Molecular Biointerface Characterisation for an Implanted Medical Device Using Cryogenic Orbitrap Secondary Ion Mass Spectrometry (Cryo-OrbiSIMS)

I m pl an te d me d i c a l d evi ce s o f t e n fa i l d ue to fo r e ig n bod y r e a ct io n (F BR ) , a p ro c e s s st il l no t f u l l y un d e r st ood . T h is s t ud y e x ami n e s s ilico ne r u bb e r p ol y ( di met hyl si lox ane) cat h e ters im pla nted i n mice for 1 a n d 2 8 d ays. F o r th e fi rs t ti m e , Cr yo-Orbi S IM S was u sed to ch ara c terise bio logica l de pos i t io n on im p l a n t s, combi nin g ToF- S IM S im a gin g an d Orbi SI M S meta b ol i te pr ofi l i n g . Mac hi n e le a rni n g a n d sta tis tical ana l ys is ide ntifie d e arl y bioi nt erfa c e m ar ke rs , i n c ludi n g ele vated s ugars a n d ita c o n at e a t d ay 1, an d inf l a mma tio n marke rs s uch as u r a t e, m yr i s t i c a c i d ( F A 1 4 : 0) , a n d p al m it ic ac id ( F A 1 6 : 0) a t da y 2 8 . D e p t h pr ofi l i n g af t er 2 8 d a y s r ev e al e d t w o d i s t i n c t m o l e c u l a r l a y e r s : l i p i d s n e a r t h e c a t h e t e r a n d fa t t y a c i d s , a m i n o a c i d s , a n d n u c l e i c a c i d s n e a r t h e h o s t tiss ue. Li pids li k e oxi di s e d PG ( 39: 1;O) an d FA 1 8:2 w e re pre s e n t at bot h ti me p oi nts , s ug ge s tin g rol e s in early i m m une r e c ogn i ti on du r in g F BR . Th e se in si g ht s adv an c e und e r sta n din g o f FBR and suppo rt th e de ve lop m e nt o f impro ved imp lant m a teri a ls . was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint

Medical d e v i c e s pla y a pi vot al r ole in m oder n me di c ine ra ngi ng fro m s t ent s, c a t h ete r s, hi p an d knee j oin ts a n d sen sors t o t reat a n d m o nit o r m e dica l co ndi tio n s or re place a nd a ugme nt phy si olo gi c a l fu ncti on s 1 . Si l ico ne - ba s ed bi o m at eria l s are com mon ly u s e d for me d ic a l de v ices, ap p l ie d a s me dical t ubi n g , tiss ue r e p la c eme n t , intraoc u lar l ense s, voice pro st hes e s , dia lysis me mbra n es, brea s t im plan ts an d ve n tr i c u l o peri t one a l sh u nt s 1,2 . T h e res p onse o f t he bo d y to im pla nts, a l so kno wn a s foreig n bo d y res p o n s e, is d esc rib e d a s co mp l ex multi st ag e process charact erise d b y a n acut e inf l a m mator y phas e w hi c h is t he res ult of a ds orp tion of biom olec u l es s uc h as chemo a t tra c ta n t, c yt o kines , gr owt h fact ors, a n d ot her bi oa c t ive age n t s lead i n g t o t h e formati o n o f pro v i s io nal m atrix 3 4 . T h i s a c u t e F B R p h a s e i s a l s o c h a r a c t e r i s e d b y t h e r e c r u i t m e n t o f i m m u n e cells s uch as ne u tr oph il s an d mo n ocyt e s t o th e s i t e of i mplant at io n . As t h e res p o n s e pr o gre s se s, t he pro tei n wit hi n t his pro v is io n a l m a trix un der g o es a dy namic a ds orp tion- d e s or p t i o n process , kn ow n as t he Vr o m an effe c t resu ltin g in t he replace m e nt of sm a ller protei ns wit h la r ger one s. In ad di t io n, t h e recruite d m o no c y te s will un der g o d iffe r ent ia t i o n int o m acrop hag e s at t h e si te of impl a n ta ti o n w h il e rel e asi ng degra d i n g e nz y m e s and r e acti ve o x yg e n s pe c ies. D u ri ng t his p e r i o d th e fu sio n o f m acr op hages i nt o p o l y nuc l ea te fore i g n bo d y gi a n t cel l s occ urs t o pha g oc yt os e t he i m p la nte d b i om a teri al . O v e rt ime, th is pro- i n fla m m at o r y s ta g e tra nsi tion s int o a chr o ni c F B R re s po nse ch ara c te r i s e d b y the pres ence o f a fibro tic c o lla gen ous l ayer betw een th e impl a n t and t he h os t tis s ue . As t h e F BR t ransi ti on in t o t he c hro n ic phase t he ma c ro p h a g es un dergo a p hen ot y p ic sw i t c h fro m pro-in flamm a t ory ( M 1 ) t o an ant i-in fl a m m at or y (M 2). Th ese M2 m acro p hage s re s ults in for m at io n of a fi b r obl a s t an d e x tra c el l ula r mat ri x -rich caps u l e th a t c o v e r s an d is olat es t h e i mplan t. The surf ace pr o pe r ties of i mplant m aterials (t op o g ra p hy, ch em i st r y, c o m plia nce) pl a y a c ritical r o le i n mo d u la t ing t h e f ore i g n b o d y r e s pon se 5 . Schreib et al h ave pr o pos e d ne w me c h a n ist i c ins igh ts in to t he process of f orei gn b o d y r e s po ns e t o i m pl ante d medical d e v ic e s 6 . U s i n g B i 3 + L iq ui d m et a l i o n g u n (L M IG)/ ToF- S IM S a nal y s is, t h e rese a rc h e rs s how ed t h at l i pi d dep osi ti on o n bi omat e r i al s after t he firs t day of im plant ati on, correla ted w i t h t he i mplant’ s i m m u n o genici t y whic h ul t im ately d ictat ed t he overa l l forei gn b od y re sp o n s e t o t he im p l a nt. T hey w ere a ble t o ide nt ify 1 1 f at t y acids, us in g LMIG/ T o F- S IMS an alysis , t h a t w ere e n r i c hed o n t he surf a c e of ex pl ante d pr o- fibro ti c im plan ts a nd u ncoate d PD MS disc s, w h ile phos p h ol i pi d an d s p hi ng omye l i n w e re enric hed on th e s u rf ace of a nti-fi br otic impla nts . T hi s correl a ti on, a nd a p oten tial rol e for s pecific lipi ds in co n trol ling F B R, off ers a no vel ad ju nct t o t he ort ho dox y of pro tei n s bein g t h e mai n de terminan t of res p ons e t o im pla nts. T h e Or biSIMS ha s b e en d e velo ped w it h s up eri or ma ss r e s ol vin g pow e r an d a c curacy, b ase d on t he Or bitra p a n a ly se r , a long wi th s o ft e r f r agm en ta ti on us in g a rg o n g a s c l us t e r i on b e am s ( G C IB) to a c h i e ve a m o r e c o mp r e hen s iv e in te rp r e ta t io n o f end oge nou s b io m ol e cu l a r p ro c e s se s, su ch a s r es pon s e o f i mp l a nt s to h o st tiss ue , us in g S I MS 7,8 . Su v a nn a p ru k et al. ap pl i ed GC I B/O rb i SIMS t o t h e m olecul ar charac te r i s a ti o n o f s e c tion s of t i s s u e proxi mal t o im pla nts t o pro vid e in format io n o n t he in vivo h ost re s pons e t o me d ic a l d e vi c e impla ntat io n , b a s ed on Orbi S IM S meta b ol i te p rofili ng of in vitro s i n g l e m a c r o p h a g e p h e n o t y p e s . I t w a s o b s e r v e d t h a t m o n o a c y l g l y c e r o l s ( M G ) a n d d i a c y l g l y c e r o l s ( D G ) d i s p l a y e d i n t h e t i s s u e w e r e s e e n a t i n c r e a s e d was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint i n t e n s i t y i n t h e t i s s u e s ≈ 1 0 µ m fr o m t h e i m p l a n t s o f r e d u c e d F B R c o l l a g e n c a p s u l e t h i c k n e s s s e e n i n h i st ol ogi c a l s e c t ion s. 8 H e r e , w e take t he a p proach of exa mini n g t h e s urface of th e re mov e d im pla nts us in g Cryo-Or biSI M S, r a t her th an an alysi n g t he h ist ol ogica l s e c ti ons, t o p r ovi de ins i g h t in to t he bioi nt e r fa c e . T h e Orbi SI M S datas ets pr o v i de a r i c h bu t c omp le x i nf orm a t io n s o urce o n t h e m ol ec ular a n d fra gme n t s fro m lipid s, ami n o aci ds, and ot h e r s mall m ole c ules. To ai d deco n vol ut i o n of t he com p l ex ma ss s p ect rome try data, artifi c ia l neural n e tw orks ( ANN) a nd de e p l ea rni n g archit ect ures u n de rp in ned b y a d v a nces in co m p u tat ion al ha r dw a re h ave be e n d evelo ped 9 . Se v eral s t u d ie s h ave rec ent ly b een p u b lis hed t ha t d e m o ns trat e t he u til i t y o f ma c hin e lea r ni n g i n vis u alis i ng and u nd e rs tan di ng hy pe rs pe c tra l datas ets 10–12 . H e r e w e ap p l y a l o g i s t i c reg r e s s ion a n a lys i s machi ne l ea rn i ng mo d e l cou pl e d wi t h a da ta - dri ven m u lt i v aria te a ppr o ach t o id e n tify a n d int e rpre t b io mole c ul e s c han ges on t h e s urface of t h e i m pl ante d s il ic on e t u b es. We pro be t h e inf l a mmat or y p ha s e by l oo k i n g a t t ubes re mo ved 1 day p ost imp l a nt atio n, an d t he e arl y fi brot i c p ha s e usi ng s a m p l es rem o v e d 2 8 da ys pos t i mp l a nt a t io n t o eluci date t h e cascad e of e v e n ts t hat t a k e s p l ace durin g a fore i g n bo d y resp o n s e t o a sil ico n e i mpla n t . A ut om a te d peak a ss ig nme nt wa s achi e v e d us in g m o le c ul ar for m ul a pre d ict o r (MF P ) t o ide nt i fy t he rang e of b i o m ol ec ule s d epos ite d in c om b i n a t io n wi t h es ta blis h e d data ba s e s 13 . It w a s fo und t h at cry o g e ni c con diti o ns a ug mente d the s ig na l of biolo gic a ll y relev ant mol e c ule s from t h e s urfa c e o f impla nts , c o n s i s t ent w i t h pre v i ous report s 14–16 . T h is me t h o do l o g y and wor kfl o w is trans lata b l e t o t he ana lysis o f n o v e l b i o m a t e r i a l s w i t h i m m u n e - i n s t r u c t i v e p r o p e r t i e s i n t h e s e a r c h f o r m a t e r i a l s c a p a b l e o f m i t i g a t i n g t h e FBR res po n s e in vivo a n d off ering new i nsi ght s i nt o t h e molec ula r b a s is o f t h e F B R. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint

