highe equency esolu ion o he CI sys em, because smalle g oups o audi o y neu ons could be s imula ed om a highe numbe o sepa a e elec odes, coming close o he si ua ion in he ‘‘no mal’’ human inne ea , whe e a ound 3,500 inne hai cells s imula e a ound 30,000 audi o y neu ons. In acous ic simula ion s udies o no mal hea ing subjec s, a highe equency esolu ion is associa ed wi h imp o ed speech and music pe cep- ion. In addi ion o his, he gapless in e ace has a high chance o cu ing down ene gy consump ion. Suppo i e e idence o he easibili y o he NANOCI aim o ec ui spi al ganglion neu on (SGN) p ocesses o he CI can be ound in he li e a u e. Di e en neu o- ophins ha e been epo ed o ha e p osu i al and guidance e ec s on SGNs. Fi s , du ing emb yonic de el- opmen , audi o y hai cells exp ess he neu o ophin p o eins b ain-de i ed neu o ophic ac o (BDNF) and neu o ophin-3, which di ec g ow h o adial ibe bun- dles om he cen ally localized spi al ganglion in o he o gan o Co i (4). Di ec ed g ow h o ne e ibe s can also be induced expe imen ally a e in ec ion o sup- po ing cells wi h adeno i al ec o s encoding o BDNF leading o i s p oduc ion in adul dea ened guinea pigs (5). Simila ly, BDNF in combina ion wi h ib oblas g ow h ac o was capable o p omo ing su i al and FIG. 1. Schema ic concep o he NANOCI p ojec . O e iew (A) and mid-modiola c oss-sec ion h ough he cochlea wi h a CI elec ode a ay inse ed in o he scala ympani ( pink,B) a dis ance om he audi o y neu on (g een,B–D). By p o iding s uc u al suppo , guidance, and a ac ion cues om an injec ed, unc ionalized nanoma ix (blue shaded ill,B–D) and om he modi ied elec ode a ay su ace (whi e coa ing o pink elec ode a ay,Cand D), a guided g ow h o he neu i e on o he elec ode su ace is induced whe e a gapless ne e-CI in e ace is o med. CI indica es cochlea implan s. NANOCI e225 O ology & Neu o ology, Vol. 38, No. 8, 2017 Copy igh © 2017 O ology & Neu o ology, Inc. Unau ho ized ep oduc ion o his a icle is p ohibi ed. inducing e-sp ou ing o audi o y neu ons a e expe - imen ally induced dea ness in adul guinea pigs (6). Mo eo e , hese au ho s obse ed ou g ow h o ne e ibe s up o he bo de o he scala ympani, whe e he neu o ophins we e eleased. The pe iphe al p ocesses o he audi o y neu ons did no , howe e , en e he pe ilymph, possibly due o lack o s uc u al suppo . In addi ion, he neu o ophic e ec o BDNF can be enhanced i combined wi h elec ical s imula ion (7). Finally, in a ecen animal s udy i was shown ha ec opic ne e ibe s obse ed in he cochlea in esponse o ch onic neu o ophin deli e y we e associa ed wi h lowe exci a ion h esholds and no inc eased sp ead o ac i- a ion compa ed wi h dea ened con ols (8). Impo an ly, speci ic neu o ophin ecep o s a e exp essed in he human cochlea neu al issue also in adul hood (9,10), making he ea men wi h exogenous neu o ophins such as BDNF by local in acochlea deli e y sys em clinically highly ele an . The e o e, based on he li e a u e i seemed clea ha neu o ophins such as BDNF could be ideal candida es o induce and e-di ec neu i e ou g ow h owa ds a modi ied CI elec- ode a ay ac ing as a sou ce o deli e y. Ne e heless, he luid illed gap be ween he neu ons and he su ace o he CI elec ode a ay would be likely o inhibi he eg ow h. To o e come his limi a ion, we hough ha a s uc u al suppo should be o e ed, along wi h guid- ance and g ow h cues o ec ui neu ons o he a ay (Fig. 1). Fo his challenging endea o , expe ize om di e en backg ounds was me ged in an in e na ional and mul i- cen ic conso ium (Table 1—o e iew o conso ium pa ne s and hei key compe ences). The