polymers Article Fabrication and Characterization of PCL/HA Filament as a 3D Printing Material Using Thermal Extrusion Technology for Bone Tissue Engineering Fengze Wang 1,2, Esma Bahar Tankus 1,2, Francesco Santarella 1,2 , Nadja Rohr 3 , Neha Sharma 1,2,4 , Sabrina Märtin 5 , Mirja Michalscheck 1,2,4, Michaela Maintz 1,2,6, Shuaishuai Cao 1,2,7,* and Florian M. Thieringer 1,2,4,* 1 MIRACLE Smart Implants Group, Department of Biomedical Engineering, University of Basel, 4123 Allschwil, Switzerland; fengze.wang@unibas.ch (F.W.); esma.tankus@unibas.ch (E.B.T.); francesco.santarella@unibas.ch (F.S.); neha.sharma@usb.ch (N.S.); mirja.michalscheck@unibas.ch (M.M.); michaela.maintz@unibas.ch (M.M.) 2 Medical Additive Manufacturing Research Group (Swiss MAM), Department of Biomedical Engineering, University of Basel, 4123 Allschwil, Switzerland 3 Biomaterials and Technology, Department of Reconstructive Dentistry, University Center for Dental Medicine Basel UZB, University of Basel, 4058 Basel, Switzerland; nadja.rohr@unibas.ch 4 Clinic of Oral and Cranio-Maxillofacial Surgery, University Hospital Basel, 4031 Basel, Switzerland 5 Biomaterials and Technology, Department of Research, University Center of Dental Medicine Basel UZB,  University of Basel, 4058 Basel, Switzerland; sabrina.maertin@uzb.ch  6 Institute for Medical Engineering and Medical Informatics, Citation: Wang, F.; Tankus, E.B.; University of Applied Sciences and Arts of Northwestern Switzerland, 4132 Muttenz, Switzerland 7 Santarella, F.; Rohr, N.; Sharma, N.; Department of Stomatology, Shenzhen University General Hospital and Shenzhen University Clinical Medical Academy, Märtin, S.; Michalscheck, M.; Maintz, Shenzhen University, Shenzhen 518071, China M.; Cao, S.; Thieringer, F.M. * Correspondence: shuaishuai.cao@unibas.ch (S.C.); florian.thieringer@usb.ch (F.M.T.) Fabrication and Characterization of PCL/HA Filament as a 3D Printing Abstract: The most common three-dimensional (3D) printing method is material extrusion, where Material Using Thermal Extrusion Technology for Bone Tissue a pre-made filament is deposited layer-by-layer. In recent years, low-cost polycaprolactone (PCL) Engineering. Polymers 2022, 14, 669. material has increasingly been used in 3D printing, exhibiting a sufficiently high quality for consider- https://doi.org/10.3390/ ation in cranio-maxillofacial reconstructions. To increase osteoconductivity, prefabricated filaments polym14040669 for bone repair based on PCL can be supplemented with hydroxyapatite (HA). However, few reports on PCL/HA composite filaments for material extrusion applications have been documented. In Academic Editor: Ali Reza this study, solvent-free fabrication for PCL/HA composite filaments (HA 0%, 5%, 10%, 15%, 20%, Zanjanijam and 25% weight/weight PCL) was addressed, and parameters for scaffold fabrication in a desktop Received: 21 December 2021 3D printer were confirmed. Filaments and scaffold fabrication temperatures rose with increased Accepted: 6 February 2022 HA content. The pore size and porosity of the six groups’ scaffolds were similar to each other, and Published: 11 February 2022 all had highly interconnected structures. Six groups’ scaffolds were evaluated by measuring the Publisher’s Note: MDPI stays neutral compressive strength, elastic modulus, water contact angle, and morphology. A higher amount of with regard to jurisdictional claims in HA increased surface roughness and hydrophilicity compared to PCL scaffolds. The increase in HA published maps and institutional