Biological properties of surface modifed 316 LVM steel

Vol.:(0123456789) 1 3 A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 h ps://doi.o g/10.1007/s43452-023-00776-7 ORIGINAL ARTICLE Biological p ope ies o su ace modi ied 316 LVM s eel Ane aDyne 1· RomanMajo 2· ŁukaszMajo 2· JanuszSzewczenko3· K zysz o Lukaszkowicz4· Ka laČechBa abaszo á5· MaciejK zywiecki6· Ma cinBasiaga3 Recei ed: 14 Ap il 2023 / Re ised: 14 July 2023 / Accep ed: 18 Augus 2023 / Published online: 20 Sep embe 2023 © The Au ho (s) 2023 Abs ac This pape aimed o in es iga e he selec ed physicochemical and biological p ope ies o i anium dioxide hin ilms depos- i ed by a omic laye deposi ion on 316LVM s ainless s eel dedica ed o ca dio ascula implan s. The main challenge in su ace modi ica ion o hese implan s is he complexi y o he p ocesses aking place in he ci cula o y sys em. The a omic laye deposi ion was ca ied ou o a numbe o cycles 500 and empe a u e 200°C o 316LVM s ainless s eel subs a e. The su ace opog aphy and su ace mic os uc u e we e examined. Mouse ib oblas s L929 and Human De mal Fib oblas s (NHDF-Ad) we e used o cy o oxici y assays. The ollowing biocompa ibili y aspec s we e in es iga ed in i o: di ec cy o oxici y, hemolysis, pla ele ac i a ion and agg ega ion, and p o-in lamma o y cy okine le els. The i anium dioxide hin ilms inhe i ed he subs a e opog aphy. The su ace mic os uc u e was amo phous wi h he ypical laye by laye g ow h. The ilm imp o ed he in i o cell esponse in e ms o cell iabili y. The cells we e also able o p oli e a e and adhe e; howe e , di e ences in he cell mo phology and he dis ibu ion o cell nuclei we e obse ed. The hos cell damage was no no ed in e ms o lac a e dehyd ogenase le els. The p oposed su ace modi ica ion educed he hemolysis index and did no signi ican ly a ec pla ele ac i a ion and agg ega ion. Acu e cy o oxici y o he hin ilms is no p edic ed basing on he in i o p o-in lamma o y cy okine assay. The esul s o he biological es s may be basis o u he biological assessmen p o ing he ull biocompa ibili y o he p oposed su ace modi ica ion dedica ed o speci ic ca dio ascula implan s. Keywo ds A omic laye deposi ion· Modi ica ion o 316LVM· Ti anium dioxide· Biological e alua ion 1 In oduc ion Fo blood con ac ing de ices, a ious in e ac ions o he bioma e ials wi h blood and su ounding issues should be conside ed. Ci cula ing blood con ac ing de ices (s en s, hea al es, a i icial hea s, and en icula -assis de ices) should be subjec o h ombosis and hema ology assays. Depending on he ype o implan , in e ac ions wi h a i- ous ypes o cells should be in es iga ed. Pla ele esponse (pla ele ac i a ion and agg ega ion) is a help ul ool o de e mining non- h ombogenic p ope ies o he implan su ace. Hemolysis induced by ci cula ing blood con ac - ing de ice may be caused by mechanical and biomechanical ac o s and induce oxic e ec s o p omo e h ombosis [1]. The assessmen o cy o oxici y is a basic assay o all issue con ac ing medical de ices and may include a ious aspec s o he cellula esponse depending on he ype o con ac expec ed. Cy o oxici y can be de e mined by e alu- a ion o cell mo phology, cell damage, cell g ow h, o cel- lula ac i i y. S ainless s eel 316LVM is commonly used in * Ma cin Basiaga [email p o ec ed] 1 Manu ac u e o Medical Ins umen s CHIRMED Ma cin Dyne , Rudniki, Poland 2 Ins i u e o Me allu gy andMa e ials Science o Polish Academy o Sciences, Reymon a 25, 30-059K aków, Poland 3 Facul y o Biomedical Enginee ing, Depa men o