Powder injection molded ceramic scaffolds: The role of pores size and surface functionalization on the cytocompatibility

Powde injec ion molded ce amic sca olds: The ole o po es size and su ace unc ionaliza ion on he cy ocompa ibili y Ma ina Ma ínko á a, ⇑ , Be enika Hausne o á a,b , Jakub Huba b , Tomáš Ma ínek c , Simona Kác ˇe o á a , Ve ˇ a Kašpá ko á a,b , Pe Humpolíc ˇek a,b, ⇑ a Tomas Ba a Uni e si y in Zlin, Cen e o Polyme Sys ems, . Tomase Ba i 5678, Zlin 76001, Czech Republic b Tomas Ba a Uni e si y in Zlin, Facul y o Technology, Va ecko a, 275, Zlin 76001, Czech Republic c Tomas Ba a Uni e si y in Zlin, Facul y o Applied In o ma ics, Nad S anemi, 4511, Zlin 76005, Czech Republic g aphical abs ac a icle in o A icle his o y: Recei ed 6 Ap il 2022 Re ised 5 Oc obe 2022 Accep ed 19 Oc obe 2022 A ailable online 20 Oc obe 2022 Keywo ds: Powde Injec ion Molding Su ace Modi ica ion Polyaniline Alumina Tissue Enginee ing Cy ocompa ibili y abs ac The alumina-based sca olds p epa ed by powde injec ion molding can be p e e en ially used o p epa- a ion o bone g a s. He e, he inal a chi ec u e o alumina sca olds was e icien ly con olled by pow- de space holde size and olume a io. The alumina is no in insically cell-ins uc i e ma e ial and hus he coa ing wi h elec ically-conduc ing polyaniline o polyaniline/biopolyme ilms p epa ed in a col- loidal dispe sion mode was used o p o ide his ad anced p ope y. The componen o he ex acellula ma ix, sodium hyalu ona e, o na u al biopolyme s (sodium algina e o chi osan) we e employed, and, subsequen ly, he cy ocompa ibili y o he na i e and unc ionalized alumina sca olds we e de e mined. Bo h he absence o cy o oxici y and he cy ocompa ibili y ha we e e ealed demons a e he applica- ion po en ial o hese composi es. The sca olds wi h po e size g ea e han 250 l m we e mo e cy ocom- pa ibili y han hose wi h po es size be ween 125 and 250 l m. The cy ocompa ibili y was con i med unde in i o-mimicking dynamic cul i a ion condi ions which u he imp o e he cell dis ibu ion and g ow h. Ó2022 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CCBY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). 1. In oduc ion Pe sonalized medical de ices such as den al p os heses, bone g a s o pe sonalised medical de ices o en equi e he p epa a- ion o p ecisely designed sca olds. While 3D p in ing echniques a e applicable o polyme -based sca olds, Powde Injec ion Mold- ing (PIM) allows he p epa a ion o p ecisely designed ce amics- h ps://doi.o g/10.1016/j.ma des.2022.111274 0264-1275/Ó2022 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). Abb e ia ions: CBS, ce amic-based sca old; CH, chi osan; SA, sodium algina e; SH, sodium hyalu ona e; AH, aniline hyd ochlo ide; APS, ammonium pe sul a e. ⇑ Co esponding au ho s. E-mail add esses: [email p o ec ed] (M. Ma ínko á), [email p o ec ed] (B. Hausne o á), [email p o ec ed] (J. Huba), [email p o ec ed] (T. Ma ínek), s_kace o- [email p o ec ed] (S. Kác ˇe o á), [email p o ec ed] (V. Kašpá ko á), [email p o ec ed] (P. Humpolíc ˇek). Ma e ials & Design 224 (2022) 111274 Con en s lis s a ailable a ScienceDi ec Ma e ials & Design jou nal homepage: www.else ie .com/loca e/ma des based p oduc s made. The p ecise design oge he wi h homoge- neous chemical composi ion [1] is conside ed as main ad an ages o PIM made ma e ials. PIM allow o ab ica e he pe sonalised medical de ices made o ce amics which can be especially ad an- ageous in bone o den al enginee ing. The esul ing po ous PIM s uc u es ha e a di e en in e