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[3D Printed Scaffold Author Manuscript.pdf](https://mdr.nims.go.jp/filesets/85b8b834-279a-4807-8687-07dcf5c1090c/download)

## Creator

[Novi Dwi Widya Rini](https://orcid.org/0000-0003-1128-7804), Adel Alshammari, Candrani Khoirinaya, Anggraini Barlian, Lia A. T. W. Asri, Glen Cooper, [Katsuhiko Ariga](https://orcid.org/0000-0002-2445-2955), [Lok Kumar Shrestha](https://orcid.org/0000-0003-2680-6291), [Arie Wibowo](https://orcid.org/0000-0002-0581-2433)

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This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1007/s42247-024-00924-6[In Copyright](http://rightsstatements.org/vocab/InC/1.0/)

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[3D printed scaffold based on polycaprolactone/self-assembled fullerene (C60) nanorod for bone tissue engineering](https://mdr.nims.go.jp/datasets/5f324851-baf5-4dfd-a1d2-58dbdb8586e6)

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3D Printed Scaffold Based on Polycaprolactone/Self-assembled Fullerene (C60) 1 Nanorod for Bone Tissue Engineering  2 Novi Dwi Widya Rini 1,2,3, Adel Alshammari 4,5, Candrani Khoirinaya 6, Anggraini Barlian 6,7, Lia 3 Amelia Tresna Wulan Asri 3, Glen Cooper 4, Katsuhiko Ariga 1,8, Lok Kumar Shrestha 1,2,* and 4 Arie Wibowo 3,7,*   5 1 Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials 6 Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan 7 2 Department of Materials Science, Institute of Pure and Applied Sciences, University of Tsukuba, 8 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573 Japan 9 3 Materials Science and Engineering Research Group, Faculty of Mechanical and Aerospace 10 Engineering, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung, 40132, Indonesia 11 4 Department of Mechanical, Aerospace, and Civil Engineering, University of Manchester, 12 Manchester M13 9PL, UK 13 5 Engineering College, University of Hail, Hail 55476, Saudi Arabia 14 6 School of Life Sciences and Technology, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 15 40132, West Java, Indonesia 16 7 Research Center for Nanoscience and Nanotechnology, Institut Teknologi Bandung, Jl. Ganesha 17 10, Bandung 40132, West Java, Indonesia 18 8 Department of Advanced Materials Science, Graduate School of Frontier Sciences, The 19 University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8561, Chiba, Japan 20 *Corresponding author: shrestha.lokkumar@nims.go.jp (L.K.S) and ariewibowo@itb.ac.id (A.W)  21  22 Abstract 23 3D printed polycaprolactone (PCL)-based scaffolds have garnered attention in bone tissue 24 engineering due to their biocompatibility, biodegradability, and precise geometry control. 25 However, it is yet challenging to tune the mechanical properties and hydrophilicity. This paper 26 reports a subtle balance between the mechanical properties and hydrophilicity of the scaffold 27 essential in bone tissue engineering using self-assembled fullerene (C60) nanorods (FNR) and 28 Pluronic 123 surface-modified fullerene nanorods (PFNR) as the reinforced filler. FNR 29 incorporated (0.013 wt%) PCL scaffold (PCL_FNR_0.013) shows enhanced compressive strength 30 (8.4 MPa) and Young’s modulus (146.2 MPa), compared to the PCL scaffold (compressive 31 strength: 3.3 MPa and Young’s modulus: 56.2 MPa) without significant changes in the 32 hydrophilicity. However, the PCL scaffolds’ hydrophilicity and mechanical properties could be 33 improved by incorporating PFNR filler. As a result, the scaffold shows excellent proliferation 34 activity of Human Wharton's Jelly Mesenchymal Stem Cells. Moreover, both the FNR and PFNR-35 incorporated PCL scaffolds show antibacterial properties essential to prevent implant-associated 36 infections. The antibacterial activity results reveal that FNR without surface modification offers 37 better antibacterial activity than PFNR, particularly against Staphylococcus aureus and 38 Escherichia coli. This study demonstrates that by adjusting the type and concentration of fillers, 39 one can tune the mechanical and wetting properties of the PCL scaffold to optimize cell 40 proliferation and antibacterial activity for potential applications in bone tissue engineering.    41  42 Keywords: 3D printing, bone tissue engineering, fullerene nanorod, hydrophilicity, 43 polycaprolactone. 44  45 1. Introduction 46 Bone grafts are the second most common medical case worldwide after blood transfusions 47 [1]. Like with other body parts, bones can regenerate and reconstruct by themselves. Many factors 48 influence the healing process, including the size of the damage gap and the amount of bone loss 49 [2,3]. Increased bone loss could limit natural healing and reconstruction, resulting in failure, 50 various complications, and, in the worst case, death [4,5]. Bone grafts bridge the gap and replace 51 the bone loss to facilitate regeneration and reconstruction [6,7]. However, autograft and allograft, 52 the gold standard, have drawbacks due to the limited supply [8,9]. Another potential approach is 53 the fabrication of synthetic scaffolds because their properties can be tuned so that the scaffold acts 54 as passive support and stimulates the growth and regeneration of the bone [10,11]. 