and Discussion Histol o gy Se c tio ns of si lico n e cat het e r s w ere imp lant e d s ubc uta neo usl y in t o male B al b / c mi c e a s s how n i n F i g u r e 1 ( a ) . Up o n im p l a n t r e m o v al a fter 1 day, t here were n o v i si ble diffe r ences i n t he s urro u ndi n g tis sue a s s e e n in Fi g u r e 1 (b ) b ut a tra n s p are n t fi l m w a s a p p arent o n t h e s urface of t he m a t eria l. A ft e r 2 8 d a y s o f i m p l a n t a t i o n , a sig n ifican t a rea of red den i n g w as ob serv ed i n t he s urro und in g t iss ue , as w ell as t he pres e nce o f va s c ula riz ati o n be l ie v e d t o b e i n d ic a ti ve of prol on ged i nfla mmati o n at th e sit e. Th is red den ed tis su e a rea w a s q u a ntif i e d i n Figur e 1 (c ) by u s i n g I mage J t o a na l ys e t he area o f i n flamm a ti o n s urro u ndin g th e cat he t e r se g m e n ts b y thres h oldi n g t he colo ur chan ge obs e r ve d in th e ski n t is s ue . Hi st ol o g y pe rfor me d on t he ti ss u e b ot h s u r r ound in g a nd w it hin th e ca t he te r t ub e ar e s ho wn in F i g u r e 1 ( d ) . The ca th ete r h a ve be e n rem ove d p ost fixati on a nd prior t o e m bed di ng d urin g sample proc e s si n g. The ti ss ue s urrou ndi n g t he imp lant s after 2 8 d a y s were s taine d u si ng 3 se parate c omp leme n t ary a p pro a c hes: haem a t ox yli n a n d e o s in ( H& E) , M a ss o n ’s tric hro me (M T ) an d Pi c ros irius red sta ins ( b ri gh t field and p ol aris e d lig ht) i n o r der to g et a f ull ch ara c terisat ion of th e tiss ue. T h e re s u l ts of all t hree s ta i n a r e s h ow n i n Fi g ur e S1 . A fibrot ic la y er i s clea rl y ind icated b y th e MT st ai n in Fi g ur e 1 ( d) a r ound the c a th et e r a t 28 d ay s co n si s t e n t w it h a F B R 17 . Th e po la ris e d P ic rosiri u s r ed st ain a l o n g wit h t he Ma ss on ’s trichro m e s tai n c l earl y reveal s a ce llul a r layer on t he im plan t sid e of t h e fibrous l a y er Fi g u r e 1 ( e ) . Pre v i ou s rep ort 18 h a v e c o n s i d e r e d t h e s e t w o l a y e r s a s o n e c o l l e c t i v e s t r a t a , i . e . t h e e n t i r e fi b r o t i c biola yer, h o w eve r t hese ima g es s h o w t h at t hi s can b e fu rt h er s u bcat egoriz ed. T he c e l lul ar laye r w a s m e as ure u s ing 7 sa mp l es a s 6 µm + 1 µm an d t he co ll a gen l a ye r w a s w h ic h w as 1 6 µ m + 5 µ m Fi g u r e 1 ( e ) . was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint Figu re 1 (a) Sc he m atic i ll u st rati ng th e subc u tan e ou s im plan tati o n of sil i co ne cat he t e r i n t o mal e Ba l b / c mi ce . (b) I m a g es o f t he i m p l a n t ed c a t h e t e r i n t h e s u b c u t a n e o u s l a y e r o f m i c e a t 1 d a y a n d 2 8 d a y s , s c a l e b a r : 0 . 2 5 m m . (c) A r e a of r e ddene d tissu e (ind i ca t ed b y ar ro w ) sh o w in g infl ammat ion surr oundin g t he cath e t er f o l lo w i ng im plan t ati o n al ong w i th t he p r e s e n c e o f v a s cul ari sat ion. D a ta ar e e xpr e sse d a s m e a n ± S D ( n = 5 f o r 1 d a y , n = 1 1 f o r 2 8 d a y s ) . (d) H& E s tain an d Mass on tri ch rom e stai nin g of F BR ti s sue s ecti o n s at 28 d a ys, s c a l e bar: 1 mm. (e) C e llu la r a n d c o ll age n la y e r t h i c k ne s s m e as ur e d f ro m hist ol ogi c al s e cti o ns . B i oin t e rf ac ial ch e m ical anal y sis T o s t u d y t h e m o l e c u l a r r e s p o n s e o f t h e h o s t t o t h e i m p l a n t a t a n i n t e r f a c i a l l e v e l , G C I B O r b i S I M S a n a l y s i s o f th e sa mple s was c on d ucte d o n t he s urfa c e of t he e x pla nt e d silic on e c a t he ter re s ul t in g in co ns u mpt io n of t he m a te ri a l a t the in t e r f a c e ov e r a 1 00 a n al ys i s s ca n s. S a m ple s n eed t o be ex amin e d eit h e r froze n h ydrat ed u si ng c ry o genics o r d e h ydrat e d at roo m tem perat ure to a l l ow t he m t o be p l ace d u nder vacu um f or S IMS an alysi s. W e c om p a r ed the s e t w o app r oa c h es ; th e p hy s i c a l e f f e c t o f t he s a mp le d r y ing und e r v a cuu m a t ro o m was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint temp erat u r e was s ee n as cr a c k s in t h e LMIG /To F -SI MS i m a ge s i n Fi g u r e 2 ( a ) , wh ere th e water evap oratio n h a s le d t o s hrin kage a n d fract ure of t he bi o m olecular lay e r . W h e n t he s ampl e s were a n al ysed u n d er c ry o g e n ic froze n-hy drat ed co nd itio ns, n o cracking w as ob ser v e d. Plot ti n g t h e i nt ensit ies of t he i o ns ac qu ired from t he roo m te m p erat ure s a m ples a g a i n s t t h o s e acq ui r ed u nder cryo gen i c con d i tio ns i n F i g u r e 2 ( b ) , cle arl y i n di c a t es th a t e m plo yin g c ryo - G CIB/Or biS I MS a ugme n t s t he i o n s i g nal i n t en sit y for a pprox ima te l y 9 0% of io ns, s o me b y as m u c h a s 10 4 . F u r t h erm ore , m ore t han 4 0 bi omol ecular s pecies w e re on l y d e t e c t e d u nder c ry o, incl u d i n g i o n s p ut at iv e l y a ss ig n ed to ; a d ia c y l -g ly c e r opho s p hoe th ano l am i n e , P E 3 6 :1 , a d i a cy l -g ly ce ro pho sph oi no si to l , P I 3 8 : 5, Gl ut a m i n e a nd t he oxi di s ed fa tt y a c y l molecu le s suc h a s N A T 3 2: 0 ;O 4 a nd N AT 32: 7;O. The au gme nte d ioniza ti o n y ield i s a ttri b u t ed t o t he p r e s e n ce of wa te r t h a t s erv e s a s a ma trix t o pr o v i de H + t r a n s fe r f o r seco nd a ry io n forma ti o n. Furt her m ore, c ryog enic a n a lys i s pre ve nts l o s s o f m olecul es t hat a re pron e t o evap o ra ti on s uc h as f att y acyls , s o fr oz en h ydrat e d acq u i sit io n is a d opt ed fo r th e res t of th e w ork in t hi s p a pe r 14,16 . T o e nabl e t h e in te r p r e ta ti on of im a g ing m a s s sp e c tral da tas e t s gen era te d in T o F -SI MS a n al ys i s , no n-n ega t i ve ma tr i x fa c toris a t io n (N MF) 19 a n a ly si s w a s e mp l oy e d as sh ow n i n Figu r e 2 (c )-( d) . T h i s r e ve a le d t h a t t h e s u r fac e o f the se gm en ts w e r e v e r y c h em i c a lly h e t e r og e n ous , a s sho w n in F i g u r e 2 ( e ) , w i t h pa t c h y bi o l og i c a l d e p os i t . NMF i mage s cate gorise d t h e l at eral c hemical va ria n c e i nt o t hre e dis ti n c t s urface c hemi str ies a l o n g t he s urfa c e of t he cat h ete r: s ec on da r y ion fra gme n t r eprese ntin g t he sil ico n e fro m t h e ca th e ter, ice an d bio logica l d epos i t at 1 day an d 28 days. I m a g e s w e re c on str uc te d for t he silic on e c a t he te r a n d m o bi l e o li go me r s usi ng m/z 73 . 0 5 (Si C 3 H 9 + ) , 147 . 06 (S i 2 C 5 H 15 O + ) a n d 2 07. 0 3 ( Si 3 C 5 H 15 O 3 + ) , i c e u s i n g t h e w a t e r c l u s t e r s H 7 O 3 + , H 9 O 4 + H 11 O 5 + fr o m H 2n+1 O n + 20 an d bi o l og i ca l d epo si ts us in g ph o sph a t i dyl ch o l in e fr a g me n t i ons a nd s a lt s ; 86 .1 0 ( C 5 H 12 N + ) an d 1 84.0 6 ( C 5 H 15 NPO 4 + ) 22. 99 (Na + ), 38. 96 ( K + ) . I t i s a p p a r e n t i n F i g u r e 2 ( e ) th at t he re was a g rea te r c o v era g e o f biol o g ic a l dep os it on t he sil ico ne surface at 2 8 da ys rel a ti v e t o 1 day. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint Figure 2 (a) L MIG /ToF-SI M S i on i m a g es of t he cat h e t er s u rf ac e s wh e n sa m pl e we r e an a lys ed und e r fr oz e n h y dra t ed a nd ro o m t em p er at ur e c o nd i t i on s . (b) C ompari so n o f GCI B/O rb i S I MS sp ectr a o f c a th e t e r surfac es analys ed u n de r f r o z en - h ydr a t e d (blu e ) and r oom t em p er at ur e ( r ed ) c o nd i t i on s . (c) S ca tt e r p l o t co mp a ri ng th e n o rma li sed io n int e nsi ti es of bi om o l ecul es de t ect e d wh e n the su rfac e o f the c at h et er s wa s a na l y s e d u nd e r r oo m t em p er a t u r e a n d f r o z en h yd r a t e d . (d) N o n- n e g at i v e m a t r i x f a c t or (N MF ) l o a di ng s s h o wi n g t h e se pa ratio n o f L M I G/ To F-S I MS da t a int o th r ee di sti nct c o mp o n e nts: i c e , bio lo gi cal d e po sitio n, and ca th e t e r w it h (e) r ep r e s e n t a t i v e t o t a l i on a n d N M F i m a g e s f o r 1 - a n d 2 8 - d a y s i m p l a n t a t i o n f o l l o w i n g 50 0 N M F i t e r a t i o n s . T h e N M F a n a l y s i s w a s p e r f o r m e d u s i n g l o a d i n g s a n d i m ag e s f ro m r a w LM IG /T o F d a t ase t s . Sc a le b ar : 10 0 µ m . Cr y o -Or bi S IM S bi om o l e cu lar d ep o sit sp e c tra l a nn o t a ti on Th e H u m an M e t abo l om e a nd c om pu t a ti on a l L I P ID MA PS ® d a ta b a se s w a s u s ed t o hel p as si gni ng t he p eaks from t he Or bi S IM S analys is . Incl u din g [M+ N a ] + , [M+ K ] + , a n d [M +Cl ] - w e r e us ed to und er t a k e an un ta r ge te d an a l ysi s of t h e biom olecul a r d e p o sit cap t ur e d in t he cryo- OrbiSIM S s p ectra usi n g th e m olecul ar for m ul a pre di c ti on (MFP) a pproac h. 13 Th e p ea k se a r ch o f t he GC IB / O rb iS I M S s pe c t r a r e s u l ti ng in i d en ti f i ca ti on o f 492 2 se co n da r y ion p ea ks in th e n e ga t iv e po la r i ty a nd 1 458 pe a k s in the p o si t iv e po la r i ty Fi g ur e 3 (b ) . F r o m t h i s , w e w e r e a bl e t o p ut a t i ve l y ass ign 90 li pid mole cula r ion p e a k s , 17 of w hich are fat t y a c i d s p e aks. It i s li k el y t hat th ese f a t t y aci d p ea k s ma y arise a s f r a gm e n t i o n s f r o m l a r g e r l i p i d m o l e c u l a r i o n p e a k . I n a d d i t i o n , w e r e a b l e to a s si gn 2 1 4 m e t ab o l ite s mol e c ular i o ns on t h e s urfa c e of th e si lico ne ca th e t ers u s ing t he H M DB f i lt er. Co nsi ste n t w it h th e gre a ter area of biolo gical dep osi t o b s e r v e d in t h e ToF-SIM S i mage s, i t was obs e r ve d t hat th ere w e r e more me t ab olite s and lipi ds were d etect ed on t h e s urface of t he t u be at 2 8 days relati v e t o 1 d a y. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint Figure 3 (a) C o m p a r is o n o f c r y o G C I B / O r b i - S I M S s p e c t r a o f s i l ic o n e c a t h e t e r s u r f a c e a t 1 d a y a n d 2 8 d a y s (b) T ab le s u m ma r is in g th e to t a l nu m b er of p ea k s d et e c t ed ac r o s s t h e c at h et er s u r f a c e at 1 da y a n d 28 d a y s al on g wi t h t h e n u m b e r o f l i p i d a n d m et a bo l i t e u ni qu e pe ak s. Iden t i f yin g s ig ni f ic a nt assi gnm e nts usi ng a Log ist i c R egr e ssi o n M achin e Le arn ing a l g o rit hm T o d ete r mi ne if we c o u l d be tter extract d a ta fr om t h e O r bi SI MS da tas e t s of t h e w h o le bi o i nt erface wit h m a c hi n e l e a rn ing , we e m pl o ye d a l ine a r l ogi s t i c r e gr e ssi on a nd ot he r n on - lin e a r ma ch i n e le a r nin g m od el s , i n c lu di n g Ra ndom Fo r e s t , Su ppo r t Ve c t o r M a c h ine a nd E xt r e me G r a di en t Bo os t (X GB o os t ) o n th e a s si g n e d met abo li t e s ill u s trat e d i n Fi g u re 4 ( a) . The mo del ai m s t o pre d ic t t he d iffe re nce b e tw e e n two clas ses ( sa mples meas u r ed a ft er 1 day o r 2 8 da ys pos t i m p l a n tatio n). The machi ne le a rni n g m o d el was ap plied t o a ll p ut ati vely ass igned bi omo le c ular s pecies i n each c o ndi tio n and fro m bo t h p o s i t iv e a n d ne gati ve po l arit y co mbi ned. T h e de ta il s of t he m e t ho d olo gy and o utp u t of a ll t he m ac hin e lea r ni n g m odels t hat w e re ra n f or t hes e d at a s ets a re de ta il e d i n t he S u pple m e n ta r y I nf o r mati on ( Fi g ur e S 2- S 6 and Ta b l e S 3 -S 5 ) . O v e r a l l, th e re s ul t s t h rou ghou t a l l was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint mod els w ere c ons i s tent, w it h t he bes t re s ult s o bta i n e d b y L o g i stic Re g r e s s ion, u s in g 5 -fo l d c ross va l i da t io n a n d 2 0 % of t he d ata as a h ol do ut te st s e t . T he work flo w hi ghli ght e d i n F i g u r e 4 ( a ) sh ow e d t hat t he s up e rv ise d ma c hin e lea r ni n g m e t ho d usi ng l o g i s t ic r e gress io n. T h e log i s t ic r e gressi o n- b as e d mac hi n e lea r ni n g mo del hig hlig ht e d t ha t t here were 54 ass ign ed s p ecies t h at were cr i t i c a l i n di ffere n tiati ng t he i nterfa c ial res p o n s e b e t w e en ex p la n t e d c at h e t er s at 1 d a y a n d 2 8 da y s . T h e s t a t is t i c a l s i g n ifi c a n c e o f t h e s e m e t a b o l i t e s w a s t e s t e d u s ing a s i m p le unp ai r e d S tud en t’ s T - t e st a nd ta bul a te d i n Fi g u r e 4 ( b ) . T h i s a n a l y s i s r e v e a l ed t h at 7 9 .6 % o f t h e speci es i d e ntified b y t h e ma c hi ne learnin g mod el were st atis tica l l y si gnif ica n t , p-value < 0 . 0 5 . T h e t a b u l a t e d m e tab ol i te s in F ig u r e 4(b ) a l s o dis pl ayed mean c oe ff icien ts val ues , w i t h neg ativ e va l u es b e in g m ore ass oc i a te d w i t h m e t a b o l i t e s t h a t a r e p r e d i c t i v e o f 1 d a y r e s p o n s e w h i l e t h e m o r e p o s i t i v e c o e f fi c i e n t a r e a s s o c i a t e d w i t h 2 8 - da y res po n se. I ntere sti ngl y , t h e t op tw o p he not y p ic m e ta bolit e pre d ictors for 1- day post -im pl a n ta tion are th e gl y c e r op ho sp ho glycerol PG 39: 1; O and t he F A 18: 2 th a t s ug gest t h at t he se lip i d s a r e s tr ong ly as soc i a te d wit h the s urface of t h e cat he t er upo n in i tia l imp l a nt atio n or m ay be c o n ti n uall y pr o d uc ed b y t he ce l ls ass ociat ed wit h t he i m p l an t s urfa c e . T hese l ipi ds may f orm t he in itial lip id la yer on t h e s ur fa c e of t h e cat he te r , for e x a m pl e fro m loca l c e ll e x tracellula r v esic le s as pr op o s ed by S c re ib e t al. 21 . was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint Fi g ur e 4 . ( a) S c he mat ic of th e d ata an al ysi s work f lo w along l ogisti c r e g r e s sio n - b as ed m ac h i ne l e ar nin g t o id e ntif y me tabol it e s t ha t a r e important in d i ff e r en ti a t ing i mpl a nt ed c athe t ers at 1 d ay a n d 28 days. (b ) Ta b l e i nd i cati ng lo gi sti c r e g r e s sio n av e r a g e c oe ffi c ie n t s a n d statisti cal si gn i fi ca nce of H u ma n M e t ab o l om e Dat abas e fi lte r ed d at as e t and compu tat i o n al L i p i d MAP S fi lt e r e d data s e t that ar e im po rt a n t i n d i f f e r e n t i a t i n g t h e s u r f a c e c h e m i s t r y o f t h e c a t h e t e r s a t 1 d a y a n d 2 8 d a y s . N e g a t i v e m e a n c o e f f i c i e n t i n d i c a t e s m e t a b o l i t e s t h a t a r e m o re a s s oc i a te d w ith ca t he te r s ur f a ce a t 1 d a y w h ile p o s i t iv e me a n co e f f ic ie n t in di ca te s me ta b ol ite s th a t a re mo re a s so c ia te d w it h c at h et er s u r f a c e a t 28 d a y s . was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint Fold chang e and s ta t i s tica l s ign ific an c e In ad diti o n t o mac hi n e le ar ni ng, w e ha ve us ed a volca n o pl o t t o h elp i nt e rpre t th e c o mp le x Or bi-S I MS d at a s et f rom t he b i oi n t e rface. Thes e are us e ful in com parin g tw o sa m p le typ es wit h a l arge n u mber of d at a com pon ent s, replica te s a n d d y n a mi c ra n ge . We dis pl a y t he m e a n i o n i nte ns it y fro m n = 30 repl ic a t e ana l y ses o n a pl o t o f lo g 10 p- va l u e d et e r m i ned bet we e n t he tw o im pla nt t i m e poi n ts v ers u s t he fo ld cha nge o ve r t ime o n a log 2 s c a l e i n Fi g u r e 5 ( a ) . It w a s obs erv e d t ha t t he r e w e r e 3 6 meta b o li tes t hat are of s ta t is ti c all y si gnif icant hig her co n c entr ati o n ( p < 0 . 