3D Printing for Cartilage Replacement: A Preliminary Study to Explore New Polymers

  Citation: Delgado, G.F.; Pinho, A.C.; Piedade, A.P. 3D Printing for Cartilage Replacement: A Preliminary Study to Explore New Polymers. Polymers 2022,14, 1044. https://doi.org/10.3390/ polym14051044 Academic Editor: Andrea Ehrmann Received: 5 February 2022 Accepted: 2 March 2022 Published: 5 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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 Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). polymers Article 3D Printing for Cartilage Replacement: A Preliminary Study to Explore New Polymers Gonçalo F. Delgado, Ana C. Pinho and Ana P. Piedade * Department of Mechanical Engineering, CEMMPRE, University of Coimbra, 3030-788 Coimbra, Portugal; [email protected] (G.F.D.); [email protected] (A.C.P.) *Correspondence: [email protected]; Tel.: +351-239-790-700 Abstract: The use of additive manufacturing technologies for biomedical applications must begin with the knowledge of the material to be used, by envisaging a very specific application rather than a more general aim. In this work, the preliminary study was focused on considering the cartilaginous tissue. This biological tissue exhibits different characteristics, such as thickness and mechanical properties, depending on its specific function in the body. Due to the lack of vascularization, cartilage is a supporting connective tissue with limited capacity for recovery and regeneration. For this reason, any approach, whether to repair/regenerate or as a total replacement, needs to fulfill the adequate mechanical and chemical properties of the surrounding native cartilage to be successful. This work aims to explore the possibility of using new polymers for cartilage total replacement approaches with polymeric materials processed with the specific 3D printing technique of fused filament fabrication (FFF). The materials studied were Nylon ® 12 (PA12), already described for this purpose, and LAYFOMM ® 60 (FOMM). FOMM has not been described in the literature for biomedical purposes. Therefore, the chemical, thermal, swelling capacity, and mechanical properties of the filaments were thoroughly characterized to better understand the structure–properties–application relationships of this new polymer. In addition, as the FFF technology is temperature based, the properties were also evaluated in the printed specimens. Due to the envisaged application, the specimens were also characterized in the wet state. When comparing the obtained results with the properties of native cartilage, it was possible to conclude that: (i) PA12 exhibits low swelling capacity, while FOMM, in its dry and wet forms, has a higher swelling capacity, closer to that of native cartilage; (ii) the mechanical properties of the polymeric materials, especially PA12, are higher than those of native cartilage; and (iii) from the mechanical properties evaluated by ultra-micro hardness tests, the values for FOMM indicate that this material could be a good alternative for cartilage replacement in older patients. This preliminary study, essentially devoted to expanding the frontiers of the current state of the art of new polymeric materials, provides valuable indications for future work targeting the envisaged applications. Keywords: cartilage tissue; 3D printing; Nylon®12; LAY-FOMM®60; mechanical properties 1. Introduction Cartilaginous tissue, or simply cartilage, is a supporting connective tissue composed of collagen, proteoglycan-rich matrix, and a single cell type, chondrocytes. This tissue differs from other human tissues due to its unique properties, especially a lack of blood vessels and nerve cells [ 1 ]. In the human body, cartilage formation is based on a process called chondrogenesis that, unfortunately, fails to enable the tissue to naturally self-repair after injury or degeneration [2]. Damage in cartilage can be induced by trauma and some clinical pathologies such as osteoarthritis [ 3 ]. Although the degeneration of cartilage is more common in the elderly due to sports activity, younger patients are increasingly being diagnosed [ 3 ]. Since this Polymers 2022,14, 1044. https://doi.org/10.3390/polym14051044 https://www.mdpi.com/journal/polymers Polymers 2022,14, 1044 2 of 19 condition strongly interferes with the patients’ quality of life, an effective solution for the repair/replacement of cartilaginous tissue is needed. As mentioned before, cartilage plays an important role in the human body, especially concerning supporting functions. Therefore, this tissue can adapt and bear mechanical loading while being able to deform and recover to the original volume, similar to a sponge with water [ 4 ]. Such demanding requirements increase the difficulty when designing devices for its substitution. Nowadays, the two most common approaches for cartilage replacement and repair are total replacement, usually with cobalt–chrome (CoCr) or