55 To successfully apply scaffolds in bone tissue engineering, the scaffold must mimic the 56 extracellular matrix of bone tissue to facilitate cell attachment and tissue formation. Furthermore, 57 the scaffold’s porosity must be interconnected to allow molecules and nutrients to penetrate the 58 interior of the scaffold and support continuous tissue growth [12,13]. The method extensively 59 explored to produce scaffolds with the required porous materials is three-dimensional (3D) 60 printing fabrication, also known as additive manufacturing [14,15]. 3D printing can construct 61 complex geometries with high control parameters, such as pore size, shape, porosity, and pore 62 interconnection. However, challenges still exist with material selection and 3D shape specificity 63 due to the specific properties of each material, such as the melting point and degradation 64 temperature, which will affect the processing parameters and the final construction [16,17].  65 Polycaprolactone (PCL) fabricated using the 3D printing method has been shown to have 66 an excellent porous structure [18]. PCL is particularly well-suited for 3D printing compared to 67 other polymers due to its low melting point of 58 – 60 °C. This melting point allows for easy 68 processing, tailored extrusion, and rapid cooling, resulting in rigid and highly precise scaffolds 69 [19]. Moreover, PCL degrades more slowly compared to other polymer-based implants, taking up 70 to four years, which makes PCL an attractive choice for long-term implants [20,21]. However, the 71 high hydrophobicity causes cell attachment to the scaffold less than optimal [22]. In addition, PCL 72 needs to be blended with other materials to get the required mechanical strength due to its low 73 mechanical properties [23]. Therefore, selecting suitable filler material is essential to improve the 74 overall characteristics of the scaffold [24]. 75 Recently, carbon-based nanomaterials such as fullerenes (C60) [25], graphene [26,27], and 76 carbon nanotubes [26], have been extensively employed as the polymer composites’ reinforcement 77 filler. Considering that fullerene (C60) has theoretically Young’s modulus of 1980 GPa for its 78 single molecule [28], it could be expected that the addition of a few fullerenes could significantly 79 increase the mechanical strength of the polymer. It has been found that the incorporation of a small 80 amount of fullerenes (C60 or C70: 0.02 to 0.08 wt%) in thermoplast-based polymer nanocomposite 81 increases Young’s modulus and tensile strength of the nanocomposites by 30-40%[29]. 82 Rajagopalan and coworkers also showed that adding 0.1 wt% of fullerene (C60) drastically 83 increases Young’s modulus of the nanocomposite membrane [30]. Moreover, previous 84 investigation has shown that the addition of a small amount of C60 nanoparticles in a 85 hydroxyapatite-chitosan composite shows an antibacterial effect [31], which is crucial in bone 86 tissue engineering due to the bacterial infections that often occur after bone surgery [32,33]. These 87 infections can lead to morbidity or even fatalities in severe cases, such as septic arthritis [34] and 88 peri-implantitis [35]. PCL does not show any antibacterial effect; therefore, it is promising to use 89 C60 as a reinforcement filler and antibacterial agent in the PCL matrix [36].  90 Nevertheless, the use of zero-dimensional (0D) C60 in a composite is not favorable due to 91 its tendency to form uncontrollable aggregates. Meanwhile, self-assembled C60-based 92 nanomaterials with higher dimensions, such as C60 nanorods, have a lower tendency to form 93 aggregates, thus, more favorable for composites [37]. Even though C60 nanorods have various 94 potential applications, their application in biomedical field is still limited due to hydrophobic 95 nature of C60 [30]. Therefore, a surface modification is required to hydrophilize its surface and to 96 promote the scaffold and cell interactions [38]. Wong et al. showed that the self-assembled C60 97 nanorod coated with Pluronic 123 (P123) exhibits hydrophilic properties compared to the nanorods 98 before the P123 coating [39]. P123 is known as a triblock copolymer consisting of polyethylene 99 oxide (PEO) as a hydrophilic segment and polypropylene oxide (PPO) as a hydrophobic segment 100 [40]. The hydrophobic C60 is encapsulated within the PPO core, while the hydrophilic PEO 101 segments form the outer shell, resulting in the hydrophilic surface of C60[41]. To our knowledge, 102 surface-modified self-assembled C60 nanorods have not been explored as a filler material in a 3D 103 PCL scaffold.  