nal s uc u e o hose ab ica ed by addi i e manu ac u ing plus allows o con ol he po osi y [2]. Aluminum oxide (alumina) has good biocompa ibili y [3], excellen co osion esis ance, high wea esis ance and s eng h, and is chemically bioine [4]. In addi ion, i s p ice is subs an ially lowe in compa ison o, o example, i anium alloys. I should also be men ioned ha he p oduc ion o po ous alumina s uc u es is no as demanding as in he case o i anium and i s alloys, which a e highly eac i e and hus mus be mixed, molded, and also debound/sin e ed ca e ully unde p o ec i e a mosphe es. Bo h, he bulk and su ace p ope ies mus be conside ed when he cy ocompa ibili y o sca olds a e conside ed. F om he bulk p ope ies, he po e shape is known o a ec cell beha io , as in hebone issue egene a ionp ocess, he size and in e connec ion o po es a ec he cell mig a ion, p oli e a ion, and ing ow h in o he sca old [5]. Ano he bulk p ope ies such po osi y [6], he mal conduc i i y [7], and elas ici y [8] is no limi ed he ce amic-based sca olds (CBS) applica ion, bu he su ace cha ac e is ics a e. Thus, he composi es combining he app op ia e bulk cha ac e is ics wi h s imuli- esponsi e su ace coa ings seems o be he mos app op ia e way o p epa a ion o desi ed cell-ins uc i e sca olds. One o he mos sui able ma e ials o he p epa a ion o s imuli- esponsi e, and hus cell-ins uc i e coa ings is polyaniline (PANI) [5]. PANI is a widely s udied conduc ing polyme ha pos- sesses some unique p ope ies. Fo example, he coa ing by PANI i sel is no adequa ely cy ocompa ible, bu can be combined wi h a a ie y o biomolecules, by he p epa a ion o hin ilms based on colloidal dispe sions, o p oduce cy ocompa ible ilms [9,10]. PANI also in insically combines elec onic and ionic conduc i i ies, which is ad an ageous o ma e ial/cell communica ion. As men- ioned, PANI can be s abilized by biopolyme s ha in luence he cellula physiology [11,12]. The ollowing compounds can be used as s abilize s: sodium hyalu ona e, which can p o ide inal ma e ial-speci ic bioac i i y, especially changes in s em cell gene exp ession [13]; chi osan, which is well known o i s an ibac e ial p ope ies [14]; and algina e, which is widely used in a a ie y o biomedical applica ions including issue enginee ing [15]. The e ha e been s udies de o ed o he space-holde -assis ed PIM o me al powde s such as 316 L s ainless s eel and se e al i anium alloys, bu he e has hi he o been no epo on aluminum oxide. Conside ing he shape o he po es, polyme hyl me hac yla e (PMMA)wasusedasasphe icalspace-holde o 316 L s ainless s eel [16,17] and Ti6Al4V [18], while sodium chlo ide (NaCl) and po as- sium chlo ide (KCl) ha e been employed in cases whe e an i egula shape o pa icles o ibe s wi h a high L/D a io is desi ed. Thus, one o he no el ies o p esen ed s udy is he use o KCl as space holde o aluminium oxide. Ano he inno a i e ea u e o he app oach used in he p esen s udy is he colloidal-based coa ing o su aces. Conc e ely, he su aces o a subs a e we e coa ed wi h a ious ilms based on polyaniline (PANI) s abilized by biopolyme s – in his case, sodium hyalu ona e, chi osan, and sodium algina e. Using hese biopolyme s, cell compa ibili y, o mo e gene ally he biocompa ibil- i y o ma e ials, migh be con olled. 