complex esea ch p ojec was subdi ided in o se e al smalle uni s (called wo k packages [WPs]) wi h de ined goals and miles ones, which a e discussed along wi h he esul s ob ained in mo e de ail below. The main aim o he p esen e iew a icle is o summa ize he his o ical backg ound o he NANOCI p ojec and o gi e an o e iew o i s achie emen s. WP1—Nanoma e ials and Bioac i e Compounds o Imp o ing he In e ace The main objec i es o his wo k package we e: 1) o de elop a biocompa ible and bio- unc ionalized, h ee- dimensional sca old, o nanoma ix, o p omo e neu al ou g ow h o ne e ibe s h ough he scala ympani on o he su ace o he CI elec ode pad, 2) o de elop bio- mime ic analogues o BDNF, and 3) o in oduce nano- su ace modi ica ions o he elec ode con ac s o s ably lock neu ons while imp o ing he elec ode conduc i e p ope ies. In he i s place, comme cially a ailable ibe o ming gel ma ices we e assessed. Pu aMa ix TM was selec ed as he bes candida e as i ul illed he basic equi emen s o being injec able as solu ion, gela inizing in esponse o ionic changes (i.e., con ac wi h he pe ilymph) wi hou swelling o mechanical s ess o su ounding issues. In addi ion, i had been shown o p omo e neu i e g ow h (11). Pa ne EMC mic ocollec ions GmbH (EMC) esyn he- sized he published gel o ming Pu aMa ix TM pep ide sequence and addi ionally modi ied i wi h laminin- de i ed sequences o neu on adhesion. Addi ionally, linking s a egies we e included o bio unc ionalize he nanoma ix wi h neu o ophic ac o s such as BDNF (12). As ou lined in he in oduc ion, neu o ophic ac o s a e impo an o media e su i al and sp ou ing o audi- o y neu ons in i o and in i o. Howe e , BDNF is no an ideal molecule o clinical inne ea applica ions because o i s sho in i o hal -li e ime. Small biomi- me ic compounds could o e an in e es ing way o sol e his p oblem. Howe e , o ou knowledge, only ew biomime ic analogues o BDNF ha e been published, TABLE 1. Lis o NANOCI conso ium pa ne ins i u ions wi h key compe ences Role A ilia ion Key Compe ences Coo dina o Uni e si y o Be n, Swi ze land Inne ea egene a ion, human and mu ine bioassays, in i o elec ophysiology (mul i-elec ode a ays) Pa ne 1 Uni e si y o Tu ¨bingen, Ge many In i o elec ophysiology, his ology, mu ine bioassays Pa ne 2 Uni e si y o Uppsala, Sweden Human bioassays, ime-lapse ideo Pa ne 3 Uni e si y o Tampe e, Finnland Nano oxici y assays in i o, d ug elease s udies, CB-CT and MRI imaging Pa ne 4 Uni e si y o applied sciences, HES-SO, La Chaux-de-Fonds, Swi ze land Nanos uc u iza ion by solid on liquid deposi ion, i adia ion; d ug-elu ion solu ions, modeling o elease Pa ne 5 Ba -Ilan Uni e si y, Rama Gan, Is ael Nanos uc u iza ion by sonochemis y, bac e ia bioassays, p oduc ion o nanopa icles (e.g., ca bon nano ubes) Pa ne 6 EMC Mic ocollec ions, GmbH, Tu ¨bingen, Ge many P oduc ion o BDNF mime ics, pu i ica ion and unc ionaliza ion o hyd ogels Pa ne 7 MEDEL, Innsb uck, Aus ia P oduc ion o animal- and human g ade nanoCI p o o ypes wi h su ace unc ionaliza ions; modelling Pa ne 8 Scip om Sa` l, S -Sulpice, Swi ze land P ojec managemen , websi e design, o ganiza ion o mee ings BDNF, b ain-de i ed neu o ophic ac o ; CB-CT, conebeam compu ed omog aphy; MRI, magne ic esonance imaging. e226 P. SENN ET AL. O ology & Neu o ology, Vol. 38, No. 8, 2017 Copy igh © 2017 O ology & Neu o ology, Inc. Unau ho ized ep oduc ion o his a icle is p ohibi ed. and hei e ec s on he inne ea neu ons a e as ye unknown (13). Pa ne EMC, he e o e, se ou o design and p oduce small molecule biomime ic compounds deduced om he di e en s uc u al pa s (loops) o