0 5, fo l d c h a n g e ≥ 2 ) at 1 da y . T h es e i n c l u d e m y o - in o s i t o l a n d s ev er al s u g a r i s o me r s . Fo r 28 days i m pl a n ta t i o n w e o bs erv e d 4 5 met abo lit e s t hat a re ex presse d a t a st atis ti cal ly s igni fican t h igh e r conce ntra tion t hat i ncl u de k now n ma r kers o f infla mmat i o n s uch a s FA 1 4 : 0, FA 1 6 : 0 an d ura t e 22 . A s i g n ifica n t i n c r e a s e i n u r a t e l e v e l s o n t h e s u r fa c e o f t h e c a t h e t e r s fr o m 1 d a y t o 2 8 d a y s w a s s e e n i n Fi g u re 5 (a ) -( b), su g g es ti n g a n i ncreas e i n infl ammat ion i n t h e tis s ues s urr ou n din g t he cat h e ter. Ura te is k now n e nd pro duc t for th e me tab olis m of purine s, t he ma i n co n sti t ue n t s of n ucle otid es 23 . D uring t h e F B R c a s cade, urat e pa r t i c ularly in it 's cr y s t a ll ine fo r m has be en re p o r ted to prom ote a p r o -i nfl amm a t ory re s p o n se t hrou g h t he inn at e im m u n e s y st em , l e a d in g to in fla mma t ion a nd pot e n t ia l ly ti ssue d a m ag e 24 . S u g a r s s u c h a s fr u c t o s e a n d g l u c o s e a s w e l l a s m e t a b o l i t e s t h a t p l a y k e y r e g u l a t o r y r o l e i n g l y c o l y s i s s u c h a s itaco n a te an d m es aco na t e 25,26 a r e fo u n d t o b e s i g n i fi c a n t l y h i g h e r o n t h e s u r fa c e o f t h e s i l i c o n e c a t h e t e r o n day 1 rela t ive t o 2 8 da y s i n Fi g ur e 5 ( a) - ( b ) . T h es e b i o m o lec ules were al so ide n ti fied b y t he l ogis tic regres si on - ba s ed m a c h i ne l ear nin g a l gorit h m t o b e predict iv e of p hen ot ypic res po ns e t o w ar d s s il ic one cat heter s urfaces at 1 d ay as s h o w n i n Fi gur e 4 ( b) . Th is o b s e rva tion is co nsi ste n t w it h t he k i n eti c s of an i nf lamma tor y re sp o ns e 27 . Duri ng th e earl y p ha s e on an inflammat o ry re s pon se t he d amag e t iss ue a n d i ts p erip h er y, in t his case t he surf ace of t he c a t het er, w ill ex p erie n c e a b urst of it aco n a te. Thi s mo l ecul e h a s be e n e xte nsi vely r eport e d t o p l a y a r o l e i n r eg u la t i n g a n d l i mi t i n g t h e l o c a l in fl a m m a t or y r e s p o n s e w h i c h i n t u r n p r e v e nt f u r t h e r d am ag e t o th e tis su e s hou ld th e re b e an y s ust aine d inflamma tion 28,29 . This eff ect is a c h ie ved by it aconat e t hro ugh t he activat io n of t h e anti- i n fla m m at ory tra n s cript ion f a c tor Nrf 2 i n macro pha ge s le a d ing to an i n c rea s e th e expres si on o f d ow n strea m ge nes i n vol v e d i n an a nt i-inf la mma t o r y res p ons e 30 . A s t h e i nfl a m m a t i on s u b s i d e s , th e le vel o f i taco nate al s o decreas e, a s s h ow n i n o ur anal y s is at 2 8 days in Fi g u r e 5 ( b ) , a s t he r e i s no n e ed fo r furt he r i m m u n e s up press io n. Th e su ga r m o le c u le s id e nt i fi ed in t he an a lys i s a r e li nke d to in f l a m m a to r y p ro ce s s es p a r t i cu l a r ly i n m a cr op h ag e s th r o ug h th e p e nt os e ph os ph a t e p ath wa y o r via gl y c o ly si s 31 . Viola et al and Li u et al have disc uss e d ho w macro pha ge s swi tc h t o g l y c olysi s a n d pen to se p hos p hat e pa t hwa y t o p rod uce energ y w h ic h he l p s a u gment c el l ula r pr o-i nfl a m m at or y res po nse s d uring a n i nf lamm a t ory res pons e 26,32 . In ad di tion , i t has al s o be e n s h o w n rece n tly h o w bo t h it aco nate an d me sa c o n a te exert anti- in fla m m ator y e f fe c ts i n pr o - inflam m a t ory m acr op hages as wel l a s pla y i n g a r ole i n i nh ibiti n g g l y c o l y sis 25 . A s ig nific a nt decrease in L- glut amin e o n th e s urfa c e of t he cat heter f rom 1 da y t o 28 days wa s se e n in Fi g ur e 5 ( b) , s u g g e s t i n g t h e uti l is ati o n of t he amin o a c ids via c a t a bolic pr ocesse s t ha t d r i v es infl ammat io n. L-glu ta m i n e w h i c h i s o ne of th e mos t ab un dan t a m ino aci ds in t h e h um an b od y, h as a ls o b een link ed to infl ammat ion via gl u t am i n ol y s i s 33 . An increa s e i n sa tu r ate d fatt y a c i d s d e p o s i tio n, F A 1 4 : 0 an d FA 1 6: 0 , w as hi ghlig ht ed at 2 8 d a y s relati ve t o 1 d a y. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint T h e s e fa t t y a c i d s h a v e b e e n s t u d i e d e x t e n s i v e l y a n d h a v e b e e n s h o w n t o b e l i n k e d t o a n i n c r e a s e i n inflam m a ti on 34,35 as w e l l as l i p i d-ind uce d c el l ula r a po pto sis 36,37 . FA 1 6 : 0, a l so know n a s palmit ic aci d, i s n o t i n h e r e n t l y a T L R a g o n i s t . H o w e v e r , u p o n e n t e r i n g c e l l s F A 1 6 : 0 c a n b e c o n v e r t e d i n t o p h o s p h o l i p i d s , dia c yl g l ycero l a n d cera mi d e s w hic h t hen l eads to acti vati on of v ari ous s i g n al l in g pa t h w ays th at are c o m m o n fo r L P S - m e d i a t e d T L R 4 a c t i v a t i o n , c u l m i n a t i n g i n a n i n fl a m m a t o r y r e s p o n s e . I n p a r t i c u l a r , m e t a b o l i c p r o d u c t s of pa l mitic a c i d affect th e a c tiv atio n of v ari ous p rot ei n k i na s es C , e ndo pl a s mic retic ul u m s tre ss a n d c ause a n increa s e i n R O S gen era t io n th a t pr omot es fu rther infla m m atio n 38 . I n a d d i t i o n , F A 1 4 : 0 w h i c h i s a l s o k n o w n a s my ri st i c acid h ave b een re porte d t o ex acerb ate a nd pro mote furth e r i nf lam m a ti on partic ula rl y i n th e pres en c e of FA 16: 0 39,40 . was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint Fi g ur e 5 . ( a) Vo l c a no pl o t of bio m o l ecula r sp e ci es d e t e ct e d on the su rfa c e o f c a th e t e r s a t 1 day a nd 2 8 d a ys. Highl i ght e d in th e p l o t a r e so m e of the s p e ci e s that dis play e d a two-fold d i ffer e n c e in s peci e s conc en tr ati o n w it h a statisti cal si gnifi ca nc e of p < 0. 0 5. F o r s p e c i e s p lo t te d , th e da ta a re e xp re s se d a s th e a ve r a ge o f a l l te ch n ic a l re p lica te s , n = 30 ( b ) Ba r ch ar t s o f se le c te d b io m o le c u l e s t h a t a re s t a t is tic a l ly s i gn if ic a n t a n d d i sp l a y e d s ign i f i c a n t c ha nge s in f o l d c h an ge ( > x 2) . D a t a are pl o t te d a s n o rm a lise d io n i n te n s it ie s t o t o ta l io n count. D a t a ar e di s pl ay ed as n = 3 0, m e an ± S D . C omp a ris o n o f dat a min i n g me th o ds was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint A co mparis o n of ut ili t y of t he m achin e l e arn i n g a n d volca n o plot a pproac he s i n i d ent i f yi ng t he s pe c ies ass ociat ed wi t h t he cat h e ter at 1 d ay a n d 28 days , s how s t ha t u p to 52 % of th e m et aboli te s i dent ified b y logis tic regres si o n - bas ed mac hine lear nin g overlap p e d wit h th e co n v e nt iona l un i v ariate a pproac h in Fig ur e S2 . T h e e ns embl e of b o t h met h odol o g i es, b y mach ine lea r n i n g and u niva r i a te st ati stic al anal y s is increa s e s t he confi dence i n t h e re s ults , as m ulti pl e p ersp ec tiv e s of im p ortanc e of t h e v a ria b le s are t ak e n i n t o co ns id e r a ti o n, b ot h l oo k i n g a t i ndi vidu al i m po rt ance b ut a ls o h ow v a ria b le s c a n be im p o r tant s yne r g is tic a ll y . Ea c h me t h o dolo g y i d e n tifi e d uni q u e s pe c ies , w ith t h e ma c hine learnin g hi ghli ght i ng a n a rrower sel ect io n of me t a bo l it es re la ti v e t o vo l c a n o pl ot a nal y s i s . T h e narro wer me tab olit e l i st is attri b ut ed t o Lea st Ab so lu te Sh ri n k a ge a nd S e le c tio n O perat o r (L ASSO) fea t u re s e lecti o n prior to ma c hine l e ar nin g. LASSO z e r os t he b eta coefficient s of t hos e v ariabl e s t ha t a r e n ot s i g ni f ic a n t t o t he o utco me a f ter w h ic h lo gis tic regre s s io n - ba se d ma c hin e le a r n i n g w a s performed on t he wi t h t h e 55 va ri ables to e n a b le th e m e t ab olite t o b e rank ed b a s ed o n th ei r c o ntr ib ut ion at 1 d ay a n d 28 day. Th is fea t ure remo va l obv ia t es t he co n t rib uti o n of me t a bolit es t ha t ha d ve r y li ttl e i n f l uenc e in t h e r an k i n g. S i nc e there are p oten tia l l y useful s pe c ie s ide ntified u ni quely b y ea c h appro ach, w e pro pos e t hat t h ey s h ou ld be u s ed i n co ncer t t o help de c o nv olute t h e co m p l ex Orb iSIMS d at aset . Mo l e cu la r de pt h pro fi l in g an d bio i n te rf a ci a l ch a r a c te ri s a t io n u si ng Or bi S I MS T o el uci date t h e i de n t it y of t his b i oi n t e rfaci al de pos it a s a f unct io n of de pt h, a GCIB/ Or biSIM S d e p t h profi l es were co n duct ed s h ow n for s elect ed i o ns in Fi g u r e 6 ( a ) . M e t a b olite s t h at s how ed t h e hi gh est si gnal i ntens i t y a t t h e s t a r t o f t h e p r o f i l e a n d d e c r e a s e s a s a fu n c t i o n o f d o s e o f p r i m a r y i o n b e a m a n d d e p t h a r e c a t e g o r i s e d a s s p e c ie s wh i ch we r e n e xt to th e ce l l s i n th e H &E se ct i on s be fo re r emo v a l fro m th e ti s sue ( Fi g ure 1 d ). Sp e ci e s th a t d i s pl a y h i g he r i n te nsi t y la t er in t he d epth p rofi le a r e categ orise d t o ori g i nate fr o m t he ma t e rial- hos t int e rface a t t h e impl ant s urface. N on-l ipi d m et a bo l it es s uch a m i n o acid s an