International Journal o f Molecular Sciences Article Directional Submicrofiber Hydrogel Composite Scaffolds Supporting Neuron Differentiation and Enabling Neurite Alignment Lena Mungenast 1,* , Fabian Züger 2, Jasmin Selvi 2, Ana Bela Faia-Torres 1, Jürgen Rühe 3, Laura Suter-Dick 1 and Maurizio R. Gullo 2,* 1 Institute for Chemistry and Bioanalytics, University of Applied Sciences FHNW, Hofackerstrasse 30, 4132 Muttenz, Switzerland 2 Institute for Medical Engineering and Medical Informatics, University of Applied Sciences FHNW, Hofackerstrasse 30, 4132 Muttenz, Switzerland 3 Department of Microsystems Engineering, University of Freiburg–IMTEK, Georges-Koehler-Allee 103, 79110 Freiburg, Germany * Correspondence: lena.mungenast@fhnw.ch (L.M.); maurizio.gullo@fhnw.ch (M.R.G.) Abstract: Cell cultures aiming at tissue regeneration benefit from scaffolds with physiologically relevant elastic moduli to optimally trigger cell attachment, proliferation and promote differentiation, guidance and tissue maturation. Complex scaffolds designed with guiding cues can mimic the anisotropic nature of neural tissues, such as spinal cord or brain, and recall the ability of human neural progenitor cells to differentiate and align. This work introduces a cost-efficient gelatin- based submicron patterned hydrogel–fiber composite with tuned stiffness, able to support cell attachment, differentiation and alignment of neurons derived from human progenitor cells. The Citation: Mungenast, L.; Züger, F.; enzymatically crosslinked gelatin-based hydrogels were generated with stiffnesses from 8 to 80 kPa, Selvi, J.; Faia-Torres, A.B.; Rühe, J.; onto which poly(ε-caprolactone) (PCL) alignment cues were electrospun such that the fibers had Suter-Dick, L.; Gullo, M.R. a preferential alignment. The fiber–hydrogel composites with a modulus of about 20 kPa showed Directional Submicrofiber Hydrogel the strongest cell attachment and highest cell proliferation, rendering them an ideal differentiation Composite Scaffolds Supporting support. Differentiated neurons aligned and bundled their neurites along the aligned PCL filaments, Neuron Differentiation and Enabling Neurite Alignment. Int. J. Mol. Sci. which is unique to this cell type on a fiber–hydrogel composite. This novel scaffold relies on robust 2022, 23, 11525. https://doi.org/ and inexpensive technology and is suitable for neural tissue engineering where directional neuron 10.3390/ijms231911525 alignment is required, such as in the spinal cord. Academic Editor: Thaqif Keywords: neural cell guiding; neurite alignment; electrospinning; fiber-hydrogel scaffold El Khassawna Received: 25 July 2022 Accepted: 26 September 2022 Published: 29 September 2022 1. Introduction Publisher’s Note: MDPI stays neutral Tissue engineering for regenerative medicine aims to achieve the physiological perfor- with regard to jurisdictional claims in mance of engineered tissue by mimicking the native architecture. Scaffolds are often used to published maps and institutional affil- bridge tissue loss and support host and proliferating cells in laying down new extracellular iations. matrix, necessary for the formation of functional tissue structures [1]. Anisotropic native neural tissues, such as spinal cord or brain, have been shown to require a sophisticated scaffold design for cell attachment, proliferation, differentiation and guidance [1–3]. Ap- proaches for efficient cell guidance and alignment often include (bio)chemical cues combined Copyright: © 2022 by the authors. with a topography consisting of structures such as microgrooves, channels or nano-and Licensee MDPI, Basel, Switzerland. microfibers [1]. The microfibers’ resemblance to the three-dimensional microstructure of This article is an open access article extracellular matrix (ECM) can be accomplished by electrospinning of polymers—a simple, distributed under the terms and versatile and cost-effective technology. Electrospun fiber networks are characterized by high conditions of the Creative Commons porosity, large surface area, submicron diameter and strong interconnection of the fiber, Attribution (CC BY) license (https:// thereby closely mimicking the 3D fiber organization of the natural extracellular matrix, in creativecommons.org/licenses/by/ particular that of collagens, ranging from single collagen fibrils of 20–500 nm to collagen 4.0/). Int. J. Mol. Sci. 2022, 23, 11525. https://doi.org/10.3390/ijms231911525 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 11525 2 of 14 bundles of several microns [4–6]. To engineer hierarchically structured scaffolds, the con- trolled deposition of fibers is crucial and several techniques for the alignment of electrospun fibers have been developed, including a rotating drum [7–9], micro patterned collectors [10], electrospinning and photolithography [11] or near-field electrospinning [12]. It has been shown that axonal alignment of neurons can be triggered and neurite outgrowth can be promoted on submicron electrospun poly(ε-caprolactone) (PCL) fiber scaffolds [9,13–17]. PCL has been shown to be biocompatible, suitable for electrospinning, capable to guide neural cells in vitro, and is approved by the FDA [18,19]. In addition to topographical features, the design of microenvironment mimicking native tissue properties is of particular importance in supporting the differentiation of (human neural) progenitor cells [1,20,21]. Since the tissue microenvironment is also determined by the stiffness, influencing many cellular processes such as adhesion, proliferation and differentiation [20,22], hydrogels can be utilized to adapt the stiffness of cell culture substrates. The elastic modulus for spinal cord tissue is considered one of the lowest found in the human body and is considered relevant below 100 kPa [20,23–26]. Mismatching