104 In this contribution, we report the fabrication of porous scaffolds composed of PCL and 105 P123 surface-modified C60 nanorods using 3D printing. The filler materials were self-assembled 106 fullerene C60 nanorods (FNR) and P123 surface-modified fullerene nanorods (PFNR). FNR was 107 prepared using the liquid-liquid interfacial precipitation (LLIP) method [42], and its surface was 108 modified with the different concentrations of P123. The mechanical properties, hydrophilicity, cell 109 viability, cell proliferation, and antibacterial activities of the 3D-printed PCL-FNR or PCL_PFNR 110 scaffolds were then evaluated. In this study, human Wharton's jelly mesenchymal stem cells (hWJ-111 MSCs) were chosen as precursor cells for cell viability and cell proliferation of the prepared 112 scaffold because hWJ-MSCs offer several advantages, including source availability, providing 113 high cell yield with non-invasive method, demonstrating excellent proliferation, and exhibiting a 114 fibroblast-like morphology [43,44]. Furthermore, our prior work demonstrated that by day 21 of 115 treatment, hWJ-MSCs had differentiated into mature osteoblasts on a PCL-based 3D scaffold [44]. 116 Another study also indicated that hWJ-MSCs are suitable for evaluating initial cellular responses 117 to materials in bone tissue engineering applications [45]. Based on our evaluation, we have found 118 that the addition of FNR enhances the mechanical properties, while the addition of PFNR enhances 119 the hydrophilicity and cell proliferation of the scaffolds. In addition, all the scaffolds with the 120 addition of FNR and PFNR filler show antibacterial activity against E. coli and S. aureus.  121 2. Materials and Methods 122 2.1. Materials 123 Pristine fullerene C60 was purchased from MTR Ltd, USA (purity 99.5%). Mesitylene 124 (99.8%), methanol (99.7%), and isopropyl alcohol (99.7%) were purchased from Wako Chemicals 125 Corporation, Tokyo, Japan and were used as received. Polycaprolactone pellets (CAPA 6500, MW 126 = 50,000 g/mol, melting point = 58 – 60 °C, density = 1.146 g/mL) provided by Perstorp 127 (Warrington, UK) were used as received to prepare composite blends with self-assembled C60 128 nanorods. Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol: 129 Pluronic 123) was purchased from Sigma-Aldrich, USA. Staphylococcus aureus (S. aureus; ATCC 130 3658) and Escherichia coli (E. coli; ATCC 8939) bacterial strains were obtained from the 131 Microbiology Laboratory, School of Pharmacy, Institut Teknologi Bandung (ITB). 132 2.2. Preparation and characterization of hydrophilic self-assembled fullerene nanorod 133 Fullerene C60 solution (1 mg/mL) was prepared in mesitylene by dissolving a required 134 amount of pristine C60, and fullerene C60 nanorod (FNR) was synthesized using the static liquid-135 liquid interfacial precipitation (LLIP) method [37]. In a typical synthesis, C60/mesitylene solution 136 (1 mL) was taken in a 13.5 mL glass vial, and anti-solvent methanol (3 mL) was then added slowly 137 on top and the system was left undisturbed for 30 min avoiding mechanical disturbance. The 138 system was then sonicated for 1 min and incubated for 24 hours at 25 °C. The following 24 hours 139 later, the FNRs were isolated by repeated centrifugation and washed with isopropyl alcohol (IPA) 140 three times. Finally, the precipitate was dried in a vacuum oven for 24 hours. 141 The surface of FNR was coated with Pluronic 123 by adding FNR powder to a solution 142 containing 0.5 wt% Pluronic 123 in DI water [39]. After 24 hours of resuspension, the supernatant 143 was removed by centrifugation, and the precipitate was dried for 24 hours by freeze-drying. 144 The morphology of FNR and PFNR were observed by scanning electron microscope (SEM, 145 Hitachi model S-4800, Japan). Fourier-Transform Infrared (FTIR) was carried out using attenuated 146 total reflection (NICOLET iS20). Raman spectra were acquired using an NRS-3100 Raman 147 spectrometer. The charge on the surface of FNR and PFNR was measured utilizing a Horiba SZ-148 100 Nano Particle Analyzer. 149 2.3. Fabrication of PCL/FNR and PCL/PFNR 3D-printed scaffold 150 A composite premix was prepared using the melt blending approach [46]. The variants of 151 the composite premix developed in this study are as follows: pure PCL (without fillers); 152 PCL_FNR_0.013 (PCL with 0.013 wt% FNR); PCL_PFNR_0.0013 (PCL with 0.0013 wt% 153 PFNR); PCL_PFNR_0.013 (PCL with 0.013 wt% PFNR); and PCL_PFNR_0.13 (PCL with 0.13 154 wt% PFNR). Each composite premix was subsequently fabricated utilizing screw-assisted 155 extrusion-based 3D printing equipment (3D Discovery, regenHU, Villaz-St-Pierre, Switzerland). 