3D-printed alginate-hydroxyapatite aerogel scaffolds for bone tissue engineering

Ma e ials Science & Enginee ing C 131 (2021) 112525 A ailable online 27 Oc obe 2021 0928-4931/© 2021 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). 3D-p in ed algina e-hyd oxyapa i e ae ogel sca olds o bone issue enginee ing Ana Iglesias-Meju o, Ca los A. Ga cía-Gonz´ alez * Depa men o Pha macology, Pha macy and Pha maceu ical Technology, I+D Fa ma g oup (GI-1645), Facul y o Pha macy and Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Uni e sidade de San iago de Compos ela, E-15782 San iago de Compos ela, Spain ARTICLE INFO Keywo ds: 3D-p in ing Supe c i ical d ying Ae ogel Bone sca old Hyd oxyapa i e ABSTRACT 3D-p in ing echnology allows he au oma ed and ep oducible manu ac u ing o unc ional s uc u es o issue enginee ing wi h cus omized geome ies and composi ions by deposi ing ma e ials laye -by-laye wi h high p ecision. Fo hese pu poses, he p oduc ion o bioac i e gel-based 3D-sca olds made o biocompa ible ma e- ials wi h well-de ined in e nal s uc u e comp ising a dual (mesopo ous and mac opo ous) and highly in e - connec ed po osi y is essen ial. In his wo k, ae ogel sca olds o bone egene a ion pu poses we e ob ained by an inno a i e s a egy ha combines he 3D-p in ing o algina e-hyd oxyapa i e (HA) hyd ogels and he su- pe c i ical CO 2 d ying o he gels. BET and SEM analyses we e pe o med o assess he ex u al pa ame e s o he ob ained ae ogel sca olds and he dimensional accu acy o he o iginal compu e -aided design (CAD) design was also e alua ed. The biological cha ac e iza ion o he ae ogel sca olds was also ca ied ou ega ding cell iabili y, adhesion and mig a ion capaci y. The ob ained algina e-HA ae ogel sca olds we e highly po ous, biocompa ible, wi h high ideli y o he CAD-pa e n and also allowed he a achmen and p oli e a ion o mesenchymal s em cells (MSCs). An enhancemen o he ib oblas mig a ion owa d he damaged a ea was obse ed in he p esence o he ae ogel o mula ions es ed, which is posi i e in e ms o bone egene a ion. 1. In oduc ion Bone de ec s esul ing om umo s, in ec ions, auma, biochemical diso de s and abno mal skele al de elopmen s gene a e se e e heal h p oblems and ep esen high sociosani a y cos s o he na ional heal h se ices [1]. Biological g a s (au og a s, allog a s) and non- deg adable g a s (me allic and ce amic implan s) a e he mos com- mon solu ions o hese pa hologies. Howe e , hei low a ailabili y ( o biological g a s) and equen pos -su gical complica ions such as he lack o osseoin eg a ion o he gene a ion o immune esponse ( o non- deg adable g a s) a e among he impo an d awbacks ha may occu [2]. Regene a i e medicine aims o ana omically and unc ionally es o e damaged issues using a combina ion o (i) ad anced biode- g adable 3D-sca olds as empo a y ex acellula ma ices (ECM), (ii) cells, and (iii) g ow h ac o s [2,3]. The p ocessing me hod used o ob ain hese bone sca olds should p o ide an op imum design o p o- mo e he new issue o ma ion. These bone sca olds should ha e a well- de ined in e nal s uc u e, dual in e connec ed po osi y and bioac i i y. Gi en he inc easing complexi y in mo phology and composi ion o hese s uc u es and he high sensi i i y o p ocessing en i onmen s (e. g., high empe a u e, shea s ess, pH, use o o ganic sol en s), inno- a i e and benign s a egies a e sough o gene a e hese new biomed- ical p oduc s [4]. 3D-p in ing is a cu ing-edge echnology ha allows he au oma ed and ep oducible ab ica ion o unc ional, scalable and cus omized a i icial s uc u es o pe sonalized medicine [5]. In gene al, his echnology uses a laye -by-laye deposi ion o biocompa ible ma e ials o achie e s able 3D-cons uc s. Speci ically, ex usion-based bio- p in ing is an addi i e manu ac u ing echnique commonly used o c ea e hyd ogel sca olds [6,7]. I is a p essu e-based me hod ha em- ploys a obo ic sys em linked o a bioink dispensing sy inge. A compu e -aided design (CAD) is used o deposi he bioink in a 3D-p e- de ined shaped s uc u e. This echnique is able o p in di e en issues wi h he gene a ion o ana omically co ec mac opo ous s uc u es [8]. A limi ed (bu g owing) numbe o bioma e ials a e sui able o bioink o mula ions o gene a e 3D-p in ed sca olds as hey mus be p in able, biocompa ible and p esen a ac i e s uc u al and mechanical p op- e ies [9]. Ne e heless, s uc u es ob ained by 3D-p in ing ha e he cus omized ex e nal mo phology essen ial o pe sonalized medicine, bu hey usually lack he high p ecision equi ed o gene a e * Co esponding au ho . E-mail add ess: [email p o ec ed] (C.A. Ga cía-Gonz´ alez). Con en