d n ucleic a c id b a s e s were conce ntra ted at t h e at t he s urfa c e of t he d e p osit w h ich a re i n clos e c ont a c t wi t h t h e ho st tis su e i n t e rface. I n contra st, li p i ds s uc h diac y l glycero p hos ph o g l ycerol, PG 39:1 ;O an d ox id ise d gl y c os p h i n g o li p i ds, S He x C e r 4 1 : 5 ; O 5 a r e b u r i e d d e e p e r i n t h e d e p o s i t a n d a r e l o c a t e d m o r e c l o s e l y w i t h t h e s u r fa c e o f t h e i m p l a n t i t s e l f. T h e p utat i v e struc t u r e s of t h e s e m e t ab olite s iden tifi e d in t he d ept h profi l e a re sh ow n in Fi g u r e 6 ( c ) . Th e su r fa c e o f t he e xp l an te d tub e s, w h ic h w as n e xt to th e c e l lu l a r l a y e r se en i n t he h is to lo g y , wa s characteris ed by a n u mber of me t a bo l it es, t h y m in e , uracil, a deni ne, c yto si ne, g u a n i ne , L -p roli ne a n d i n os it ol cyclic p hos p hate, w hi c h w e a ss u m e a re rel a t ed to cells. So m e lipi ds w ere a l so fo u nd at t hi s i n terf ace, i nc lu de icosa tetraen oi c aci d a nd th e gl y cero p hos ph oi n o s i t ol PI ( 3 8 : 4). T he s e l i pi d s cla sse s h a ve b een s h o w n t o be i n v o l v e d i n a v a r i e t y o f b i o l o g i c a l p r o c e s s e s a s w e l l a s b e i n g a k e y m a r k e r fo r p l a s m a m e m b r a n e a n d end o pl as mic retic ul u m 41,42 . I t w a s se en in Fi g u re 4 ( b) t hat o x i d i se d d i acy l g l y c eroph os p hog ly c e r ol, P G 3 9: 1 ;O and fa tt y a c i d , FA 18: 2 s pecies were ra n ked b y lo g i stic re gre s s ion- ba se d ma c hin e l earni ng alg o r i t h m as biom olec u l es t hat a re mos t ly as s o c iate d w i t h t he s urface of t h e im plan ts a t 1 da y. I t c a n t h eref ore be p ost ulate d t ha t t hes e lipi d s pe c ies may f o r m an i ni ti al li pid dep osi t t h a t is cl ose ly a s soci at e d wit h t he s urface of t h e c a t h e t e r u p o n i m p l a n t a t i o n . T h e p r e s e n c e o f t h e s e i n i t i a l l i p i d s o n t h e s u r fa c e o f t h e c a t h e t e r m a y a r i s e from th e d e p osi tion of li pid fro m s u r rou n din g t i s s ue d u e t o i n i tia l i mpla n t ati o n proce d u r e. Ov e rt ime ot her li pi d speci es an d m e ta boli tes are dep osi te d o n t o t he li pi d l a ye rs res ult ing i n t he f ormati o n of a s tr a tifi ed l i p id was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint de pos it la y ers. O ve rall , t his w ork s h owe d t h at t he use o f c ry o-Or biSIMS a n d machi n e learni ng pro v i d e s us t h e to ols ne e d e d to unc over t he differe n t layers of lipi ds a nd me tab olit e s t hat m ake u p t h e bi oint erface wh ich reflects th e m ole c u lar his t o r y of t h e im plant. Figure 6 (a) D e p t h pr o f i l e a t s i l i co ne c a t he te r su r fa ce s v e r s u s pr im a r y i o ns d o se ( io ns /c m 2 ) and e sti mat ed de p th d i splayi n g li pi d s, ami no a ci d an d nuc l e i c ac id ba s e s m ol ecu l a r i ons a t 28 d ays. M ol ecu l e s pr e s e nt o n the s u rf ac e o f th e d e posit (cl os e to th e i n t e rfa c e) i s ca t egor is ed as i nt e rfa cia l w hil e th o s e p r es e n t d ee p i n t he depo s i t is c at eg o ris ed as d i sta nt fr om int erfac e (b) S c h e mat i c ill u str ati ng an d su m ma risi ng the pr e s enc e of biol o g i ca l d e p osit for m e d on the su rfac e o f m e d ic al de vi c e foll owin g imp lanta t i o n (c) C h e m i cal stru ctur e of speci e s d e t e ct e d by c ry o-O r b i S I M S . C omp a ris o n with S IM S lit e r atur e on i m pl a nts . I n t h i s s t u d y , i t w a s d i s c o v e r e d t h a t m o r e b i o m o l e c u l e s w e r e d e t e c t e d i n t h e n e ga t i v e p o l a r i t y , 2 4 7 met abo li t e s , rel a t iv e t o th e pos i t i ve p ola ri t y whic h onl y det ecte d 45 m e t ab olite s. T h is c or roborate s w ha t w as pre v i o usl y o bser v e d b y T oub o u l et al . a nd Angerer et al. w h o d e t e c t e d m o r e l i p i d s p e c i e s s u c h a s p ho sp hoi n o s i tols, fa tt y aci ds a n d tri gl yceri d e s in m urin e a nd h uma n t iss u e secti o ns i n neg ati v e pola r it y rel a ti v e to posi ti ve polari t y analy sis 43,44 . The c omplet e l ist of MF P filtere d a nd as si gne d peak s wit h re f ere nce t o was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint a s s i g n m e n t d a t a b a s e s a r e p r e s e n t e d i n Tab le S1- S2 . Th e h ig h e r s pe c t ra l an d m a ss re sol v i ng po w e r o f c r yo - OrbiS I MS an d t he GCI B primary beam h a s ena b l ed u s t o de tect a nd assi g n more bio mol ecula r s pe c i es on t he s u r fa c e o f im p l an ts f o l lo wi ng e x t ra c ti on r e l ati v e to th e s tudy co ndu ct ed by S ch r e ib et al 6 . Mac hi ne learni ng i n com bina tion wit h u niva r iate sta tis tical an a l y sis ha s ena b le d us t o n arrow d ow n t he c on t rib utio n of i m p ortan t of b i om o l ecul a r s pecies at t h e t wo ti m e p o i n t of i m pla nt a n alysi s. W e s how ed th a t s ug ar-ba s e d mol e c ules at 1 day were ke y feature s id e n tifi ed o n th e surf ace of th e i m plan t wh ile li p i d s pe c ies s u c h as fatt y acids a n d ura te were m ore im port ant feature s a t 28 da ys tha t i ndicat e t he p r e s e nce of i nf l a m mati on a t th e si te o f i mpla nt. P r e v iou s wo r k by S ch re ib et al. di s s ecte d an d ret ri e ved PD MS d iscs im plan te d in t he int rape r i t o ne a l sp ace o f C57 BL/ 6 m ice, k n o w n t o h ave a di ff eren t i m mu ne pr o fil e c ompared t o B ALB/c mice. The B AL B / c mous e str a i n , use d in t he c urrent w ork, d i s pl a ys a more T h 2 b ias ed res po ns e in co ntra st t o th e C5 7 b l /6 st ra i n, t h at is m ore s k e w e d t o w a r d s a T h 1 r e s p o n s e . 45 F o r t h e i r P D M S i m p l a n t , S c h r e i b et al. re porte d a fibr otic c ollag en c aps ule of 33 + 6 µm i n th i ck ne s s 28 day s a ft e r i m p la n t a ti on ( n=3 ) int o th e in tr a p e r i ton ea l sp a c e. I n th e sub c u ta ne o u s site fro m w h ich our imp lant s w e re retrieve d , the c olla g e n t h ic kn ess wa s fo u nd to be t hi nner, 16 µ m + 5 µm at 2 8 days i m plan tat ion in B AL B/c mice. Th ese di ff erence s in c olla g e n c aps ul e t hi c kn e ss meas ure d m a y be attri bute d t o t he d ifferent s ha pe a n d s iz e of i m pl a n ts u se d, d iffe r ent i m p lant ati on site as wel l as t he diff e rences i n fi brot ic resp o n s e ex press ed b y t h e C 57 BL/ 6 mou s e str a i n r e la ti v e t o t he BA L B / c mi c e use d i n t he current w or k. Th ese d iff ere n c e s ma y r e s ult i n t h e f orm a ti o n o f a di ff eren t FB R ca ps ul e wit h dist i n c t str u c ture al o n g w it h diff ere n t l e v e l o f cell i nfi l trati o n i n t o th e ca p s ul e r e la tive to our curre nt w o r k. Schre ib et al. rep o rt ed n o a d h e si on o f t he colla g e n ous cap s u l e t o t he e x pl a nt s, a n d a s parc e cell d i st rib uti o n o n the i mplan t surf ace a s v is ua l ise d b y mi c rosco p y . O ur Ma sso n 's trichr om e s t a i nin g a ll ow e d us t o i d e n tify t he pre s e nc e of a cellu l ar la yer at t he i n te rface be twe e n i m p la n t s ite an d t he fi b r ous ca p s ule. D ue to com plex i m m u n e a n d cellu l ar c a s cade t hat ta k e pl a c e betw e e n s ilico ne - bas e d impl ants a n d t h e h os t tis s ue , t hese c e ll u l ar la y ers sh ow n i n Fi g ur e 1( d) ma y co nsis t o f a v ar iet y o f cells ran g i n g fro m mon ocy tes , ma c ropha ge, T-cel ls a n d fibrob las t 46 . Schreib et al. ob ser v e d t h at th e cells w e re s urro un ded by pa tches of m olec u le s t hat c on t ai ned th e s am e seco nd a ry i on si gna t ures a s t h e pl a sm a me mbra ne . T h e y s ugg e s t ed t hat t his o bser v a tion may b e a ttri b u te d t o th e c e lls leav ing parts o f t h eir mem b ra ne o n th e s urface of t he d isc u p on co nt act a nd/or extr a c e ll ular vesic l e s secrete d fro m th e s e s p ars el y d is t ribu te d cell s . Wi t h cry o -O r biSIMS, w e ha v e b een a b le t o i den tif y i c o s a t e t r a e n o i c a c i d a n d g l y c e r o p h o s p h o i n o s i t o l P I ( 3 8 : 4 ) w h i c h a r e k e y m a r k e r fo r p l a s m a m e m b r a n e a n d e n dop l a sm i c r e t i c ul um 41,42 . The prese nce o f t his li pid ma y aris e from ho st c e ll laye r , as ill ustra ted i n Fi g u r e 6( b ) de p o s it ing p art of t he ir me mbra ne u p on co n tact w i t h t he i m pla nt. I t is w