156 During the 3D printing process, the composite premix was placed into a reservoir and melted at 157 90 °C. After the premix had melted homogeneously in the reservoir, it was pumped into the screw 158 chamber via air pressure in the tool. Next, the premix was extruded using a 500 µm needle. The 159 premix that exited the extruder was liquid and created filaments with layer orientations ranging 160 from 0 to 90 degrees. The extrusion process adjusted the deposition speed and screw rotation speed 161 parameters. Finally, the scaffold was cooled layer by layer, resulting in a solid 3D scaffold. 162 2.4. Characterization of PCL/FNR and PCL/PFNR 3D-printed scaffold  163 For the 3D scaffold, the morphology was characterized by SEM, Hitachi model SU-3500 164 at an accelerating voltage of 10 kV. Scaffold porosity represents the percentage of measured 165 scaffold density (scaffold mass divided by apparent volume, neglecting the pores) and the initial 166 density of material (measured using the psychometry method, considering the pores) [47]. The 167 compressive strength of the scaffold was measured using a Universal Testing Machine, Tensilon 168 RTF-1310, A&D Company. The sessile drop method (Contact Angle Meters, Kyowa) was used to 169 measure the contact angle by dropping 2 µL of water on the surface of the scaffold. The droplet 170 was recorded 1 second after dropping. All measurements were carried out with a 5 mm x 5 mm x 171 3 mm (triplicate) scaffold.  172 Cell viability and cell proliferation on the scaffold were determined using the colorimetric 173 method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) [48,49]. 174 Before the MTT test, Human Wharton's Jelly Mesenchymal Stem Cell (hWJ-MSC) cells were 175 cultured on scaffolds at a concentration of 1 x 105 cells/well in microplates with a flat bottom using 176 Dulbecco's Modified Eagle media (DMEM). Cell culture incubation was carried out at 37 °C, with 177 CO2 levels ranging from 5-6.5% in the cell culture incubator. The MTT test was performed on 178 each 3 days of incubation. To perform the MTT test, 10 µL of reagent was added to each well. The 179 cells were then incubated for another 4 hours in a cell culture incubator. Next, 100 µL of PBS was 180 applied to each well. The absorbance of the dissolved purple formazan crystals was then measured 181 using an ELISA reader, Bio-Rad iMark, at a wavelength of 595 nm. 182 The scaffolds’ antibacterial properties were determined by the total plate count (TPC) [50]. 183 The bacterial suspension (E. coli and S. aureus) was prepared by adding one culture cycle of 184 bacteria to Mueller Hinton Broth (MHB) media and incubated at 37 °C for 24 hours. After that, 185 the turbidity of the bacteria suspension was measured to be equivalent to 0.5 Mc Farland. The 186 suspension was diluted 1:20 with MHB medium. The test bacteria were then decimally diluted by 187 pipetting 1 mL into the first vial containing 9 mL of 0.9% NaCl with a 10-6 dilution. Then, 1 mL 188 of the 10-6 dilution was placed in a sterile petri dish, and 20 mL of Mueller Hinton Agar (MHA) 189 was added to the petri dish. The diffusion method was obtained by putting the sample on MHA 190 media and pouring another Agar covering the sample. The incubation was done for 24 hours at 191 37 °C. Using a caliper, the diameter of the encountered obstruction was determined. Next, 100 µL 192 of top agar from the inhibition region was mixed with 900 µL of 0.9% NaCl solution (dilution 10-193 1). The dilution was increased to decimal 10-8. The TPC was then performed in triplicate and 194 incubated for another 24 hours at 37 °C. Following that, the number of colonies that grew was 195 counted. 196 2.5. Statistical analysis 197 Each experiment was tested with a minimum of three replicates. Statistical analysis was 198 conducted with Origin 9 (Originlab, USA) and Prism 10 (GraphPadm USA), and its statistical 199 significance was ascertained when P < 0.05. One-way ANOVA was used to make statistical 200 comparisons for studies with more than 2 groups, while hWJ-MSC’s cell viability was assessed 201 using Two-way ANOVA repeated measures. Post-hoc analysis was performed via Tukey’s 202 multiple comparison test. All values are expressed as mean ± standard deviation (SD) or mean ± 203 standard error of the mean. 204 3. Results and Discussion 205 3.1. Preparation and characterization of hydrophilic self-assembled fullerene nanorod 206 Self-assembled fullerene nanorods (FNRs) with a uniform size distribution were prepared 207 using the static LLIP method [51]. The hydrophobic surface of the FNRs was modified with P123 208 coating to obtain hydrophilic PFNR [39]. The schematic of the preparation of FNR and PFNR is 209 illustrated in Fig. 1.  210  211 Fig. 1 (a) Scheme of self-assembled fullerene nanorod (FNR) preparation using the static LLIP 212 method and (b) surface-modified hydrophilic self-assembled FNR (PFNR) preparation by coating 213 the FNR with P123. 