s lis s a ailable a ScienceDi ec Ma e ials Science & Enginee ing C jou nal homepage: www.else ie .com/loca e/msec h ps://doi.o g/10.1016/j.msec.2021.112525 Recei ed 30 July 2021; Recei ed in e ised o m 18 Oc obe 2021; Accep ed 23 Oc obe 2021 Ma e ials Science & Enginee ing C 131 (2021) 112525 2 nanos uc u ed sca olds, wi h a con ol in he mac o and mic os uc- u al le els. Fo his eason, he combina ion o 3D-p in ing wi h o he echniques is needed o yield cus omized nanos uc u ed bioma e ials. Polyme ic nanos uc u es wi h ad anced p ope ies can be ob ained using supe c i ical luid echnology based on he use o supe c i ical ca bon dioxide (scCO 2 ) [10]. Namely, scCO 2 -assis ed d ying is adequa e o he ex ac ion o he sol en o a gel while p ese ing he in e nal gel s uc u al p ope ies, such as high po osi y and high speci ic su ace a eas, in he so-called ae ogels. This echnology can be used o ob ain ae ogel-con aining g a s and implan s [11], among hem ae ogel bone sca olds o issue enginee ing applica ions [12–14]. Ae ogels p esen a high mesopo osi y compa able o ha o he na i e ECM, which a o cell g ow h, and can be ob ained om polysaccha ides and p o eins [15]. Algina e is a na u al biopolyme widely used o bone sca olds and o bioink o mula ion because i is biocompa ible, biodeg adable, non- oxic and non-immunogenic, and suppo s cell g ow h [16]. Algina e gene a es a s able hyd ogel in he p esence o low concen a ions o di alen ca ions, namely Ca 2+ which p esen s he ad an age o i s low oxici y wi h espec o o he di alen ions. Finally, algina e composi ion esembles glycosaminoglycan (GAG) s uc u e, one o he majo com- ponen s o he na u al ECM in human issue. Howe e , algina e-based s uc u es usually lack bioac i i y and should be combined wi h o he admix u es o bone issue enginee ing pu poses [17]. Hyd oxyapa i e (HA) is he majo ino ganic componen in mamma- lian ha d issues and also an excellen bone subs i u e ma e ial because i is bioac i e, os eoconduc i e, non- oxic and highly biocompa ible due o i s chemical and s uc u al simila i y o na u al bone mine als [18,19]. This chemical simila i y is essen ial o apa i e deposi ion and subse- quen bone egene a ion. HA may also ha e a ole as a c oss-linking agen and as an ino ganic mechanical ein o ce [20]. Mo eo e , HA and i s composi es a e sui able o a achmen , p oli e a ion and di - e en ia ion o mesenchymal s em cells (MSCs). All hese p ope ies ende HA a o able o os eoin eg a ion [21–23]. The sou ce and size o HA pa icles is an impo an ac o on algina e-HA gel sca olds, because he HA g ain size and speci ic su ace a ea a ec he gelling ime and heological p ope ies o he hyd ogels. Mic oHA has been p oposed o applica ions equi ing apid gela ion kine ics and imp o ed mechanical p ope ies, while nanoHA is app op ia e o ende homogeneous hyd ogels unde mo e con olled gela ion kine ics [20]. O e all, algina e-HA composi es a e p omising candida es o bone issue engi- nee ing whe e HA also igge s he mine aliza ion p ocess and s imu- la es calcium phospha e and HA nanoc ys als nuclea ion and g ow h [19,21]. S a egies o he gene a ion o a complex 3D-shaped s uc u es wi h di e en unc ionali ies and a chi ec u es anging om he nanoscale o he mac oscale a e unde de elopmen o gene a e s uc u es wi h his po e hie a chy [24]. The design and op imiza ion o he inal po e s uc u e (po e size hie a chy and p opo ions) is also essen ial o he ma e ial pe o mance in issue enginee ing applica ions. The use o ae ogel echnology can sol e some o he cu en 3D-p in ing limi a ions ega ding he nanos uc u a ion o 3D-sca olds, bu he p oduc ion o ae ogels wi h a cus omized ex e nal mo phology is a emaining ema kable challenge [25]. Recen ly, se e al 3D-p in ed ae ogels ha e shown an excellen p in abili y in he we s a e and he p ese a ion o hei ex u al p ope ies like a high speci ic su ace a ea [25–27]. Ne e heless, ae ogels usually lack mac opo osi y and me hods o con e i and o con ol he mac opo e size a e unde de elopmen [28,53–55]. In gene al, ae ogels wi h a ious po e size anges a e highly desi able o applica ions like bone issue enginee ing [29]. The unexplo ed combina ion o 3D-p in ing and ae ogel echnolo- gies o bone issue enginee ing is he ein p oposed as a win-win s a egy o ob ain bioma e ials wi h imp o ed pe o mance and a chi ec u e. Finally, new s a egies o imp o e he ae ogels s eng hening and he p in ing p ocess necessa y o ob ain mo e ine and in ica e s uc u es a e s ill a challenge nowadays. In his wo k, 3D-p in ed algina e ae ogel sca olds con aining hyd oxyapa i e we e p ocessed by a combina ion o 3D-p