ultrahigh molecular weight polyethylene (UHMWPE)-based structures, and the scaffold implantation, in the case of tissue regeneration approach [ 5 – 7 ]. In the case of total replacement, the major challenges are related to the materials used and their mechanical performance, namely tensile strength and Young modulus. Even though CoCr is considered as a biocompatible and non-degradable material, its stiffness leads to the mechanical shielding of the bone induced by mechanical loading [ 7 ]. Although UHMWPE presents mechanical properties similar to those of native cartilage, it can be structurally unstable under loading, hindering its mechanical and tribological performance [ 6 ]. Moreover, due to wear, metal ions can be released, leading to genotoxic effects such as carcinogenicity and DNA damage [8–10]. For repair or regeneration purposes, biomedical scaffolds are often indicated as an advantageous solution as they can provide a 3D framework to enable cell proliferation, matrix deposition, and consequent tissue regeneration [ 11 ]. From work published in this area, the most common natural materials used in such devices are collagen, agarose, chitosan, hyaluronic acid, fibrin, and alginate [ 12 – 14 ]. Of the synthetic polymers, poly(ethylene glycol) (PEG) [ 15 , 16 ], poly(lactic acid) (PLA) [ 17 , 18 ], poly(vinyl alcohol) (PVA) [ 19 , 20 ] and polyurethane (PU) [21,22] are the most described and the produce best outputs. For the design and architecture of the scaffolds and biomedical devices already reported for cartilage regeneration, structures such as membranes, hydrogels, and nanofibers produced by phase inversion [ 23 ], solvent-casting particle leaching [ 24 ], freeze–drying [ 25 ], and electrospinning [ 26 ] are among the approaches providing the most promising results. Nonetheless, the outcomes are still insufficient. Additive manufacturing (AM) or 3D printing is a technology that enables the preparation of fully customizable scaffolds [ 27 , 28 ] with intricate shapes that can be designed using computer-aided design (CAD) or computed tomography (CT) data [ 29 – 31 ]. Due to the ease of processing and geometric freedom, this has been investigated for cartilaginous tissue regeneration purposes. Indeed, She et al. published a work describing the preparation of a scaffold prepared by 3D printing using two different materials [ 32 ]. The outside was a printed PCL hollow ring with a collagen sponge inside to mimic the anatomy of the native trachea of white rabbits. In vitro tests proved the growth of tracheal cartilage within the scaffold. The production of a silk fibroin-based scaffold with a 3D printed PCL mesh filling was also reported elsewhere [ 33 ]. 3D printed PCL/graphene composite scaffolds were also reported showing improved lubricity and drug-releasing properties [34]. In a different approach, a scaffold combining 3D printed polycarbonate–urethane (PCU) and UHMWPE was studied for the purposes of native lubrication mechanisms [ 35 ]. Unfortunately, surface roughness and consequent high friction coefficient have jeopardized its performance. In order to investigate the role of inner scaffold architecture, Jung et al. developed a 3D-printed PU tracheal scaffold with microscale design, which proved to be beneficial for cell infiltration and biological integration of the device [ 36 ]. Despite the number of publications and studies, each material and design approach needs to be directed to a specific type of cartilage and local implantation, which impairs the agreement and standardization of which route (material/processing technique) to follow. PA12 is a semicrystalline polymer with excellent impact resistance at low temperatures, low water absorption, resistance to stress cracking, and fatigue under high-frequency cyclical loading conditions, which is frequently used in AM due to the feasibility of the Polymers 2022,14, 1044 3 of 19 process [ 37 , 38 ]. In addition, it is commonly used for applications related to the biomedical field, including for cartilage [39]. Therefore, it was used as a control material. FOMM is a new commercially available material, and constitutes a mixture of two polymers, one of which is PVA. FOMM becomes flexible and porous when immersed in water due to the removal, by dissolution, of the PVA content. This characteristic may be of the main interest when applying this polymeric material for cartilage replacement. As cartilage does not contain blood vessels or nerves, and is supplied with nutrients through the compression and flexion of the tissue, it needs to have a porous structure to allow these interactions. Pitaru et al. published a work in which they use FOMM in an attempt to match the mechanical properties of native anterior cruciate ligaments, with promising results [ 40 ]. Nevertheless, the cited article is the only one concerning the consulted bibliography that presents research with FOMM material. The present work describes a preliminary study exploring the possibility of using PA12 and FOMM for the preparation of structures, by 3D printing, for cartilage repair. To the best of our knowledge, this is the first time that such materials have been proposed for this specific application. 