affil- content improved the compressive strength and elastic modulus. The obtained data provide the basis iations. for the biological evaluation and future clinical applications of PCL/HA material. Keywords: polycaprolactone (PCL); hydroxyapatite (HA); material extrusion; three-dimensional printing; scaffold; hydrophilicity; mechanical testing Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 1. Introduction Attribution (CC BY) license (https:// Oral and maxillofacial tumors and trauma often lead to different degrees of jaw defects, creativecommons.org/licenses/by/ and their reconstruction remains a challenging task [1]. Autogenous or allogeneic bone 4.0/). Polymers 2022, 14, 669. https://doi.org/10.3390/polym14040669 https://www.mdpi.com/journal/polymers Polymers 2022, 14, 669 2 of 14 transplants are commonly used to rehabilitate jaw defects [2–4]. Due to the limited avail- ability of bone material, various biomaterials have been applied to generate scaffolds using three-dimensional (3D) printing [5]. Among the biocompatible materials, polycaprolactone (PCL) has attracted much in bone tissue engineering and is widely used in 3D printing, enabling the fabrication of complex patient-specific and biomimetic structures [6]. PCL is a resorbable polymer with a low production cost, and it can be combined with osteocon- ductive materials such as hydroxyapatite (HA) [7–9]. Composites comprise two or more materials, and the goal is to create more efficient scaffolds by combining the regenerative properties of more than one biomaterial [10]. Furthermore, composites containing HA and polymers combine good mechanical properties with good biocompatibility, yielding a 3D substitute that mimics the heterogeneity and hierarchical structure of the native extracellular bone matrix [11,12]. Fabrication methods for PCL/HA porous biodegradable polymer scaffolds include thermally induced phase separation (TIPS), salt leaching, gas forming, freeze-drying, and 3D printing technology [13,14]. However, compared with the above methods, only 3D printing technology enables the control of the internal structures of complex implants [15]. This technique is currently being explored for surgical bone replacement [16]. Three types of 3D printers have been considered for bone reconstruction: stereolithography (SLA), material extrusion, and selective laser sintering (SLS). SLA uses only photoactivatable biopolymers [17], whereas the SLS method suffers from a limited choice of materials [18]. Compared with other 3D printing technologies, material-extrusion is a scalable industrial route that does not require a solvent and optimizes the filler (HA) distribution [19]. With material extrusion printing, a prefabricated filament is loaded and extruded from a hot nozzle to a workbench. The extruded filament hardens at room temperature with cool- ing fans to form a solid structure in a layer-by-layer pattern [20]. Material extrusion printing is suitable for printing patient-specific implants and easy-to-scale manufacturing technol- ogy, and constitutes the vast majority of the 3D printer market. However, the material extrusion method has not been extensively investigated for scaffold fabrication in max- illofacial reconstruction [21,22]. Melting material for the fabrication of a single composite filament is an affordable and cost-effective method. The procurement cost of PCL powder is about USD 2 per gram, whereas HA powder is USD 4 per gram [23]. Hence, PCL/HA filament fabrication material costs were around USD 2.1 to 2.5 per