in ing o hyd ogels and supe c i ical CO 2 d ying o bone egen- e a ion pu poses. The e ec s o algina e, HA and Ca 2+ concen a ions on he 3D-p in ed hyd ogel sca olds p ocessabili y we e assessed. Then, he 3D-p in ed ae ogels ob ained a e supe c i ical d ying we e e alu- a ed ega ding hei ex u al (speci ic su ace a ea, po osi y, densi y, sh inkage) and biological (cy o- and hemocompa ibili y, adhesion, mig a ion) p ope ies. The ideli y o he di e en gel o mula ions wi h espec o he o iginal CAD ile was also e alua ed. 2. Ma e ials and me hods 2.1. Ma e ials Alginic acid sodium sal om b own algae wi h medium iscosi y (gulu onic acid/mannu onic acid a io o 70/30, Mw 403 kDa, 3170 cps) and calcium chlo ide (CaCl 2 ; M w 110.98 g/mol, 99.99% pu i y) we e p o ided by Sigma Ald ich (Mad id, Spain). Hyd oxyapa i e (HA; M w 502.31 g/mol, eagen g ade pu i y, mic opowde ) was p o ided by Fluidino a (Mo ei a da Maia, Po ugal). CO 2 (pu i y >99.9%) was supplied by Nippon Gases (Mad id, Spain) and absolu e e hanol (E OH) by VWR (Radno , PA, USA). Wa e was pu i ied using e e se osmosis ( esis i i y >18 MΩ⋅cm; Milli-Q, Millipo e®, Mad id, Spain). 2.2. 3D-p in ing o hyd ogel sca olds Aqueous bioinks wi h h ee di e en algina e concen a ions (6, 8 and 10 w % wi h espec o wa e ) we e p epa ed using milliQ wa e as a sol en . Fu he mo e, di e en HA concen a ions (0, 8, 16 and 24 w % wi h espec o wa e ) we e added o he 6 w % algina e solu ions. All algina e solu ions we e p epa ed unde igo ous agi a ion (600 pm) employing a homogenize (VWR os 60, Pennsyl ania, USA) o a leas 1 h a oom empe a u e (RT). The hus ob ained algina e solu ions we e degassed in a sonica ion ba h (B anson 3510 Eme son, Fe guson, MO, USA) o 10 min o elimina e ai bubbles. Hyd ogels we e ob ained by p in ing he algina e inks wi h a Cellink BIOX Biop in e (Bos on, MA, USA) a RT using an ex usion p in head wi h a 3-mL sy inge and a 410- μ m nozzle a he p in ing p essu e o 50 kPa and a p in ing eloci y o 12 mm/s. 3D-p in ed algina e hyd ogels wi h dimensions o 20x20x1 mm ( o physicochemical es s, c . Sec ions 2.4 and 2.5) and 10x10x1 mm ( o biological es s, c . Sec ions 2.6 o 2.8) we e ob ained using a g id pa e n and 3 laye s. A e he p in ing p ocess, all sca olds we e pu di ec ly in con ac wi h CaCl 2 aqueous solu ions (0.1, 0.5 and 1 M) o gela ion (Fig. S1 o Supplemen a y In o). 2.3. Supe c i ical d ying o 3D-p in ed gels Algina e hyd ogels u n alcogels by imme sion in absolu e E OH. Two sol en exchanges o e hanol we e ca ied ou wi h an exchange equency o 24 h. The alcogels we e w apped in il e pape and d ied by supe c i ical d ying o ob ain he ae ogels. B ie ly, gels we e placed in o a 100-mL s ainless s eel au ocla e (Tha P ocess, Pi sbu g, PA, USA) and imme sed in 25 mL o absolu e e hanol o p e en hem om sh inkage be o e being in con ac wi h comp essed CO 2 . scCO 2 was supplied using a dual pis on pump and in oduced om he op o a essel hea ed a he cons an empe a u e o 40 ◦C. Fi s ly, a con inuous CO 2 low a e (5–7 g/min) a 120 ba ook place o 4 h. Ae ogels we e hen ob ained a e CO 2 dep essu iza ion a a a e o 2 ba /min, collec ed om he au ocla e and s o ed o cha ac e iza ion. The ob- ained sca olds we e deno ed as Alg x%,HA y%,CaCl 2 zM (x =6, 8, 10 w %; y =8, 16, 24 w %; z =0.1, 0.5, 1 M). 2.4. Physicochemical cha ac e iza ion o algina e ae ogel sca olds The skele al densi y o he ae ogels ( ρ skel ) was de e mined using a helium pycnome e (Quan ach ome, Boyn on Beach, FL, USA) a 25 ◦C A. Iglesias-Meju o and C.A. Ga cía-Gonz´ alez Ma e ials Science & Enginee ing C 131 (2021) 112525 3 and 1.01 ba . Values we e ob ained om i e eplica es. The appa en densi y o he ae ogel sca olds o mula ions ( ρ app ) was de e mined ollowing he Eq. (1): ρ app =Ae ogel mass/Ae ogel olume (1) whe e he olume o he ae ogels was ob ained by measu ing he h ee dimensions o he ae ogel ob ained wi h a digi al calipe (Fowle ™, New on, MA, USA). The po osi y o he s uc u es ( ε ) was ob ained om Eq. (2) om he speci ic olume conside ing he ex e nal dimensions o he ma e ial (V ) and he speci ic olume occupied by he oids in he ull olume (V oid, ). ε =(1−( ρ app/ ρ skel)) x100 =(V oid, /V )x100 (2) The inne po osi y o he ae ogel ibe s ( ε ) was ob ained om Eq. (3) om he speci ic olume occupied by he ibe s (V ibe s ) and he speci ic olume occupied by he oids (i.e. po es) in he ibe s (V oid, ibe s ). ε =(V oid, ibe s/V ibe s)x100 (3) V oid, ibe s =V ibe s − (1/ ρ skel)(4) The olume ic sh inkage (in pe cen age) o he ae ogel sca olds was calcula ed om he ex e nal dimensions o he ma e ial be o e and a e supe