ort h not in g t he dep th prof ile o f th e i m pl a nt s urface a t 1 d ay s h owe d t ha t mos t o f t he n uc leic aci d b as es are locat e d de e p er i n t h e d epo siti o n awa y fr o m t he i nt e r fa c e la y er co mpared t o 28 da ys . The pre se n c e o f t he nucleic aci d may arise fro m t he dep osi ti on of cell u l ar resi d u e s uch as t hro ug h e x trace llular v es icle-a s soci a t ed DN A fro m the s urrou n d i n g cel ls comi ng i n cl ose co ntact w i t h t h e surface d uri n g t he ea rl y phas es of th e i mpla ntat io n 47 . A l so , th e in te r fa ce layer a t 1 da y c on sis te d mostl y of s u ga r is o m e rs a n d i ts res p ecti ve meta b ol i tes as s how n i n Fi g ur e S 8 an d Tabl e S6 . The enrich ment of s u gars a nd m e ta b ol i te s at t he i n t er fa c i a l la y e r s m a y b e a t tr i bu t ed to in cr e a se i n de m an d was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint for e ne r g y pa r t i c ularly by macro p h a ges an d ne ut rop hils a t th e im p l ant sit e d uri n g t h e ea rly i nflam m a tor y r e s pon se 48 . T h is e a r l y i n fla m m a t i o n i s a l s o a r e s u lt o f t h e d i s r u p t i o n o f c e l l s t r u c t u r e s d u e t o t h e i m p l a nt a t i o n proce dure of t h e cath e t er int o t he su bcu tane ous p oc k et. We h a ve hi ghli ght ed th a t t here is a n i nc re a se d i n ita c o n a te at t he s u r fa c e of the i m p lant at 1 d a y vs 28 d a y s . Th e dept h profile da ta fr om Fig u re S 8 an d Tabl e S 6 sh owe d t hat th e m olecul e is s pa tiall y m os tl y locat e d o n t he o ut ermos t la y er of t he depo si t, near t h e i nterfacial layer. T he pres ence of t his a nti-i nflam m a tor y m ol e c u l e a t t he outer mos t lay e r of t he de pos i t s m a y s erv e as a n to l i mit t h e infl amma tion at t h e s ite o f im plant an d m it i g at e f u rt h er dama ge t o t he peri phery tis s u e . Our GCI B/Orb iS I MS d e p t h prof il e a ls o s ho we d t hat t he o ut e r most l ayer d et ecte d on t h e s u r fa c e of t he impl ant i s e n r i c h e d i n l i p i d s m o s t l y o f w h i c h a r e fa t t y a c i d s , F A 1 6 : 0 , F A 1 8 : 0 , F A 1 8 : 1 a n d i c o s a t e t r a e n o i c a c i d a t 2 8 days. T his o bs ervati on i s c on sis te nt w it h Sc h r e ib et al . w ho r e po rte d t h e en r i c hm ent o f f at ty a c i d on th e surface of PD MS d i s k u p on i mpla nt a ti o n int o C 5 7 BL / 6 m ice a t 1 da y 6 u s i n g s t a t i c L M I G T o F - S I M S a n a l y s i s t o a n a ly se the s u r fa ce li pi d la y e r o n th e i r im pl an t . H o w ever, due t o li mit ed s pectral re s o l ut i on of ToF-S I MS, t h e y were u nable t o id entif y an y mo l ecul a r i on s fro m ot h er l i pi d an d n o n -li pid s pe c ie s o n t he surfa c e of t heir impla nt. In t his s t ud y , wit h th e a i d of t he O rbitra p an alyser w e als o a b le t o s how t hat t his int e rfacia l l a yer is n ot o nl y e nri c h ed wit h fatt y aci d s b u t al s o c o n ta in s nuc l eic aci ds bas es s uc h as g u a nine , a d enin e a n d c y t osi n e. T h e D N A b a s e a d e n i n e h a s b e e n w i d e l y u s e d a s a n u c l e a r c e l l m a r k e r fo r L M I G / T o F - S I M S a n d G C I B / O r b i - S I M S anal y s is 7,49 . Th e prese nce of uracil w hich is o nly pre sent o n t h e s urfa c e i n di c a t es t h e pr ese n c e of R NA w hile th e prese nce of t hymin e is an i n d icat or t hat D N A is als o pres ent o n t he surf ace of t he t u be s in Figur e 6( a) . I n addi ti on, d u e to in he r ent dep th pro fili ng cap a bi li t y o f t he Or bi -SI MS , w e a re als o able t o pro be th e pre se nce o f addi ti onal li pi d s pe c i e s t ha t m ay be prese nt be neat h th i s fat t y acid e nric he d s urfa c e. B eneat h this fa tt y a c id e n ri ch l a y e r , we a l so sh o we d th e p r es en ce o f o th e r l ip i d spe ci e s su ch a s d ia cy lgl y c e roph os phog ly c e r o l ( P G 3 9 : 1;O), ox idis ed glyc os p hing o l i pids ( SH exCer 4 1: 5 ;O 5) , g l ycerop ho sp h o i n osi to l s m on op h os p h at e ( P IP 39: 5) a s well a s unsa t urated fatt y a c i d s (FA 1 8:2). T his in de e d b u il d u pon t he lip id h y pot he sis pr o pos ed Sc hreib et al th a t lip ids de p o s it i o n on t he s urfa c e of b iom ate r i a l can p lay a c riti c al rol e i n b i om a teri al -i n du c ed f oreig n b o d y rea c tion a n d fibro sis. H o w ever, t hi s lip id d e p osit ion i s n ot a h omo g e n eous li pid la y er b ut i ns te a d c on sist o f layers of stra tified lip id s pe c i e s. T he a b i lit y t o id e n tif y t his s t ra t i fi e d li pid layer was o n l y a c h ie vab l e b y t he u s e of cryo GCI B / Orbi- S IM S an a l ysi s th at ena ble the pres e r vat i o n of t hi s lipi d laye r i n a n e a r native st at e durin g data a c q u i siti o n. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint

Cryo-Or biSI M S us ing an ar g o n GCIB to obtai n hi gh m a s s res olvi ng p o w er has b een u ti li se d to de p t h pr ofile th e biol o g ic a l dep os it f o rm e d o n t h e s urface of si lico ne ca th et e r im pla n t ed in mi c e at 1 day a n d 28 d a y s. S I MS - MF P and lo g i s ti c re g r e s sion m achi n e le a rni n g m o de l h a v e al l owe d t he ide ntifi c a ti o n a nd c haracterisa ti o n of t h i s com pl ex bio mo l ecu lar layer t h at c ons is t of bi omol e c ules of di ff e re nt c o m p osi ti on a t 1 an d 2 8 d a y s. T w o dis ti n c t li pidic l a ye rs , w it h t he o u te rm os t layer cont a i ni ng n ucle i c acid b a s e an d fa tt y a c i ds ha s als o b e e n iden tifi e d a t t h e interface be tw ee n i mpla nt ed si li c o n e c at h e ter a nd hos t t iss ue pr ovid i ng molecu la r i nsi ght i nt o th e fo re i g n b o dy rea c ti on a t 2 8 da y s . T his n u c l eic aci d rich lipi d i n t erface f orms th e brid g e t hat l i n k bet wee n th e b io m a teri a ls an d th e cel l ul a r la y ers t hat surro u nds t he bio materia l s . It w a s s how n t h at t he el e v a t ed levels of ura t e, F A 14: 0, FA 1 6: 0 c o upled w it h t he pres en c e o f i c o s atetra e n o ic acid at t h e i m plan t ti ss ue i n terfa c e pro v i d e i ndic a t or for inc re as ed inflamm a t ory re s p onse a t t he im plan tat i o n s ite. Suc h f in di n g w as c orro borate d b y t h e o bs e rv at i o n of a h i g hly i nfl a m ed s u bcut ane ous t iss u e a t 2 8 da y w hic h dis pl aye d t he pre s e nc e o f c o l l a g e n a s s h o w n via h is t olo gi c al a na l y s i s . Ov er all , t h ese fi n d i n g s s h ow t ha t cryo- GCI B/Orbi S IM S analy tical workflow pr o v i des us wit h t he n ecess a ry to ols to de c onv olu te a n d u n d ersta n d t h e com pl ex bi o i nt erf a c e formed o n s i lic on e im p l a nt duri n g a n F B R re sp o nse. S uc h m e t ho dol o gy could be tran slat abl e t o n ove l im mun e ins tructi v e s urfaces ena b l i ng u s t o gain in s i g h ts o n t h e p erfor manc e o f bio mat e rial s in vivo . I ndee d, t h e exp a n s ion of th is c oncep t may pr o v i de an i mp e t us t o pa ve t h e w ay f or adva n c eme n t s in dev e l op i n g no vel immu n e -i nstr uc tive m ate r ials wi th a u g m e n t ed func ti onalit y a n d b ioc om pati bi l it y . Ackn owl edg eme nts T h e Or biSIMS facilit y wa s fu n ded b y t he EPSR C S tra te g ic Eq uip me n t Grant ‘ 3 D Orb iS I M S:Label free c h e m ical im a gin g o f ma t e rials , c el ls a n d tis s ues’ EP / P0 2 986 8/ 1 an d t he w ork i s als o fun de d by th e E P SRC D esi gni n g b io - ins tructi v e m at e r i als for tr ansla tio n-read y medica l de v ic e s EP/X 0 01 1 56/1 2 0 23-2 02 7. Ian Gi lmore i s t ha nke d fo r h i s s u g g e s t i o n t o e x p l o r e t h e u s e o f a V o l c a n o P l o t f o r O r b i S I M S d a t a f o r t h e fi r s t t i m e . D r E l i s a b e t h I s o n i s th a n k e d for her co ntri b u t io n in pe rformi ng p ro f il ometry an alysis on t he cat h e ter s urf ace. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint

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M olecul a r o r i e nta tio n a nd st ra t ificati o n revealed in RN A-lip id nan o p articl es u si n g C r yoge nic O r b itr ap Se c o n d ary I o n Mass Sp e c trom e tr y ( Cry o-Or bi SI MS ). Preprint a t h t tps ://do i .or g/1 0 . 2 6 4 3 4/ch emrxi v - 2 025-z x kf1 ( 2 02 5). 53 . D a n ie l Le a et al . Bi omateri a ls -for-Me di c a l- D e v ic e s - AI / Helix . Prepri n t at h t tps ://git hu b.co m/Bio ma t e rials -for-M edical -Devices- AI/H eli x .

