The Potential of Polyethylene Terephthalate Glycol As Biomaterial for Bone Tissue Engineering

Article The Potential of Polyethylene Terephthalate Glycol as Biomaterial for Bone Tissue Engineering

Mohamed H. Hassan 1,* , Abdalla M. Omar 1, Evangelos Daskalakis 1, Yanhao Hou 1 , Boyang Huang 1 , Ilya Strashnov 2, Bruce D. Grieve 3 and Paulo Bártolo 1 1 Department of Mechanical, Aerospace and Civil Engineering, University of Manchester, Manchester M13 9PL, UK; [email protected] (A.M.O.); [email protected] (E.D.); [email protected] (Y.H.); [email protected] (B.H.); [email protected] (P.B.) 2 Department of Chemistry, University of Manchester, Manchester M13 9PL, UK; [email protected] 3 Department of Electrical & Electronic Engineering, University of Manchester, Manchester M13 9PL, UK; [email protected] * Correspondence: [email protected]

Received: 4 December 2020; Accepted: 15 December 2020; Published: 18 December 2020

Abstract: The search for materials with improved mechanical and biological properties is a major challenge in tissue engineering. This paper investigates, for the first time, the use of Polyethylene Terephthalate Glycol (PETG), a glycol-modified class of Polyethylene Terephthalate (PET), as a potential material for the fabrication of bone scaffolds. PETG scaffolds with a 0/90 lay-dawn pattern and different pore sizes (300, 350 and 450 µm) were produced using a filament-based extrusion additive manufacturing system and mechanically and biologically characterized. The performance of PETG scaffolds with 300 µm of pore size was compared with polycaprolactone (PCL). Results show that PETG scaffolds present significantly higher mechanical properties than PCL scaffolds, providing a biomechanical environment that promotes high cell attachment and proliferation.

Keywords: biomaterial; polyethylene terephthalate glycol; tissue engineering

1. Introduction Tissue engineering is an interdisciplinary field that applies the concepts of engineering and life sciences for the development of biological substitutes that maintain, restore or improve tissue or organ functions, and comprises two main strategies: scaffold-based and cell-laden approaches [1–6]. The scaffold-based approach, the most common strategy for bone tissue engineering applications, is based on the use of three-dimensional (3D) biocompatible and biodegradable porous structures that provide the substrate and the biomechanical environment for cells to attach, differentiate and proliferate [7–12]. Bone scaffolds must be produced using non-toxic materials (the materials must also degrade into non-toxic products), the degradation rate of the scaffold must be adjustable in order to match the rate of tissue regeneration, must present appropriate porosity, pore interconnectivity and pore structure (large number of pores may be able to enhance vascularization, while small pore sizes are preferable to provide large surface per volume ratio), should encourage the formation of the extra cellular matrix (ECM) by promoting cellular functions, should present sufficient strength and stiffness to withstand stresses in the host tissue environment and present adequate surface finish, guaranteeing that a good biomechanical coupling is achieved between the scaffold and the tissue [13–17]. Cell seeding on printed scaffolds depends on fast attachment of cell to scaffolds, high cell survival and uniform cell distribution, and these are strongly dependent on the scaffold material, architecture, surface stiffness and surface energy [2,18].