c i ical d ying ollowing he Eq. (5): Volume sh inkage = [(Alcogel olume–Ae ogel olume)/Alcogel olume ]x100 (5) Low- empe a u e N 2 adso p ion/deso p ion analysis (ASAP 2000 Mic ome i ics Inc.; No c oss, GA, USA) was pe o med o assess ae ogel ex u al p ope ies. Be o e measu emen s, samples we e degassed unde acuum a 40 ◦C o 24 h. Speci ic su ace a ea (A BET ) o ae ogels was e alua ed employing he BET (B unaue –Emme –Telle ) me hod. Spe- ci ic po e olume (V p ), po e size dis ibu ions and mean po e diame e (d p ) we e de e mined applying he BJH (Ba e –Joyne –Halenda) me hod. The mo phology o he ae ogels was e alua ed by scanning elec on mic oscopy (SEM, EVO LS15, Zeiss, Obe kochen, Ge many). Ae ogel samples we e i idium-spu e ed p io o imaging o minimize cha ging and o imp o e he image quali y. 2.5. Dimensional accu acy es s Two indices (p in ing accu acy and shape ideli y ac o –SFF–) we e employed o assess he ideli y o he gel s uc u es ( o alcogels and ae ogels) wi h espec o he o iginal p in ing ile p ocessed by 3D-p in - ing in he i s s ep o he dual p ocessing s a egy he ein p oposed. The p in ing accu acy and SFF we e calcula ed om Eqs. (6) and (7), espec i ely: P in ing accu acy (%) = [1− ((ǀAA–Aiǀ)/Ai ) ] x100 (6) SFF =Ai/A(7) whe e A i is he alcogel o ae ogel p in ed a ea espec i ely, and A is he CAD a ea (p in ing ile). 2.6. Cell iabili y es s The cy ocompa ibili y o he di e en algina e-HA ae ogel sca olds was de e mined by assessing he iabili y o mouse emb yo ib oblas s (BALB/c3 T3) a e 24 and 48 h o cul u e in he p esence o he ae ogel o mula ions using he WST-1 es and in iplica e. This es is based on he deg ada ion o WST-1 in o o mazan and is di ec ly co ela ed wi h he numbe o me abolically ac i e cells. BALB cells (6500 cells/cm 2 ) we e seeded in 24-well pla es in DMEM supplemen ed wi h 15% e al bo ine se um, penicillin 100 U/mL and s ep omycin 100 g/mL. Cells we e incuba ed a 37 ◦C in a humidi ied a mosphe e en iched wi h 5% CO 2 . Sca olds (1 ×1 ×0.1 cm) we e UV-s e ilized o 1 h and hen placed in he wells wi h cells con aining 1500 μ L o DMEM supplemen . Posi i e con ols o cells wi h 1000 μ L o medium and blanks o 1000 μ L o medium (bo h in iplica e) we e main ained a he same condi ions. A e 24 and 48 h o cul u e, sca olds we e emo ed, 250 μ L o medium was le in he wells and 25 μ L o WST 1 eagen was added. The pla e was incuba ed o 2 h a he same condi ions and hen shaken ho - oughly o 1 min. Finally, 110 μ L we e ans e ed o a 96-well pla e o measu e he abso bance a λ =450 nm in a mic opla e eade (In ini e® M200, Tecan G oup L d., M¨ annedo , Swi ze land). 2.7. Cell a achmen es s The sp eading o human bone ma ow mesenchymal s em cells (MSCs) on o he sca olds was e alua ed by DAPI s aining o isualize he cellula nuclei a e 6 and 13 days o cul u e [30]. MSC-seeded sca olds we e ixed wi h pa a o maldehyde (4% w / ) o 10 min and washed wi h PBS. Then, hey we e incuba ed in T i on/PBS (0.2% / ) solu ion o 5 min. Finally, sca olds we e washed wi h PBS, placed on glass slides, and one d op o he DAPI-con aining P oLong gold an i ade moun an (Molecula P obes Inc., Eugene, OR, USA) was added o each sca old be o e hei s o age a −20 ◦C. Finally, cell a achmen in he sca olds was e alua ed using a Leica TCS-SP2 spec al con ocal mic o- scope (Leica TCS-SP2, Leica Mic osys ems Heidelbe g GmbH, Man- nheim, Ge many) [31]. An open sou ce so wa e (ImageJ 1.5i, US Na ional Ins i u es o Heal h, Be hesda, MD, USA) was used o analyze he cap u ed images. 2.8. Mig a ion assay Cell mig a ion was e alua ed by a sc a ch wound healing assay [32,33]. A e he con luence, BALB cells monolaye was sc a ched by a s aigh line using a s e ile pipe e ip and mimicking a wound in i o. PBS was hen employed o wash cells and emo e cell deb is. The di e en ae ogel sca old o mula ions we e placed in he wells wi h esh cell cul u e medium a 37 ◦C. Pho og aphs o he sc a ch wound we e ob ained wi h an in e ed mic oscope (Olympus, Japan) immedi- a ely ( =0 h) and a 24 h o analyze he cell mig a ion. The mig a ion a ea (%) was assessed as ollows (Eq. (8)): Mig a ion a ea (%) = (A0–An)/A0×100 (8) whe e A 0 ep esen s he ini ial wound a ea ( =0 h) and A n ep esen s he esidual wound a ea a 24 h measu ed by ImageJ so wa e. 2.9. S a is ical analysis Resul s o cell iabili y es s o each ae ogel sca old ype (n =3) we e epo ed as mean alue ±s anda d de ia ion. - es s we e ca ied ou o de e mine he s a is ical signi icance o he di e ences among he g oups, and alues o p <0.05 we e conside ed as s a is ically signi ican . 