he BDNF molecule wi h high s abili y, biocompa ibili y, and bioa ailabili y in he inne ea . P omising candida es we e es ed in an in i o bioassay using an audi o y neu on explan assay. Resul s o his sc een a e cu en ly p epa ed o publica ion. Fo s able locking o audi o y neu i es on he pla inum elec ode su ace and op imiza ion o elec ode con- duc i i y, di e en nano unc ionaliza ions we e de el- oped and es ed by pa ne Ba -Ilan Uni e si y (BIU). F om a pool o se e al candida es, hyb id nanocompo- si es o conduc i e polyme s and mul iwall ca bon nano- ubes we e ound o subs an ially oughen he pla inum su ace and he eby o imp o e conduc i i y o s anda d cochlea implan pla inum pads in elec ochemical measu emen s by pa ne MEDEL (14). In addi ion, hese nanocomposi es subs an ially educed s imula ion h esholds o audi o y neu onal explan cul u es on mul i-elec ode a ays compa ed wi h s anda d g ey pla inum su aces. WP2—Nano unc ionaliza ion o he Elec ode A ay Su ace This wo k package was designed o s uc u ally op i- mize he non-conduc i e silicone ca ie su ace o he CI o d ug deli e y and o implemen an ibac e ial ac i i y. Pa ne HES-SO e alua ed wo di e en echnologies o elease o p o eins om ese oi s on he su ace. Fi s ly, he solid on liquid (SOLID) nano echnology was e alua ed. To his end, he silicone ca ie o he elec- ode was u he coa ed wi h a pa ylene laye con aining mic oscale ese oi s wi h 60 mm holes c ea ed by lase (15,16). This allowed o elease o luo escen mol- ecules ( luo escine), used as epo e s in hese s udies. In addi ion, nanos uc u iza ion me hods such as local- ized i adia ions o Pa ylene-coa ed su aces passed bio- compa ibili y es ing wi h an audi o y neu on su i al assay, sugges ing ha such echnologies migh be used o p ecise local e ching o a pa ylene-coa ed su ace o c ea e wells o nanopo es o con olled elease o g ow h ac o s (17). Finally, a simple app oach was e alua ed, whe ein luo escein c ys als, in powde o m, we e encapsula ed in a hin laye o pa ylene, hence c ea ing andom nano- me e -sized pinholes. This was ound o be e ec i e o con olled d ug- elease om he elec ode su ace (18). The e o e, he app oach was selec ed, as i would acili- a e he s o age o p o eins on he CI silicone su ace in a solid o m (powde ), g ea ly imp o ing handling and shipping p ope ies. To diminish he isk o in ec ions and coloniza ion o he elec ode a ay, pa ne BIU u he modi ied he non- conduc i e silicone pa s wi h an imic obial nanopa - icles. ZnO-MgF 2 and CuZnO we e inco po a ed in o he silicone a ay sonochemically. These nanopa icles p o ed o be s ably locked on he su ace wi h minimal o absen leaching o in o a i icial pe ilymph and wi h subs an ial inhibi ion o bio ilm o ma ion (19). WP3—Modeling, Coding, and In o ma ion T ans e This wo k package add essed he unc ional exploi a- ion o he gapless elec ode–audi o y neu on in e ace and looked in o op imized design o u u e cochlea implan sys ems wi h inc eased bi-di ec ionali y and highe channel numbe s. In e ace-dependen s imula ion pa e ns we e s udied using spi al ganglion neu on explan cul u es on mul i- elec ode a ays (MEAs) by he Uni e si y o Be n (20). We we e able o demons a e a educ ion in he ene gy needed o elici a esponse as a unc ion o he dis ance o he s imula ing elec ode o he neu onal cul u e. This p o ed he key hypo hesis ha he gapless in e ace allows o educ ion o s imula ion ene gy. In addi ion, op imiza ion o s imula ion pulses using in e phase gaps, ains o pulses, and long biphasic pulses was assessed. The la e we e also shown o allow o a educ ion o he ene gy needed o s