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A variety of biodegradable materials have been used to produce the scaffolds, including a wide range of organic, inorganic and composite materials [15,19–21]. Among these materials, polycaprolactone (PCL), an aliphatic polyester, was extensively used by our group for bone tissue engineering applications. PCL scaffolds with different porosities and pore sizes were successfully used to support the attachment and proliferation of osteoblasts and mesenchymal stem cells and osteogenic differentiation [22–25]. In vivo studies, using PCL scaffolds to treat critical size calvaria defects in rats, also showed the ability to support new tissue formation [26]. However, PCL presents limited bioactivity, long degradation times and low mechanical properties not suitable for load-bearing applications [8]. In order to improve the bioactivity nature of the scaffolds, new surface modification strategies such as selective plasma modification, acetone vapor annealing surface treatment and dopamine grafting, and silica nanoparticles coating, were explored [22]. Bioactivity was also improved by mixing PCL with ceramic materials such as hydroxyapatite (HA) and tricalcium phosphate (TCP), which also allowed to increase mechanical properties and to accelerate the degradation process. PCL/HA/multi-wall carbon nanotubes, mimicking the structure of bone, were successfully produced using extrusion-based additive manufacturing [27]. Carbon nanomaterials such as graphene and carbon nanotubes were also used to improve mechanical properties and to allow the fabrication of electro-active scaffolds. In vivo studies showed that these scaffolds together with electrical stimulation significantly accelerate the formation of new bone and the production of more organized new bone [21]. However, in all these cases, as PCL is the main material, the overall biological and mechanical properties of the scaffolds were constrained by the individual PCL properties. Therefore, the rationale of this research is to identify a biocompatible and biodegradable synthetic polymer that can be used as an alternative to PCL, allowing to produce scaffolds with significantly better mechanical and biological properties. Polyethylene Terephthalate (PET) has been used in the medical field, for example for prosthetic vascular grafts, due to its good mechanical properties and biocompatibility [28]. Moreover, PET is also biodegradable, making it an interesting material for tissue engineering, but due to its high crystallinity, it presents significant printability limitations [29]. One route to overcome this printability limitation is through a glycol modification process, which allows to reduce the crystallinity level by disrupting the order of the polymer chain without compromising the biological characteristics of the initial polymer [24]. This strategy allows to obtain Polyethylene Terephthalate Glycol (PETG), investigated for the first time in this paper, as a potential material for tissue engineering applications. Thus, this research work addresses the following research questions: (1) is it possible to create PETG scaffolds, with significantly improved mechanical properties compared to PCL scaffolds, presenting compressive modulus in the range of trabecular bone? (2) Is it possible to create PETG scaffolds with superior biological performance compared to PCL scaffolds without any surface modification?

2. Materials and Methods

2.1. Materials

PETG, with a printing temperature between 195 and 220 ◦C, was purchased from RS components (Northants, UK). PCL pellets (Mw 50,000 Da, CAPA 6500) were supplied by Perstorp Caprolactones (Malmo, Sweden). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) matrix was prepared using 2,5 Dihydroxybenzoic acid (DHB), Phenol and Chloroform (HPLC (High Performance Liquid Chromatography) grade), which were purchased from Sigma Aldrich (Dorset, UK). Biological analysis was carried out using Human adipose-derived stem cells (hADSCs), MesenPRO RSTM basal media, 2% (v/v) growth supplement, 1% (v/v) glutamine and 1% (v/v) penicillin/streptomycin, purchased from STEMPRO Invitroge, Thermo Fisher (Glasgow, UK) 2.2. Chemicals Analysis (MALDI-TOF MS). PET is a common polymer used in many applications due to its high melting point, but it presents low printability. To overcome these problems, manufacturers such as RS components modified PET by Polymers 2020, 12, 3045 3 of 12 adding Cyclohexanedimethanol (CHDM), producing PETG. MALDI-TOF MS was used to confirm the CHDM presence. This technique is based on the use of short, high-intensity laser pulses to form gas ions. In this case, the analyte molecules are not exposed to the laser but are added to a matrix of small organic molecules. The matrix shows strong absorption of the laser wavelength, improving efficiency and energy transfer [30]. Therefore, the matrix must have high electronic absorption for the used wavelength, stable under vacuum, low vapor pressure, good solubility in solvents that dissolve the analyte and good miscibility with the analyte in solid state. The PETG sample was dissolved in phenol/chloroform 1/1 (v/v). 2,5 Dihydroxybenzoic acid (DHB) was used as a matrix. A dried droplet sample was prepared by putting 1 µL of a mixture of 1 mL of 2,5 DHB in Phenol/chloroform, 1/1 (v/v), as a matrix and 1 µL of polymer in phenol/chloroform, 1/1 (v/v), on the target plate [30,31]. In order to assess the chemical composition of PETG, the mass spectrometer Plus MALDI-TOF/TOFMS analyzer Axima Confidence (Shimadzu Biotech, Kiyamachi-Nijo, Japan) equipped with a Nitrogen laser emitting at 337.1 nm was used to achieve data acquisition in positive ion mode. Data acquisition and processing were carried out using the Kratos analyzer software (Kratos, Manchester, UK). The acceleration voltage was set to 20 KV and the extraction delay time used was 15,000 in reflector MS mode. All mass spectra were collected by averaging the signal of 1620 laser shots.