2. Materials and Methods 2.1. Materials In the present work, polymeric filaments with a diameter of 1.75 ± 0.03 mm were used. Nylon ® 12 (PA12) filament was supplied by DoWire ® (Seixal, Portugal) and LAYFOMM ® 60 (FOMM) filament was acquired from Filament2print ® (Nigrán, Spain). For comparative purposes, and prior to some characterization techniques, PA12 and FOMM filaments and printed parts were immersed in deionized water for four days (wPA12 and wFOMM, respectively). 2.2. Processing by 3D Printing All specimens were printed using a FlashForge TM Creator 3 3D printer (Ílhavo, Portugal) with a dual extruder, each with a 0.4 mm diameter nozzle. PA12 filament was printed at 260 ◦ C with a bed temperature of 110 ◦ C, while FOMM was printed at 230 ◦ C with a bed temperature of 70 ◦ C (Figure 1). The printing parameters for FOMM were previously optimized by varying a set of parameters that included: printing temperature from 220–250 ◦C , bed temperature from 30–80 ◦ C, and printing speed from 15 to 30 mm · s −1 . Both materials were printed at the same speed, 25 mm · s −1 , with a 50% hexagonal infill pattern and 180 µ m layer thickness. Two bottom and upper layers (100% infill with a linear pattern) were used to support and facilitate the specimen printing. The geometry of the printed specimens was chosen according to the requirements of the characterization technique, as discussed in the following sections. Polymers 2022, 14, 1044 3 of 19 frequency cyclical loading conditions, which is frequently used in AM due to the feasibility of the process [37,38]. In addition, it is commonly used for applications related to the biomedical field, including for cartilage [39]. Therefore, it was used as a control material. FOMM is a new commercially available material, and constitutes a mixture of two polymers, one of which is PVA. FOMM becomes flexible and porous when immersed in water due to the removal, by dissolution, of the PVA content. This characteristic may be of the main interest when applying this polymeric material for cartilage replacement. As cartilage does not contain blood vessels or nerves, and is supplied with nutrients through the compression and flexion of the tissue, it needs to have a porous structure to allow these interactions. Pitaru et al. published a work in which they use FOMM in an attempt to match the mechanical properties of native anterior cruciate ligaments, with promising results [40]. Nevertheless, the cited article is the only one concerning the consulted bibliography that presents research with FOMM material. The present work describes a preliminary study exploring the possibility of using PA12 and FOMM for the preparation of structures, by 3D printing, for cartilage repair. To the best of our knowledge, this is the first time that such materials have been proposed for this specific application. 2. Materials and Methods 2.1. Materials In the present work, polymeric filaments with a diameter of 1.75 ± 0.03 mm were used. Nylon® 12 (PA12) filament was supplied by DoWire® (Seixal, Portugal) and LAYFOMM® 60 (FOMM) filament was acquired from Filament2print® (Nigrán, Spain). For comparative purposes, and prior to some characterization techniques, PA12 and FOMM filaments and printed parts were immersed in deionized water for four days (wPA12 and wFOMM, respectively). 2.2. Processing by 3D Printing All specimens were printed using a FlashForgeTM Creator 3 3D printer (Ílhavo, Portugal) with a dual extruder, each with a 0.4 mm diameter nozzle. PA12 filament was printed at 260 °C with a bed temperature of 110 °C, while FOMM was printed at 230 °C with a bed temperature of 70 °C (Figure 1). The printing parameters for FOMM were previously optimized by varying a set of parameters that included: printing temperature from 220–250 °C, bed temperature from 30–80 °C, and printing speed from 15 to 30 mm·s−1. Both materials were printed at the same speed, 25 mm·s−1, with a 50% hexagonal infill pattern and 180 μm layer thickness. Two bottom and upper layers (100% infill with a linear pattern) were used to support and facilitate the specimen printing. The geometry of the printed specimens was chosen according to the requirements of the characterization technique, as discussed in the following sections. Figure 1. Macrograph of FOMM printed test specimen for flexural tests. 2.3. Characterization 2.3.1. Chemical Characterization The infrared (IR) spectra of the studied filaments were acquired with FTNIR/MIR equipment (PerkinElmer, Frontier model, Waltham, MA, USA), equipped with an Figure 1. Macrograph of FOMM printed test specimen for flexural tests. 