Bioma e ials andMedical De ices Enginee ing, Silesian Uni e si y o Technology, Roose el a 40, 41-800Zab ze, Poland 4 Facul y o Mechanical Enginee ing, Depa men o Enginee ing Ma e ials andBioma e ials, Silesian Uni e si y o Technology, Kona skiego 18A S ., 44-100Gliwice, Poland 5 Nano echnology Cen e, CEET, VŠB-Technical Uni e si y o Os a a, Os a a, CzechRepublic 6 Ins i u e o Physics—CSE, Silesian Uni e si y o Technology, Kona skiego 22B S ., 44-100Gliwice, Poland A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 1 3 237 Page 2 o 14 biomedical applica ions as co ona y and pulmona y s en s. As a e y needs mechanical suppo o i s healing wi hou isk o ecoil, s ainless s eel is conside ed o such appli- ca ions. Howe e , compa ed o o he me allic bioma e ials (e.g., Ti-based alloys), co osion esis ance o biomedical- g ade o s ainless s eel 316LVM is signi ican ly low. Passi e laye does no p o ec he s eel su icien ly when subjec ed o ex emely co osi e en i onmen s. The e o e, p o iding coa ings o co osion p o ec ion which does no change he mechanical p ope ies o he ma e ial a e subjec o cu en esea ch, as co osion o a me allic ma e ial is one o he mos impo an issues o be conside ed when i is designed o implan s. Taking in o accoun he abo e equi emen s, low- empe - a u e su ace modi ica ion echniques wi h abili y o co e geome ically complex su aces (minia u ized implan s) on all sides should be conside ed. The ALD echnology, which is a a ia ion o he CVD me hod, allows o he p oduc ion o ul a- hin, homogeneous and epea able coa ings wi h a wide ange o po en ial biological applica ions. In ALD, al e na ing pulses o p ecu so and eac an gases a e sepa- a ed by pu ging s eps wi h an ine ca ie gas (N2 o A ) o achie e sel -limi ing su ace eac ions. The ALD eac- ion ends when all a ailable si es on he eac i e su ace a e occupied. This ea u e is e e ed o as sel -limi ing, which enables ALD o deposi uni o m laye s o high con o ma ion and p ecisely con olled hickness on all ypes o subs a es [2]. Nume ous es s o coa ings using he ALD me hod show an imp o emen in he co osion esis ance o he subs a e depending on chemical composi ion (SiO2, Al2O3, SiO2/ TiO2, ZnO, and Z O2–Al2O3) o he laye s and ALD p ocess pa ame e s [3–7]. Some single aspec s o biological p op- e ies we e also s udied, indica ing he in luence o ALD p ocess pa ame e s on a ious aspec s o biocompa ibili y [8–18]. The hi d-gene a ion s en s cu en ly used in clinical p ac ice a e he esul o many yea s o esea ch aimed a minimizing he isk o es enosis and h ombosis. While he p oblem o es enosis has been e ec i ely sol ed by he use o mode n an i-p oli e a i e d ugs and ma ices o biodeg adable d ug- eleasing polyme s, he p oblem o la e h ombosis has no been ully sol ed. The use o dual an i- pla ele he apy is s ill he s anda d a e s en ing, which in some cases inc eases he isk o bleeding. The op imal s en su ace should p e en he adso p ion o ib inogen, p e en he adhesion, agg ega ion, and ac i a ion o pla ele s, inhibi he adhesion and p oli e a ion o smoo h muscle cells, and p omo e he adhesion and g ow h o endo helial cells. Va i- ous su ace modi ica ion echniques based on deposi ion o i anium dioxide, has been widely used o biomedical appli- ca ions, showing a posi i e in luence on a ious aspec s o biocompa ibili y. The p esence o hyd oxyl g oups on he su ace p omo es osseoin eg a ion in bone implan s. The same p ope y can be also used in su ace modi ica ion o s en s, consis ing o su ace immobiliza ion