2.2. Scaffolds’ Fabrication PETG and PCL scaffolds were produced using a filament-based additive manufacturing system (Flashforge Creator pro, Jinhua, China), considering a 0/90◦ lay-down pattern. PCL scaffolds were designed considering a pore size of 300 µm, and PETG scaffolds were designed considering three different pore sizes (PETG-300: 300 µm of pore size; PETG-350: 350 µm of pore size; PETG-450: 450 µm of pore size). Each scaffold was printed considering a total of 12 layers. The printing conditions are listed in Table1.

Table 1. Scaﬀold printing parameters.

Parameters Values Layer thickness (mm) 0.33 Filament diameter (mm) 0.35 Nozzle temperature (◦C) 230 Bed temperature (◦C) 60 Printing speed (mm/s) 20

2.3. Morphological Analysis The scaffolds’ morphology was characterized using the Scanning Electron Microscopy (SEM) FEI ESEM Quanta 200 system (FEI Company, Hillsboro, OR, USA). The EMITECH K550X sputter coater (Quorum Technologies, Lewes, UK) was used for gold-coating the scaffolds prior to imaging. Imaging was carried out using 15 KV acceleration voltage. The obtained images were analyzed using the ImageJ software (Laboratory for Optical and Computational Instrumentation, University of Wisconsin, WI, USA), allowing to determine pore size, filament diameter and layer thickness. For each scaffold type, a total of nine measurements were considered.

2.4. Mechanical Compression Tests Uniaxial compression tests were performed using an INSTRON X testing system (High Wycombe, UK) equipped with a 100 N load cell according to the ASTM D695-1. The scaﬀold samples were cut into 3 3 mm2 blocks. Samples (n = 6) were tested in a dry state at the displacement rate of 0.5 mm/min × until the strain reached 0.3 mm/mm. The obtained strain-stress data were further processed and plotted using the software Origin (Origin Lab, Northampton, MA, USA). Polymers 2020, 12, 3045 4 of 12

2.5. Biological Analysis

2.5.1. Cell Culture Human adipose-derived stem cells (hADSCs) were used to investigate the cytotoxicity of the scaffolds. MesenPRO RSTM, Thermo Fisher (Glasgow, UK),1 basal media, 2% (v/v) growth supplement, 1% (v/v) glutamine and 1% (v/v) penicillin/streptomycin were used for cell culture. hADSCs (passage = 5) were cultured in an incubator (37 ◦C, 5% CO2 and 95% humidity) until an appropriate cell density (90%) was achieved before cell seeding. Scaffolds were sterilized in an 80% Ethanol solution for 4 h, followed by washing with Phosphate Buffered Saline (PBS) solution twice. The scaffolds were left to dry overnight in a sterile lamina flow cabinet. The sterilized scaffolds were transferred into 24-well plates and kept inside the incubator before the cell seeding. An 89 µL cell suspension containing 50,000 cells was added into each scaffold at day 0. The samples were then transferred to the incubator for 2 h, allowing hADSCs to attach to the scaffolds followed by adding 900 µL of cell culture media to cover the scaffold. The scaffolds were transferred into new 24-well plates on the following day and the cell culture media was replaced.

2.5.2. Alamar Blue Assay Cell metabolic activity was investigated using the Alamar Blue assay, Sigma Aldrich (Dorset, UK), which can be used as an indicator of cell proliferation as it quantifies the metabolic activity of cells. Tests were performed at days 1, 7 and 14 after cell seeding. At each time point, 90 µL of Alamar Blue solution was added to each scaffold and the scaffolds were incubated for 4 h. Then, 200 µL solution from each sample was transferred into a 96-well plate and tested using a microplate reader at 530 nm excitation and 590 nm emission. After the measurements, the scaffolds were washed three times with sterilized PBS to remove the residual Alamar Blue solution and new media was added. Cell culture media was changed every two days.