imula ion o SGNs (20). The close p oximi y o he elec odes o he neu ons allows many elec odes o be ac i e a he same ime, hus imposing less s ic empo al es ic ion on coding s a - egies compa ed wi h cu en CI sys ems. Based on his da a, sound coding s a egies o an implan ea u ing a gapless in e ace and mo e han 30 independen channels we e modeled using a cus om-made, mul icompa men model o he audi o y ne e by pa ne MEDEL (da a no published). To inc ease bi-di ec ionali y o u u e CI-sys ems, a Fab y–Pe o ibe ip senso was de eloped o measu e- men o empe a u e and p essu e sui able o inco po a- ion in o he elec ode a ay. The sys em includes a low-bending loss ibe equipped wi h a solid on liquid deposi ed e minal (21). The sensi i i y o he sys em heo e ically allows o he de ec ion o con ac o he ip wi h he su ounding s uc u es in he cochlea, in e es ing o measu ing inse ion o ces in obo ic su ge y, bu also po en ially o use as an implan able mic ophone o p essu e senso . WP 4—Bioassays A a ie y o bioassays we e used in NANOCI o es g ow h ac o s, g ow h ac o mime ics, nanoma e ials, elec ode su aces, d ug elease, an ibac e ial ac i i y, and s imula ion pa e ns, among o he en i ies. The pa - ne Uni e si y o Tu ¨bingen es ed he injec able, pep ide- based hyd ogel de eloped by pa ne EMC o i ’s pe missi e p ope ies o acili a e neu i e g ow h owa ds he CI in a spi al ganglion explan assay (12). In addi ion, he pa ne Uni e si y o Uppsala s udied neu al p ogen- i o cell-de i ed spi al ganglion-like neu ons (22). They also s udied he egene a i e capaci y o human su ge y- de i ed es ibula neu ons (23) as well as he possibili y o a ac ing and guiding hese p ima y es ibula neu ons in a 3-D nanoma ix (Li e al., Unpublished da a, cu en ly unde e ision). In addi ion, his ological s udies we e unde aken o be e unde s and he spi al NANOCI e227 O ology & Neu o ology, Vol. 38, No. 8, 2017 Copy igh © 2017 O ology & Neu o ology, Inc. Unau ho ized ep oduc ion o his a icle is p ohibi ed. ganglion ana omy and i s ele ance o cochlea implan- a ion oday as well as o po en ial u u e sys ems wi h a gapless in e ace o audi o y neu ons (24–26). These esul s show, o he i s ime ha he human audi o y ne e has a egene a i e capaci y and ha audi o y neu ons can g ow h ough a 3-D nanoma ix. The his opa hological s udies also e eal po en ial unde lying mechanisms ha may allow human audi o y neu ons o, unlike hei animal coun e pa s, pe sis a e hai cell loss, making he use o cochlea implan s possible. WP5—Imaging, Biocompa ibili y, and Release Dynamics o he Modi ied CI Elec ode The aims o his wo k package we e 1) o assess he neu o ophin elease om hyd ogels and om implan su aces, 2) o measu e and isualize he gap be ween he elec ode a ay and he medial cochlea wall using cone- beam compu ed omog aphy (CBCT), and 3) o assess he biocompa ibili y and sa e y o he modi ied elec ode a ay a e coa ing wi h nanoma e ials. The pa ne a he Uni e si y o Tampe e, in collabo a ion wi h o he s, mainly led his WP. I was demons a ed ha he elease o neu o ophins o small molecules in a phan om model wi h a i icial pe ilymph (da a no published) co esponds o he in i o dis ibu ion o labeled Gadolinium nanocomposi es o T kB-ligands in he a cochlea a e ans ympanic injec ion, as measu ed by MRI (27,28). Based on hese expe imen s, he inne ea dis ibu ion o neu o ophins eleased om he hyd ogel and om he cochlea implan su ace was p edic ed. Fo he second aim, imaging pa ame e s o cone- beam CT we e op imized yielding highe esolu ion a lowe i adia ion doses. Cada e ic human empo al bones we e implan ed wi h di e en