3. Resul s and discussion 3.1. E ec o algina e and CaCl 2 concen a ion on sca olds The easible ope a ing egion o algina e ink o mula ion composi- ion and Ca 2+ concen a ion anges o 3D-p in ing was i s ly de e - mined. The ollowing c i e ia we e ollowed: (i) algina e bioink mus be liquid-like in he p in head o a oid he ip clogging, bu (ii) i mus be solid-like in he p in bed o main ain he cus omized 3D-geome y du ing he deposi ion p ocess and a oid he s uc u al in eg i y loss. Homogeneous 3D-s uc u es o med by di e en ia ed ilamen s a - anged in laye s we e acco dingly ob ained wi h algina e concen a ions in he 6–10 w % ange and a CaCl 2 concen a ion o 0.5 M (Fig. 1). Inks A. Iglesias-Meju o and C.A. Ga cía-Gonz´ alez Ma e ials Science & Enginee ing C 131 (2021) 112525 4 below o abo e his algina e concen a ion ange we e disca ded due o he non-homogeneous appea ance ob ained o he 3D-p in ed hyd o- gels. The 3D-s uc u e and po osi y, wi h mesopo es and mac opo es, was clea ly ecognized in all ae ogels p oduced in he easible egion. Tigh e ibe in e ac ions appea o be p esen a algina e concen a ions o 10 w %. A highly in e connec ed s uc u e wi h mesopo es and mac opo es was obse ed. An imp o emen in he ex u al pa ame e s (A BET and V p ) in all ae ogels was also obse ed (Table 1) wi h espec o o he algina e ae ogels p e iously desc ibed [34]. Ionic c osslinking o algina e gels p o ided long- e m s abili y o inal s uc u es in he 0.1–1 M CaCl 2 ange. Algina e sca olds c oss- linked a his CaCl 2 concen a ion ange had a highly po ous s uc u e h oughou he sca old wi h a high ae ogel su ace a ea (Fig. 2 and Table 1). Finally, algina e sca olds c osslinked wi h CaCl 2 1 M solu ion had mo e homogeneously p in ed s ands, which could be a ibu ed o mo e Ca +2 ions being in ol ed in he ionic c osslinking o he algina e [35]. In gene al, ae ogel sca olds ob ained a algina e concen a ion 6 w % and geli ied wi h CaCl 2 1 M exhibi ed non-wo en ibe s wi h igh e junc ions i compa ed o he es o o mula ions s udied and esul ing in less b i le cons uc s. High alues o ex u al p ope ies we e ob ained o all he es ed ae ogel o mula ions, al hough he composi ion and c osslinke con- cen a ion had an in luence in he esul s (Table 1), being he highes alues ound in ae ogels ob ained om inks using an algina e concen- a ion o 6 w % (Table 1). A simila end in e ms o ex u al p ope ies was ob ained in ae ogels om algina e and algina e composi es, like algina e-pec in mic osphe es gene a ed wi h di e en algina e- o-pec in p opo ions [36]. 3.2. E ec o hyd oxyapa i e concen a ion on sca old mo phology Algina e hyd ogel p in ing esul ed in s able single ilamen s (Fig. 1), bu wi hou signi ican binding be ween laye s. Mo eo e , algina e ae ogel sca olds s a ed o dissol e jus a e hei imme sion in aqueous solu ions losing hei in eg i y a e 24 h. The addi ion o HA o he algina e ink was hypo hesized o p ese e he s ands geome y while imp o ing s uc u e s abili y and bioac i i y o sca olds yielding an imp o ed 3D-s uc u e o bone issue enginee ing. HA is hyd ophobic and he sca olds would dissol e mo e g adually unde physiological condi ions and main ain hei shape, a he expense o a educ ion in he po osi y o he s uc u e [37,38]. To compensa e his, algina e ae ogels we e he ein c osslinked a high concen a ions o he di alen ca ions solu ion (CaCl 2 1 M), so ha mo e ca ions we e cap u ed inside he sca olds and b idged he gel ne wo k. Finally, HA is in ol ed in p o ein adso p ion and his p ocess causes cells anspo a ion in o he sca old, which is posi i e because i may p omo e cell adhesion and p oli e a ion on sca old su aces [39]. Homogeneous 3D-s uc u es we e ob ained o he HA concen a ion ange es ed (0–24 w %) wi h ibe s and laye s consis en ly a anged. The mac opo ous and mesopo ous s uc u es o he sca olds we e main ained as well. A highe HA concen a ions, mo e HA g anules appea o ming he ilamen s s acked oge he wi h he algina e (Fig. 3). The easy handling o he syn hesized algina e-HA sca olds and he SEM images showing binding be ween s ands appea o con i m he s uc- u al in eg i y and s abili y o he ae ogels a e 3D-p in ing. N 2 adso p ion-deso p ion analysis o algina e-HA ae ogel sca olds show an impo an dec ease in he speci ic su ace a ea and o al po e olume, while he po e diame e is almos unal e ed wi h espec o sca olds ab ica ed wi h algina e only (Table 2). The dec ease in he o al po e olume could be a ibu ed o he in e ac ions be ween –NH 3+ g oups o algina e and –OH g oups and Ca 2+ ions o HA ha lead o he inal deposi ion o HA o e he sca old c ea ing an i egula su ace s uc u e [16]. In gene al, he HA addi ion dec eased