In vi vo stud y Ma l e Ba lb / c mice (a ged 5 6- 62 da ys, a ppro x . 25g) w e r e u s e d in t h is s tu d y . Mi c e w ere p urchas ed fro m a recog n is e d s up p l ier (Charles R iver Laborat or y, U K) a nd w e r e i nit i al ly be ho use d in gro up s of 5 (cag e s i z e 5 0 0 cm 2 wit h s aw dus t b e d din g an d siz zl e n e s t) a c cord i n g t o co nd itio ns sti p ul a te d b y t he H ome O ffi c e . T o acclima t ise t he a nimals t o th ei r s urr oun din gs , p r ior to e x pe ri menta ti on, ani mals w ere ho u sed for a perio d of at le as t 7 da y s w it hou t di st urb anc e , ot h er th an t o refresh th e ir b ed d i n g an d t o re pl e n is h th eir fo o d an d w a t e r p r ov is io ns . E a c h an i ma l wa s r ando m ly a s si gn e d a cod e wh i ch w il l d i ct a t e the im pl an t (or i m pl a n ts ) th at i t w i ll receiv e an d t h e form of p roc e s si ng t o be un d ert ake n at t h e ti m e of harv e s t ing. A nim a ls w il l be a naest h etiz e d usi n g in h a la ti o n of is o flura ne an d ai r, a n d th e dors u m o f ea c h mo us e w ill b e s ha ve d and w as he d ( 0. 05% aque ous Chl orh e x i d i ne Gluc ona te). The e x trem e s o f a 1. 0 c m l i ne, d ors o - ve n tr al i n ori e nt atio n, wil l b e “ dot- ma rk e d ” o nt o t he s haved s k i n of each fl an k u si ng a flexi bl e trans p are n t pl a stic te mpl ate an d a n ind el i bl e ma rk er pen. S e v e n ty perce nt alc oho l wil l b e a p plie d t o t h e s ki n i m m e diatel y pri or to inc isio n. A full- t hi c knes s i n c i s i o n a l w o u n d , 1. 0 c m i n l e ng t h , w as c r e at ed b y i nc i s i n g t h e s ki n b e t w e e n t h e s e do t - m a r ks . T h is in c is io n w i l l i n c lu de the p ann i cu lus ca r no su s an d hyp ode r m i s. Th e fr e e e d ge of th e sk in w il l the n b e l i fte d , and on e s ub - cuta neo us poc ke t w ill b e c re ate d fro m e ac h i ncisi on w it h t he aid of b lun t-n os e d sciss o r s. A si n gle p ie c e o f c a t h et e r wi t h an 12 m m e x t e rn a l d ia m e te r a nd a len gth o f 5 m m w as we t ted wit h Ph a rm a g r ad e s te r i le w a te r and w ere i nsert ed i nt o each poc k e t. Care w i ll be t aken no t t o t ouc h t he face o f i mp l a nt ma t eria l s . F ol l ow in g ins e r tio n, si tes w e re i ns pe c ted to e ns ure t hat s a m ples are l o c a t ed a p pr o pria t el y . Aft er sam ple i ns e r tion , t h e incisi ona l w ou nd s w ere c l o s ed u si ng non-re s orb a b le s u t ures. Aft er bi oma teri a l i mpla n t ati o n, ani m a ls w er e hou se d i ndi v i d uall y . A nim a ls were m ai nt a in ed a t a n a mbie nt temp erat u r e of 2 3 o C wi t h 1 2- hour lig ht/ dark cy c le s a nd were pro v i ded wi t h fo o d a nd wa te r a d libit u m. Fo l lo wi ng al l a n ae st he ti c e v e n t s, animals we re pl ace d in a warm en v ir o nmen t a nd were moni t ored u nt il t he y have full y re c o v ere d f r o m th e proce dure. A ll ani m al s were pr ovid ed w it h a p propriat e a n alges ia ( Vet ergesic , [b up re n orp h i n e ]) a fter s urg ery a n d were p ro vi ded ad dit iona l a nal ge s ics as re q ui r ed. The mi c e were admi ni s te r ed wit h p r o ph y l actic a nt ibi otics (i n t he form of Enrof l oxaci n [ Ba ytr i l], su bcu tan eo u s ( s.c)) o n th e d a y o f w oun d ing a nd sub se qu e nt ly o n ev e r y 7 th p o st - ope ra t ive d ay . A l l a n im a l p ro ce du r e s we r e c a r r i e d o u t in a Ho me O ffi c e licen se d es tab lis h m e nt u n der th e f ollow in g H o m e Off ice L i c e nc e s ( P C D: 5 0/2 50 5; PPL: P P 287 0359 ; P I L : I B C E F DF5 5 ; P I L : I 3 4 817 249 ) . T h e h e a l th st a tu s o f an i m a l s wa s mo n i to r ed wi th i n tw o hours o f impla ntat io n a nd s u bs eq uen tl y d a il y for th e first 7 d a ys a n d w eekly t h ere a ft e r. A nimals w ere m onit ore d dail y for th e first 7 d a ys t o e ns ure t hat s ut ures r ema i n i n pla c e ; s u tures will be r e m ove d on d ay 7 . An i m a l s wi l l th en b e m a in t a in e d fo r a fu r th e r 2 1 d ay s unt i l p o st - im pl a nt a t i on da y 2 8 . A t 1 day or 2 8 d a ys p os t-i mplant ati o n , a nimals will b e h u manely k ill ed b y a U K Home Off ice com pli a n t meth o d. Fo l lo wi ng c onfirm a ti o n of d e at h, e ach a n i m a l w ill be r a p id ly diss ecte d, a n d al l im p l a nt a ti o n si tes h a r vest e d. Excis ed impl a n t s wi ll be sn ap-fr ozen i n liqui d n i tro gen a n d sh i p p ed on dry -ic e (- 8 0°C). Imp lant ati on s i t es for his to l o g ical i nves ti ga t io n w e r e fixe d ( 1 0% neutral buffere d f ormali n, S i g ma, U K) an d proc e s se d t o p araffi n w a x . Excis ed tiss u e wi ll be s an dwich e d bet wee n t w o pi eces of f o a m, prior t o bei ng p la c ed i n fixati ve, t o red uce t he exte nt o f t is s ue c u r l i n g . Fi x e d s pecim e n s w er e tr i m med a nd dis secte d i n a cranio-ca ud a l di r e c t ion, g e ne r a t i ng tw o h a lf sit es (fi gure 3). T h e dors al hal f w e re proces se d a n d em b e d ded i n para ffi n wax ( t he re main i n g h a lf will be st ore d i n 7 0% E t O H). Sp e c i me ns w i l l b e o r i e n tat ed in suc h a fas hio n as t o e ns ure th at a ppr opriat e was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint tran svers e s ecti o ns of t he i m p lant ati on si tes were t ak en. 6µ m sect io n s w e r e ta k e n, proce ss ed an d s tain ed w it h H&E. I n a ddit ion, t he sect io ns were s tain ed u sin g Picrosiri us R ed Stai n (P olysc i e nces, Hirsc hberg, Germ an y) a n d Ma s s o n’ s Tri c hrome S ta i n (P o l yscie nces, Hir sc h b erg, Ge rm a n y ) ki t s. T h e i mage o f t h e s t a i ned s e c ti ons were acq ui re d usi ng Axi osca n 7 f r om Zeis. T h is w a s d o n e us in g t il e d s e q u e ntial a c quisi ti o n w i th flas h brig ht fie l d a t 2 0 x o bject ive. For s e c tion s t aine d us in g Picrosiri us R e d, the imag e was a l so acq u ire d usi n g p o l arise d l i g h t . Inflam e d area meas ureme nt w as d o n e b y t aking ma c rosc op ic image s of ex p l a n t ed s k i n ti ss ue w h ich were pin ne d d ow n t o flat te n o ut t h e s k i n. Ima g e J was u ti li se d t o an a l ys e t h e a r e a of in flamm a ti o n s urro u n d ing t he cathe te r s egme nts by t hres h oldi n g th e c o lo u r c hang e o bser v e d i n the s kin ti ss ue. Sample pr ep a rati o n and Cr y o-O rbi S I M S anal ysis T h e cat het e rs were s t ore d a t -80 O C u n t i l a n a l y s i s . P r i o r t o c r y o - O r b i S I M S a n a l y s i s , t h e s a m p l e w e r e t h a w e d a t r o o m t e m p e r a t u r e fo r 2 m i n u t e s . T h i s i s t o e n a b l e t h e s a m p l e t o b e s l i c e d i n h a l f a n d l a i d fl a t o n a c r y o - mani pula tio n st a ti o n. The s a m p le s were a d hered to t he cr yo-m ani p u lat i o n st ati o n u s in g a t h i n l a y er of OC T glue. U si n g a pa i r of forcep s th e s am pl es w ere then s na p fr e eze u s i ng l i q uid n itro gen. Be fore me a s urem ent by cryo-Orb iS I MS, s a m pl e s were pla c ed i n a cryo- m a ni pul a t io n st ati o n, Lei c a EM VC M (L e ic a , German y), fro m where t he y w e re tra n s fe r re d t o t he cry o-Or b i SIM S us in g a s hu ttl e c hamb e r L e ica EM