leading o gene a ion o ni ic oxide o p omo e eendo helializa ion [18]. The ongoing in ensi e esea ch in o he c ea ion o biocompa ible implan su aces in con ac wi h he ci cula - ing blood indica es he lack o sa is ac o y solu ions in his ield, mainly due o he complexi y o he physiological p o- cesses occu ing in he ca dio ascula sys em. In his case, he biological es ing p og am should co e many aspec s o biocompa ibili y. The combina ion o biological p ope ies wi h he app op ia e physicochemical p ope ies o implan s is also an impo an ac o in luencing he selec ion o app o- p ia e p ocess pa ame e s o implan su ace modi ica ion. The aim o his s udy is o in es iga e he biological p op- e ies o i anium dioxide hin ilms deposi ed by a omic laye deposi ion on 316 LVM s ainless s eel dedica ed o ci cula ing blood con ac ing medical de ices. The p ecu so s used in he ALD p ocess (TiCl4/H2O) a e he mally s able up o high empe a u es, bu he co osi e byp oduc HCl may be ha m ul o he deposi ion ins umen s; he e o e, deposi- ion empe a u e 200°C was p oposed o he ALD p ocess. The mos impo an aspec s o he biocompa ibili y we e s udied, including: di ec cy o oxici y, pla ele ac i a ion and agg ega ion, hemolysis, and p edic ion o acu e oxici y. 2 Ma e ials andme hods 2.1 Ma e ials The es specimens a e desc ibed in Table1. 5 pcs. o each specimen we e used o es ing. A e age alues a e p esen ed in he es esul s. The es sample o 316LVM was subjec o p elimina y su ace inishing ypical o s en ab ica ion and deposi ion o TiO2 by he means o ALD. Table 1 Specimen desc ip ion Specimen code Specimen desc ip ion 316LVM S ainless s eel 316LVM, annealed, elec opolished (H3PO4/H2SO4), passi a ed (40% HNO3), au ocla ed 316LVM-TiO2S ainless s eel 316LVM, annealed, elec opolished (H3PO4/H2SO4), passi a ed (40% HNO3) ALD-TiO2- coa ed (500 cycles, 200°C), au ocla ed, pu ging gas ni ogen A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 1 3 Page 3 o 14 237 The es ma e ial was 316LVM s ainless s eel in he annealed s a e in he o m o discs wi h diame e d = 14mm and hickness g = 3mm. Modi ica ion o he su ace o he analyzed bioma e ial was ca ied ou using such p ocedu es as: elec ochemical polishing, passi a ion, applica ion o a TiO2 coa ing using he low- empe a u e A omic Laye Depo- si ion (ALD) me hod, as well as medical s e iliza ion. To ensu e he equi ed physical and chemical p ope ies o he 316LVM s eel su ace, condi ions o i s su ace ea men ha e been de eloped. The i s su ace ea men was elec- ochemical polishing, which was ca ied ou in a ba h based on a mix u e o o hophospho ic acid and sul u ic acid (VI). This p ocess was ca ied ou un il he su ace oughness o Ra < 0.16µm, ecommended o blood con ac ing implan s, was ob ained. Then, he samples we e chemically passi a ed in 40% HNO3 acid. These a e he basic s ages o shaping he su ace o me al bioma e ials used o implan s wi h minia- u ized geome ic ea u es. Then, a laye o TiO2 was depos- i ed on he p epa ed samples using he ALD me hod. TiCl4 (Ti anium (IV) chlo ide) + H2O was used as a p ecu so . The pa ame e s o he deposi ion a e desc ibed in Table2. The las s age o he p oposed su ace ea men included medical s e iliza ion, which was ca ied ou using he s eam me hod in an au ocla e a empe a u e T = 135°C and p es- su e p = 2.1ba . 