he ex u al p ope ies in all algina e-HA o mula ions, due o he low mesopo osi y o he HA powde (2 m 2 /g). Despi e ha , he ex u al p ope ies o he HA-con aining algina e ae ogels may s ill be enough o a o cell seed- ing and nu ien di usion h oughou he sca old s uc u e. The esul s ob ained in e ms o ex u al pa ame e s a e he addi ion o HA a e cohe en wi h p e ious s udies using o he polyme ic sca olds [16,40]. Appa en densi y alues inc eased g adually wi h he addi ion o HA esul ing in a e e se e ec on po osi y, al hough all ae ogel sca old o mula ions es ed eached alues abo e 80% (Table 2). The po osi y o Fig. 1. SEM pic u es o 3D-p in ed algina e ae ogels p epa ed a CaCl 2 concen a ion o 0.5 M and om aqueous algina e inks o di e en concen a ions: a) 6, b) 8, and c) 10 w %. Ae ogels a e obse ed a wo di e en magni ica ions. Table 1 Tex u al p ope ies o ae ogel-based sca olds om inks a di e en algina e (6, 8, 10 w %) concen a ions and c osslinked in gela ion ba hs o di e en CaCl 2 (0.1, 0.5, 1 M) concen a ions. No a ion: A BET : speci ic BET su ace a ea, d p : BJH-mean po e diame e , V p : BJH-speci ic po e olume. Ae ogel sca old A BET (m 2 /g) d p (nm) V p (cm 3 /g) Alg 6%, HA 0%, CaCl 2 0.1 M 438 ±22 22 ±1 3.14 ±0.16 Alg 6%, HA 0%, CaCl 2 0.5 M 277 ±14 22 ±1 2.00 ±0.10 Alg 6%, HA 0%, CaCl 2 1 M 183 ±9 19 ±1 1.16 ±0.06 Alg 8%, HA 0%, CaCl 2 0.1 M 180 ±9 17 ±1 0.95 ±0.05 Alg 8%, HA 0%, CaCl 2 0.5 M 177 ±9 22 ±1 1.00 ±0.05 Alg 8%, HA 0%, CaCl 2 1 M 147 ±7 21 ±1 0.97 ±0.05 Alg 10%, HA 0%, CaCl 2 0.1 M 172 ±9 18 ±1 1.04 ±0.05 Alg 10%, HA 0%, CaCl 2 0.5 M 158 ±8 16 ±1 0.87 ±0.04 Alg 10%, HA 0%, CaCl 2 1 M 219 ±11 14 ±1 1.11 ±0.06 A. Iglesias-Meju o and C.A. Ga cía-Gonz´ alez Ma e ials Science & Enginee ing C 131 (2021) 112525 5 Fig. 2. SEM pic u es o 3D-p in ed algina e ae ogels p epa ed om 6 w % aqueous algina e inks and a di e en CaCl 2 concen a ions: a) 0.1, b) 0.5 and c) 1 M. Ae ogels a e obse ed a wo di e en magni ica ions. Fig. 3. Images and SEM pic u es o 3D-p in ed algina e-HA ae ogels p epa ed om 6 w % algina e inks wi h di e en HA concen a ions: (a) 0, (b, e) 8, (c, ) 16, and (d, g) 24 w %. In all cases he gela ion was pe o med using 1 M CaCl 2 solu ions. Ae ogesl a e obse ed a wo di e en magni ica ions. Table 2 Tex u al p ope ies o ae ogel-based sca olds p ocessed om 6 w % algina e inks wi h di e en HA concen a ions (0, 8, 16, 24 w %) and c osslinked wi h CaCl 2 1 M solu ion. No a ion: A BET : speci ic BET su ace a ea, d p : BJH-mean po e diame e , V p : BJH-speci ic po e olume, ρ app : appa en densi y, ρ skel : skele al densi y, ε : sca old po osi y, ε : ae ogel ibe s po osi y. Ae ogel sca old A BET (m 2 /g) d p (nm) V p (cm 3 /g) ρ app (g/ mL) ρ skel (g/ cm 3 ) ε (%) ε (%) Alg 6%, HA 0%, CaCl 2 1 M 183 ±9 19 ±1 1.16 ±0.06 0.14 ±0.01 1.18 ±0.16 88.56 ±1.29 86.58 ±2.26 Alg 6%, HA 8%, CaCl 2 1 M 118 ±6 24 ±1 0.99 ±0.05 0.24 ±0.03 1.72 ±0.09 85.85 ±1.59 85.25 ±4.02 Alg 6%, HA 16%, CaCl 2 1 M 67 ±3 26 ±1 0.60 ±0.03 0.29 ±0.04 1.93 ±0.04 85.06 ±2.40 79.78 ±3.92 Alg 6%, HA 24%, CaCl 2 1 M 29 ±2 31 ±2 0.21 ±0.01 0.34 ±0.02 1.72 ±0.14 80.33 ±1.19 76.68 ±3.54 A. Iglesias-Meju o and C.A. Ga cía-Gonz´ alez Ma e ials Science & Enginee ing C 131 (2021) 112525 6 he ibe s also dec eased wi h he addi ion o HA. The o al po osi y alues o he sca olds (88.56 o 80.33%) a e sligh ly highe han he ae ogel ibe s po osi y alues (86.58 o 76.68%) as he sca olds a e o med by a 3D-mesh o e ically and ho izon ally aligned ae ogel mic o ibe s sepa a ed by mac opo ous gaps. These a e good esul s since he addi ion o HA ha e s ongly a ec ed o he ex u al pa ame e s (A BET ) bu no po osi y. Ne e heless, highe po osi y (80–99%) and lowe densi ies (0.02 g/ cm 3 as bulk densi y) alues we e p e iously epo ed o algina e ae ogel in he o m o mic opa icles [41–43]. Consis en ly, he inco po a ion o HA has ad an ages (bioac i i y, sim- ila i y o human bone mine al phase) [18,19] a he expense o a sligh dec ease in ex u al pa ame e s. The olume ic sh inkage o ae ogel sca olds a e pe o ming he supe c i ical d ying was es ed (Fig. 4) o un eil he in luence o his p ocess in he end ae ogel sca olds. The di ec sol en exchange o e hanol esul ed in ae ogels wi hou high olume ic sh inkage alues and wi h educed o e all p ocessing ime. S uc u es con aining HA had a lowe olume sh inkage wi h espec o he o mula ion wi hou HA (Alg 6%, HA 0%, CaCl 2 1 M). These a e good esul s since highe olume sh inkage alues (42–65%) ha e been epo ed o algina e ae ogels ob ained by sequen ial sol en exchanges [44]. 