human g ade coch- lea implan elec ode a ays (Fig. 2) and s udied wi h he op imized CBCT se ings o inse ion dep h, scala loca ion, and he dis ance o he a ay om he modiola wall (29). Finally, he biocompa ibili y and oxici y o nano ech- nologically modi ied elec ode componen s we e e al- ua ed in i o using he BALB/c3T3 and NR8383 cell lines ollowing he ISO 10993–5 and OECD GD-129 guidelines. No in i o o speci ic o o oxici y es s we e pe o med o he bulk o nanoma e ial candida es and oxici y was p edic ed based on issue equi alen s a his s age. Some o he ea lie candida es o an ibac e ial nanocoa ing in he p ojec (ZnO and MgF 2 ) we e la e abandoned due o oxici y in hese assays. The nano- composi es used o coa he pla inum elec ode pa s (mul iwall ca bon nano ubes) we e ound o be no oxic o BALB/c3T3 cells, while sligh ly dec easing iabili y o NR8383 cells (30). WP6—Pilo , Animal G ade nanoCI Mul icomponen Valida ion The inal goal o wo k package 6 was o de elop an animal-g ade p o o ype o in i o es ing inco po a ing he di e en nano- and bio- unc ionaliza ion discussed abo e. The pa ne MEDEL p oduced a 4-channel guinea pig elec ode a ay (Fig. 3), which was hen unc ionalized wi h di e en componen s. Namely 1) conduc i e polyTh copolyme (pEDOT)/oxidized mul iwall ca bon nano ubes bound on pla inum con ac s, 2) pa ylene-coa ed elec ode a ay ca ie doped wi h an i ouling agen (CuZnO nanopa icles), and 3) 7,8,3- ihyd oxy la one, a BDNF analogue shown o ac as a high a ini y agonis FIG. 2. P o o ype o he NANOCI elec ode a ay ea u ing 36 elec odes on able (A) and a e inse ion in o a cada e ic human empo al bone, as isualized h ough cone-beam compu ed omog aphy (Band C). This p o o ype yields p oo o concep , ha channel numbe s can be ipled wi h oday’s manu ac u ing me hods. No in acochlea damage and an in ac basila memb ane we e obse ed (D). Howe e , due o inc eased s i ness o he de ice, a ull inse ion was no possible. e228 P. SENN ET AL. O ology & Neu o ology, Vol. 38, No. 8, 2017 Copy igh © 2017 O ology & Neu o ology, Inc. Unau ho ized ep oduc ion o his a icle is p ohibi ed. o he T kB ecep o , deposi ed in solid s a e in he su ace o he elec ode a ay ca ie . Fu he mo e, MEDEL p oduced a ca he e o in a- cochlea applica ion o he pu i ied and laminin-loaded hyd ogel (p oduced by EMC unde WP2, Fig. 3D). The mul icomponen alida ion o hese p oduc s included i s he ypical quali y and pe o mance es ing a MEDEL, ollowed by in i o expe imen s pe o med a he Uni e si y o Tu ¨bingen using a dea ened guinea pig animal model. Dea ened guinea pigs we e implan ed FIG. 3. Speci ica ions (A) and pho og aphs o he p o o ype on able (B) and inse ed in o he cochlea (C) o he guinea pig cochlea implan wi h ou elec odes (a ows in B). The leng h o he in acochlea a ay is 4 mm, as indica ed wi h a s oppe (blue o al) loca ed be ween wo da k ma ke s a dis ance 3 and 5 mm om he ip o be e es ima e o achie ed inse ion dep h. Ca he e s o in acochlea injec ion o he nanoma ix in luidic o m (D). The ca he e s we e manu ac u ed in wo a ian s wi h di e en diame e s o he silicone ubing (0.3 mm wi h yellow s anda d sy inge needle and 0.6 mm wi h whi e s anda d sy inge needle). The black do indica es he 3-mm inse ion dep h as a e e ence poin (a ows in D). CI indica es cochlea implan s. FIG. 4. P oo o concep o he gapless in e ace be ween audi o y neu ons (g een) and he CI-elec ode a ay in scheme (A) and in he dea ened guinea pig inne ea in i o (B). Audi o y neu ons a e s ained wi h a neu on-speci ic ma ke o b-III ubulin (g een, TUJ), cell nuclei a e s ained in blue (DAPI nuclea s aining), pho