2.5.3. Statistical Analysis Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s test. Diﬀerences were considered statistically signiﬁcant at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA) was used in this research.

3. Results and Discussion

3.1. Chemical Analysis The MALDI-TOF MS spectrum presented in Figure1 shows a wide peak in the high mass range, and as a consequence, cannot provide structural information. Therefore, to identify the polymeric material under analysis, the repeating units is the only possible method [32,33]. In this case, the oligomers with low mass are considered to obtain the relevant structural information, such as repeated units and possible end groups [30,31]. In Figure1, two lines were drawn to highlight the terephthalic acid (TPA) + ethylene glycol (EG) (black line), and TPA + CHDM (blue line). Besides the TPA + EG and TPA + CHDM peaks, the MALDI spectrum contains other peaks that correspond to fragment ions. The main constituent compounds and corresponding molecular weight values before the condensation reaction are presented in Table2[34]. Polymers 2020, 12, 3045 5 of 12

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FigureFigure 1. 1. TheThe Matrix-assisted laserlaser desorptiondesorption/ionizat/ionizationion time-of-ﬂight time-of-flight mass mass spectrometry spectrometry (MALDI-TOF (MALDI- TOFMS) MS) spectrum spectrum of Polyethylene of Polyethylene terephthalate terephthalate glycol glycol modiﬁed modified (PETG). (PET ItsG). characteristic Its characteristic repeated repeated units unitsare highlighted: are highlighted: 192.8 192.8 g/mol g/mol for the for Terephthalic the Terephtha acidlic +acidEthylene + Ethylene Glycol Glycol (TPA (TPA+ EG) + unitEG) andunit 274.3and 274.3g/mol g/mol for Terephthalic for Terephthalic acid + acidCyclohexanedimethanol + Cyclohexanedimethanol (TPA (TPA+ CHDM). + CHDM).

TableTable 2. 2. ChemicalChemical constituents constituents for for PETG. PETG.

CompoundCompound Molecular Molecular Formula Formula Molecular Molecular Weight Weight (g/mol) (g/mol)

TerephthalicTerephthalic acid acid (TPA) (TPA) C C88HH66OO4 4 166.13166.13 EthyleneEthylene Glycol Glycol (EG) (EG) (CH (CH22OHOH2)2 )62.07 62.07 CyclohexanedimethanolCyclohexanedimethanol (CHDM) (CHDM) C88HH1616OO2 2 144.21144.21

ItIt is is also also important important to to notice notice that that the the addition addition of of CHDM CHDM to to PET PET disturbs disturbs the the structure structure of of the the polymer,polymer, thus thus reducing reducing the the crystallinity, crystallinity, melting melting temperature temperature and and mechanical mechanical properties, properties, therefore therefore increasingincreasing printability printability [35]. [35].

3.2.3.2. Scaffold Scaffold Morphology Morphology FigureFigure 2 shows the SEM micrographsmicrographs of thethe printedprinted scascaffolds.ffolds. As As obse observed,rved, scaffolds scaffolds present present well-definedwell-defined geometries geometries with with regu regularlar distributed distributed squared squared pores. pores. The The cross-section cross-section images images (Figure (Figure 2B,2B, D)D) show show a a uniform uniform distribution distribution of of adjacent adjacent laye layers.rs. Moreover, Moreover, the the presence presence of of micro-pores micro-pores on on the the surfacesurface of thethe PCLPCL filaments filaments can can be be observed observed from from Figure Figure2A,C, 2A,C, which which can be can attributed be attributed to the material to the materialrheological rheological characteristics characteristics and to the and presence to the ofpresence brittle crystallineof brittle crystalline zones on the zones filament on the surface filament that surfacecollapse that during collapse the during printing the process printing [8]. process This can [8]. alsoThis explain can also the explain better the surface better quality surface of quality PETG ofsca PETGffolds scaffolds as it is known as it is thatknown PETG that is PETG less crystalline is less crystalline than PCL than [35 ].PCL However, [35]. However, further studiesfurther muststudies be mustconducted be conducted to confirm to confirm this. this. ContraryContrary to to PETG PETG scaffolds, scaffolds, which which present present pore pore size size (350 (350 µm)µm) and and filament filament diameter diameter (370 (370 µm)µm) valuesvalues similar similar to to the designed ones, aa deviationdeviation waswas observedobserved for for PCL PCL sca scaffoldsffolds (filament (filament diameter diameter of of340 340 ±20, 20, and and pore pore size size of of 360 360 ±10). 10). ± ±