2.3. Characterization 2.3.1. Chemical Characterization The infrared (IR) spectra of the studied filaments were acquired with FTNIR/MIR equipment (PerkinElmer, Frontier model, Waltham, MA, USA), equipped with an attenuated total reflectance (ATR), an FR-DTGS detector, and a KBr beam splitter, at 20 ◦ C. For the data acquisition, the resolution was 4 cm −1 , a constant force of 80 N, and 16 accumulation interferograms. PerkinElmer also supplied the ATR module with a diamond/ZnSe Polymers 2022,14, 1044 4 of 19 crystal. After the data collection, the spectrums were analyzed through the SPECTRUM 10 STD software. 2.3.2. Thermal Characterization The thermal stability of filaments and printed parts was studied using a TGA Q500 V20.13 equipment by TA instruments (New Castle, DE, USA), with a heating rate of 10 ◦C·min−1 , between 25–600 ◦ C, with a nitrogen flux of 50 mL · min −1 . The results were analyzed using the TA Instruments Universal Analysis 2000 software supplied by the manufacturer. The thermal events of the studied filaments and printed specimens were assessed using a DSC Q100 V9.9 equipment by TA instruments, with a heating rate of 10 ◦ C · min −1 and a 50 mL · min −1 constant flux of nitrogen. The analysis of the results of the first heating cycle and the determination of the crystallization and enthalpies ( ∆Hcc and ∆Hm , respectively) were performed using TA Instruments Universal Analysis 2000 software, supplied by TA Instruments. The percentage of crystallinity (Xc) was calculated using Equation (1): Xc(%)=∆Hm−∆Hcc ∆H∞ ×100 (1) where ∆H∞ is a characteristic value of each material, corresponding to the melting enthalpy variation considering 100% of crystallinity [41]. The weight of the samples used for both thermal characterization techniques was kept constant at 8 mg. 2.3.3. Morphological Characterization The scanning electron microscopy (SEM) technique was used to observe the morphological dissimilarities between FOMM and wFOMM. The equipment used for the filament characterization was a ZEISS ® Merlin 61–50 Microscope (Carl Zeiss, Oberkochen, Germany), Gemini 2, with an accelerating voltage of 2 kV. Using a sputtering technique, all samples were coated with a 3 nm layer of gold. Samples were coated for 60 s with the help of EDWARDS EXC 120 sputtering equipment (Crawley, UK), with a power source Huttinger PFG 1500 DC (Schwaig bei Nuremberg, Germany). The sputtering conditions were: power, 0.11 kW; voltage, 1000 V; current, 1.83 A. The surface and cross-section morphologies of the printed specimens were characterized using an FEI Quanta 400FEG ESEM (FEI, Hillsboro, OR, USA). For the cross-sectional observation, the samples were immersed for 90 s in liquid nitrogen. This allowed for a clean fracture of the samples by mechanical impact. The printed samples were observed without any metallic coating. 2.3.4. Swelling Capacity The water uptake of the filaments studied at the present work was assessed by swelling capacity tests (SC). Five test samples of PA12, FOMM, and wFOMM filaments were dried at 50 ◦ C until weight equilibrium and their initial weight collected. Then, all samples were immersed in 15 mL of ionized water at room temperature for seven days. The weight of the samples was collected every 24 h or 48 h and the water was substituted. The SC of the materials was determined through Equation (2): SC (%)=WS−W0 WS ×100 (2) where WSrepresents the swollen weight and W0is the initial dried weight [42]. 2.3.5. Mechanical Characterization The tensile strength of the studied materials was determined using a Shimadzu apparatus, more specifically the Autograph AGS-X model (Tokyo, Japan), with a 5 kN load cell and a grip speed of 5 mm · min −1 . All materials (PA12, wPA12, FOMM, and wFOMM) were Polymers 2022,14, 1044 5 of 19 tested at both filament (100 mm segments) and printed specimen ( 100 mm ×20 mm ×2 mm , according to ASTM D3039) configurations. Figure 2shows a representative filament test. Polymers 2022, 14, 1044 5 of 19 The tensile strength of the studied materials was determined using a Shimadzu apparatus, more specifically the Autograph AGS-X model (Tokyo, Japan), with a 5 kN load cell and a grip speed of 5 mm·min−1. All materials (PA12, wPA12, FOMM, and wFOMM) were tested at both filament (100 mm segments) and printed specimen (100 mm × 20 mm × 2 mm, according to ASTM D3039) configurations. Figure 2 shows a representative filament test. Figure 2. Representative macrograph of tensile test of the FOMM filaments. Five samples of each material and form were considered for the study. For all tested materials, the distance between opposite ends, span, was 50 mm, and the obtained results were analyzed on Trapezium X software (Tokyo, Japan). The results were displayed in stress–strain curves, from which the calculation of Young’s modulus (E) was performed, according to Equation (3) [43], E=σ ԑ (3) where σ refers to stress and ԑ is the strain. Three-point bending (3PB) tests determined the flexural strength of the printed specimens. Five samples of each