og aph p o ided by Ma cus Mu ¨lle and Hube Lo ¨wenheim. CI indica es cochlea implan s. NANOCI e229 O ology & Neu o ology, Vol. 38, No. 8, 2017 Copy igh © 2017 O ology & Neu o ology, Inc. Unau ho ized ep oduc ion o his a icle is p ohibi ed. wi h s anda d and modi ied guinea pig cochlea implan elec ode a ays (Fig. 3) in combina ion wi h unc ion- alized nanoma ix gels applied h ough he a o emen- ioned ca he e (Fig. 3D). The in i o expe imen s yielded p oo o p inciple ha neu i es can g ow unde neu o ophin s imula ion and h ough s uc u al suppo om he nanoma ix in o he scala ympani and on o he su ace o he elec ode a ay, o ming a gapless in e ace in he dea ened and implan ed cochlea (Fig. 4). Func- ionally, he gapless in e ace led o lowe s imula ion h esholds and a la ge dynamic ange in hese animals in i o and he de ailed esul s a e cu en ly being p epa ed o publica ion. Finally, MEDEL es ed he limi s o oday’s manu- ac u ing echnologies and buil a human-g ade p o o ype elec ode a ay ea u ing 36 pla inum con ac s (Fig. 2A). The human-g ade p o o ype elec ode a ay was inse ed in o a cada e ic human empo al bone a he Uni e si y o Tampe e o imaging (and no used in i o) (Fig. 2B). No in acochlea damage was obse ed (Fig. 2B and C), howe e due o inc eased s i ness ela ed o he highe pla inum con en s o he a ay, i could no be ully inse ed in o he cochlea. DISCUSSION AND CONCLUSION In conclusion, he NANOCI p ojec yielded p oo o concep ha a gapless in e ace be ween audi o y neu ons and cochlea implan elec ode a ays is easible in i o. Func ionally, he gapless in e ace led o lowe s imu- la ion h esholds and a la ge dynamic ange in i o compa ed wi h un ea ed animals and o an up o i e old educ ion in equi ed s imula ion ene gy in an in i o model using audi o y neu ons cul u ed on mul i-elec- ode a ays. The ele ance o his p omising obse a ion o po en ial u u e clinical applica ions emains unknown a his poin in ime. Pa icula ly, i canno be o eseen, how many eg own neu i es will be unc- ionally ele an and how many pa allel channels o he cochlea implan can be e ec i ely used o s imula ion in he u u e. In addi ion, he o al ene gy consump ion o a u u e CI sys em ha could be based on he NANOCI p inciple canno be p edic ed based on ou expe imen s, as many ac o s ele an o clinical CI sys ems we e no aken in o accoun in he expe imen al models. Se e al o he issues such as sa e y and unc ional s abili y o e ime and beyond e-implan a ion p ocedu es need o be se iously add essed. Ne e heless, we emain op imis ic ha some o he de elopmen s made du ing he NANOCI p ojec may help o imp o e cochlea implan sys ems in he u u e in one way o ano he . I is impo an o no e ha all o he esea che s expe ienced a subs an ial bene i om he in e disciplina y collabo a ion o he conso ium pa ne s in ol ed in he p ojec . Acknowledgmen s: The au ho s would like o hank he ol- lowing NANOCI collabo a o s o hei aluable con i- bu ions: Emanuele Ma coni, Anne Tsche e , Ju ¨ g S ei , Hans Ruedi Widme , S e ano di San o, Ma ija Bos o¨m, Sebas ien B un, F ancois Feu ie , Thie y Allen, Ha y Wi hlow, Rena e Spohn, Raphael Ki an, Roland Hessle , F ede ik Klause , Jochen Tillein, Giulia Ma ini, So¨ en Schilp, Te esa Melchionna, Raimund Naschbe ge , Ingebo g Hochmai and Ki s en Leu gen. Fu he , hey would like o hank S e an Helle and Daniel Bodme o being pa o he ad iso y boa d and Silke K ol (p ojec echnical assis an ) and Heico F ima (p ojec o ice ) o hei aluable wo k o he p ojec a he EU. 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NANOCI e231 O ology & Neu o ology, Vol. 38, No. 8, 2017