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Figure 2. ((AA)) Top Top view view of of a a polycaprolactone polycaprolactone (PCL) (PCL) scaffold, scaffold, ( (BB)) cross-section cross-section view view of of a a PCL PCL scaffold, scaffold, (C) top viewview ofof aa PolyethylenePolyethylene terephthalateterephthalate glycol glycol modified modified (PETG) (PETG) sca scaffold,ffold, (D ()D cross-section) cross-section view view of ofa Polyethylenea Polyethylene terephthalate terephthalate glycol glycol modified modified (PETG) (PETG) scaffold. scaffold. All scaffolds All werescaffolds designed were considering designed consideringa pore size of a 350poreµ m.size of 350 µm.

3.3. Mechanical Mechanical Compression Compression Test Mechanical compression test results are presented in Figures3 3–5.–5. FigureFigure3 3 presents presents the the stress stress versus strainstrain curves curves for for all all considered considered sca ffscaffolds,olds, showing showing that, eventhat, foreven large for pore large sizes, pore PETG sizes, sca PETGffolds scaffoldsexhibit higher exhibit mechanical higher mechanical properties. properties. These curves These were curves also usedwere to also calculate used bothto calculate compressive both compressivemodulus and modulus compressive and compressive strength. From strength. Figures From4 and Fi5,gures it is possible 4 and 5, to it observeis possible that to compared observe that to comparedPCL scaffolds, to PCL PETG scaffolds, scaffolds PETG with the scaffolds same pore with size th (350e sameµm) pore exhibit size significantly (350 µm) exhibit higher significantly compressive highermodulus compressive and compressive modulus strength, and indicatingcompressive that, st fromrength, a mechanical indicating perspective, that, from and a consideringmechanical perspective,the same sca andffold considering architecture, the PETG same sca scaffoldffolds arearchitecture, more suitable PETG for scaffolds bone tissue are more engineering suitable than for bonePCL scatissueffolds. engineering For PETG scathanffolds, PCL the scaffolds. results alsoFor showPETG that scaffolds, by increasing the resu thelts pore also size show from that 300 by to increasing450 µm, the the compressive pore size modulusfrom 300 of to the 450 PETG µm, sca theffolds compressive decreases frommodulus 213 of12.2 the to PETG141 8.20scaffolds MPa. ± ± decreasesResults also from show 213 that ± 12.2 by increasing to 141 ± the8.20 pore MPa. size, Resu compressivelts also show strength that by decreases increasing from the 5.44 pore0.19 size, to ± compressive3.55 0.17 MPa strength. The averagedecreases compressive from 5.44 ± modulus 0.19 to 3.55 and ± strengths0.17 MPa. are The presented average compressive in Table3. Results modulus also ± andshow strengths that the are PETG-300 presented and in PETG-350 Table 3. Results scaffolds also have show the that same the orderPETG-300 of magnitude and PETG-350 of mechanical scaffolds propertieshave the same as human order trabecularof magnitude bone of (compressive mechanical pr modulusoperties meanas human value trabecular of 194 MPa, bone and (compressive compressive modulusstrength meanmean valuevalue ofof 3.55194 MPa)MPa, [and36– 39compressive]. strength mean value of 3.55 MPa) [36–39].