printed material were considered for the calculations. The dimensions of the testing specimens (60 mm × 10 mm × 2 mm) were chosen according to the ASTM Standard D790 recommendations. Tests were conducted using an Autograph AGS-X equipment from Shimadzu, with a 5 kN load cell and a displacement rate of 2 mm·min−1. The flexural strength (σ) was determined as the nominal stress in the middle span section obtained using the maximum load value, according to Equation (4), σ  = 3PL 2bh (4) where P refers to the maximum load, h and b are the thickness and the width of the specimen, respectively, and L represents the span length, which was kept constant at 40 mm. Flexural modulus (E) was determined following the linear elastic bending beams theory relationship, which can be expressed by Equation (5), E  =∆PL 48∆uI (5) where ∆P is the load range, ∆µ is the flexural displacement range, and I refers to the moment of inertia. E was acquired by linear regression of the obtained load–displacement curves contemplating the interval in the linear segment with a correlation factor greater than 95%. Figure 2. Representative macrograph of tensile test of the FOMM filaments. Five samples of each material and form were considered for the study. For all tested materials, the distance between opposite ends, span, was 50 mm, and the obtained results were analyzed on Trapezium X software (Tokyo, Japan). The results were displayed in stress–strain curves, from which the calculation of Young’s modulus (E) was performed, according to Equation (3) [43], E=σ ε(3) where σrefers to stress and εis the strain. Three-point bending (3PB) tests determined the flexural strength of the printed specimens. Five samples of each printed material were considered for the calculations. The dimensions of the testing specimens (60 mm × 10 mm × 2 mm) were chosen according to the ASTM Standard D790 recommendations. Tests were conducted using an Autograph AGS-X equipment from Shimadzu, with a 5 kN load cell and a displacement rate of 2 mm·min−1 . The flexural strength ( σf ) was determined as the nominal stress in the middle span section obtained using the maximum load value, according to Equation (4), σf=3PL 2bh2(4) where P refers to the maximum load, h and bare the thickness and the width of the specimen, respectively, and L represents the span length, which was kept constant at 40 mm . Flexural modulus ( Ef ) was determined following the linear elastic bending beams theory relationship, which can be expressed by Equation (5), Ef=∆PL3 48∆uI (5) where ∆P is the load range, ∆µ is the flexural displacement range, and I refers to the moment of inertia. Ef was acquired by linear regression of the obtained load–displacement curves contemplating the interval in the linear segment with a correlation factor greater than 95%. Ultra-microhardness characterization results were recorded by Fischerscope H100 equipment (Sindelfingen, Germany). Three printed specimens of PA12, wPA12, FOMM, and wFOMM were submitted to 5 indentation runs in 2 different areas. The test cycles Polymers 2022,14, 1044 6 of 19 consisted of a load–hold–unload function. The load rate was tuned so that each run would last about 60 s, with 30 s hold period at the maximum load for thermal drift correction. Six indentations were performed, in each run, at maximum load using 0.525 root increments, and 19 measurements in 30 s were made to access the creep value. The load increased from 0.4 mN to 1000 mN. 3. Results and Discussion 3.1. Filament Characterization 3.1.1. Chemical Composition The polymeric filaments were used as received. As is usual, suppliers do not share factual information on several aspects, including the percentage and type of additives mixed in the main polymeric material. For this reason, the chemical composition of PA12 and FOMM filaments was evaluated by FTIR. Poly(vinyl alcohol) (PVA) and wFOMM filaments were also analyzed and compared with the original FOMM spectrum to confirm the existence of PVA in the original formulation and its dissolution by immersion in water, as stated by the supplier. Figure 3displays the obtained spectrum for each tested filament. Figure 3a confirms the chemical structure of PA12, as it displays similarity with other spectra already reported in the literature [ 44 ]. The presence of the stretching vibration of N–H, CH 2 , and C=O at 3286 cm −1 (a), 3000–2800 cm −1 (b), and 1633 cm −1 (c), respectively, are highlighted; the overlapping of the bands corresponding to the bending vibration of C=O and the stretching vibration of C–N at 1537 cm −1 (d); and finally, the bending vibration of CH 2 at 1447 cm −1 (e) [ 44 ]. Therefore, if any additives have been added to PA12, they are present in a residual concentration that will not affect the chemical properties of the polyamide. For the analysis of FOMM results, it must be reminded that the literature lacks information concerning this material’s chemical composition. In addition, the