the stiffness of a scaffold/implant and that of the host tissue will alter the migration behavior and differentiation cycle of neural progenitor cells into neurons [18,25–28]. The most commonly used hydrogels in neural tissue engineering, hyaluronic acid and poly (ethylene glycol), require crosslinking usually involving the use of expensive, toxic chemicals or time-consuming pre-modification of the monomer to enable UV-crosslinking, which in turns makes it more challenging to use those hydrogels for cell encapsulated hydrogel scaffolds [27–30]. Due to their availability, good biocompatibility and low cost, gelatin-based hydrogels have often been used as cell culture substrates [31]; however, thermally crosslinked gelatin hydrogels have low stability and a high degradation rate at physiological conditions [32]. To provide mechanical and proteolytic stability, gelatin hydrogels enzymatically crosslinked with transglutaminase (TG) offer a low cost, fast, environmentally friendly, non-toxic alternative for gelatin hydrogels. By varying the gelatin and enzyme concentrations/ratios, the elastic modulus of the hy- drogels can be tuned to achieve the desired tissue-relevant stiffness. Gelatin-methacryloyl (GelMa) hydrogels with 35 kPA elastic modulus have been proven to support adhesion and proliferation of PC12 neuronal model cells, and to further enable cell spreading and neurite outgrowth [33,34]. TG-crosslinked gelatin hydrogels patterned by microcontact printing enabled the adhesion and proliferation of Schwann cells while maintaining healthy morphology, adhesion and extensions of axons [35]. In this work, we present the combination of topographical and microenvironmental features leading to a novel gelatin-based hydrogel–fiber composite scaffold tunable in stiffness, fiber alignment and fiber density, specifically adjusted to support the attachment, differentiation and alignment of neurons derived from a human progenitor cell line (ReN VM cells), capable to differentiate into neurons, astrocytes and oligodendrocytes. While all these cells are of utmost importance and key players of the central nervous system, neurons remain the main cell type responsible for signal transmission from the brain to the peripheral nervous system. Combining the benefits of tunable hydrogel stiffness and electrospun microfibers into a hybrid cell culture substrate, we produced a scaffold with tissue-relevant elastic modulus and topographical guidance cues that support neuronal differentiation and cellular alignment. Such a composite material can be developed as an implantable scaffold to support the regeneration of spinal cord in patients after traumatic injury. Moreover, scaffolds featuring directionality and alignment cues may also be im- plemented as in vitro neuronal models and serve the investigation of therapies’ efficacy promoting neural regeneration. 2. Results For the hydrogel–fiber composite serving as the scaffold for the culture of neural cells, gelatin was selected as the base material for the hydrogel with stiffnesses ideal for the support of neural cells. As illustrated in Figure 1, for the second layer, located on top of the hydrogel, a sparse layer of electrospun fibers representing the topographical cue was Int. J. MoInl.t. SJ.c Mi. o2l.0 S2c2i., 220322, ,1 2135, x2 5FOR PEER REVIEW  3 of 14  3 of 14   the support of neural cells. As illustrated in Figure 1, for the second layer, located on top  doef pthoes hityeddr.ogFeol,r at hspeargsee nlaeyreart ioof neleocftrtohsepufinb feibresr,st hreeprseysenntthinegt itchep otolpyomgrearphpiocally c-uεe- caprolactone was cwhaoss deenp.osTithedis. Fpoor tlhyem genrerwataiosnu osf ethde ftiobeprsr, othdeu sycnethaetfiicb peorlylmayere prowlyh‐εi‐cahprwolasctothnei n enough so that twheasg cehloasteinn. Twhiass pnoloytmceor vwearse udsecdo tmo pprloedteuclye .aT fihbeerc loaymerb winhaicthio wnaso tfhtihn eesneoutgwho sos tarting materials atlhlaotw theed getlhaetinp wroads nuoctt icoovneorefdh cyomdrpolegteelly–.fi Tbhee rcosmcabfifnoaltdiosn, owf thhiecshe twweo rsetacrthinagr amcateter‐ized using optical rials allowed the production of hydrogel–fiber scaffolds, which were characterized using  mopictircoals mcoicproyscaonpdy annadn noainnodinednetnatatitioonn,, pprrioior rtot aopapplicpaltiicoan tiino inn‐ivnitrion c-evlil tcruoltcuerel.l  culture. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW  4 of 14    day 14, which demonstrates the superior cell supporting nature of the engineered hydro‐ gels. As no significant differences in cell viability could be found between 7 and 21 kPa  hydrogels, the latter was selected for the creation of the hybrid fiber–hydrogel structures.  As expected, the plain hydrogels could not provide sufficient alignment cues for neu‐ ronal cells. The ReN VM cells did not spontaneously grow with a preferred directionality  but were randomly distributed on the hydrogel scaffold (Figure 2c). To promote the emer‐ gence of a directional alignment, directionally aligned PCL fibers and randomly aligned  fibers as a control were spun directly on top of the hydrogels by electrospinning (Figure  3a). The electrospinning process was carried out such that only very thin mono layers (<1   μm) of oriented fibers were produced. The degree of alignment was assessed by the ori‐ enFitgautiroen 1 o. Pf rtohdeu fcibtieorns p arloocnegss t ohfe f irbeefre–rheyndcreo agxeils s caatf 0fo°l wdsh aincdh  urespe rine sceenllt ceul ttuhree  aexxipse arilmonengt ws. Figure 1. Production process of fiber–hydrogel scaffolds and use in cell culthuirceh experiments. the moSstrt uficbtuerrsa lw seurpep oorrite notfe cdu.l Btuyr eo,p vtiiambiizliitnyg a tnhde  dspififnenreinngti aptaioranm oef theursm wainth n efoucruasl  porno pgoenl‐i‐ ytmore rc efSllolt wlriun recastt ueis–r