2.2 Mic os uc u e Cha ac e iza ion o he mic os uc u e o he su ace laye was pe o med using ansmission elec on mic oscopy (TEM). The mic os uc u e was cha ac e ized by he TEM echnique in he so-called b igh - ield obse a ion BF TEM, scanning ansmission elec on mic oscopy (STEM), and high- esolu ion TEM (HRTEM). Quali a i e chemical analysis was pe o med by X- ay spec oscopy wi h ene gy dispe sion (EDS). Comp ehensi e TEM analysis was pe - o med using TECNAI G2 F20 (200kV) FEG (FEI) and THEMIS (200kV) FEG (The mo Fishe Company) mic o- scopes. Thin ilms o TEM obse a ion we e p epa ed by he ocused gallium ion beam (FIB) echnique on an SCIOS II DualBeam (The moFishe Company) equipped wi h an EasyLi insi u mic omanipula o . 2.3 Su ace opog aphy Su ace opog aphy es ing was pe o med using an op i- cal p o ilome e 3D Su ace Me ology Mic oscope Leica DCM8. Su ace scans we e p ocessed in Leica Map so wa e, esul ing in a e age Sa alues and 3D su ace isualiza ions. 2.4 Cell iabili y assay The cell iabili y assay was pe o med by he di ec me hod acco ding o he PN-EN ISO 10993-5:2009 s anda d [19] on ib oblas s (L929 ATCC). The supplemen ed P omoCell Fib oblas G ow h Medium 2 en iched wi h an ibio ics (SIGMA ALDRICH An ibio ic An imico ic Solu ion) was used as a medium. Cell iabili y was assessed a e a 24-h incuba ion using luo escence mic oscopy. FDA ( luo escein diace a e) and PI (p opidium iodide) we e used in cell s ain- ing. The esea ch was ca ied ou using a Ca l Zeiss Exci e 5 scanning lase con ocal mic oscope. Image analysis and cell coun ing we e pe o med using ZEN 2008 so wa e and AxioVision 4 Module Au oMeasu e. The cy o oxici y s udies we e complemen ed by he di ec me hod o es ing he le el o lac a e dehyd ogenase (LDH), a subs ance eleased om damaged cells. Human ib oblas cells (L929 ATCC) we e used o he s udy, and Fib oblas G ow h Medium 2 (P omoCell) en iched wi h supplemen s and an ibio ics was used as he cul u e medium. A e incu- ba ion, he medium was aken om he abo e samples and cen i uged, and hen, he supe na an was aken om he cen i uged solu ion o he analysis o LDH le el using he colo ime ic Roche Cy o oxici y Assay Ki . 2.5 S udies o cell g ow h, p oli e a ion, andadhesion The p oli e a ion s udy was pe o med on Human De mal Fib oblas s in he adul skin e sion (NHDF-Ad). The s udy quali a i ely assessed cell mo phology, g ow h, adhesion, and di ec ion o elonga ion p ocesses based on he appea - ance o he cy oskele on, as well as he appea ance and dis ibu ion o cell nuclei and hei abili y o p oli e a e. Table 2 Technological condi ions o he ALD me hod TiCl4Ca ie gas low a e (N2/A ) [sccm] 200 P ecu so in oduc ion ime [s] 0,1 Chambe pu ging ime [s] 4,0 H2O Szybkość p zepływu gazu nośnego (N2) [sccm] 200 Reagen in oduc ion ime [s] 0,1 Chambe pu ging ime [s] 5,0 Chambe empe a u e [°C] 200 Numbe o cycles 500 A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 1 3 237 Page 4 o 14 Fig. 1 TEM BF esul s o he ALD o TiO2 coa ing Fig. 2 STEM + EDS esul s o he ALD o TiO2 coa ing A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 1 3 Page 5 o 14 237 The cul u e was ca ied ou in a dedica ed supplemen ed medium in an incuba o a 37°C, 99% humidi y and 5% CO2. Cells we e ixed wi h o maldehyde, pe meabilized wi h T i onX-100 and blocked in 0.1% bo ine se um albu- min (BSA) solu ion in T is-bu e ed saline (TBS). To assess cell p oli e a ion, In i ogen™ DAPI D1306 by The mo Fig. 3 STEM + EDS esul s o he ALD o TiO2 coa ing Fig. 4 HRTEM esul s o he ALD o TiO2 coa ing Fig. 5 Example mo phology o he su ace o uncoa ed 316LVM samples, op ical p o- ilome y, × 50 A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 1 3 237 Page 6 o 14 Fishe was used o s ain cell nuclei, allowing o nuclea and ch omosomal coun e s aining. DAPI emi s blue luo escence when bound o AT egions o DNA. Alexa Fluo 488 om The mo Fishe (a abbi IgG seconda y an ibody) wi h g een luo escence was used o F-ac in ( ib illa ac in) s aining. Ac in is an elemen o he cy oskele on in ol ed in cell di i- sion, main enance, and changes in he shape and mo emen o he cell. Fluo escen ly labeled ac in is an impo an ool o s udying he s uc u al dynamics o he cy oskele on in he li ing and ixed cells. 