3.3. Dimensional accu acy es s P in ing accu acy and SFF indices we e employed o assess he ep oducibili y and he ideli y o he dual p ocessing s a egy he ein p oposed. The alues ob ained o bo h pa ame e s a e highe when assessed in he alcogels han when assessed in he ae ogels (Table 3). This ea u e was expec ed since he olume sh inkage epo ed in Sec- ion 3.2 in luences he dimensional accu acy indices. None heless, he alcogel p in ing accu acy and SFF alues ob ained a e close o he alues p e iously epo ed o 3D-p in ed algina e gels and o o he poly- saccha ide gels [37,45]. 3.4. Biocompa ibili y es s o ae ogel sca olds BALB cells we e cul u ed in he p esence o algina e-HA ae ogel sca olds o cell cy o oxici y e alua ion. Fig. 5 ep esen s he cell iabili y a e cul u e o 24 and 48 h wi h alues eaching ca. 100% o algina e ae ogels, which means ha hey had no nega i e e ec on cell p oli e a ion. Simila esul s on BALB cells iabili y a e 48 h we e ound o algina e-HA ae ogels, ega dless o he HA concen a ion. In gene al, hese iabili y esul s indica e ha nei he he sca old composi ion no he dual p ocessing s a egy comp omised he cell iabili y. The high cell iabili y obse ed a e he es s con i med he non- oxici y o he ae ogel s uc u es and co ela es wi h sca olds con aining he same componen s [46]. Indeed, he biocompa ibili y o he ini ial componen s o he sca olds (algina e and HA) was al eady epo ed [47]. O e all, hese a e p omising esul s since cy ocompa i- bili y and a sui able mic os uc u e (po e size and po osi y) go e n he p ope ies o an ideal sca old. 3.5. Cell a achmen and p oli e a ion es s on ae ogel sca olds A challenge in issue enginee ing is he gene a ion o bioma e ials wi h an adequa e s uc u e o ac as a subs a e o cell bonding, mul iplica ion, de elopmen and ep oduc ion [48]. MSCs a achmen and p oli e a ion in ae ogels we e he ein es ed as his cell line is a ac i e o issue enginee ing applica ions aiming a he gene a ion o a i icial bone subs i u es. MSCs p esen sel - enewal po en ial, hey can di e en ia e in i o and ex i o in o di e en cell ypes like chond ocy es o os eoblas s and hei isola ion is no ela ed o dono -si e mo bidi y [49]. In he case o os eoblas s, hei a achmen o sca olds depends on 0 10 20 30 40 50 60 Alg 6%, HA 0%, CaCl₂ 1M Alg 6%, HA 8%, CaCl₂ 1M Alg 6%, HA 16%, CaCl₂ 1M Alg 6%, HA 24%, CaCl₂ 1M Volume sh inkage (%) Fig. 4. Volume sh inkage (in pe cen age) o di e en algina e-HA o mula ions. Table 3 P in ing ideli y indices o ae ogels p epa ed om algina e-HA o mula ions. Ae ogels we e manu ac u ed a an algina e concen a ion o 6 w %, a di e en HA concen a ions (0, 8, 16 and 24 w %) and a a CaCl 2 concen a ion o 1 M. Ae ogel sca old Alcogel P in ing Accu acy (%) Ae ogel P in ing Accu acy (%) Alcogel SFF Ae ogel SFF Alg 6%, HA 0%, CaCl₂ 1 M 77.73 ±11.03 43.56 ±12.94 0.82 ±0.08 0.64 ±0.05 Alg 6%, HA 8%, CaCl₂ 1 M 75.51 ±8.54 39.06 ±10.70 0.81 ±0.05 0.62 ±0.04 Alg 6%, HA 16%, CaCl₂ 1 M 83.51 ±2.29 50.18 ±6.05 0.86 ±0.02 0.67 ±0.03 Alg 6%, HA 24%, CaCl₂ 1 M 83.35 ±6.21 48.14 ±4.84 0.86 ±0.05 0.66 ±0.02 0 20 40 60 80 100 120 140 160 Posi e Con ol Alg 6%, HA 0%, CaCl₂ 1M Alg 6%, HA 8%, CaCl₂ 1M Alg 6%, HA 16%, CaCl₂ 1M Alg 6%, HA 24%, CaCl₂ 1M Cell Viabili y (%) Fig. 5. Cell iabili y es s o algina e-HA ae ogels. Viabili y (exp essed in %) o BALB cells a e 24 (g ey ba s) and 48 h (black) o con ac wi h sca olds de e mined by he WST-1 es . The e was no s a is ically signi ican di e ences among g oups ( - es ; p <0.05). A. Iglesias-Meju o and C.A. Ga cía-Gonz´ alez Ma e ials Science & Enginee ing C 131 (2021) 112525 7 he su ace a ea and he po osi y o he ma e ials. No di e ences in MSCs a achmen and sp eading we e obse ed be ween ae ogel o mula ions in e ms o nuclei s aining (Fig. 6). The p esence o HA in he sca olds did no in luence MSCs a achmen as p e iously obse ed o s uc u es wi h simila composi ion [46,49]. Ne e heless, o highe HA concen a ions (16 and 24 w %) he a achmen dec eases a 6 and 13 days p obably due o he high su- pe icial oughness and i egula i y ha gene ally a o cell a achmen (HA concen a ion o 8 w %) bu a such highe le els i could make i ha d o MSCs o p oli e a e ully a ached o he ae ogel. A p oli e a ion endency is obse ed o all ae ogel o mula ions, since mo e nuclei we e s ained a e cul u ing sca olds o 13 days han a e 6 days (Fig. 6). This ea u e indica es ha sca olds we e non- oxic, biocompa ible, sui able o he a achmen and g ow h o MSCs and able o p omo e cell adhesion and p oli e a ion, as well as he cell coloni- za ion o he sca old. Small po e diame e s and high su ace a eas as ob ained o he ae ogels o his wo k a e sui able o os eoblas a achmen because hese condi ions