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Figure 3. Stress vs. strain curves for PCL scaffolds and PETG scaffolds with different pore sizes. FigureFigure 3.3. Stress vs.vs. strain curves for PCL scascaffoldsffolds andand PETGPETG scascaffoldsffolds withwith didifferentfferent porepore sizes. sizes.

Trabecular bone Trabecular bone

FigureFigure 4. 4.Compression Compression modulus modulus for for the the PCLPCL scascaffoldffold andand PETGPETG scaffolds scaffolds with with diffe differentrent pore pore size. size. *** ## ### ***Figurepp << 0.0010.001 4. Compression comparedcompared with with modulus control control for (PCL), (PCL), the PCL ## p pscaffold< <0.010.01 and and ### PE p

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Figure 5. Compression strength values for the PCL scaffold and PETG scaffolds with different pore size. *** p < 0.001 compared with control (PCL), ## p < 0.01 and ### p < 0.001 compared with different poreFigure size. 5. Compression The *** Statistical strength evidence values (p < for0.001) the isPCL the sca one-wayffold and analysis PETG of scaffolds the mechanical with different compression pore testsize. with *** p the < 0.001 use of compared GraphPad with Prism control software (PCL), and ## it p is < used 0.01 toand show ### p the < 0.001 difference compared be-tween with the different results. Thepore * size. is small The *** diff Statisticalerence and evidence while more (p < 0.001) * are is added the one-way the diff erencesanalysis betweenof the mechanical the results compression are higher. *test compared with the with use PCL of GraphPad scaffolds, #Prism compared software with and diff erentit is used pore sizeto show of PETG the scadifferenceffolds. be-tween the results. The * is small differenceTable and 3. Compressive while more * mechanical are added properties.the differences between the results are higher. * compared with PCL scaffolds, # compared with different pore size of PETG scaffolds. PCL PETG-300 PETG-350 PETG-450 Compressive Modulus (MPa)Table 3. Compressive 76.1 7.53 mechanical 213 12.2 properties. 196 6.53 141 8.20 ± ± ± ± Compressive Strength (MPa) 2.56 0.26 5.44 0.19 5.09 0.21 3.55 0.17 ± PCL PETG-300± PETG-350± PETG-450 ± Compressive Modulus (MPa) 76.1 ± 7.53 213± 12.2 196 ± 6.53 141 ± 8.20 3.4. Biological TestCompressive Strength (MPa) 2.56 ± 0.26 5.44 ± 0.19 5.09 ± 0.21 3.55 ± 0.17 Alamar Blue Assay was used to assess the metabolic activities of hADSCs seeded on PETG 3.4. Biological Test and PCL scaffolds at days 1, 7 and 14 of cell culture (Figure6). Results show that the fluorescence intensitiesAlamar for Blue all scaAssayffolds was increase used to by assess increasing the metabo the celllic activities culture time, of hADSCs indicating seeded that on all PETG scaffolds and arePCL cytocompatible scaffolds at days and 1, able 7 and to support 14 of cell cell culture attachment (Figure and 6). spreading. Results show At day that 1, the fluorescencefluorescence intensityintensities of for the all PCL scaffolds scaffold increase is statistically by increasing higher the than cell PETGculture sca time,ffolds. indicating However, that the all fluorescencescaffolds are intensitiescytocompatible of PETG-300 and able and to PETG-350 support scacellff oldsattachme significantlynt and increasespreading. and At are day statistically 1, the fluorescence higher than thoseintensity of PCL of the sca PCLffolds scaffold at day 7is of statistically cell culture. higher Moreover, than thePETG diff scaffolds.erences of However, the fluorescence the fluorescence intensities forintensities all PETG of scaPETG-300ffolds from and PETG-350 day 1 to day scaffolds 7 of cell significantly culture are increase significantly and are higher statistically than those higher of PCLthan scathoseffolds, of PCL suggesting scaffolds a rapidat day colonization 7 of cell culture. of hADSCs Moreover, on PETGthe differences scaffolds. of At the day fluorescence 14, all PETG intensities scaffolds presentfor all PETG statistically scaffolds higher from fluorescence day 1 to day intensities 7 of cell thanculture PCL are sca significantlyffolds. Although higher the than diff erencesthose of of PCL the fluorescencescaffolds, suggesting intensities a from rapid day colonization 7 to day 14 decrease of hADSCs for all on sca PETGffolds, allscaffolds. PETG sca Atff oldsday present14, all higherPETG valuesscaffolds than present PCLsca statisticallyffolds. These higher results fluorescence show that PETGintensities scaff oldsthan presentPCL scaffolds. better cell Although affinity than the PCLdifferences scaffolds. of the In termsfluorescence of pore size,intensities PETG-350 from sca dayffolds 7 to show day 14 overall decrease higher for fluorescenceall scaffolds, intensities all PETG thanscaffolds other present PETG scahigherffolds values at days than 1 and PCL 7. scaffolds. However, These PETG-450 results sca ffshowolds exhibitthat PETG a fast scaffolds increase present on the cellularbetter cell metabolic affinity activitythan PCL from scaffolds. day 7 to In day terms 14 comparedof pore size, to PETG-350 other scaff olds.scaffolds This show might overall be attributed higher tofluorescence low cell seeding intensities efficiency than other at day PETG 0. At scaffold day 14,s bothat days PETG-350 1 and 7. and However, PETG-450 PETG-450 scaffolds scaffolds present similarexhibit fluorescencea fast increase intensity on the results. cellular Moreover, metabolic at dayactivity 14,these from results day 7 seemto day to indicate14 compared higher to cellular other activityscaffolds. in This scaff oldsmight presenting be attributed larger topore low sizes,cell seeding which mightefficiency be attributed at day 0. At to theday fact 14, thatboth larger PETG-350 pore sizesand PETG-450 allow better scaffolds cell infiltration present andsimilar reduce fluorescence the risk of intensity cell aggregation results. [40Moreover,]. at day 14, these results seem to indicate higher cellular activity in scaffolds presenting larger pore sizes, which might