supplier only refers to the presence of PVA and does not provide any more details concerning the other polymer. For this reason, a PVA filament spectrum was overlapped with FOMM to identify the peaks referring to PVA. From the comparison the spectra of PVA and FOMM, it is possible to identify the well-defined PVA peaks located between 3500–3000 cm −1 (f) related to the stretching vibrations of the O-H group and the stretching vibration of the C=O group between 1750–1650 cm−1(c), even though they are slightly shifted. These variations were already expected since the mixture of PVA with another polymeric material, as reported by Alireza Kharazmi et al. obtained a similar outcome when ZnS nanoparticles were incorporated into PVA [45]. To identify the remaining FOMM peaks, Pitaru et al. proposed that FOMM is composed of a mixture of PVA and flexible thermoplastic polyurethane (TPU) [40]. This is the only published work that analyzes the other polymer present besides PVA, to the best of our knowledge. For this reason, the obtained wFOMM spectrum (with no PVA due to dissolution in water) was compared with TPU spectra from the literature. It is possible to observe the typical bands associated with TPU, such as the stretching vibration of the N–H group at 3350–3250 cm −1 (a), the band corresponding to CH 2 between 2950–2850 cm −1 (b), the stretching vibration of C=O at 1750–1650 cm −1 (c), and the stretching vibration of C–N between 1260–1230 cm −1 (g). Since only the N–H stretching band is not common to PVA, it is not possible to fully conclude, at this stage, that TPU may be the other polymer mixed with PVA. However, considering the literature and the obtained results, this is a strong possibility. Polymers 2022,14, 1044 7 of 19 Polymers 2022, 14, 1044 7 of 19 Figure 3. FTIR spectra of the polymeric filaments: (a) PA12, (b) FOMM, PVA and wFOMM. The letters (a–g) identify the characteristic bands discussed in the text. 3.1.2. Thermal Characterization Thermogravimetric analysis (TGA) was used to assess the thermal stability of the PA12 and FOMM filaments. The resulting thermogravimetric curves are plotted in Figure 4. Figure 3. FTIR spectra of the polymeric filaments: ( a ) PA12, ( b ) FOMM, PVA and wFOMM. The letters (a–g) identify the characteristic bands discussed in the text. 3.1.2. Thermal Characterization Thermogravimetric analysis (TGA) was used to assess the thermal stability of the PA12 and FOMM filaments. The resulting thermogravimetric curves are plotted in Figure 4. The thermal stability of materials is especially important when processing by 3D printing since it is a temperature-based process. For this reason, it is important to ensure that materials are extruded without jeopardizing their integrity. From the observation of Figure 4a it is possible to conclude that the decomposition of PA12 occurred within a single step between 375 ◦ C and 500 ◦ C, which was an expected result and in agreement with other results [46]. Polymers 2022,14, 1044 8 of 19 Polymers 2022, 14, 1044 8 of 19 Figure 4. Weight loss and derivative of weight loss (DTG) thermogravimetric curves of (a) PA12 and (b) FOMM, as received. The thermal stability of materials is especially important when processing by 3D printing since it is a temperature-based process. For this reason, it is important to ensure that materials are extruded without jeopardizing their integrity. From the observation of Figure 4a it is possible to conclude that the decomposition of PA12 occurred within a single step between 375 °C and 500 °C, which was an expected result and in agreement with other results [46]. In the case of FOMM, once again, no direct comparisons can be established with the scientific literature due to the lack of studies of this polymer. Nonetheless, the obtained thermogravimetric curves exhibited three weight loss stages: around 100 °C, between 250– 360 °C, and 360–475 °C. The first stage (100 °C) is assigned to the loss of water. In turn, the second and third stages (250–340 °C and 340–450 °C) match the decomposition stages of urethane bonds and polyol chains, respectively, and are usually found in TPU decomposition profiles [47]. One can then assume that TPU seems to be one of the counterparts that constitute the FOMM filament. However, one cannot exclude the information concerning the chemical composition of FOMM provided by the supplier which indicates that PVA is part of the composition of FOMM. For this reason, the profile obtained for FOMM was compared with pure PVA decomposition profiles found in the literature. Herein, three decomposition stages were found, and their temperatures also matched with FOMM. However, in the case of PVA, the second stage refers to the decomposition of bound water, which is water that is directly bonded to the polymeric structure, not only absorbed on the surface, and the third stage is assigned to the decomposition and consequent carbonization of the PVA network [48]. Since TPU