npaerloesdvuildep‐ecpdo olbleryct ctoourfs tdcoiumstlaitznuecdree –h,dyvedpiraoobsgiietliliost.ny A tai mkneedy,  tfdheaeift uffierbere ero nfd ttehinaestsiietoy g nfeolosr  fiesah tcouh  mmdeaaptnc‐hn  eural progenitor octsehitleilo Ynlio nwuenassg ’asids Mjuposrtdoeudvl utiods  2eo5fd  t±hb 3ey. 0c%eclu lfsis botefor t mhcoeiv zceernadtgreah loy nnde trhvoeog uhesy ldsyr.osgtAeeml.k ,T ewhyihsi fceehna aitbsu lienrde  taho erf etgytuhplieacrsa el  gels is to match msetiafnfn sepsas crianngg oef  2200  ±t o5  μ10m0  bkePtaw. eTeon  tthheis f ipbuerrps’o mseo, naoimlayinegr  (tFoi ggurraed 3uca)l, layn  idn car feiabseer  tthickness  itnh  teheY mouicnrogn’ sraMngoe.d Tuhleu  fsiboerf dthiaemecteelrl sobotafinthede wceas  itnr athl en seurbv‐mouics systemh,e welhastic rometer range  foicr h is in the typicalmoduli,  bsottihff rnaensdsthrraeen hydrogels witom (0.g86e ±2 00.08to μm10) 0h diankdPfferen alaig. nTto cotnhciesntprautiropnos soef ,gaeilamtinin  (g10t%o, 1g5r%a,d 2u0% w/w), were produced. For crosslinking, transgedlu (t0a.m84i n±a 0s.e0 w6 μithm a)  fciobnecres.n Ttrhaetisoen p oafr a5m0 metger ally increase the elastic /ms wL ewrea s  cmhadoosdedendu s ltooi,  athtsh eto rg eoeelfafethirn ys sudofrfluioctigieoennlt  satonwpdo iignthrcaupdbhaiitfcefadel  rcfeornll  1tg2uc hiod anatn c3ce7en  °cCtur.e aIsnt wi tohintihss opuortof acelgtsesr,li aanctgyi ntlh ter(a 1snu0sb%fe‐r,  15%, 20% w/w), swtorfae taree  lsyptsifirnfonede bsuso cnoedr d al.ectaFidnosg r taocs r iano tsbrsaalr‐i rnaienkrd it noin gct,eelrtlmr maonolevscgeumlluaertn act rmboysis noliavnsekreilnowga diotifnh pgar tohcteeo ihnysc dtehrnortogruealg twhio iftnohr ‐of 50 mg/mL was faimbdeadrtsie.o dAn sto odf eatphni ecistgeodpe elianp tFidnigeus boroel nu3dbt.i,  oTthnhee a vanovdleurmiangece vu sibeawmat poelfde t htfheoi crfikb1ne2ers–hsh wyadtars3o 72g.e0◦l  mscmaff. oAlds  dshisopwlany sin   the 3D orga C. In this process, acyl transfer of aFilgyusrien 2eab, a nti zaatond te iomn of the fiberlepaedrsattuorei noft 3 s7 o °nC top of ra- an thde isnti th tfefn e ehsysdrormo olfe a glel lh. Thculayrdr eo gheyldcrosss  r(o6glin.8 e lk skinP earg  v±eo 0 s f.  5a;s 2 t0h.e7  bkaPsae  ±  m0a.4te; r7i9a.l0 a knPda  p±r e0s.8e)n wtsa asn w eivtheinn  stuhref adcees ifor the fibers. In this three‐dimensiona pl vroietwei, nthset hrough formation doienfpgao gnsietliaostnoin po fce otphnteci edfnibeterabrstoi oonnnd .t Ih.neT  cshoumerfpacaver iewsr raegd erasnagme (p20le–1t0h0i kcPkan) easnsd winacrsea2s.e0s mwimth increas‐ointh toou tth siisg, nthifeic ealnats teimc mbeoddduilnugs  ionft oa  tshtaen hdyadrdr.o Atgisessl uisseh ‐own in Figure 2a, sahctuowlatuntr.ee T mphoepl ycersrotyasrste‐usnerece tfiloonfal3 ◦ ask i sv7 aierwoCu cnltedha 3re.l5y s GitnPidfafi cn[a3et6es]s.s  thoaft tahlel fhibyedrsr aorge edlespo(s6i.t8edk aPs aa m±on0o. 5; 20.7 kPa ± 0.4; l7ay9e.0r oTknoP  teahve±a tlou0pat .oe8 f)t htwhee a ehsffyewdcrti otoghfe ilhn ysudtrhrfoaegceedl,  eosstfififerfrneiendsgs r iaonnd igcveidll( ua2ad0l h–toe1sp0ioo0gnr kaapnPhdai )cpaarlo ncliudfeesria nwtcioirtnhe, a atsnh es  with increasing avperoraggeen ihtoeirg Rhet Nof  V8M60  c±e 8ll0s  nwmer aeb soevede etdhe o snu prflaacine.  gTehlaet irne shuyltdinrogg feilbse wr liathy etrh ed itdh rneoet d inifdfeucgela re en t  anelya ss t tig inicfiocanncte dnitfrfearteinocne .oInn  tchoem  elpasatrici smonodtuoluths iosf,  tphlae elastic modulus of a standard tissue-culture polyisct myroednueli fl(7a ksPka,i s21a krPoau, n80d kPa) described above. In in a dhdyidtiroong,e tlhs e( Fcuigltuurree  3odn) ,h  tyhdurso ‐ mgaeilnst wainitihnogu tth teo tpiossgurea‐prheliecavla ncut esstiff 3n.e5ssG. Pa [36]. served as a positive control to investigate the influence  of topographical alignment cues in later experiments. All three hydrogel concentrations  enabled the adhesion and proliferation of the cells, as determined by the increased num‐ ber of viable cells and depicted in the optical micrographs in Figure 2b,c, respectively. The  cells displayed a spread morphology especially in the case of the lower stiffness hydro‐ gels,  indicating  that  the  laminin‐coated  hydrogels  provided  sufficient  cell‐anchoring  points on the surface. The cell viability even after two weeks of culture, as evidenced by  the increased cell numbers on the hydrogel, as well as the spread morphology of the cells,  are strong indicators of the biocompatibility of the scaffold. Moreover, based on the Ala‐ mar blue assay, the number of viable cells on all tested hydrogels was significantly higher  compared to the reference material (laminin coated tissue‐culture polystyrene: TCPS) at      FFigiguurer 2e. 2G.eGlateinla htiyndrhoygedlsr oogf evalsriaobflev astrififanbelses ssutipfpfnorets asdshuespiopno arntda dprhoelisfieorantioann dof pnreourliafle prraot‐ion of neural progen- genitor  cells.  (a) Elastic modulus  [kPA] of  transglutaminase‐crosslinked gelatin hydrogels  (10%,  1i5to%r, 2c0e%ll sw./w(a) a)tE 3l7a °sCt,i cnummobedru olfu hsyd[kroPgAels] Nof =t 9r;a (nb)s Mgleutatbaomlici nvaiasbeil-ictyr o(dsestleirnmkineedd gbey lAatlainmahry  drogels (10%, 15%, B2l0u%e aswsa/yw) o)fa Rte3N7 V◦MC ,cenllus mprboleifreroaftehdy fodrr 1o4g dealyssN on= ge9l;at(ibn )hMyderotageblos;l uicnpvaiiarebdil ti‐ttyes(t d(αe t90% of fibers distributed within ±10◦. In comparison, the fibers deposited randomly showed a slightly coiled structure, often found for randomly distributed electrospun fibers, and they were deposited without a significant main axis. When a sample with random deposition is taken and the angle with highest fiber deposition is viewed (here −22◦), only 15% of fibers are distributed within ±10◦ of this angle. To ensure the integrity of the fiber–hydrogel constructs during the experimental period, their long-term stability was tested in cell culture medium at physiological temper- ature (37 ◦C) for 28 days. The condition of the incubated fiber–hydrogel constructs was assessed by optical micrographs (Figure 3e, 28 days-time point). Although the fibers were Int. J. Mol. Sci. 2022, 23, 11525 5 of 14 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW  5 of 14    not chemically bound to the surface of the hydrogel, neither delamination of fibers from the hydrogel, nor PCL degradation nor loss of hydrogel integrity were observed.   