2. Ma e ials and me hods 2.1. P epa a ion o na i e ce amic-based subs a es The powde componen s o he PIM compound we e aluminum oxide (Ma inswe k – Hube Co po a ion, USA) ( q = 3.98 g/cm 3 , size ange 0.1–3.0 l m) and a powde space holde (PSH), po as- sium chlo ide (KCl, Sigma Ald ich, Ge many) ( q = 1.98 g/cm 3 , size ange 125–500 l m). The powde s we e admixed in o a pa ially wa e -soluble binde (Licomon EK 583, q = 1.08 g/cm 3 , iscosi y 1.5 mPa.s a 130 °C) in a ba ch mixe (Plas i-Co de , B abende , Ge many) wi h coun e - o a ing blades. The powde con en was kep a 60 ol%, he powde : PSH a io a ied om 20:40 o 50:10, and wo size anges o he PSH we e es ed, see Table 1 (samples ma ked as CBS – Ce amic Based Subs a es). Injec ion molding was pe o med on an injec ion molding machine (All ounde 370S, A bu g, ARBURG GmbH + Co KG, Löss- bu g, Ge many) wi h a uni e sal ooling ame. The inse s o es samples we e made by machining EN AW 7022 aluminum alloy. The es geome y consis ed o a ound pla e (diame e 45 mm) wi h a highly-d a ed angle due o he absence o an ejec o sys- em. The molding p essu es we e op imized o ob ain de ec - ee samples, (see Table 1). A e wa ds, he wa e -soluble binde componen and pa o he PSH we e emo ed by imme sion in dis illed wa e (60 °C) o 24 h. The emaining binde ( he backbone) was debound he - mally (280 °C) a a mosphe ic p essu e. Sin e ing was ca ied ou in a PIM u nace (CLASIC CZ s. .o., Re nice, Czech Republic) up o a maximum empe a u e o 1670 °C and o a holding ime o 1 h. The su ace o CBS we e inspec ed using SEM mic oscopy (VEGA, Tescan). 2.2. Su ace unc ionaliza ion o na i e subs a e The su ace o na i e CBS was u he coa ed o become bioac- i e. Fou di e en composi ions o he coa ing we e es ed. Fi s ly, polyaniline (PANI) was used o coa ing. This coa ing can p o ide elec oac i i y bu acco ding o p e ious s udies does no p o ide adequa e cy ocompa ibili y. Ano he h ee coa ings we e he e o e p epa ed ia he inno a i e in-si u polyme iza ion o aniline hyd ochlo ide in he p esence o s abilize s – conc e ely, sodium hyalu ona e (SH), sodium algina e (SA), and chi osan (CH) [11]. The inal composi e coa ing is denomina ed ei he as PANI/SH, PANI/SA, o PANI/CH. In all cases, aniline hyd ochlo ide (AH, Sigma Ald ich, Ge many) and ammonium pe sul a e (APS, Sigma Ald ich, Ge many) we e used o he p epa a ion o each coa ing. P epa a ion o PANI coa ing. PANI ilms we e p epa ed by mixing 0.2 M AH and 0.25 M APS solu ions and hen pou ing he eac ion mix u e o e samples and in o Pe i dishes. A e 1 h o polyme - iza ion, he su aces we e washed wi h 0.2 M HCl (Pen a, Czech Republic) and insed wi h me hanol (Pen a, Czech Republic). P epa a ion o PANI/SH coa ing. Fo he p epa a ion o PANI wi h SH, 0.2 M AH, 0.1 M APS, and 1 % SH (Con ip o a.s., Czech Republic) we e used. SH in demine alized wa e was shaken a 55 °C o e - nigh . Then, he AH solu ion was added, ollowed by he APS solu- ion. The eac ion mix u e was pou ed o e samples and in o Pe i dishes, and polyme iza ion ca ied ou o 4 h.. As be o e, his s ep was ollowed by washing wi h 0.2 M HCl and insing wi h me hanol. P epa a ion o PANI/SA coa ing. PANI ilm s abilized wi h sodium algina e (IPL, Czech Republic) was p epa ed using 0.2 M AH, 0.25 M APS, and 2 % SA. The solu ion o SA was made in demine alized wa e and shaken a 37 °C o e nigh . A e wa d, AH was added, ol- lowed by APS; he ea e , samples and he Pe i dishes we e coa ed wi h he PANI/SA mix u e. The polyme ilms we e allowed o polyme ize o 4 h. The las s ep was he ixa ion o ilms wi h 0.2 M HCl and me hanol. P epa a ion o PANI/CH coa ing. This su ace modi ica ion was made wi h 0.2 M AH, 0.01 M APS, and 2 % chi osan (Sigma Ald ich, Ge many). Fi s ly, a solu ion o CH was p epa ed by dissol ing i in 1 M HCl and shaking he solu ion a 55 °C o e nigh . A e il a ion o he solu ion, AH was added, ollowed by APS. The su aces we e M. Ma ínko á, B. Hausne o á, J. Huba e al. Ma e ials & Design 224 (2022) 111274 2 co e ed wi h he esul ing mix u e and he ilm was le o poly- me ize o 12 h. Subsequen ly, he ilms we e washed wi h 0.2 M HCl and insed wi h me hanol. 