2.6 E alua ion o  hee ec o bioma e ial on ed blood cells The aim o he in i o s udies was o e alua e he e ec o ex ac s con ac ed wi h ma e ials on p ese ed whole blood componen s. The es s we e ca ied ou in acco dance wi h he PN-EN ISO 10993-4:2018-02 [20] and ASTM F 756–00 [21] s anda ds. The es s we e ca ied ou on human whole blood (KPK) ob ained om he Regional Cen e o Blood Dona ion and Hemo he apy in Ka owice. The blood was an icoagula ed wi h CPDA p ese a i e luid. The esea ch included h ee s udy g oups: • Blank es : CCP ees anding a oom empe a u e, no subjec ed o he condi ions o he expe imen , • Nega i e con ol: CCP wi hou con ac wi h he es ma e ial, subjec ed o expe imen al condi ions (slow mixing). • S udy g oup: ma e ials con ac ed wi h CCP in expe i- men al condi ions. The KPK was subjec ed o a 3-h con ac wi h he es ed ma e ials and ex ac s a a empe a u e o 37°C, in a slow- mixing sys em. The impac o he es ed ma e ials on ed blood cell pa ame e s was de e mined by assessing he deg ee o hemolysis, de e mined by calcula ing he hemoly- sis index o he es ed ma e ials. Fig. 6 Example mo phology o he su ace o samples wi h TiO2 laye , op ical p o ilom- e y, × 50 Table 3 A e age alues o su ace a i hme ic mean high and ough- ness Modi ica ion o su ace Sa, nm Ra, nm 316LVM 11.63 9.34 316LVM-TiO211.52 9.89 Fig. 7 Con ocal mic oscopy o uncoa ed 316LVM samples—all channels A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 1 3 Page 7 o 14 237 2.7 S udies o  h ombogenic p ope ies Me hods de eloped by Sanak e al. [22] we e used o he s udy. P-selec in ac i a ing ma ke s and he ib inogen an i- gen-binding si e in he GP IIb–IIIa assembly (PAC-1) we e used as indica o s o pla ele ac i a ion and CD 61 ma ke was used o agg ega ion assessmen . Posi i e and nega i e con ols we e p epa ed o analyze he amoun o agg ega es. Nega i e con ol was ob ained om human blood d awn on sodium ci a e. A posi i e con ol was p epa ed by mixing blood wi h adenosine di-phospha e (ADP). 3 Resul s 3.1 Mic os uc u e The esul s o he conduc ed mic os uc u e es s a e p e- sen ed in Figs.1, 2, 3, 4. S udies using di ac ion con as showed on he c oss-sec ion he g ow h o a omic laye by a omic laye , co ec o he ALD echnique, a a dis ance o abou 5nm om he subs a e (Fig.4). Fu he on, he s uc- u e was amo phous. In addi ion, undesi able blis e s we e also no iced, which may indica e a poo e quali y o adhe- sion o he coa ing o he subs a e (Fig.1). The amo phous na u e o he coa ing was also con i med by scanning ans- mission elec on mic oscopy (STEM) and high- esolu ion HRTEM images (Fig.3 and 4). The esul s o STEM + EDS Fig. 8 Con ocal mic oscopy o uncoa ed 316LVM samples—sepa a ed channels Fig. 9 Con ocal mic oscopy o TiO2-coa ed samples—all channels Fig. 10 Con ocal mic oscopy o TiO2-coa ed samples—sepa a ed channels; ed—dead cells, g een—ali e cells, and g een + ed—all channels A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 1 3 237 Page 8 o 14 con i med he p esence o Ti a oms in