Figure 3. Scaffolds of eFliegcutreo3s.pSucanff oPldCsLof feilbecetross opunn gPeClLatfiibne rhsyondgreolgateinlsh. y(dar)o gOelps.t(iac)aOl pmticicalrmogicrraogprhapsh osfo frraan‐dom dom and aligned electraonsdpaulingn PedCeLle fcitbroesrpsu onnP C2L1 fikbPears honyd21rokgPaelhsy, dsrcoagleels b, sacra l=e b1a0r0= ; 1(0b0);  (3bD) 3 Dvovloulummee viewa nd and cross section of concfrooscsasle mctiiocnroogf rcoanpfhocsa ol mf PicCroLg rfaipbhesrso f(iPnC rLefidb)e orsn( i2n1r ekdP)ao nhy21dkrPoagheylsd (roing eglsre(ienng)r,e secna),lesc ale bar = 200 μm; (c) Histogbarra=m2s0 0wµmith; ( cd) iHsitsrtiobguratmiosnw oithf ddiisrtericbtuitoionnaolfitdyir μecmtio noafl iPtyCµLm foifbPeCrLs fiobne r2s1o nk2P1AkP hAyhdydroro‐gels gels (left: random with( lnefut:mrabndeorm ofw fitihbenrusm Nbe r=o f2fi0b0e,r sriNgh=t2: 0a0l,irgignhet:da lwignitehd wniuthmnbuemrb eorfo ffifibbeerrss NN= =5 5000)0; ()d; )(Edl)a stic Elastic modulus [kPA] mofo dtrualunss[gklPuAt]aomf itnraansseg‐luctraomssinliansek-cerdo sgsleinlakteidng helyadtinrohgyedlrso,g helisg,hhligighhligtehdte dwwitihth rree◦ d d  bbooxx1 5% gelatin with and without PCL fibers at 37 C, number of hydrogels N = 9; (e) PCL fibers on 21 kPa 15% gelatin with and wgeitlahtoinuhty PdrCogLe lfiabfteerrs2 8adt a3y7s a°tC3,7 n◦Cu/m5%beCrO ofi nhycedllrcouglteulrse mNe d=i u9m; (,esc)a PleCbLar f1i0b0eµrms .on 21 2 kPa gelatin hydrogel after 28 days at 37 °C/5% CO2 in cell culture medium, scale bar 100 μm.  As established in the previous experiments, the hydrogels supported the adhesion and The deposition porfo ltihfeera ftiiobnerosf nineu rtahlep rcoogpenpietorr tcuelblse;  hionwsteavlelra, tthioense lpeldai ntoh ysdtrroagieglshwt efirbeenrost cdaep‐able to guide and orient neural cells. To investigate the influence of topographical cues on the posited in a highly pbaerhaalvleiolr foafsnheiuornal; phroegreen sithoor wcelnls iann dFcigomurmei t3tead, ntheuer ofnibs,eRrseN arVeM acliegllns ewder aelcounltgur ed the main axis horizonontahlylyd.r oAgsel sdceopnitcatienidn ginr atnhdeo mhisatnodgarlaigmne idne Fleicgtruorspeu 3nc,P tLhCefi fbiberesra dndepcomsiptiaorend  to had a very narrow dthiestprliabinuthiyodnr oogfe lsd.iCreeclltivoianbailliittyya swsaiytshs h>o9w0%ed noof nfiebgeatrisv edeifsfetrctibouf ttheedfi wbeirtshoinn the ±10°.  In comparisonc, etlhlse’ m  fetabolic activity and cell numbers withinFigure 4iab,eprrso ldifeerpaotiosniteodf R reaNncdeollsmwlyas sshimoiwlare t od he n a 14 da a lisglnig yhs tclued fiby lt ucrerso e oi pleerdion hy ds dt. As srorguecl,t huorar w ned, n in o  m often found for randofimberlsyo dnihstyrdirbougetel adn edloenctprloaisnphuynd rfoigbeelrssc,a fafonlds t.hey were deposited without  a significant main axis. When a sample with random deposition  is taken and the angle  with highest  fiber deposition  is viewed  (here  −22°), only 15% of  fibers are distributed  within ±10° of this angle. To ensure the integrity of the fiber–hydrogel constructs during  the experimental period,  their  long‐term stability was  tested  in cell culture medium at  physiological temperature (37 °C) for 28 days. The condition of the incubated fiber–hy‐ drogel constructs was assessed by optical micrographs  (Figure 3e, 28 days‐time point).  Although  the  fibers were not chemically bound  to  the surface of  the hydrogel, neither  delamination of fibers from the hydrogel, nor PCL degradation nor loss of hydrogel in‐ tegrity were observed.  As established in the previous experiments, the hydrogels supported the adhesion  and proliferation of neural progenitor cells; however, these plain hydrogels were not ca‐ pable to guide and orient neural cells. To investigate the influence of topographical cues  on the behavior of neural progenitor cells and committed neurons, ReN VM cells were    Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW  6 of 14    cultured on hydrogels containing random and aligned electrospun PLC fibers and com‐ pared to the plain hydrogels. Cell viability assays showed no negative effect of the fibers  on  the cells’ metabolic activity and cell numbers within  the 14 days culture period. As  Int. J. Mol. Sci. 2022, 23, 11525 shown in Figure 4a, proliferation of ReN cells was similar on aligned fibers on hydr6oogfe1l4,  random fibers on hydrogel and on plain hydrogel scaffolds.    Figure 4.. Neurall progeniitor celllls and commiitted neurons on PCL-‐gellatiin scaffollds.. Cellll viiabiilliity  (determined by Alamar Blue assay) of (a) ReN VM cells proliferated for 14 days and (b) committed  neeurroonss ffoorr 2211 daayss oon hydrroogeellss aand hybrriid ((aalliigneed oorr rraandoomlly diissttrriibutteed)) ffiibeerrss oon hydrroogeell  ssccaaffffoollddss;; nnuummbbeerr ooff hhyyddrrooggeellss NN == 99..  