2.3. Cy o oxici y de e mina ion In he es s, a mouse emb yonic ib oblas cell line (ATCC CRL- 1658 NIH/3T3, USA) was used. The cul i a ion medium consis ed o Dulbecco’s Modi ied Eagle’s Medium (PAA Labo a o ies GmbH, Aus ia) con aining 10 % bo ine cal se um (BioSe a, F ance) and 1 % o Penicillin/S ep omycin (GE Heal hca e HyClone, Uni ed Kingdom). The es was epea ed wice, each wi h i e epe i ions pe sample. Cells we e incuba ed a 37 °Cin5%CO 2 in humidi ied ai . Cy o oxici y o he na i e subs a e. Na i e CBS we e c ushed and ex ac ed acco ding o ISO s anda d 10993–12 in media wi h a concen a ion o 0.2 g/mL. The es ed samples we e ex ac ed in a cul u e medium o 24 h a 37 °C wi h s i ing. The pa en ex ac s (100 ol%) we e hen dilu ed in he cul u e medium o achie e inal concen a ions o 75, 50, 25, 10, and 1 ol%. All ex ac s we e used wi hin 24 h. Cy o oxici y es ing i sel was pe o med acco ding o ISO p o- ocol 10 993–5. Cells we e p eincuba ed in 96 well pla es (TPP, Swi ze land) a a concen a ion o 10 5 cells pe mL. The ex ac s we e added o p e-cul i a ed cells o ano he 24 h. All es s we e pe o med in quad uplica es. The e alua ion o cell iabili y a he end o exposu e was pe o med using Te azolium (MTT cell p oli - e a ion assay ki , Duche a Biochemie, Ne he lands). The abso - bance was measu ed a 570 nm wi h an In ini e M200 P o NanoQuan ins umen (Tecan, Swi ze land) and he e e ence wa eleng h was adjus ed o 690 nm. The esul s a e p esen ed as he cell iabili y (%) in NIH/3T3 cul u e compa ed o ha in med- ium wi hou PIM ex ac s ( e e ence cell iabili y co esponds o 1). The mo phology o cells om he cul u e pla es was assessed a e hei cul i a ion in CBS ex ac s by using an in e ed Olympus IX 81 phase con as mic oscope (Olympus, Ge many). 2.4. Cy ocompa ibili y de e mina ion The cy ocompa ibili y s udy began wi h he de e mina ion o cell adhesion, g ow h, and p oli e a ion on (1) na i e CBS and (2) he ilms used o i s unc ionaliza ion. The esul s om hese ini- ial s eps allowed us o design and pe o m (3) bioac i i y s udies in which cell g ow h and ing ow h unde s a ic o dynamic condi- ions wi h elec ical s imula ion we e in es iga ed. The cul i a ion condi ions o expe imen s 1 and 2 we e he same: he cells we e incuba ed a 37 °C in humidi ied ai wi h 5%CO 2 o 2 days. The di e ences we e in he concen a ions o seeded cells: 2 10 5 pe mL o expe imen 1, and hal ha con- cen a ion (10 5 pe mL) o expe imen 2. The expe imen al se up and cul i a ion condi ions o expe imen 3 we e adjus ed as ol- lows: A concen a ion o 2 10 5 cells pe mL was seeded on sub- s a es wi h a unc ionalized su ace. A e 3 days o p oli e a ion, samples we e ans e ed o a bio eac o enabling elec ical s imu- la ion. The bio eac o was un o 6 h pe day o a o al un- ime o 72 h, each successi e hou -long pe iod al e na ing be ween elec i- cal s imula ion and no s imula ion. The medium low was 54 RPM. The pulse had a ec angula wa e o m wi h a wid h o 3000 ms, and he ol age was se a 0.1 V. Cells we e isualised h ough nuclei coun e s aining by Hoechs 33,258 (In i ogen, USA) and ac in ilamen s we e isualized by coun e s aining wi h Ac inRed TM 555 (The mo Fishe Scien i ic, USA). 