214 Owing to the intrinsic hydrophobic properties of fullerene, the FNRs are not dispersible in 215 water (Fig. 2a). On the other hand, after the surface modification, the PFNR could be dispersed in 216 water (Fig. 2b) [42]. SEM observations reveal different surface morphology of the FNR and PFNR. 217 The PFNR (Fig. 2d) has a rougher surface than the FNR (Fig. 2c), confirming successful P123 218 coating on the FNR surface [39]. It is noting that the SEM images of the PFNR reveal some 219 inhomogeneous surface coating, showing the variations in surface coverage. The histograms of 220 length and diameter distributions of the FNR are shown in Fig. 2e and 2f, respectively. Based on 221 the SEM images, the average length and diameter of FNR are ca. 2,540 ± 751 nm and 160 ± 50 222 nm, respectively. Due to the rough surface and inhomogeneous surface coatings, we did not plot 223 the histograms of the length and diameter distributions of the PFNRs.  224   225 Fig. 2 (a) Digital image showing poor dispersibility of FNR in DI water due to its hydrophobic 226 properties; (b) the corresponding digital image of the PFNR in DI water showing its excellent 227 dispersibility in water due to the surface modification; (c) SEM image of FNR with a smooth 228 surface morphology and (d) PFNR with a rough surface morphology indicating the surface 229 modification by P123 coating; (e) histogram of length and (f) diameter distribution of FNR. 230 Fig. 3a shows the FTIR spectra of FNR, PFNR, and P123. The FTIR peaks at 575, 1183, 231 and 1428 cm−1 present both in FNR and PFNR correspond to the C-C bonds of the C60 molecule 232 [30,52]. Aside from this, the new peaks have appeared at 1100 and 2970 cm-1, corresponding to 233 C-O stretching from hydrophilic PEO (1100 cm-1) and antisymmetric CH3 stretching from 234 hydrophobic PPO (2970 cm-1) [39,53], demonstrating the successful coating of P123 on the FNR 235 surface. The P123 coating was further validated by the surface charge (zeta potential) 236 measurements. The PFNR has a higher negative zeta potential (-57.0 ± 1.6 mV) than the FNR (-237 3.6 ± 0.3 mV), validating P123 coating [39]. Fig. 3b shows Raman spectra of FNR, PFNR, and 238 pristine C60. All the spectra exhibit prominent peaks corresponding to Ag (1), Ag (2), Hg (1), Hg 239 (2), Hg (3), Hg (4), Hg (7), and Hg (8) bands with no apparent Ag (2) peak shift, indicating that free 240 molecular rotation of fullerene molecules persist both in FNR and PFNR without the 241 polymerization of C60 molecules [39]. 242  243 Fig. 3 (a) FTIR spectra of FNR, PFNR, and P123; (b) Raman shift of C60 pristine, FNR, and PFNR. 244 3.2. Fabrication of PCL/FNR and PCL/PFNR 3D-printed scaffold 245 Fig.4 shows the digital images of the 3D-printed scaffolds (pure PCL, PCL_FNR_0.013, 246 PCL_PFNR_0.0013, PCL_PFNR_0.013, PCL_PFNR_0.13) with dimensions of 25 mm x 25 mm 247 x 3 mm. The scaffold with pure PCL is white, the same as the PCL pellet color. However, FNR 248 and PFNR-incorporated scaffolds have different colors, and the color depends on the concentration. 249 As can be seen in Fig. 4, the PCL_FNR_0.013 scaffold has a yellowish-brown color. Due to very 250 low concentration, the color of the PCL_PFNR_0.0013 scaffold (Fig. 4c) is similar to the pure 251 PCL scaffold (Fig. 4a). The color became intense and brownish at higher concentrations of PFNR 252 in the PCL_PFNR_0.13 scaffold (Fig. 4e).  253  254 Fig. 4 Top view optical images of the 3D-printed scaffold (a) pure PCL; (b) PCL_FNR_0.013; (c) 255 PCL_PFNR_0.0013; (d) PCL_PFNR_0.013; and (e) PCL_PFNR_0.13. 256 3.3. Characterization of PCL/FNR and PCL/PFNR 3D-printed scaffold 257 3.3.1. Scaffold Morphology 258 The scaffold’s surface morphology, filament size, pore size, and porosity were studied by 259 SEM analyses. The illustration of the top view and cross-section view of the scaffold during SEM 260 observation is provided in Fig. 5a and SEM images of the top view and cross-sectional view of the 261 scaffolds are presented in Fig. 5b and Fig. 5c respectively. As can be seen in SEM images from 262 the top view of the scaffolds that were taken at low magnification (Fig. 5b), all the scaffolds were 263 well-aligned with relatively uniform in their shapes, filaments and pores size. SEM images from 264 the top view (Fig. 5b) and cross section (Fig. 5c) of the scaffolds that were taken at medium 265 magnification showed that all the scaffolds have rough surfaces regardless the filler and its 266 concentration. The rough surface could be attributed to the rheological characteristics of the 267 material, which influence the extrusion flow in the 3D printing process [54].  