gram, depending on the proportion (0–25%) of HA added. Filament fabrication favorably uses a thermal procedure, having the advantage of producing solvent-free and eco-friendly filaments with no harmful effects (such as cell death) derived from solvent residuals [24,25]. Although solvent-casting techniques are commonly used to fabricate PCL composite filaments, these techniques suffer from inherent limitations, such as the challenging fabrication process associated with the leaching of solvent residues in the fabricated scaffolds; thus, it might not be suitable for scaffold fabrication in hospital settings [26–29]. A hot-melt filament extrusion machine would include HA and fabricate the composite filament in a cost-contained situation [30,31]. Nonetheless, after obtaining a successful filament, the optimum build orientation, layer thickness, nozzle diameter, infill pattern, and bed temperature are necessary to fulfill this need [32]. Hot-melt PCL/HA filament fabrication and the scaffolds fabricated from this process are understudied; to date, only scant information is available on the fabrication settings for machines in research environments [33]. Additionally, further studies should be conducted to assess the optimal HA percentage for osteoconductive applications. This study aimed to test the feasibility of filament fabrication for an osteoconductive material (PCL-HA) and printing parameters with an affordable material on an extrusion- based 3D printer. Filaments of PCL with different HA percentage contents were used to test the effect on printing parameters. Compressive strength, elastic modulus, water contact angle, and morphological analysis parameters were obtained as quality indications for implantability. Polymers 2022, 14, x FOR PEER REVIEW 3 of 14 P olymers 2022, 14, 669 3 of 14 22.. MMaatteerriiaallss aanndd MMeetthhooddss 22..11.. PPCCLL aanndd HHAA CCoommppoossiittee FFiillaammeenntt FFaabbrriiccaattiioonn SSiixx ddififfefererennt tw weiegihgthpt rporpooprotirotinosnosf oHf AH(Aav (earvaegreapgoe wpdoewrdseizre s: i1z5e:µ 1m5 ,μNman, jNinagnEjimngp eErmor- Npearnoor MNaatneori Mal aCtoer.,iaLlt dC.o, .N, Latndji.n, Ng,aCnhjiinnga,) C0%hi,n5a%) 0, %10,% 5%, 1,5 1%0%, 2,0 1%5%an, 2d0,%25 a%ndw,/ w25w%e wre/wm wixeerde wmiitxhePdC wLit(hm PolCeLcu (lmarowleeciuglhatr: w50e,i0g0h0t:g 5/0m,0o0l0, pgo/mwdole,r psoizwed: 1e2r 0siµzme: ,1ρ20= μ1.m14, 6ρg =/ 1m.1L4,6G ga/omJLu, mGaatoe rJiua lmCaote.,rLiatld C.,oG.,u Latndg.,z Ghouua,nCgzhhinoau),. 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Fuorutre mtepmepraertuatruerge rgardaideinetnst(sT (4T–4T–1T,1w, whhereereT T44i sist thhee fifirrsstt meellttiinnggs sppoottc lcolosessetstto toth tehheo hpoppepr,efro, llfowlloewd ebdy Tb3y, T32,, tTh2e,n thTe1n, tThe1,e txhitem exeiltti nmgepltoiningt )p, owinerte), awdejurest eadjtuost1e0d% toh i1g0h%er hthigahnetrh tehPaCn Lt’hsed PeCclLar’se ddemcelaltriendg mteemltpinegra teumrep(e6r0a◦tuCre→ (6606 °◦C )→an 6d6 t°hCe) eaxntrdu tdheer eroxtrautidoenra lrostpaeteiodnoafl tshpeeesidn golfe t-hscer esiwngelxet-rsucrdeewr wexatsruinditeira lwlyaas dinjuitsitaeldlya atd5juRsPtMed taot e5n RsuPrMe tthoa etnmsuarte rtihaal tc amnabteerieaxl tcraund ebde. eFxitlraumdedn.t sFitlhaamt ednidts nthoattc domidp nlyotw coitmhpalyc iwrciuthla ra schiracpuelawr esrheaepxec wluedreed e.xFcilluadmeedn. tFciolaomlinengt wcoaoslpinegrf owrams epdewrfoitrhmaed uwailt-hfa na sdyusatle-mfan(c soyosltienmg f(acnooslpinege dfaant s1p0e0e%d) atto 1a0c0h%ie) vtoe ascohliidevifiec saotiloidni.ficTahtieofin.n Tahl efo