3. Resul s and discussion 3.1. Space holde size and olume de e mine he a chi ec u e o CBS Subs a e composi ion, a chi ec u e, po e size, and po osi y all play impo an oles in he con ex o designing ma e ials o use wi hin biomedicine. He e, we used di e en sizes o space holde , di e en space holde s powde a ios, and di e en p ocessing pa ame e s. Sca old po osi y inc eases wi h inc easing addi ions o he powde space holde . In addi ion, he shape o he po es can be in luenced by he shape o he PSH g ains. As can be clea ly seen in Fig. 1, he c i ical pa ame e de e mining he ma e ial p ope ies is he space holde size and i s a io o powde , while p ocessing pa ame e s seems o be less impo an . Using hese wo componen s, he a chi ec u e, po osi y, and especially he po e size can be easily con olled. 3.2. Na i e CBS do no induce cy o oxici y The le el o cy o oxici y p ede e mines any applica ion o a ma e ial in biomedicine. He e, he ISO p ocedu e was used o de e mine he cy o oxici y o he na i e subs a e. The cy o oxici y e alua ion is based on he de e mina ion o e ec o soluble impu- i ies leaching om he ma e ial, hus he cy o oxici y o subs a es wi h unc ionalized su aces was no de e mined, as he olume a io o coa ings was e y low and could no hus induce cy o ox- ici y. The su ace unc ionaliza ion can howe e in luence he cy o- compa ibili y due o ecep o -based in e ac ion wi h cells. This issue is de e mined and discussed u he . As p esen ed in Fig. 2, he na i e CBS did no induce cy o oxic- i y. Only in he case o wo samples, CBS 250_B, CBS 250-F ma ked as B and F, he cell iabili y sligh ly dec eased bellow he 70 %, and hus app oach he cy o oxici y h eshold. I he s anda d de ia ions and cy o oxici y o highe concen a ions (in case o sample B) a e conside ed, he cy o oxici y o hose concen a ions was no clea ly p o ed. I can hus be concluded ha na i e CBS a e appli- cable in biomedicine and can be u he unc ionalized o become bioac i e. Table 1 Composi ion o mix u e and pa ame e s o powde injec ion moulding p ocess. Two anges o PSH g ain sizes we e used in his s udy, i s ly g ains in he size ange o 125–250 ( 250 ) l m and g ains o 250–500 l m( 250 ). Abb e ia ion Powde Space Holde Powde P essu e [ba ] size [ l m] [ ol.%] [ ol.%] Injec ion Holding CBS 250_A 250–500 20 40 2100 1650 CBS 250_B 30 30 1500 1200 CBS 250_C 40 20 1900 1500 CBS 250_D 50 10 2100 1650 CBS 250-E 125–250 20 40 2100 1650 CBS 250-F 30 30 1500 1200 CBS 250-G 40 20 1900 1500 CBS 250-H 50 10 2100 1650 M. Ma ínko á, B. Hausne o á, J. Huba e al. Ma e ials & Design 224 (2022) 111274 3 3.3. Bioac i e coa ings p epa ed in colloidal dispe sion mode a e cy ocompa ible The conduc i i y is one o he c ucial p ope ies when he bioac i i y o bone sca olds and g a s a e conside ed [19]. The conduc i i y can be achie ed by inco po a ion o conduc ing poly- me s. I was, howe e , p e iously de e mined ha na i e PANI does no p o ide an adequa e cell esponse, which was also con- i med by his s udy (see Fig. 3B). Func ionaliza ion was he e o e pe o med by means o an inno a i e p ocedu e combining he Fig. 1. SEM images o he s uc u e o na i e CBS p epa ed using di e en composi ions and p epa a ion pa ame e s. The e ec o he size o he space-holde as well as he a io be ween space and powde olume on he inal s uc u e o he subs a e can be clea ly seen. The po e size is highly co ela ed o he size o space holde , while he space holde / powde olume a ion p ede e mine he po osi y and po e in e connec ion. A = CBS 250_A B = CBS 250_B , C = CBS 250_C , D = CBS 250_D , E = CBS 250-E , F = CBS 250-F , G = CBS 250-G , H = CBS 250-H. Fig. 2. The cy o oxici y o na i e CBS de e mined by a dec ease in cell iabili y compa ed o he e e ence (da a we e con e ed o a pe cen age o he con ol and exp essed as he mean ± s anda d de ia ion, n = 5). The ISO 10–993 p ocedu e, conc e ely he es ing o ex ac s, was used. The cy o oxici y h eshold is ma ked by dashed lines; when he iabili y alls below 70 %, samples a e conside ed o induce cy o oxici y. None o he es ed na i e subs a es induced cy o oxici y. A = CBS 250_A ; B = CBS 250_B , C = CBS 250_C , D = CBS 250_D , E = CBS 250-E , F = CBS 250-F , G = CBS 250-G , H = CBS 250-H. M. Ma ínko á, B. Hausne o á, J. Huba e al. Ma e ials & Design 224 (2022) 111274 4 syn he ic conduc ing polyme (p o iding s imuli- esponsi i y) wi h biopolyme s (p o iding cy ocompa ibili y). The p ocedu e is based on he p epa a ion o hin ilms in colloidal dispe sion mode [10]. The inal composi e coa ing can hus p o ide bo h bioac i i y and cy ocompa ibili y. To con i m he cy ocompa ibili y o he su ace unc ionaliza- ion employed he e, cells we e seeded on o su aces and hei p o- li e a ion and mo phology we e de e mined. The quan i ica ion o cells on he indi idual su aces was no de e mined due o bo h he in e ac ion o ilms wi h eagen s used o cell iabili y e alua ion (especially he MTT assay esul s can be biased) and due o he ac , ha cell quan i y does no p o ide ele an in o ma ion abou he eal cy ocompa ibili y which depends on cell physiology and mo - phology. I is clea ly shown ha cells on he PANI ilms we e no capable o p oli e a ion (see Fig. 3B). F om he s udy [11],i is known ha he addi ion o biocompa ible polysaccha ides o col- loidal ilms changes he su ace ene gy and su ace opog aphy, which can also lead o a change in cy ocompa ibili y. Fig. 3C show ha he numbe o cells on he su ace modi ied wi h PANI/SA was smalle han he numbe s o cells on he su aces modi ied wi h PANI/SH (Fig. 3E) and PANI/CH (Fig. 3D) ilm. Also, on PANI/SA (Fig. 3C), ound cells could be obse ed ha we e capable o adhe - ing on o he su ace, bu hey could no p oli e a e. The numbe o cells on he su ace s abilized by algina e was smalle in compa - ison o he o he s abilized ilms. Fig. 3E shows he cell on he su - ace coa ed wi h PANI/SH a e p esen in highe quan i y and especially hei mo phology is mo e physiological. This could be because o SH, which can suppo no only cell adhesion and p o- li e a ion bu mainly hei physiological s a e acco ding o he ype o adhe ed cells. The ac in cy oskele on o cells on his ilm was mo e ib ous compa ed o he e e ence sample (Fig. 3A). The p o- li e a ion a e on he PANI/CH ilm (Fig. 3D) was simila o ha on he PANI/SH ilm and also he mo phology o ac in ibe s was sim- ila in hese wo cases. I can be clea ly seen ha he bes cy ocom- pa ibili y was o e ed by he PANI/SH and PANI/CH coa ings. F om hose, he PANI/SH was la e chosen as he mos p omising ea - men o he de e mina ion o bioac i i y unde dynamic cul i a- ion condi ions. Sodium hyalu ona e was chosen wi h ega d o bioac i i y ela ed o s em cell di e en ia ion [20]. 