268  269 Fig. 5 (a) The illustration of the top view and cross-section view of the scaffold; the SEM images 270 of the scaffold from (b) the top view and (c) the cross-section view. 271 The filament diameter and pore size and porosity of the scaffolds are summarized in Table 272 1. The pore diameters of all the scaffolds are higher than 300 µm, which are suitable for promoting 273 angiogenesis and osteogenesis, enhancing the production of new bone in bone tissue engineering 274 applications [55].  275 Table 1. The filament diameter, pore size, and porosity of each scaffold. 276 Scaffold Filament diameter (µm) Pore size (µm) Porosity (%) PCL 506.8 ± 30.7 422.2 ± 28.7 54.6 ± 6.5 PCL_FNR_0.013 613.1 ± 28.6 407.6 ± 26.3 47.7 ± 3.1 PCL_PFNR_0.0013 536.1 ± 39.7 481.4 ± 26.8 53.5 ± 3.2 PCL_PFNR_0.013 617.6 ± 48.1 448.6 ± 21.8 56.7 ± 5.2 PCL_PFNR_0.13 537.6 ± 40.3 450.9 ± 48.7 57.4 ± 5.0 3.3.2 Mechanical Properties 277 The scaffold is supposed to endure compressive loads, including the body weight and 278 external load when implanted in patients [13]. Young's modulus of healthy human cancellous bone 279 has been reported to range from 100 to 5000 MPa, and the compressive strength ranges from 1 to 280 12 MPa [56]. Fig. 6 shows the representative compressive stress-strain curves of the 3D-printed 281 scaffolds. In this study, Young’s modulus was determined as the gradient in the linear elastic 282 region, and the compressive strength was specified as a stress in 10% strain (ISO 844-2014).  283  284 Fig. 6 Representative compressive stress-strain curves of 3D-printed scaffolds. 285 Table 2 summarizes the mechanical properties of 3D-printed scaffolds. The pure PCL 286 scaffold has the lowest Young’s modulus (56.2 ± 4.2 MPa), which increased drastically (260%: 287 146.2 ± 5.5 MPa) upon the addition of 0.013 wt% FNR. Young’s modulus increases by over 200% 288 in the PFNR system. Our previous report showed that a PCL-based 3D scaffold with 2 wt% 289 polyaniline (PANI) increased the compressive Young’s modulus only up to 120% (82.61 ± 6.94 290 MPa) [15]. Another study using 3 wt% multi-walled carbon nanotube (MWCNT) filler showed 291 170% (88 ± 3 MPa) increase in Young’s modulus [57]. These results highlight that FNR and PFNR 292 provide superior reinforcement compared to PANI and MWCNT. Moreover, Young’s modulus of 293 scaffolds with the addition of FNR and PFNR filler is within the range of healthy human cancellous 294 bone [56]. This alignment is crucial since a mismatch of Young’s modulus can result in stress 295 shielding and failed bone regeneration [58].  296 Table 2. The compressive Young’s modulus and compressive strength of 3D-printed scaffolds. 297 Scaffold Compressive  Young’s Modulus (MPa) Compressive  Strength (MPa) PCL 56.2 ± 4.2 3.3 ± 0.1 PCL_FNR_0.013 146.2 ± 5.5 8.4 ± 0.1 PCL_PFNR_0.0013 119.9 ± 5.7 3.7 ± 0.4 PCL_PFNR_0.013 128.0 ± 5.2 4.7 ± 0.5 PCL_PFNR_0.13 138.4 ± 2.5 5.3 ± 0.4  298 PCL with FNR filler has a higher modulus value than PCL with PFNR filler (Table 2) due 299 to the strong hydrophobic interfacial interaction between the PCL matrix and FNR, which inhibits 300 the movement of polymer chains during compression [59]. Meanwhile, hydrophilic PFNR has no 301 specific interfacial interaction with hydrophobic PCL. As a result, PFNR in the PCL matrix is less 302 rigid and has a lower inhibitory effect on polymer chain movement during compression.  303 The compressive strength follows the same trend as Young’s modulus results (Table 2). 304 Based on the compressive stress-strain curves (Fig. 6), the scaffolds have reached the plastic area 305 at 10% strain. Due to the presence of hydrophobic interfacial interaction between PCL and FNR, 306 more significant load is required to compress the scaffold, resulting in a higher compressive 307 strength [59]. On the other hand, due to the lack of specific interactions between PCL and PFNR, 308 compression occurs independently, which lowers the required compressive load, and hence, low 309 compressive strength is observed in the PFNR system. These results highlight that surface 310 modification leads to different reinforcement results.  311 3.3.3 Hydrophilicity 312 The hydrophilic surface is preferable for cell attachment compared to the hydrophobic 313 surface [60]. We tried to tune the hydrophilicity of the PCL_FNR scaffold by modifying the FNR’s 314 surface with P123 coating. Fig. 7 shows the contact angles of the prepared scaffolds. Since the 315 measurements were taken immediately (1 second) after water dropping on the scaffold's surface, 316 the surface properties played a more significant role in determining the contact angle than the 317 porosity, as there was no time for the water to penetrate the pores.  318  319 Fig. 7 Contact angles of PCL and FNR and PFNR added PCL scaffolds along with the water 320 droplet images (1 second) on each 3D-printed scaffold. The red horizontal dash line at 90° indicates 321 the threshold to distinguish between hydrophilic and hydrophobic. The values of the contact angle 322 are as follows: PCL (95.4° ± 0.8°), PCL_FNR_0.013 (104.2° ± 7.8°), PCL_PFNR_0.0013 (93.3° 323 ± 6.6°), PCL_PFNR_0.013 (76.5° ± 4.4°), PCL_PFNR_0.13 (89.4° ± 3.2°). 