fuinraml feolutirn mg ezlotinnegt zeomnpee treamtuprers- (aTt4u–rTe1s )(oTf4e–aTc1h) gorfo euapchw geroeudpe tweremrein deedtebrymdiencerdea bsyin dgetchreetaesminpge trhateu treembpye1ra◦tCuirne ebayc h1 c°yCc lien aenadchd ceyccrelea saindg dtheecreexatsruinsgio tnhesp eexetdru(sRioPnM s)puenetdil (tRhPeMdi)a umnettiel rthseta dbialimzeedteart s1t.a7b5il±ize0d.0 a5tm 1.m75, a±n 0d.0w5 emremc,o annsdid wereerde caofnusnidcteiroenda al dfuianmcteiotenraflo driammaeteterria floerx mtrautseiroinal 3eDxtrpursiniotnin 3gD[ 3p4r]i.nTtihneg fi[3la4m]. eTnhtew fialsamtheent lwinakse dthteona lipnuklelder ttoo ac oplulelcletrt htoe pcorolldecutc tth(eF ipgruordeu1c).t A(Ffitgeur rteh e1)p. uAllfeterrh tahde sptaubllielirz headdt hsteabdiilaizmeedt ethreo df itahme efitlearm oef nthtse wfiiltahminenthtse wtaitrhgient tdhiea mtaergteert rdainagmeeftoerr r1a0nmgei nfo, rth 1e0 wmiinnd, ethr ew wasinsdeetrt wo assp oseotl ttoh sepfioloalm theen ftisl.amFielanmts.e Fnitlsamweenretst wheenres tthoerend stionreZdip inlo Zc ibpalogcs baangds darnided dfroierd1 5fomr 1in5 mwiitnh waivtha cau vuamcudurmye drr(yPeirs t(oPnisptounm ppum40p6 G40, 6RGec, iRpercoitporro, tDoer,n Dmeanrmk)abrke)f obree- pforirnet ipnrgin. tEinxgt.r uEsxitornussipoene dspreeefde rrsetfoertsh teo stchree wscrreowta triootnaatliosnpaele sdp(eRePdM (R)PcMon)t rcoolnletrdolbleydt hbey 3tdhee v3odmevaoc hminaceh, winhee, rweahsertheaesfi tlhaem feilnatmeexntrtu esxiotrnusspioene dspweeads cwalacsu claatlecduloantetdh oene xthtreu edxetdruPdCeLd fiPlCamL efinlatmleenngtt hlepnegrthse pceorn sdec(monmd/ (sm).m/s). FFiiggurree 11.. Fiillameentt ffabrriiccaattiioon aand maatteerriiaall eexxttrruussiioonn--bbaasseedd3 3DDp prriinnttiinngg.. 44,, 33,, 22,, 11:: sseeparrattee zzoneess iin tthhee fifillaammeenntt--mmaakkeerr. .T Thheen neewwc coommppoossiitteefi fillaammeenntti issc coolllleecctteedda anndd uusseedd ffoorr 33DD pprriinnttiinngg ffoolllloowwiinngg tthhee rreepprreesseenntteeddG G-c-cooddeefi fliele, ,g giviviningga ap prordoduuctcot fo1f 515× ×1 155× × 22 3 mmmm3.. TThhee pprriinntteedd ssccaaffffoolldd iiss tteesstteedd ffoorr SSEEMM ((ssccaannnniinngg eelleeccttrroonnm miiccrroossccooppyy)),,c coommpprreessssiioonn,,a annddw weettttaabbiilliittyyp prrooppeerrttieiess.. TThhee ssaammee fifillaammeenntt iiss uusseedd for potential application in the bone replacement model. for potential application in the bone replacement model. 22..22.. 33D Prriinttiingg ooff Sccaaffffoollddss A 3D ssccaffffolld modell wass develloped ((Tiinkerrccad,, Auttodessk,, San Frranciissco,, CA,, USA,, aand PrusaSlliicer,, Versiion 2..3..1,, PPrraagguuee, ,CCzzeecchh RRepepuublbilci)c. )T. hTeh peapramraemteertse orsf tohfet hGe-cGod-ceo fdilee fiinlecliundceludd tehde tlhaeyelra yheerighheti g(1h0t0( 1μ0m0 )µ wmi)thw aitnh iannfililn dfiellndsietyn soitfy 9o0f%9. 0T%h.e Tinhfeilli npfialtlteprant twerans wchaosscehno asse na garsida wgriitdh awni tihnfailnl aingfilel annagmledn ianm tehde sinoftwhearseo fatsw “a0r/e12a0s°”“ 0to/ 1a2v0o◦i”d tiontaevrfoiliad- imnteenrfitoluams deinstoanucsed cilsotasunrce dcluoes utor emdeulteintgo. mSuebltsienqgu. eSnutblys,e tqhuee snixtl yd,itfhfeeresnixt gdrifofuerpesn’ tscgarfofuolpdss’ Polymers 2022, 14, 669 4 of 14 scaffolds were printed using a printer (Prusa Mini, Prague, Czech Republic) with a nozzle of 400 µm (Figure 1). After loading the filament into the Prusa Mini printer, parameters, such as the printing speed, nozzle temperature, heat bed temperature, and nozzle flow factors (amount of material flowing