During the differentiation of ReN VM cells to neurons, the cell viability on all tested  scaffolds plateaued from day 7 to day 21 (Figure 4b).. Thiiss ssttagnaattiion ooff cceellll prrolliiffeerraattiioon,,  combiined wiitthh ththee fafactc thtahta thteh oepotpictaicl aml imcriocgroragprahps h(Fsig(Fuirgeu 5rea) 5ian)dincadtiec ahteealhtheayl tahnyd aandd‐ haedrheenrte ncetlclse,l lssu,gsguegsgtes stthset hineiitniaitioanti oonf ocef lcl edllifdfeifrfeenrteinatioatni opnropcreoscse. sTs.heT hdeifdfeirfefenrteianttiioanti oonf  porfopgreongietonri tcoerllcse  lwlsasw caosncfoirnmfiermd ebdy bimymimumnousntaoisntianing inogf sopfescpifeic imficarmkaer kaetr daatyd 1a yan1da n1d4  (1a4d(daidtidointiaol nimalaigmeas gines siunpSpulepmpelenmtaernyt ianrfyorInmfaotrimona)t.i oImn)m. uImnomflunooreflsucoernecsec erenvcearlevde a lveedrya  lvoewry elxopwreesxspiornes lseivoenl loefv tehleo fntehueranle sutreaml  sctelml mcealrlkmera nrkeesrtin,e satnind, eaxnpdresxspiorens soifo tnuobfutluinb aunlidn  cahnodlicnheo alicneetyalc‐tertaynl-stfrearnasfee (rCasheA(TC)h, AhaTl)l,mhaarlklms oafr knseuofranle duirfafelrdeinffteiarteinotni aitni oanll isncaflfloslcdasf f(oFlidgs‐ u(Frieg 5ubr)e. 5Inb )a.dIndiatidodni,t tihone ,etlhoengealotinogna atinodn alnigdnamliegntm oef nteoufrintesu writaess ewviadseenvti odnen htyobnrihdy sbcraifd‐ fsocladffso (lFdisg(uFrieg u5rae,b5)a. ,bT)h.eT  ihneteintseitnys iotyf oexfpexrepsrseisosnio onf otfutbuubluinli nis iscocmompapraarbalbel efofor rbboothth ffiibbeerr–– hyydrrooggeell ssccaaffffoolldss,, aass weellll aass  tthee pllaaiin hyydrrooggeell ((FFiiggurree 55d)),, whiicch  iindiiccaatteess aa ssiimiillaarr  eeffffiiccaaccyy ooff ffiibbeerrss aanndd hhyyddrrooggeell aatt tthhee iinndduuccttiioonn ooff tthhee ddiiffffeerreennttiiaattiioonn pprroocceessss ooff hhuumaann  nneeuurraall pprrooggeenniittoorrc  eclellslsi nitnoton enueruonrosn. Ns. oNtaobtlayb, ltyh,e  tehxep reexspsiroenssoiofnC hoAf TCshhAoTw esdhotowbeed htiog hbeer  hinigthheera ilnig tnheed aslciagfnfoedld ,scpaofifnotlidn,g ptooiwntairndgs taopwraerfedrse nat piarlemfeoretonrt-inael umroontolri‐kneeduirfofenr elnikteia dtiiofnfeirn‐ ethnetisactaioffno lidns twhei tshcaalfifgonldeds wfibitehr sa,ltihgenreedb yfiibmerpsl,i ctahteinregbay pimotepnlitciaaltiinngfl uae pnocteeonftitahle itnofpluoegnrcaep hoyf  tohne tthoepdogifrfaerpehnyt ioanti othnep daitfhfweraeyn.tiation pathway.  NNeevveerrtthheelleessss,, hhyybbrriidd ffiibbeerr––hhyyddrrooggeell ssccaaffffoollddss sshhoowweedd tthheemmsseellvveess ttoo bbee eesssseennttiiaall ffoorr  iinn vviivvoo‐-lliikkee ddiirreeccttiioonnaall aalliiggnnmmeenntt ooff tthhee ddiiffffeerreennttiiaatteedd nneeuurroonnss ((FFiigguurree 55aa––cc)),, vviissiibbllee aafftteerr  tthhee 1144tthh ddaayy ooff cceellll ccuullttuurree.. MMoorree tthhaann 5500%% ooff tthhee cceellllss wweerree ppoossiittiioonneedd wwiitthhiinn aann aannggllee ooff  ddeevviiaattiioonn ooff <<1100%% wwiitthh rreeggaarrdd ttoo tthhee mmaaiinn aaxxiiss ooff tthhee ffiibbeerr ssccaaffffoolldd..  The differentiated neurons clearly followed the fiber pattern on the hydrogel and built a well oriented network at day 14 for both random and aligned fibers (Figure 5a). Neuronal bodies were slightly elongated, and neurites extended (Figure 5b). The narrower distribu- tion of neurons on the aligned PLC fiber scaffolds, in comparison to that on random fibers or hydrogel (Figure 5c) where the neurons built completely random networks, suggests that topographically aligned cues were essential for neuronal orientation. Additionally, “nerve bundle”-like arrangements, only observed along the aligned PCL fibers, imply not only that this fiber–hydrogel scaffold influences the directionality of individual neurons and their neurites, but also that it enables tissue-like association of neurons (Figure 5e). This was particularly observed when the space between PCL topographical cues was extended and at longer cultivation periods (post day 14).   Int. J. Mol. Sci. 2022In, t2.3J,. xM FoOl.RS cPi.E2E0R2 2R,E2V3,I1E1W52 5 7 of 14  7 of 14     Figure 5. Alignment of committed neurons on PCL‐gelatin scaffolds. (a) Optical micrographs and  (b) confocal micFrioggurraep5h.s−Ailmigmnmuneonsttoafinceodm wmiitthte  tdunbueulirno, nnseostninP, CCLh-AgeTla atinnds cDaAffPolId  (sg.r(eae)nO, pyetilcloalwm,  icrographs and red, blue) −of ne(bu)rocnosn foonc aallimgnicerdo,g rraanpdhosm−liym dmisutrniobsuttaeidn ePdCwL-igtheltautibnu slicna,ffnoeldstsi na,nCdh 2A1 TkPaan dgeDlaAtiPnI  (green, yellow, hydrogels at dayr e1d4,; b(cl)u He)is−toogfrnaemusr oonf saloignnamligennte dof, rnaenudriotmesl oynd fiisbterirb-uhyteddroPgCeLl -sgcaeflafotilndss,c nauffmolbdesra onfd  21 kPa gelatin cells N = 100; (dh) yIndtreongseiltsy aatndaalyysi1s4 o; f( cn)eHstiisnto agnrdam tusbouflianl igofn mneeunrtoonfs noenu ariltiegsneodn PfiCbeLr--gheyldatriong seclasfc‐affolds, number folds, number oof fimcealglsesN N= =1 090; ;(e()d )NIenutreon-sbituyndanlea layrsriasnogfemneesntitn onan adligtunbeudl iPnCoLf-gneluartoinn sscoanffoalldigsn, ed PCL-gelatin immunostained swcaitfhfo tludbs,unliunm, nbesrtionf, iCmhaAgeTs aNnd= D9;A(eP)I N(geruereon-,b yuenldlolwe a, rreadn, gbelmuee)n atto dnaayl i1g4n, eadll PsCcaLle-g  elatin scaffolds, bars = 100 μm.  immunostained with tubulin, nestin, ChAT and DAPI (green, yellow, red, blue) at day 14, all scale bars = 100 µm. The differentiated neurons clearly  followed  the  fiber pattern on  the hydrogel and  built a well ori3e.nDteidsc nuestswioonrk at day 14 for both random and aligned fibers (Figure 5a).  Neuronal bodies wNereeu rsalilgthistlsyu eesl,oinngcaluteddi,n gantdh ensepuirniatelsc oerxdte,nadreedan  (iFsoigtruorpe ic5ba)n. dThcoem npalre‐x tissues. Scaf- rower distributfioolnd sotfh naetuprroonvsi doen bthioec ahleimgniecadl PaLnCd  tfoibpeorg srcaapfhfoicldals,c iune csormespemarbisloi ng toth tohsaet soene n in the native random fibers toisrs uheysdsrtorgoenlg (lFyigsuprep o5rct) twisshueeree nthgein neeurirnognsa nbduirletg ceonmerpalteivteelym readnidcionme  [n37e]t.