324 Fig. 7 shows that adding 0.013 wt% FNR to the PCL scaffold increases the contact angle, 325 making the scaffold more hydrophobic. This is because of the intrinsic hydrophobic properties of 326 PCL and FNR [55,61]. The hydrophobic nature likely strengthens the non-polar interactions 327 between the scaffold and water, resulting in higher contact angle values [62]. In contrast, the 328 addition of 0.0013 wt% and 0.013 wt% PFNR considerably reduces the contact angle, giving the 329 scaffold hydrophilic properties. SEM images that were taken at high magnification from the top 330 view of the scaffolds with FNR 0.013 wt% and PFNR 0.013 wt% (Fig. 5b) revealed that the 331 presence of P123 as surface modifier in PFNR could reduce aggregation of the filler in the 332 fabricated scaffold. P123 likely introduced hydrophilic functional groups to the surface of the filler, 333 promoting better interaction with water molecules and thus lowering the contact angle [63]. 334 However, when the PFNR concentration is increased further to 0.13 wt%, the contact angle 335 unexpectedly increases. This unexpected increase in contact angle may be attributed to several 336 factors. The aggregation of PFNR particles might occur in higher concentrations, reducing their 337 effective surface area for hydrophilic effect [64]. As shown in SEM images that were taken at high 338 magnification from the top view of the scaffolds with PFNR as filler, aggregation became more 339 pronounced as the concentration of PFNR in scaffold increased from 0.0013 wt% to 0.13 wt% 340 (Fig. 5b). This phenomenon likely occurs because PFNR particles tend to cluster together at higher 341 concentrations, limiting their dispersion and reducing their effective surface area in the fabricated 342 scaffolds, which in turn affects hydrophilicity. Another possible reason is that 0.13 wt% might be 343 a saturation point where PFNR does not further enhance hydrophilicity and instead leads to a more 344 heterogeneous surface, causing inconsistent interactions with water [65]. These results indicate 345 that the balance concentration, 0.013 wt% PFNR, is optimal for achieving the best hydrophilic 346 scaffold.  347 3.3.4 Cell Viability 348 The viability of human Wharton's Jelly Mesenchymal Stem Cell (hWJ-MSC) on each 349 scaffold was determined using the MTT assay after 72 hours of cell incubation. Fig. 8 depicts a 350 graph of cell viability percentages. Notably, the PCL_PFNR_0.013 demonstrated the highest cell 351 viability (90.4 ± 4.3%), surpassing the viability observed with pure PCL (84.9 ± 9.7%). This 352 suggests that PFNR enhances the scaffold’s surface properties at this concentration, promoting 353 better cell adhesion. Conversely, both PCL_PFNR_0.0013 and PCL_PFNR_0.13 resulted in 354 decreased cell viability. However, the cell viability of all scaffolds is above 70%, which meets the 355 standards of ISO 10993-5:2009 for in vitro cytotoxicity. This signifies that the scaffolds prepared 356 in this study do not exhibit substantial toxicity towards hWJ-MSC.  357  358 Fig. 8 Cell viability diagram of hWJ-MSCs after 72 hours grown on various 3D-printed scaffolds 359 was assessed by MTT (n=5). *Denotes a significant difference in cell viability (P < 0.05). 360 ***denotes a significant difference in cell viability (P < 0.001). The values of the cell viability are 361 as follows: PCL (84.9 ± 9.7 %), PCL_FNR_0.013 (78.1 ± 2.5 %), PCL_PFNR_0.0013 (81.7 ± 362 1.9 %), PCL_PFNR_0.013 (90.4 ± 4.3 %), PCL_PFNR_0.13 (74.3 ± 4.2 %). 363 3.3.5 Cell Proliferation 364 The proliferation of hWJ-MSC cells on each scaffold was assessed using the MTT assay 365 (absorbance at 595 nm) up to 12 days of incubation. Fig. 9 shows an increase in cell proliferation 366 for all scaffolds from day 3 to day 12 of incubation.  367  368 Fig. 9 Graph of hWJ-MSC cell proliferation from day 1 to day 12 on each 3D-printed scaffold 369 which was assessed by MTT (n=3). *denotes a significant difference in cell viability (p < 0.05). 370 **denotes a significant difference in cell viability (p < 0.01).  371 Notably, the PCL scaffold with a filler of PFNR has a higher cell proliferation than the 372 pure PCL scaffold and PCL scaffold with a hydrophobic FNR. Among the PFNR-filled scaffolds, 373 the PCL_PFNR_0.013 showed the highest cell proliferation, followed by PCL_PFNR_0.13 and 374 PCL_PFNR_0.0013. These results indicate that the presence of hydrophilic PFNR, particularly at 375 the optimal concentration of 0.013 wt%, enhances the scaffold’s capacity to support cell growth 376 more effectively than pure PCL or FNR-filled scaffolds. This trend is consistent with the contact 377 angle results, suggesting that the lower contact angle leads to better cell attachment [44]. A 378 hydrophilic surface increases the binding of the adhesive molecules on the substrate, making the 379 substrate favorable for cell growth and proliferation [38]. Consequently, the scaffold with 0.013 380 wt% PFNR, which has the lowest contact angle, provided the most hydrophilic surface, leading to 381 the highest cell proliferation.  