3.4. Cells can g ow on he su ace and wi hin he po es o CBS unde s a ic cul i a ion condi ions In addi ion, cell g ow h on he su ace o he sample, as well as cell ing ow h in o he po es we e in es iga ed on na i e as well unc ionalized CBS. Cells we e able o a ach o, and subsequen ly g ow on all men ioned samples. Fig. 4 p o ides a de ailed iew o he su ace o he na i e CBS wi hou any su ace ea men and wi h di e en po es sizes (A 250 l m and mo e, B 125–250 l m). O e all, he g ow h o cells was be e on samples wi h a po e size g ea e han 250 l m han on samples wi h a po e size be ween 125 and 250 l m. The lowes iabili y was obse ed o CBS unc ionalized wi h PANI. In con- as , unc ionaliza ion by ilms p epa ed in colloidal dispe sion mode imp o ed cell g ow h on PIM samples in he case o all s a- bilize s. Also, he e we e no signi ican di e ences in cell g ow h be ween CBS PANI/SH , CBSP PANI/SA , and CBS PANI/CH . Mo eo e , he cell Fig. 3. Cell p oli e a ion on e e ence – he cell cul u e polys y ene wi hou su ace modi ica ion (A); PANI (B), PANI/SA (C); PANI/CH (D); and PANI/SH (E). Cells we e seeded in a concen a ion o 10 5 cells pe mL and cul i a ed o 2 days. Cell nuclei we e isualized by coun e s aining wi h Hoechs ; ac in ilamen s we e isualized by coun e s aining wi h Ac inRed TM 555 The app op ia e cy ocompa ibili y was obse ed in he case o he PANI/CH and PANI/SH coa ing. Fig. 4. Cell g ow h on he su ace and wi hin he po es o na i e CBS 250_B (A) and CBS 250_F (B) p epa ed wi h 30 ol% o space holde . The cells we e seeded in a concen a ion o 2 10 5 cells pe mL and cul i a ed o 2 days. Indi idual cells we e isualised h ough nuclei coun e s aining by Hoechs (whi e). The e we e no signi ican di e ences be ween any o na i e CBS. The CBS 250_D we e chosen as he mos app op ia e o o he expe imen s. M. Ma ínko á, B. Hausne o á, J. Huba e al. Ma e ials & Design 224 (2022) 111274 5 quan i y was e y simila o ha obse ed o pu e PIM samples. Based on mic oscopic obse a ions i can be concluded, ha cells we e able o ing ow h deepe in o he po es in all o he samples. Due o he bes cy ocompa ibili y o CBS wi h po es highe han 250 l m and mos p omising p ope ies o PANI/SH based coa ing he only CBS 250_D_PANI/SH was used o u he expe imen s. 3.5. Dynamic condi ions and elec ical s imula ion imp o e cy ocompa ibili y The inal goal o he he e p esen ed s udy was o p epa e a CBS which is no only cy ocompa ible bu also possess s imuli- esponsi e and hus e en cell-ins uc i e po en ial. The impo - ance o in i o occu ed dynamic condi ion is o en omi ed wi hin he cy ocompa ibili y s udy. In his con ex , he mechano ansduc- ion which in eg a es a ious physical cues om a cell’s su ound- ing mic oen i onmen and con e s hem in o biochemical in acellula signaling esponses a e mos impo an . The low o cul i a ion medium (shea s ess) is main pa o mechano ans- duc ion when he bone sca olds a e conside ed. He e we apply he media low 54 RPMI. Excep o mechano ansduc ion, he ins uc i e ole o elec oconduc i i y is ob iously o g ea impo - ance. Especially in case o bone issue, he ole o elec oconduc- i i y o ma e ials is discussed [21]. The e a e no gene ally accep ed p o ocols used o he elec ical s imula ion o cells, he se -ups a y no only in he applied ol age ( om 4–5 mV o 150 mV) bu also pulse du a ion (mos ly om 2 ms o 200 ms), equency (mos ly be ween 2 and 6 Hz), and wa e o m (e.g. monophasic, squa e) [19,22,23]. In he e p esen ed expe imen s he comme cial equipmen was used and he elec i- cal s imula ion pa ame e s we e as ollows: ol age 0.1 V, pulse wid h 3000 ms, a angemen on squa e-wa e. As men ioned, he CBS wi h a po e size abo e 250 l m was cho- sen o he expe imen s unde dynamic condi ions. The p e e en- ial cy ocompa ibili y o ma e ials wi h po e size o abou 200 o 300 l m is gene ally conside ed as ideal o bone issue eplace- men s [24] and was con i med e en on he CBS (see Fig. 4A). The coa ing wi h PANI/SH was chosen as he mos p omising, mainly due o he bioac