Polymers 2020, 12, x 9 of 12 be attributed to the fact that larger pore sizes allow better cell infiltration and reduce the risk of cell aggregation [40]. SEM images of cells attached and spreading on PETG-350 scaffolds are shown in Figure 7. From thisPolymers figure,2020, 12it, 3045is possible to observe that cells are homogeneously distributed on the filaments,9 of 12 establishing cell–cell networks and bridging adjacent layers.

Figure 6. AlamarAlamar Blue Blue results results for for both both PCL PCL and and PETG PETG scaf scafffoldsolds at at days days 1, 1, 7 7 and and 14 14 post-cell-seeding. post-cell-seeding. * Statistical Statistical evidence evidence (p ( p< <0.05)0.05) analyzed analyzed by one-way by one-way analysis analysis of variance of variance (ANOVA) (ANOVA) and Tukey’s and Tukey’s post- test.post-test. The * Statistical The * Statistical evidence evidence (p < 0.05),**, (p < 0.05), *** is the **, ***one-way is the one-wayanalysis of analysis variance of (one-way variance ANOVA) (one-way andANOVA) Tukey’s and post Tukey’s hoc post test hocwith test the with use the of use GraphP of GraphPadad Prism Prism software software and and it is it isused used to to show show the differencedifference be-tween the results. The The * * is is small small di differencefference and while more more * are added the differences differences between the results are higher.

SEM images of cells attached and spreading on PETG-350 scaffolds are shown in Figure7. From this figure, it is possible to observe that cells are homogeneously distributed on the filaments, establishing cell–cell networks and bridging adjacent layers.

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FigureFigure 7. Cells 7. Cells on PETG-350 on PETG-350 scaff oldsscaffolds after after 14 days 14 days of cell of seeding. cell seeding. (A) Top (A) viewTop view image image of the of PETG the PETG scaffold,scaffold, (B) cross-section (B) cross-section image image of PETG of PETG scaff old,scaffold, (C) magnified (C) magnified image image showing showing cells coveringcells covering the the PETGPETG filament, filament, (D) cells (D) bridgingcells bridging adjacent adjacent layers. layers.