he coa ing, which was also homogenously dis ibu ed along he hin ilms (Fig.2). The absence o subs a e o igina ed a oms in he coa ing was con i med. 3.2 Su ace opog aphy Example su ace opog aphies o he subs a e and TiO2-coa ed specimens a e p esen ed in Figs.5 and 6. Sa (a i hme ic mean heigh ) and Ra (a i hme ic a e age alue o il e ed oughness) alues a e p esen ed in Table3. Simila geome ical ea u es o he su ace a e clea ly isible, which indica es he endency o he laye o inhe i he subs a e su ace opog aphy. 3.3 Cell iabili y assay The esul s o he conduc ed cell su i al s udies a e shown in Figs.7, 8, 9, 10, 11. Analyzing he con ocal mic oscopy pho o, weak g ow h on he 316LVM s eel subs a e was obse ed. The cells had a cha ac e is ic ound shape, which indica es he poo iabili y o cells wi h ac i e mi ochond ia and a la ge numbe o nec o ic cells. In u n, su ace modi ica ion by applying a TiO2 coa ing imp o ed he biological p ope ies. A dec ease in cy o oxici y was obse ed. The cells on he su ace o he coa ing ha e an elonga ed shape and a educed numbe o nec o ic cells was ound. Fib oblas cells belong o he g oup o adhe en cells. In he case o he co ec , om he poin o iew o subs a e biology, memb ane pola i- za ion occu s and he ac in cy oskele on is ac i a ed. The cells elease he so-called phyllo- and lamellipodia, which is a posi i e phenomenon. Based on he s udies, su i al a es o 95% and 100% we e ound o he 316LVM and 316LVM wi h TiO2 samples, espec i ely. Acco ding o ISO 10993, educ ion o cell iabili y by mo e han 30% is conside ed a cy o oxic e ec , bu his was no obse ed in he analyzed samples. Figu e12 shows a g aph o LDH lac a e dehyd oge- nase le els. The lowe he alue o he es ed sample, he smalle he amoun o sec e ed isoenzyme, which means less cy o oxici y. Fo he es ma e ials, all alues a e in he same ange as he LDH alue o he con ol ma e ial, which was polys y ene (PS) in his s udy. The posi i e con ol was p esen ed as a c i ical alue o 0.6, which indi- ca es a 50% p obabili y o cell memb ane damage. 3.4 S udies o cell g ow h, p oli e a ion, andadhesion The esul s o he pe o med cell p oli e a ion s udies a e shown in Figs.13 and 14. Analyzing he con ocal Fig. 11 Numbe o iable cells s. all cells 316LVM 316 LVM + TiO2 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 2000 4000 6000 8000 1000012000 1400016000 180002 0000 Viable cells All cells 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 316LVM 316LVM+TiO2 PS (nega e con ol) Posi e con ol LDH [O.D.] Cy o oxici y in di ec con ac Fig. 12 LDH le el o es ed samples in di ec con ac A chi es o Ci il and Mechanical Enginee ing (2023) 23:237 1 3 Page 9 o 14 237 mic oscopy images, bo h he 316LVM e e ence sample and he TiO2 coa ing show a ypical no mal appea ance o ib oblas s wi h a spindle elonga ed shape, ocal adhesion (b igh elonga ed spo s), and a ied di ec ion o elonga- ion. In bo h cases, nume ous ound o o al cell nuclei can be seen, some in he di iding phase (close o each o he o pa ially o e lapping blue spo s). In he case o he TiO2 coa ing, a g ea e di e si y o he cy oskele on and a di e en densi y and leng h o ac in ilamen s can be obse ed. Places a e also isible wi h inc eased densi y o cell nuclei. In case o uncoa ed samples, in e up ions o he ac in ibe s con inui y in he o m o da k lines we e obse ed. On bo h he 316LVM e e ence sample and he TiO2 coa ing, ound da k spo s a e obse ed. Fig. 13 Example pho os o p oli e a ion s udies aken wi h a scanning lase mic oscope o sample 316LVM