‐ Previous stud- works, suggestise sthfaotc tuospinogroapnhniceaulrlyal aplirgongeedn cituoers cwelelrseu epsspeonrttiianlg fosrc anfefuorldonsarle oproiertnetdattiohne. importance of Additionally, “hnyedrrvoeg eblupnrdolpee”r‐tliiekses uarcrhaansgelmasetnictsm, odnulylu osbosnertvhedir adlioffnegre nthtiea taiolingnsuedcc ePsCsLa nd the suitabil- fibers, imply niotty oonfltyo pthoagtr tahpish ifcibalerc–uheysdfororgthele sscuacffcoelsds fiunlflauliegnncmese tnhteo df inreeuctriaolncaelliltsy [o1f7 ,i3n8‐,39]. For many dividual neuroinmsp alnedm tehnetierd nneueurirtoens,a blumt oadlseol st,htahte itu esneadbslceas ftfioslsduse‐eliitkhee arslsaocckiatotipoong orfa npehuic‐al cues [40–42] rons (Figure 5et)o. Trehpisl iwcaatse ptharetiacnuilsaortlryo opbicsenravteudr ewohfesnp tihnea lspcoacrde ,boertwtheeens tPifCfnLe tsospfoogrrtaispshu‐e-relevant sub- ical cues was esxttreantdesed[1 a6n,4d3 ]a.tO lounrgceorm cuplotisvitaetifiobne pr–erhiyoddrso (gpeolsst cdaaffyo 1ld4s). provide the appropriate stiffness as well as alignment cues to support both the differentiation of the neural progenitor cell 3. Discussion  line ReN cell VM into neurons and their directional alignment. The human progenitor cell Neural tislsiuneesu, sinedcluhderiengca tnheb espdiinffaelr ceonrtdia, taerde saimniusolttarnopeoicu aslnydo crosmelpecletixv etilsysuinetso. Snceaufr‐ons, astrocytes folds that provaidned boiloicghoedmenicdarlo acnydte tso,paovgoridapinhgictahle cuusees orefseexmpbenlisnigv eth, loasbeo sre-iennt einn stihvee naantdivlei mited primary tissues stronglyc eslulsp.pTohret tsiusscucee sesnfugilnineeterignrga tainond rweigtehntehreathiveree mperdesiceinntee d[37fi]b. ePrr–ehvyioduros gsteuldsc‐affolds will be ies focusing onb neenuerfiacli aplrofogrecneitllocr oc-eclul lstuuprepsoartsisnagy sscianfffoulrdths errepstourdteides t.he importance of hy‐ drogel propertpies su The fabri roducchin ags cerloass cation of fibe stliicn mkeoddguelulast  ro–hydroinnh tyhderiro  gdeiflfceogels wre mnphit o ciha stiiotens scuacfcfoelsds was successfully achieved byserved as ths eancodl ltehceti sounitsaubbisl‐trate for electro- ity of topograpshpiucanl PcCuLesfi fboerr tsh. eF usurtchceersmsfourle a, ltihgenmmeecnht aonfi cnaelusrtaalb cileitlyls o[f1t7h,3e82,319k]P. Faohry mdraongye l would enable implemented nceounrvoennaile nmtohdaenlsd,l itnhge ufosredsu srcgaefofonlsdsin eaithcelirn liacackl  steotptionggr.aTphheicsatli fcfuneess s[4o0f–o4u2]r gelatin-based to replicate theh ayndirsoogtreolspwic ansaitnurthe eorf asnpgineaolf ctohradt, toyrp itchael lsytioffbnseesrsv efodr ftoisrsluoew‐rpeleervcaenntt asguebp‐ orcine Type A strates [16,43]. Our composite fiber–hydrogel scaffolds provide the appropriate stiffness    Int. J. Mol. Sci. 2022, 23, 11525 8 of 14 gelatin crosslinked by TG and other comparable GelMa based hydrogels [33,35]. Likewise, with PCL fibers added, our composite scaffolds showed the elastic modulus of neural tissue, e.g., spinal cord with 20–100 kPA [23–25,44], and provided a suitable substrate for cell proliferation and differentiation of human progenitor cells. The additional laminin coating guaranteed cell adhesion since, in non-coated scaffolds, neural cells tend to detach very fast due to the lack of adhesion motifs [35,45]. For anisotropic scaffold design, the PCL fiber deposition was performed in an aligned fashion directly on the hydrogel. The typical electrospinning-engineered submicron PCL fiber diameter was achieved for aligned as well as randomly distributed fibers. Our homemade gap-based electrospinning setup yielded highly parallel fiber distribution, comparable to alignment distributions achieved using a rotating mandrel [46,47], with the advantage of being amenable to a broad range of substrates as collectors. In comparison, the use of hydrogels in a rotating drum is typically challenging due to (1) the limited mechanical stability of hydrogels when used as substrates upon the high rotation speed of the drum required for sufficient alignment, and (2) the diffi- cult transfer of a fragile fiber mesh without destruction of the fibers themselves, and of the generated aligned pattern onto the designated hydrogel, when fibers are spun on a collector plate and not directly on the hydrogel [48,49]. The technical challenge of depositing highly aligned fibers on a not perfectly flat surface, such as that of a hydrogel, was overcome by optimizing the needle-collector distance, placement of collection substrate and polymer flow rate. This resulted in PCL fiber–hydrogel scaffolds with more than 90% of fibers within ±12◦, superior to those found on 3D collectors substrates achieving more than 80% of fibers within ±20◦ deviation to a reference line [9,50]. The topographical sub-micrometer cues provided by the engineered scaffolds were in the same range as observed in collagen fibers (20–500 nm) [6] of the native extracellular matrix and acted as a trigger of in vivo-like cell alignment [51,52]. Our fabrication process also allowed control of the fiber density in order to avoid highly dense fiber meshes that can hinder the contact of cells to the underlying hy- drogel substrate and therefore diminish the positive influence of in vivo-relevant hydrogel stiffness [46,53]. Hence, with the optimized setting, we can manipulate the balance between number of topographical cues and inter-fiber spacing on the hydrogel, an advantage for the production of micro topographies by molding or hot embossing where a new expensive mask is required for every new casting. The tuning of alignment sites in our scaffolds is leading on the one hand to an alignment of individual neurites and, on the other hand, to an aligned growth of several neurons. The individual neurite growth clearly follows the undulating shape of the PCL fibers (Figure 5a,b) and is therefore a clear indication of to- pography being the main alignment cue. In addition to the topographical cues, the aligned neuronal and neuron bundle growth could also be triggered by an anisotropic elasticity in the hydrogel, eventually induced by the deposition of aligned PCL fibers [54–56]. Further