allow cell in e pene a ion. 3.6. Cell mig a ion es Fig. 7a– shows he e olu ion o he wound closu e p ocess a e he sc a ch es wi h BALB cells in he p esence o di e en ae ogel o mu- la ions. Fig. 7g shows he inc ease in BALB cell mig a ion in he p esence o ae ogel sca olds wi h espec o he posi i e con ol. These a e good esul s since he well-known bioine p ope ies o algina e make i ha d o be deg aded in i o and his ea u e could ha e es ic ed cellula adhesion and mig a ion as p e iously epo ed [50]. In gene al, s uc- u es ha a e s able, wi h sa is ac o y mo phology and po e size allow signi ican human ib oblas mig a ion in wound closu e in i o assays [51]. Ne e heless, inding he bes mic oa chi ec u e o cell mig a ion in issue egene a ion is s ill challenging. In p e ious s udies, human de mal ib oblas mig a ion wi h algina e composi es showed a high closu e o he sc a ch [51]. Fu he mo e, algina e composi es combined wi h Ca 2+ ca ions esul ed in an enhancemen on ib oblas s mig a ion like he he ein obse ed, p obably due o changes in he egula ion o genes in ol ed in he wound healing p ocess ca ied ou by ib oblas s. Finally, as chemo axis is an inhe en esponse o cells i was hypo he- sized o be in ol ed in he cell mig a ion p ocess since he highe con- cen a ion o biochemical ac o s sec e ed by inju ed issues ac i a e he su ounding cells and s imula e he mig a ion o cells [52]. This bio- logical esponse could also play a signi ican ole in he inc ease o he mig a ion o ib oblas s in he p esence o algina e-based ae ogels he ein obse ed. 4. Conclusions Algina e-HA ae ogel sca olds we e success ully ab ica ed wi h a p ecise and cus omized nanos uc u e by he combina ion o 3D-p in - ing and ae ogel echnologies. This echnological combina ion esul ed Fig. 6. Con ocal mic oscopy images o DAPI-s ained MSCs seeded on ae ogel sca olds and cul u ed o (a) 6, and (b) 13 days. (c) Cell a achmen and p oli e a ion es s o algina e-HA ae ogels a e 6 (g ey ba s) and 13 (black) days o cul u e in MSCs. A. Iglesias-Meju o and C.A. Ga cía-Gonz´ alez Ma e ials Science & Enginee ing C 131 (2021) 112525 8 in nanos uc u ed and dual po ous algina e ae ogels o in e es o issue enginee ing applica ions. 3D-s uc u e is conse ed a e all p ocessing s eps and algina e, HA and CaCl 2 concen a ions de e mine sca old ex u e. An op imized sca old o mula ion mus p o ide a long- e m s abili y and he equi ed biocompa ibili y o issue enginee ing ap- plica ions. Cell iabili y es s e ealed no oxici y e ec o nega i e impac on BALB cells on no mal cell en i onmen because o he p es- ence o he sca old o mula ions. Fu he mo e, ib oblas s also suc- cess ully accomplished he mig a ion p ocess when cul u ed wi h he ae ogel o mula ions he ein p oposed. MSCs we e able o a ach and p oli e a e on algina e-HA ae ogels upon cul u e o 6 and 13 days. All hese ea u es a e highly desi able o sca olds o ac as empo a y suppo s and ha bo he bone egene a ion p ocess. Finally, he ideli y o he end ae ogel sca olds wi h espec o he o iginal CAD is high. Consis en ly, he mac opo ous algina e-HA ae ogel sca olds ob ained by his no el echnological combina ion o 3D-p in ing and supe c i ical d ying me hods ep esen a p omising al e na i e o bone egene a i e applica ions opening up new possibili ies o pe sonalized medicine. O he equi emen s o accomplish bone issue o ma ion like he deg ada ion a e o he 3D-s uc u es and hei mechanical p ope ies will be op imized in a u u e wo k. Supplemen a y da a o his a icle can be ound online a h ps://doi. o g/10.1016/j.msec.2021.112525. Acknowledgmen s This wo k was suppo ed by Xun a de Galicia [ED431C 2020/17], MICINN [PID2020-120010RB-I00], Agencia Es a al de In es igaci´ on [AEI] and FEDER unds. Wo k ca ied ou in he amewo k o he COST Ac ion CA18125 “Ad anced Enginee ing and Resea ch o ae oGels o En i onmen and Li e Sciences” (AERoGELS) and unded by he Eu o- pean Commission. A.I.-M. acknowledges o Xun a de Galicia o he p edoc o al esea ch ellowship [ED481A- 2020/104]. A. Alonso is commended o his echnical con ibu ion o his wo k. Decla a ion o compe ing in e es Au ho s decla e no con lic o in e es . Re e ences [1] S. Yue, H. He, B. Li, T. Hou, Hyd ogel as a bioma e ial o bone issue enginee ing: a e iew, Nanoma e ials 10 (2020) 1511, h ps://doi.o g/10.3390/nano10081511. [2] N. Ami yaghoubi, M. Fa hi, N.N. Pesyan, M. Samiei, J. Ba a , Y. Omidi, Bioac i e polyme ic sca olds o os eogenic epai and bone egene a i e medicine, Med. Res. Re . 40 (2020) 1833–1870, h ps://doi.o g/10.1002/med.21672. [3] Z. Wang, W. Kapadia, C. 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