VC T5 0 0 (L eica, Ge r ma ny). T h e cryo - O rbiSIM S i s e q u i p ped wit h a fully p r op orti ona l– i nt e g ral–deri vati ve (PID ) t emp e rat ure c ontro lle r , whic h con trols resis tive heati n g . T h i s set u p als o i ncor porat e s a direct liq ui d nitr oge n (L N2) c lose d l oo p circ u lati on c o o li n g s tag e t o ena ble t h e s ample t o be t h er moreg ulat ed und er cr y o g e n ic c o n di tio n w i t hi n th e load loc k an d mai n c hamb e r. T h e i n s tru m e nt i s i ns t a l led wit h a cr yo g e nic st orage ta nk w h e re LN2 was p umpe d for c irculati ng t he c ooli ng me diu m thr o ugh vacu um feed thr o u g h s t o a c ool ing f in ger be l ow t he sa mpl e, al l owi n g fa s t cool i n g t o − 180 ° C w it h a st abil it y of ± 1– 2 °C for at le a s t 7 d ays. Ma ss c a libratio n of t h e Q Exacti ve ins tr umen t was pe r fo r m e d o nce a d ay usi n g sil ver clust er io n s . Ele c tro ns w it h an e ner g y of 2 1 e V a n d a curren t o f −1 0 μ A a n d a rg o n g as flo odi n g w e r e use d f or char g e c om pens ati o n . For all Orbitra p data , mass spectra l infor ma t io n w a s col l ecte d fro m a m ass ra nge from 8 0 t o 120 0 Da. The O r bi t rap anal y z er was op e rat e d in pos iti ve - io n an d nega tive- i o n m o d e at t h e 2 4 0, 00 0 a t m / z 2 0 0 mas s-res ol uti on s e t t in g ( 51 2 ms tra nsie nt tim e ). Th e GCI B Orbi SI M S res ult ing i n co n s ump ti on of t he materia l on t he s urface of t he cat h e ter o ver 1 0 0 sc a ns I o n T o F S u r fa c e L a b 7 . 1 . 1 1 6 1 8 2 w e r e u s e d t o p r o c e s s t h e r e s u l t s a n d c r e a t e t h e p e a k l i s t s fo r e x p o r t . C h e m i c a l fi l t e r i n g v i a S I M S - m o l e c u l a r fo r m u l a p r e d i c t o r ( M F P ) w a s d o n e via cal c u la ti ng t he poss ible c he m ical formul a pe r mu tat ion b ased o n e l e ment al re s trictio n s from sec ond ary io n data, i n c ludi n g t h at o b t ai ned fro m a de pt h profile an alysi s. T h e e l eme nta l li m it ati on ap p lied for s e c o ndar y io n ass ig nme nt : C [ 4–1 5 0 ], H [2–2 5 0 ], N [ 0–3 0], O [ 0 – 2 0 ] , S [ 0 – 1 ] , P [ 0 - 2 ] , N a [ 0 – 1 ] , K [ 0 – 1 ] a n d C l [ 0 - 1 ] . T h e d o u b l e b o n d e q u i v a l e n c e w a s s e t b e t w e e n - 1 0 t o 5 0 . All p os si ble che mi c a l for m u las were w i t hi n t he m ass de v ia ti on of ± 5 p p m below m/z 9 5 an d ± 2 p pm a b o v e m/z 95 . All t he p re dict e d form ul a f iltere d b as ed o n t h e H uma n Met abo lo m e an d LIP I D M A PS® d a ta b a ses. Put a t iv e a ss ig nme nts w e r e ma d e usi n g H uma n M e t ab o l o me D ata ba s e (H M D B) fil ter b a s ed up o n s eco ndar y ions a c curat e m a s s es, mz . L i pi d pu t a t iv e a s si gn m e nt s we r e m a de u s i ng c om pu t at io na l ly g en e r at ed b ul k li pi d s data ba s e fro m L IP I DM AP S b ase d u p o n seco n d ar y ions a c curat e ma sses , mz . T h es e a s s i g n m e n t s a r e u s e d t o pro v i d e a s u b c l as s ass ign m e n t, b u t t he exact s truct ures a r e n ot q u ot e d si nce we d o n o t have MS d a ta. So m e o f th ese as si g nmen ts m a y al so r e s u l t fro m frag m e nt a t io n o f lar g er s peci e s . No n - ne g a t i ve ma t r ix f a c to ri s a t io n T h e I O N T O F S u r fa c e L a b ® s o ft w a r e w a s u s e d t o e x p o r t s t h e d a t a i n t o a B I F 6 fi l e f o r m a t . F o r e a c h d a t a s e t , Su rface La b 7. 2 (I ON T OF GmbH) w as use d t o perf o r m a n a u t o m ated p e a k s earch on t he t otal s p e c tra r e s trict e d o nly t o p e a k s wit h int ens it y ab o v e 1 0 0 a. u. P eak i ntens iti es w e re th e n e x porte d for e ac h obs erv a ti o n. N o n - nega tive matrix fact orisa tion (NM F) w as p e rformed us in g t h e si m s MVA s oft ware. P r ior t o N MF , d at a were Pois s on s cal e d t o accou nt for n on- u nifo r m n oise a c ros s t h e ma s s s pectra. NM F wit h 3 f act o r s was ach ieve d usi n g a Poiss o n- ba s ed multi plicati ve u p da t e ru le al gorit h m. The nu mber o f fa c t ors was c hos en b ase d o n pri n c ip a l co mp one nt anal ysis o f a co mpres s e d ma trix of t he ori g i na l da ta set. A to tal o f 5 0 0 ite r ati o ns w as con d u c te d per d atas e t . was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint Depth Pr o fi l e A na l ysis Estim ation Inf o rma ti o n fr om p ubli sh ed de pt h profili ng of or ganic m ol ecular st and ard (Ir gan ox 1 01 0) 50 w a s u s e d t o esti mate t he de p t h of GCI B dept h profili ng . T h is w a s b as e d on inf orm a t io n from primary i on dos e of t he GCI B, whic h is dis pl a y ed as a pri m ar y X-a x i s i n F i g u r e 6 ( a ) an d t he d e p th is pre se n te d as a s econ dary x axis. Th e sp ut ter y i eld fro m 2 0 k e V Ar 3000 + on Irga nox 1 0 10 w as adj us te d for t h e cry o g e n ic c o ndit ion (-1 70° C) us ed i n t his s t udy . Th i s wa s don e by co m pa ri n g t o th e l ite r a t u re v a lu e s u s ing th e c a l cu l a t ion s d ev el op e d b y S e a h et al . 51 a s u s ed p r e v i ou sly by Ko t o w s k a et al 52 . Ma c h in e Le a rn i ng of C ryo - O r b i S IMS da t a Ma c hin e l e arn ing w as e x plore d to aut om a t e t he d i s co v er y of dep osi te d b i o molecu la r spe c ies a n d t o be t t e r u nderst an d t h e dep osi ti on wit hi n 1 d ay a n d 28 d a y s of im plan tat i o n. T w o d at a s ets o f bi omo le c ules dep osi tio n from t h e ne ga t i ve a nd pos i t i ve i o n s pect r a were anal y se d . T h eir biom o l ecul a r s pe cies int ens iti es w ere iden tifi e d usi n g s e c o ndar y io n mas s sp ectro metr y c oupl e d t o an Orbi trap m ass an alyse r. T he s am pl e s w ere sc a le d t o i ntens it y va l u e s be t w een 0 a n d 1 to av oi d num e r i c a l i nst a bil i t y i n t he m ach ine l ear nin g trai n i n g proces s, w here 0 re pres ents t he l ow est i nt e n sit y i n t he da tas et a n d 1 re prese nts th e hi ghe st i n t en sit y. Ma c hin e lear nin g ap pro a c h e s ( R and o m F ores t, S V M, X G B o ost ) a nd lo gistic r egre ssi o n w ere trai n e d us in g H e lix 53 . Logi s tic reg r es si on pr oduc ed o veral l t he b est res ult s. It is a s t a t is tical met ho d for p r ed ictin g t he proba bilit y o f a b i n a r y o u t c o m e , s u c h a s t h e p r e s e n c e o f 1 d a y a n d 2 8 d a y s b i o m o l e c u l a r s p e c i e s , b a s e d o n p r e d i c t o r va ri ables, s uch as th e int e nsi tie s o f b io molec ul ar s pecie s. It emp loys a mult ivaria te l in e a r reg r e s s ion fu ncti o n to le a r n t he relati on s hip betw een t he o ut come a n d predict ors , fol l o w ed by a lo gis t ic f unc tio n ( S i g m oi d f u ncti on) t o ma p t h e learn ed rel a ti ons hi p t o a pro babi l it y ra n g e o f 0 t o 1 . This met ho d is va l u a b le for int e rpre ti n g t he i mpact of pre di c t or variabl es, i n d icate d b y reg re ss io n coeff icie n ts , es p ecial l y i n c a s e s wh ere th e ou tc o m e is bin ary. A d ditio na l d et ai ls o n th e machi ne lea r ni n g m et h odo logi e s an d o u t put are det ailed in th e s up pleme ntary i nformati o n. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.09.652996doi: bioRxiv preprint