i i y o SH. Bo h desc ibed cell-s imuli ex e nal ac o s, shea s ess and ex e nal elec ical s imuli, we e applied oge he , and he esul s a e p esen ed in Fig. 5. I is ob ious ha applica ion o shea s ess and ex e nal elec ical s imula ion has an impo an e ec on ew cellula pa ame e s (please compa e he Fig. 5A and 5B e sus 5C and 5D). Fi s ly, he cell quan i y is highe on he su aces exposed o dynamic condi ions, and mo e impo an ly he cell dis ibu ion is mo e homogeneous. This is c i ical o he sca old accep ance a e implan a ion [25]. In addi ion, a sligh ly di e en cell mo - phology and s uc u e o cy oskele on can be obse ed unde dynamic condi ions wi h elec ical pulses han unde s a ic condi- ions. This can be connec ed o bo h he applied shea s ess and elec os imula ion [26,27]. 4. Conclusion Ce amic-based sca olds p epa ed by Powde Injec ion Molding a e p omising candida es o use as medical sca olds, especially in bone egene a ion and es o a ion. The composi ion o he PIM mix u e as well as he p ocessing pa ame e s we e es ed o e eal hei impac on he a chi ec u e o he po ous ma e ial. I was ound ha he inal a chi ec u e can be e icien ly con olled by he powde space holde size and he olume a io. Howe e , alu- mina i sel does no p o ide adequa e cy ocompa ibili y, o , espe- cially, bioac i i y. None o he p epa ed ce amic-based sca olds induced cy o oxici y, and mo e impo an ly, cells we e able o g ow on hei su ace and ing ow h in o he po es. The sca olds su aces we e he e o e subsequen ly unc ionalized by s imuli- esponsi e polyaniline-based ilms. To imp o e he cy ocompa i- bili y, coa ings we e inno a i ely p epa ed in colloidal dispe sion mode and combined he syn he ic conduc ing polyme wi h biopolyme s abilize s (sodium hyalu ona e, chi osan, and sodium algina e). This unc ionaliza ion u he signi ican ly imp o ed he cy ocompa ibili y o he ce amic-based sca olds. The bioac i i y o he p epa ed sca olds was con i med by an imp o emen in cy o- compa ibili y when dynamic cul i a ion condi ions and elec ical impulses we e applied. The in i o mimicking condi ions imp o e he cy ocompa ibili y o sca olds, especially in con ex o cell dis- ibu ion and g ow h. Da a a ailabili y Da a will be made a ailable on eques . Decla a ion o Compe ing In e es The au ho s decla e ha hey ha e no known compe ing inan- cial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . Acknowledgemen s The wo k was suppo ed wi hin he p ojec OP RDE Junio G an s o TBU in Zlin, Reg. No. CZ.02.2.69/0.0/0.0/19_073/001694 1 (JUNG-2020-001), Czech Science Founda ion (20-28732S) and Minis y o Educa ion, You h and Spo s o he Czech Republic – DKRVO (RP/CPS/2022/001 and RP/CPS/2022/003). Fig. 5. Cell dis ibu ion and cell mo phology on CBS 250_D_PANI/SH unde s a ic (A,B) o dynamic (C,D) cul i a ion condi ions. Cells we e seeded in a concen a ion o 2  10 5 cells pe mL and p ecul i a ed in s a ic condi ions o 3 days and subsequen ly cul i a ed in dynamic condi ions o ano he 3 days. Cell nuclei a e isualized by coun e s aining wi h Hoechs , ac in ilamen s a e isualized by coun e s aining wi h Ac inRed. Pa ame e s o dynamic condi ions: Media low 54 RPMI, pulse wid h 3000 ms, squa e wa e o m, ol age 0.1 V. M. Ma ínko á, B. Hausne o á, J. Huba e al. Ma e ials & Design 224 (2022) 111274 6 Appendix A. Supplemen a y ma e ial Supplemen a y da a o his a icle can be ound online a h ps://doi.o g/10.1016/j.ma des.2022.111274. Re e ences [1] G. Ma ula, A. Sza kowska, K. Ma us, B. 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