detailed local elasticity measurements would, however, be necessary to reveal the presence of an elastic anisotropy in the hydrogel and assess its mechanotransduction pathways in the alignment process. The results presented here confirm the importance of scaffold design as previously shown for in vivo PCL-based scaffolds by Wong et al. [21], indicating that the association and orientation of neurons into “nerve bundles” for successful spinal cord regeneration is influenced by topographical submicron cues. The biocompatibility of our fiber–hydrogel scaffold for ReN VM cells is in accordance with plain gelatin-based scaffolds [35] but offers the additional feature of topographical cues. When we compare cells on scaffolds based on random PCL fibers on glass, which have been shown to promote differentiation but no alignment [16], our scaffolds led to a comparable expression profile of neuronal differentiation markers, in addition to the successful spatial alignment. The alignment of cells along the fibers, quantified by the deviation angle of the cells’ main axis to the fiber scaffold, is similar to PCL scaffolds previously reported in the literature for other neural progenitor cell lines [46,57]. The differentiation of neural progenitor cells into neurons on the scaffold was verified by confocal imaging of tubulin ß III, an early differentiation marker in the cytoskeleton. Significant tubulin ß III expression was observed from day 14 on. These results are in accordance with previously published data showing Int. J. Mol. Sci. 2022, 23, 11525 9 of 14 that the differentiation of ReN VM cells leads to significantly increased levels of tubulin ß III in the neural cell population over 14 days [16,58]. Choline acetyltransferase (ChAT), the enzyme producing the neurotransmitter acetylcholine in motoneurons [59], was pre- dominately expressed from day 7 on in the differentiated ReN VM cells, indicating that they preferentially followed a motoneuronal differentiation pathway. This was particularly observed in the aligned scaffolds, implicating a potential influence of the topography on the differentiation pathway. This is of particular interest in tissue engineering due to the demand for models of traumatic injuries like spinal cord injury, since the intrinsic regrowth ability of motoneurons is higher than for sensory neurons, and thereby offers an attractive target to regenerate functional circuits within the central nervous system [60]. Here, and for the first time, the neural progenitor ReN VM cell line was differentiated into directionally aligned motor neuron-like cells on an optimized fiber–hydrogel scaffold, displaying tissue-relevant stiffness and oriented fibers serving as topographical cues for cell alignment. This novel approach for scaffold manufacturing provides an optimized set of cues that allows differentiation of the neural progenitor cells in vitro. 4. Materials and Methods 4.1. Production of Gelatin Hydrogels Gelatin (from porcine skin, Sigma G1890, St. Louis, MI, USA) was dissolved at concentrations of 10%, 15% and 20% w/w in PBS (Sigma, 806552) and the pH adjusted to 7.4. Transglutaminase (Ajinomoto) with a concentration of 50 mg/mL was added 1/9 v/v to the gelatin solution and incubated for 12 h at 37 ◦C for enzymatic crosslinking. The gels were produced in a 24-well plate format with an average thickness of 2.0 mm ± 0.2 mm and stored in PBS at 4 ◦C until further use. All work involving cell culture was conducted under sterile conditions. 4.2. Fiber Spinning Prior to electrospinning, polycaprolactone (PCL, Sigma, 440744) was dissolved as a 20% w/w solution in 30/70% v/v acetic acid/acetone (Sigma, 695092/Sigma, 34850). The electrospinning setup was custom made and included a high voltage generator (AIP Wild, FUG HCB), a syringe pump (TSE systems, 540080) and a copper collector plate. The following optimized electrospinning parameters were employed for the spinning solutions: 20 kV voltage, 15 cm distance from needle to collector, 1.0 mL/h polymer flow rate, and 1-5 min spinning time. For the aligned deposition of fibers, the distance between the copper rods, placed on either side of the collector glass slide, was between 3 and 5 cm. The spinning was performed on thin pre-crosslinked gelatin-transglutaminase hydrogels. For cell culture experiments, the fiber–hydrogels were punched out with biopsy punchers to the required size and sterilized (20 min UV) prior to use. All hydrogel fiber scaffolds were coated with 10 µg/mL laminin (Merck, L2020, Kenilworth, NJ, USA) for 2 h at 37 ◦C. 4.3. Characterization of Hydrogel–Fiber Scaffold The stiffness of the hydrogels was determined using a nano indenter (Optics11, Pi- uma, Amsterdam, The Netherlands). Hydrogel samples, gelatin only and PCL-gelatin fiber–hydrogels, were both immersed in PBS and kept at 37 ◦C during the measurement. Indentations were performed using a cantilever (Optics11, P210388M), with geo factor 2.52 in air, 0.28 N/m spring constant and a spherical tip (r = 53 µm), in displacement mode. Each sample was indented in scan mode 3 × 3 with (dx, dy = 2000 µm) thrice. The data fitting and calculation of the elastic modulus was based on the Hertzian-contact model. Optical micrographs were taken with an Olympus IX73 inverted microscope. The fiber diameter and alignment were analyzed on optical micrographs with ImageJ using the directionality plug-in. The “Fourier components” analysis method with number of bins Nbin = 90 from −90◦ to 90◦ was applied on 8-bit images and the data plotted as fre- quency distributions. Different fiber densities resulting from varying spinning parameters Int. J. Mol. Sci. 2022, 23, 11525 10 of 14 (time = 1 min, 3 min, 5 min; distance = 15, 20 cm) were determined from segmented bimodal images (N = 9) by using ImageJ [46,47]. Three-dimensional images of fiber–hydrogel composites were taken with a confocal microscope (Olympus, FV3000, Tokyo, Japan). The gelatin hydrogel was stained with 1/100 flamingo protein gel stain (Bio-Rad, 1610491, Hercules, CA, USA) and then used as base material for electrospinning. PCL was mixed with 1 µg/mL Nile red (Sigma, N31013) solution and used as previously described for electrospinning. 