4. Conclusions 4. Conclusions Scaffolds for bone tissue engineering should present appropriate mechanical properties and Scaffolds for bone tissue engineering should present appropriate mechanical properties and promote the right environment for cells to attach and proliferate. Among the different biocompatible promote the right environment for cells to attach and proliferate. Among the different biocompatible materials being investigated for bone applications, PCL is the most common one. However, PCL scaffolds materials being investigated for bone applications, PCL is the most common one. However, PCL do not present suitable mechanical properties and these properties significantly decrease by increasing scaffolds do not present suitable mechanical properties and these properties significantly decrease porosity or pore size, important parameters to allow vascularization, supplying oxygen and nutrients by increasing porosity or pore size, important parameters to allow vascularization, supplying oxygen to cells inside the scaffold. This paper investigated, for the first time, the potential use of PETG as an and nutrients to cells inside the scaffold. This paper investigated, for the first time, the potential use alternative material to PCL. This material, obtained by adding CHDM to PET (as confirmed by MALDI of PETG as an alternative material to PCL. This material, obtained by adding CHDM to PET (as analysis), was successfully printed, and the produced scaffolds exhibited a well-defined geometry. confirmed by MALDI analysis), was successfully printed, and the produced scaffolds exhibited a Resultswell-defined also showed geometry. that PETG Results scaffolds also present showed higher th compressiveat PETG scaffolds modulus andpresent compressive higher strengthcompressive µ thanmodulus PCL scaffolds, and compressive not only for thestrength reference than case PCL (350 scaffolds,m of porenot only size) for but the also reference when we case considered (350 µm of µ PETGpore scaffolds size) but with also larger when pore we sizes considered (450 m). PETG Moreover, scaffolds PETG with scaffolds larger also pore showed sizes (450 better µm). biological Moreover, behavior,PETG as scaffolds observed also from showed the Alamar better Bluebiological assay. behavi In thisor, case, as observed it was also from possible the Alamar to observe Blue highassay. In cellularthis activity case, it was in scaffolds also possible presenting to observe large high pore cellular sizes. Considering activity in sc bothaffolds mechanical presenting and large biological pore sizes. µ performances,Considering results both seem mechanical to suggest and that biological the PETG perfor scaffoldmances, with results pore size seem of 350to suggestm is, amongthat the the PETG differentscaffold scaffolds with pore considered size of in350 this µm study, is, among the most the appropriatedifferent scaffolds one. considered in this study, the most Authorappropriate Contributions: one. Conceptualization, M.H.H.; methodology, M.H.H., E.D., Y.H., and I.S.; software, M.H.H. and Y.H.; validation and formal analysis, A.M.O. and B.H.; investigation, M.H.H., A.M.O., E.D., and Y.H.; resources and dataAuthor curation, Contributions: All Authors; Conceptualization, writing—original M.H.H.; draft preparation, methodology, M.H.H.; M.H.H., writing—review E.D., Y.H., and and I.S.; editing, software, M.H.H., M.H.H. A.M.O.,and and Y.H.; P.B.; validation visualization, and E.D.formal and analysis, Y.H.; supervision, A.M.O. and B.D.G. B.H.; and investigation, P.B. All authors M.H.H., have A.M.O., read and E.D., agreed and to Y.H.; the published version of the manuscript. resources and data curation, All Authors; writing—original draft preparation, M.H.H.; writing—review and Funding:editing,This M.H.H., project A.M.O., has been and partially P.B.; visualization, supported by E.D. the Universityand Y.H.; supervision, of Manchester B.D.G. and and the EngineeringP.B. All authors and have Physicalread Sciences and agreed Research to the Council published (EPSRC) version of theof the UK, manuscript. the Global Challenges Research Fund (CRF), grant number EP/R01513/1. Funding: This project has been partially supported by the University of Manchester and the Engineering and Conﬂicts of Interest: The authors declare no conﬂict of interest. Physical Sciences Research Council (EPSRC) of the UK, the Global Challenges Research Fund (CRF), grant number EP/R01513/1.

Conflicts of Interest: The authors declare no conflict of interest.

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