through the nozzle) were adjusted, and the print fan speed was set to 255 RPM at room temperature (20 ◦C) to cool the scaffolds. Three different sizes of six groups’ scaffolds were printed—square solid specimens (15 × 15 × 2 mm3, n = 5, for surface roughness measurements and water contact angle test), square porous scaffolds (15 × 15 × 2 mm3, n = 3, for morphological analysis) and square porous scaffolds (15 × 15 × 4 mm3, n = 3, for the mechanical test)—according to the test needed. After production, scaffolds were stored in a desiccator with silica gel before use. 2.3. Surface Roughness and Pore Size Quantification Five solid specimens for each group were printed to measure the surface roughness parameters using a 3D laser scanning microscope (VK-X-1050, magnification 50×, Keyence, Osaka, Japan). Three sites of 200 µm× 200 µm per scaffold were obtained, and after surface shape correction and height cuts, a 0.8 µm Gaussian S-filter was applied. The arithmetical mean height (Sa) and the maximum height of surface (Sz) were extracted using the multi-file analyser software (Version 2.1.3.89). The pore size of porous scaffolds (n = 3) was deter- mined by considering 30 pores for each scaffold using a 3D laser scanning microscope [9]. The porosity of the scaffolds was obtained following the equation: porosity (%) = 1-scaffold density/arithmetic mean density. The scaffold density was defined as the weight of the scaffold divided by the volume of the scaffold. The mean densities of PCL and HA were 1.146 g/cm3 and 3.16 g/cm3, respectively [36]. 2.4. Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy Images of the surface morphology of the porous scaffolds were obtained using scan- ning electron microscopy (SEM, XL30, Philips, Eindhoven, The Netherlands) at an accel- erating voltage of 10 kV and 50× magnification. Additionally, energy-dispersive X-ray spectroscopy (EDX) was performed to determine the elements present on the surface of the scaffolds at 20 kV. 2.5. Water Contact Angle The hydrophilicity of the scaffold materials was tested on three solid specimens per group using a drop shape analyzer (DSA100, Krüss, Hamburg, Germany). The specimens were cleaned in an ultrasonic bath (TPC-15, Telsonic Ultrasonic, Bronschhofen, Switzerland) with 70% ethanol for 4 min. Three 2 µL drops of ultrapure water were applied to each specimen using the sessile drop technique. After drop placement, the angle between the specimen surface and the contour of the drop was analyzed as the contact angle. The mean of the right and left contact angles were calculated for each specimen. 2.6. Mechanical Test The compressive strength of the scaffolds (15 × 15 × 4 mm3) was evaluated using a universal testing machine (Z020, Zwick/Roell, Ulm, Germany) at a crosshead speed of 1 mm/min. Compressive strength (MPa) was calculated as the maximum applied load (N)/compressed area of the surface (mm2) after a 20% deformation of the scaffolds. The elastic modulus of the samples was additionally recorded between 2% and 5% deformation. 2.7. Statistical Analysis Data were expressed as the mean ± standard error of the mean (s.e.m). The data were analyzed with a one-way analysis of variance (ANOVA), and post hoc Tukey’s test was performed to compare the statistical difference. The level of significance was set to p < 0.05. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA). Polymers 2022, 14, 669 5 of 14 3. Results 3.1. Filament Fabrication Filament extrusion was successfully achieved by tuning the four melting points of the filament-maker (3devo) (Table 1). The PCL melting temperature was gradually increased by increasing the amount of HA. The first melting point (T4) allowed a pre-melting of the PCL powder; therefore, this PCL temperature plus 10% variation was set when supple- mented with HA powder. The intermediate melting points (T3 and T2) were set at higher temperatures, from 64 ◦C to 70 ◦C, to allow interspersion of HA particles into the PCL matrix. The exit melting point temperature (T1) was intended to achieve correct filament extrusion from the filament-maker nozzle. T1 was generally at a lower temperature than the other points. The extruder rotational speed was initially set at 5 RPM and reduced to a range of 2—2.9 RPM on a trial-and-error basis. The applied parameters for each 5% increase in HA content in PCL are displayed in Table 1. In general, 61 ◦C to 69 ◦C was required when HA was present in the powder mix versus 60 ◦C when pristine PCL powder was used. Table 1. Parameters of the filament fabrication. Temperature Gradients/◦C Groups Extruder RPM & T4 T3 T2 T1 Filament (mm/s) Speed PCL 60 64 64 62 2.0 (6.7) PCL + 5% HA 61 66 66 67 2.9 (10.3) PCL + 10% HA 69 70 70 69 2.5 (9.7) PCL + 15% HA 67 67 66 65 2.4 (8.8) PCL + 20% HA 65 67 67 65 2.5 (9.7) PCL + 25% HA 61 66 67 66 2.0 (6.7) RPM: revolution(s) per minute. extruder rotational speed. Filament extrusion speed (mm/s) is expressed in brackets. T1–T4: Four separate zones’ temperature in the filament-maker. 3.2. Printing Procedure Table 2 describes which temperature, speed, and flow factors can be used according to the HA content. From pure PCL filament to 25% HA content, an increase of 30 ◦C, and minor fluctuations in printing speed and flow factor were recorded. The applied parameters reported in Table 2 were used to print the six groups’ scaffolds, maintaining a mean pore size of 550 µm (Section 3.3). These parameters describe the temperature required to melt the filament, ranging from 174 ◦C to 205 ◦C. We fabricated ten scaffolds per group to verify the above parameters, and the results showed that the parameters were stable and reliable with a 100% success rate (Figure 2 and Supplementary Figure S3). Table 2. Parameters used for 3D printing for six groups’ scaffolds. Material Extrusion Printing Parameters Groups Nozzle (◦C) Speed (mm/s) Heated Bed (◦C) Flow Factor (%) PCL 174 100 30 95 PCL + 5% HA 175 100 30 95 PCL + 10% HA 175 110 30 95 PCL + 15% HA 185 100 30 95 PCL + 20% HA 198 120 30 100 PCL + 25% HA 205 110 30 100 Pollymeerrss 2022,, 14,, 6x6 F9OR PEER REVIEW 6 6ooff 14 Figure 2. Examplless ooff pprriinntteedd ssixix ggrroouuppss’ ’ssccaaffofoldlds s(1(155 × ×151 ×5 2× m2mm).m (A).)( PAC)LP.C (BL.) (PBC)LP C+ L5%+ H5%A.H (CA). (PCC)LP +C L10+%1 H0%A.H (DA). (PDC)LP +C 1L5+%1 H5%A.H (EA). P(EC)LP +C 2L0+% 2H0%A.H (FA) .P(CF)LP +C 2L5%+ 2H5A%. HA. 33..33.. SSppeecciimmeenn MMoorrpphhoollooggiiccaall CChhaarraacctteerriizzaattiioonn TThhee ppoorree ssiizzeess ooff ssccaaffffoollddss ccoonnttaaiinniinngg ddiiffffeerreenntt HHAA ccoonntteenntt rreemmaaiinneedd ccoonnssttaanntt ((nnoott ssiiggnniiffiiccaannttllyy ddiiffffeerreenntt ffrroomm 555500 µμmm)).. TThhee ppoorroossiittyy rraattee eexxhhiibbiitteedd aa ssmmaalllleerr oosscciillllaattiioonn rraannggee,, wwiitthh PPCCLL ++ 1100%% HHAA sshhoowwiinngg tthhee hhiigghheesstt ppoorroossiittyy ((6655..44 ±± 00..33%%,, ** == pp << 00..0055)) aanndd PPCCLL ++ 55%% HHAA tthhee lloowweesstt ((6600%% ±± 00.9.9%%, ,** == pp << 00.0.055) )vveerrssuuss ththee ccoonnttrrooll aanndd tthhee ootthheerr