382 3.3.6 Antibacterial Activity 383 Since PCL does not show any antibacterial effect, we added FNR and PFNR to provide 384 antibacterial activity in the scaffold [36,66]. Previous studies indicated that the addition of 385 fullerene in chitosan nano-conjugate enhances its antibacterial action specifically against S. aureus, 386 a major pathogen causing infections in the bones and joints [67]. Moreover, incorporating fullerene 387 into a phosphate-alginate composite unaffected by the normobiota (beneficial bacteria in the oral 388 cavity), suggests that this material can be safely used in medical applications [68].  389 We evaluated the antibacterial properties of the scaffolds against Gram-positive E. coli 390 (ATCC 8939) and Gram-negative S. aureus (ATCC 6538). As shown in Fig. 10a and 10b, all 391 scaffolds with the addition of FNR and PFNR show antibacterial activity against E. coli and S. 392 aureus. Compared to the control, all scaffolds significantly decreased (more than 99%) the number 393 of bacteria colonies. This is consistent with the previous study that demonstrated the addition of a 394 small amount of aggregated fullerene (0.00004 wt% nano-C60) to the culture medium, resulting in 395 antibacterial activity against E. coli [69]. A previous study showed that Gram-positive bacteria 396 tend to be more susceptible than Gram-negative due to the interaction between fullerene molecules 397 and bacterial cell walls. Fullerenes reduces the proportion of unsaturated fatty acids and increases 398 the proportion of cyclopropane fatty acids in the bacterial cell wall [70]. However, this study shows 399 that the reduction of Gram-positive and Gram-negative has a similar percentage. It might be caused 400 by unspecific filler interactions with membrane proteins and other vital molecules due to the 401 different forms of fullerene [71]. 402  403 Fig. 10 Antibacterial activity of each 3D scaffold using total plate count (TPC) method in (a) 404 Gram-positive cultures of E. coli and (b) Gram-negative cultures of S. aureus (n=3). 405 Moreover, the antibacterial activity of the scaffold against E. coli and S. aureus shows a 406 similar trend. The scaffold with the highest concentration of PFNR exhibits the fewest bacterial 407 colonies, indicating the most excellent antibacterial activity. Conversely, the scaffold with the 408 lowest concentration of PFNR showed the most minor antibacterial activity, demonstrating that 409 increasing the concentration of PFNR enhances antibacterial activity. Meanwhile, the 410 PCL_FNR_0.013 scaffold shows higher antibacterial activity than the PCL_PFNR_0.013 scaffold, 411 suggesting that non-coated FNR provides better antibacterial activity than PFNR and it can be 412 attributed to the hydrophobic surface of the material which can interact with the hydrophobic 413 membrane of the cell wall and rupture it [72].  414 4. Conclusion  415 We successfully fabricated a 3D-printed PCL scaffold reinforced with self-assembled FNR, 416 achieving superior mechanical properties compared to other filler materials used in 3D-printed 417 PCL scaffolds. We modified the surface of FNR with a P123 coating (PFNR) to introduce 418 hydrophilic functional groups on the surface of FNR. By optimizing the concentration of PFNR, 419 we effectively tuned the scaffold’s hydrophilicity, leading to increased adhesion of biomolecules 420 and enhanced cell proliferation. Additionally, both FNR and PFNR-reinforced scaffolds exhibited 421 potent antibacterial activity against S. aureus and E. coli, addressing the critical challenge of 422 preventing implant-associated infections. These findings highlight the potential of FNR and PFNR 423 as advanced fillers in PCL-based scaffolds to provide tunable properties that enhance mechanical 424 strength, biocompatibility, and antibacterial efficacy for bone tissue engineering.  425  426 References 427 1. F. Mahyudin, D. N. Utomo, H. Suroto, T. W. Martanto, M. Edward, and I. L. Gaol, Int. J. Biomater. 428 2017, 7571523 (2017). 429 2. P. V. Giannoudis, Fracture Reduction and Fixation Techniques: Upper Extremities 1 (2018). 430 3. R. Wu, Y. Li, M. Shen, X. Yang, L. Zhang, X. Ke, G. Yang, C. Gao, Z. Gou, and S. Xu, Bioact. Mater. 431 6, 1242 (2021). 432 4. M. Laubach, S. Suresh, B. Herath, M.-L. Wille, H. Delbrück, H. Alabdulrahman, D. W. Hutmacher, and 433 F. Hildebrand, J. Orthop. Translat. 34, 73 (2022). 434 5. 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