4.4. Neural Cell Culture The human neural progenitor cell line ReN VM (Sigma Aldrich, SSC08) was cul- tured in proliferation medium composed of DMEM/F12 medium (Dulbecco’s modified Eagle’s medium/Nutrient mix F-12, Gibco, 11320, Waltham, MA, USA) supplemented with B27 supplement (Gibco, 17504044), Heparin (Stemcell technologies, 07980, Vancou- ver, Canada), EGF (Sino Biologics, 10605-HNAE, Beijing, China), bFGF (Thermo Fisher, PHG0024, Waltham, MA, USA) and 1.0% penicillin-streptomycin (Gibco, 15140122). For all in vitro experiments, cells at passage 10–20 were used. Throughout the cultivation period, the cells were incubated at 37 ◦C and 5% CO2 and the medium was exchanged every two or three days. Prior to seeding of ReN VM cells, cell culture flasks were coated with 10 µg/mL laminin (Merck, L2020, Kenilworth, NJ, USA) for 2 h at 37 ◦C to promote cell adhesion. To induce neuronal differentiation of cells on the scaffold, the medium was changed to specific neuronal differentiation medium composed of neurobasal medium (Gibco, 21103049) supplemented with B27 supplement (Gibco, 17504044), GlutaMax (Gibco, 35050038) and 1.0% penicillin streptomycin (Gibco, 15140122) at day 1 [61]. 4.5. Characterization of Cell Adhesion, Proliferation and Differentiation To characterize the biocompatibility of the scaffolds and study the cell behavior on the scaffolds, ReN cells were detached from the cell culture flask using Accutase (Gibco, A1110501) and then seeded onto the scaffolds with a density of 10,000 cells/scaffold. The adhesion of ReN VM cells on the scaffolds was analyzed by optical microscopy and images taken 24 h after seeding, with different magnifications. The proliferation of cells was determined by measuring the metabolic activity with an Alamar Blue assay (Thermo Fisher, DAL1025, Waltham, MA, USA). After 2 h of incubation, the fluorescence was measured at 560 nm with a plate reader (Agilent BioTek Synergy H4). For immunofluorescence staining (after 1/7/14/21/28 days), cells were fixed at room temperature in 4% PFA solution (EM-grade, EMS, 15712), permeabilized with 0.2% Triton X-100 (Sigma, 94326) and blocked with 3% BSA (Sigma, A4503). After washing three times with PBS, the cells were incubated with the primary antibody solution overnight at 4 ◦C, followed by the secondary antibody solution at room temperature for 2 h. ReN VM cells were probed with the primary antibody anti-tubulin (Novus Bio, NB100 1612, 1/500, Centennial, CO, USA) with an anti-goat Alexa Fluor 555 secondary antibody (Abbexa, abx142427, 1/500, Cambridge, UK), anti-ChAT (Abcam, ab32454, 1/500, Cambridge, UK) with an anti-sheep Alexa Fluor 568 secondary antibody (Abcam, ab175712, 1/500) and anti-Nestin conjugated to Alexa Fluor 488 antibody (Stemcell, 60091AD, 1/500, Vancouver, Canada). After three washing steps with PBS, the cells were incubated at room temperature for 5 min with 4′6 diamidino-2-phenylindole (DAPI, Invitrogen, 1:1000, Waltham, MA, USA) to stain the nuclei. For imaging with a confocal microscope, the cells on hydrogels were mounted on a glass slide with antifade mounting medium (Thermo Fisher, P36982, Waltham, MA, USA). The quantification and intensity analysis of stained cells (Day 0, Day 14) were performed using CellProfiler [62], applying the ‘IdentifyPrimaryObjects’ and ‘MeasureObjectIntensity’ modules. 5. Conclusions Enzymatically crosslinked gelatin-based hydrogels were generated with different stiffnesses, which successfully supported the adhesion, proliferation and differentiation of Int. J. Mol. Sci. 2022, 23, 11525 11 of 14 a human neural progenitor cell line. Utilizing electrospinning as a tool to add anisotropic features to the hydrogels, a sparse monolayer of polycaprolactone fibers was spun on top of the gel in either a random or aligned fashion without significantly altering the stiffness of the hydrogel substrate. On the fiber–hydrogel scaffolds, the progenitor ReN cells attached, proliferated and—after induction of differentiation—aligned their neurites along the polycaprolactone filaments. On these fiber–hydrogel scaffolds we could observe, for the first time using differentiating ReN cells, a directed organization at the single cell level (neurite extension), as well as at the cellular network level (“nerve bundles”). The production of fiber–hydrogel scaffolds offers great potential for the fabrication of scaffolds for tissue engineering and regenerative medicine, combining the advantages of hydrogels (e.g., stiffness) with electrospun fibers replicating a native extracellular matrix and introducing topographical cues. Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms231911525/s1. Author Contributions: The research was designed by L.M., A.B.F.-T. and M.R.G.; L.M. and F.Z. performed the experimental work with the help of J.S.; L.M., M.R.G. and L.S.-D. analyzed and interpreted the data. L.M. prepared the manuscript in consultation with A.B.F.-T., J.R., L.S.-D. and M.R.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the FHNW HLS Research fund. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data supporting reported results can be found at https://drive.switch. ch/index.php/apps/files/?dir=/Institution/Paper%20Neodent&fileid=4940792059 (accessed on 24 July 2022). Acknowledgments: The authors acknowledge the help of Franziska Koch with the measurements at the nanoindenter. 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