ggrroouuppss ((FFiigguurree 33AA,,BB))..D Deessppitietet hthees tsattaistitsictiaclasli gsinginfiicfaicnacne,ceth, ethpeo rpoosriotysiotyf aollf sailxl gsirxo ugpros’uspcsa’f fsocladfsfowldass waraosu nadro6u0n%d. 6S0E%M. wSEaMs u sweadst ousoebdta tion tohbetamino rtphheo mloogryp(hFoiglougrey 3(DFi)g. Tuhree 3imDa).g Teshree vimeaalgedest hrea-t valelasliexdg rtohuatp salol fssicxa fgfroolduspds iospf lsacyaefdfohldigsh dlyisipnltaeyrceodn nheigctheldy pinotreersctoruncnteucrteesd. Apdordei tisotrnuaclltyu,rtehse. Astrdudcittuiorneaalplyp, eathreed srtoruucgthuerre thapepmeoarreedH rAouwgahseirn ctlhued emdo. rTeo HveAri fwy awsh ientchleurdtehdis. cToor revleartiefdy wwihtehththeer tihnicsr ecaosreredlaintecdo rwpiotrha tthioen inocfrHeaAse,dE DinXcowrpaosrcaotinodnu ocft eHdAf,o Er DeaXc hwgarso cuopn.dFurcotemd tfhoer eEaDcXh ,gwroeuapn. aFlryozmed thteh eEDpeXr,c wenet aagnealoyfzeHdA thoen ptehrceesnutarfgaec eofo Hf sAc aofnfo tlhdes .suTrhfaecEe DofX scaanfafolyldsiss. Trehvee aElDedXt hanataltyhseisw reeivgehat lreadti othoaft ctahlec iwumeig(Chta )raatniod opfh coaslpchiuomru s(C(Pa)) ianncdre pasheodspcohnocroums (itPa)n itnly- wcrietahsethde cionnccreoamsiintagnatlmy owuinthts tohfe HinAcrienacsliundge adm(Foiugnutrse o3fC H).A included (Figure 3C). 3.4. Specimen Roughness Characterization Surface roughness parameters Sa (arithmetical mean height) and Sz (maximum height) are displayed in Figure 4. The PCL + 25% HA group showed the highest mean Sa (ca.0.25), and the PCL + 5% HA group showed the lowest mean Sa value (ca.0.15), with statistical differences from the control PCL (* = p < 0.05). Regarding Sz, although no statistical differences were recorded between the groups (n.s. = p > 0.05), the values increased with the increased HA content. 3.5. Water Contact Angle Test Water contact angles were highest for the PCL + 5% HA group (84 ± 7.2◦, * = p < 0.05) versus the PCL control. In contrast, the PCL + 25% HA group showed the best hydrophilic performance (67 ± 3.9◦, * = p < 0.05) versus the PCL + 5% HA group (Figure 5A). Contact angles of intermediate HA content were not statistically significant from the PCL control. Water drops on all solid specimens are displayed in Figure 5. PolPyomlyemrse r2s02202,2 1, 144, ,x6 F69OR PEER REVIEW 7 of 14 7 of 14 FFigiguurere3 .3S. uSrufarcfaecceh acrhaacrtaercitzeartiizoantioofnt hoef stchaeff oscldasf.fo(Ald)sT. h(Ae p) oTrhees ipzeoroef sthizees ioxf gtrhoeu psisxo gf rsocaufpfosl dosf scaffolds (m(meeanan± ± ss.e.e.m.m););9 900m meaesausruermemenetns tosf o3f s3c asfcfaoflfdosldins einac ehagchro gurpo(uopn e(-ownaey-wAaNyO AVNAO+VTAuk +e yT’uskpeoys’ts post hoc htoecstt,e nst.,s.n .=s .p= >p 0>.005.)0. 5()B. )( BP)oProosroitsyit ywwasa scacalclcuulalatteedd bbaasseedd oonn tthheew weiegihgthot fotfh tehsec asfcfaofldfosl.dTs.h eThe graph grreapprhesreepnrtess tehnet smtheeanm ±e asn.e.±ms (.eo.nme-(wonaey-w AaNyOAVNAO V+A Tu+kTeuyk’se yp’sospto hstoch otecstte, s*t ,=* p=

0.05), the values increased with the increased HA content. PolPyomlyemrse r2s02202,2 1, 144, ,x6 F69OR PEER REVIEW 8 of 14 8 of 14 FFigiguurere4 .4S.u Srufarcfearcoeu rgohungeshsnaensasl yasnisaolyf sthise osifx tghreo uspixs ’gsrcoafufoplsd’s .sc(Aaf)fGolrdaps.h (oAn) tGheralepfths iodne rtehper elseefnt tsside repre- Ssaen(atrsi tSham (eatricitahlmetaincahl emigehatn); h(Be)igGhrta)p; h(Bo)n Gtrhaeprhig ohnt rtehper ersigenhtts rSezprveasleunests( mSza xviamluems h(meiagxhitmofum height soufr fsaucerf).aGcer)a.p Ghrsasphhows smhoeawn m±esa.en.m ±, s*.=e.pm<, *0 .=05 p, o