and PVA degradation stages overlap and match the FOMM profile, the presence of TPU in the composition of FOMM could not be wholly confirmed by TGA measurements. The onset and peak temperatures determined from the analysis of the displayed thermograms are presented in Table 1. Table 1. Reference temperatures obtained by TGA. Filament Ton (°C) T5% (°C) T10% (°C) Tp1 (°C) Tp2 (°C) PA12 433.1 416.3 426.9 454.6 - FOMM 296.5 298.7 314.4 333.5 403.1 Ton—Onset temperature; T5%—Temperature to which corresponds 5% of weight loss; T10%—Temperature to which corresponds 10% of weight loss; Tp—peak temperature. Figure 4. Weight loss and derivative of weight loss (DTG) thermogravimetric curves of ( a ) PA12 and (b) FOMM, as received. In the case of FOMM, once again, no direct comparisons can be established with the scientific literature due to the lack of studies of this polymer. Nonetheless, the obtained thermogravimetric curves exhibited three weight loss stages: around 100 ◦ C, between 250–360 ◦ C, and 360–475 ◦ C. The first stage (100 ◦ C) is assigned to the loss of water. In turn, the second and third stages (250–340 ◦C and 340–450 ◦C) match the decomposition stages of urethane bonds and polyol chains, respectively, and are usually found in TPU decomposition profiles [ 47 ]. One can then assume that TPU seems to be one of the counterparts that constitute the FOMM filament. However, one cannot exclude the information concerning the chemical composition of FOMM provided by the supplier which indicates that PVA is part of the composition of FOMM. For this reason, the profile obtained for FOMM was compared with pure PVA decomposition profiles found in the literature. Herein, three decomposition stages were found, and their temperatures also matched with FOMM. However, in the case of PVA, the second stage refers to the decomposition of bound water, which is water that is directly bonded to the polymeric structure, not only absorbed on the surface, and the third stage is assigned to the decomposition and consequent carbonization of the PVA network [48]. Since TPU and PVA degradation stages overlap and match the FOMM profile, the presence of TPU in the composition of FOMM could not be wholly confirmed by TGA measurements. The onset and peak temperatures determined from the analysis of the displayed thermograms are presented in Table 1. Table 1. Reference temperatures obtained by TGA. Filament Ton (◦C) T5% (◦C) T10% (◦C) Tp1 (◦C) Tp2 (◦C) PA12 433.1 416.3 426.9 454.6 - FOMM 296.5 298.7 314.4 333.5 403.1 T on —Onset temperature; T 5% —Temperature to which corresponds 5% of weight loss; T 10% —Temperature to which corresponds 10% of weight loss; Tp—peak temperature. By comparing the values of the two filaments, it is evident that PA12 has superior thermal stability and can withstand temperatures close to 400 ◦ C, as all decomposition and onset temperatures are above this value. The experimentally determined T on of PA12 was 433.1 ◦ C; above this temperature, PA12 starts to disintegrate and does not maintain its structural integrity. On the other hand, the T on of FOMM occurs slightly before the material loses 5% of its mass. The thermal events of the filaments were studied by DSC to correctly define the printing parameters according to the thermal transitions of the materials. The resulting Polymers 2022,14, 1044 9 of 19 curves are plotted in Figure 5and the determined transition temperatures are presented in Table 2. Polymers 2022, 14, 1044 9 of 19 By comparing the values of the two filaments, it is evident that PA12 has superior thermal stability and can withstand temperatures close to 400 °C, as all decomposition and onset temperatures are above this value. The experimentally determined Ton of PA12 was 433.1 °C; above this temperature, PA12 starts to disintegrate and does not maintain its structural integrity. On the other hand, the Ton of FOMM occurs slightly before the material loses 5% of its mass. The thermal events of the filaments were studied by DSC to correctly define the printing parameters according to the thermal transitions of the materials. The resulting curves are plotted in Figure 5 and the determined transition temperatures are presented in Table 2. Figure 5. Heat flux curves obtained for PA12 and FOMM filaments, as received. Table 2. Transition temperatures obtained by DSC of the filaments. Filament Tg1 (°C) Tg2 (°C) Tcc (°C) Tm (°C) PA12 107.6 - 143.6 246.6 FOMM −42.9 82.4 - 155.9 The heat flux curve of PA12 presents three different thermal events at specific temperatures, where the first endothermic reaction, at 107.6 °C, corresponds to the glass transition temperature (Tg). Then, at 143.6 °C, the plot displays an exothermic curve which indicates the cold crystallization (Tcc) of the polymeric structure. Finally, at 246.6 °C, the material undergoes melting (Tm). The determined values of ∆H and ∆H were 3.25 J·g−1 and 19.06 J·g−1, respectively. Assuming that PA12 ∆H is 209.3 J·g−1 [41], the calculated value for X was 6.7%. Regarding FOMM, the identification of the thermal events was first established considering the known data available in the literature for the same PVA sample used in the previous FTIR analysis. Pure PVA presents a single Tg close to around 80 °C [49], which was also observed in the FOMM curve, and thus reinforced the possible presence of PVA in FOMM. Then, the FOMM profile was compared with heat flux curves of TPU available in the literature to confirm if TPU was part of FOMM composition. As reported elsewhere [31], TPU displays two glass transitions, the first negative and the second around 70–80 °C. In addition, TPU has a melting temperature, Tm, between 150–160 °C [50], also observed in the FOMM profile. Since these three transitions can be observed in FOMM, it can be concluded that FOMM contains PVA and TPU. DSC measurements were crucial for the selection of printing temperatures since they should be higher than the Tm of the materials (246.6 °C for PA12 and 155.9 °C for FOMM). 3.1.3. Morphological Characterization Figure 5. Heat flux curves obtained for PA12 and FOMM filaments, as received. Table 2. Transition temperatures obtained by DSC of the filaments. Filament Tg1 (◦C) Tg2 (◦C) Tcc (◦C) Tm(◦C) PA12 107.6 - 143.6 246.6 FOMM −42.9 82.4 - 155.9 The heat flux curve of PA12 presents three different thermal events at specific temperatures, where the first endothermic reaction, at 107.6 ◦ C, corresponds to the glass transition temperature (T g ). Then, at 143.6 ◦ C, the plot displays an exothermic curve which indicates the cold crystallization (T cc ) of the polymeric structure. Finally, at 246.6 ◦ C, the material undergoes melting (T m ). The determined values of ∆Hcc and ∆Hm were 3.25 J · g −1 and 19.06 J · g −1 , respectively. Assuming that PA12 ∆H∞ is 209.3 J · g −1 [ 41 ], the calculated value for Xcwas 6.7%. Regarding FOMM, the identification of the thermal events was first established considering the known data available in the literature for the same PVA sample used in the previous FTIR analysis. Pure PVA presents a single T g close to around 80 ◦ C [ 49 ], which was also observed in the FOMM curve, and thus reinforced the possible presence of PVA in FOMM. Then, the FOMM profile was compared with heat flux curves of TPU available in the literature to confirm if TPU was part of FOMM composition. As reported elsewhere [ 31 ], TPU displays two glass transitions, the first negative and the second around 70–80 ◦ C. In addition, TPU has a melting temperature, T m , between 150–160 ◦ C [ 50 ], also observed in the FOMM profile. Since these three transitions can be observed in FOMM, it can be concluded that FOMM contains PVA and TPU. DSC measurements were crucial for the selection of printing temperatures since they should be higher than the T m of the materials (246.6 ◦C for PA12 and 155.9 ◦C for FOMM). 3.1.3. Morphological Characterization The morphology of FOMM and wFOMM filaments was observed by SEM, and the obtained micrographs are displayed in Figure 6. Polymers 2022,14, 1044 16 of 19 higher. On the contrary, FOMM results are closer to the human rib cartilage evaluated by the same technique. However, it is important to note that the listed values from native cartilage refer to male patients between 63 and 86 years old [ 60 ]. Chondrocytes begin to dissipate from the superficial region with increased age, and accumulate in the deeper layers. As a result, the hydration decreases, and the matrix becomes stiffer [ 65 ]. Thus, these values should be lower in younger patients. Nevertheless, FOMM presents closer results to native cartilage than PA12, indicating that this material could be a good alternative for cartilage replacement in older patients. Since the prevalence of cartilage diseases is higher in older patients, the impact of FOMM results is even more relevant. 4. Conclusions The present work aimed to produce 3D printed structures that could be used in the biomedical field, specifically for cartilage repair/substitution. This work also intended to expand the frontiers of knowledge using a polymeric material rarely reported in the literature, FOMM. The study also examined PA12. Both the dry and wet forms of each materials were subjected to filament characterization (chemical, thermal, and mechanical), confirming the chemical composition of PA12 and, in the case of FOMM, calculating the amount of PVA in its structure (15%). In addition, it was concluded that FOMM was composed of PVA and TPU. 3PB tests conducted on dry and wet specimens showed that wFOMM samples had compelling results similar to native cartilage. The SC of FOMM and wFOMM proved to be similar to native cartilage. PA12, in turn, exhibited a poor swelling rate, which could be helpful for cartilage repair/regeneration in a multimaterial approach. In the ultra-microhardness test, as expected, all test pieces had higher Er compared with the 3PB test. However, wPA12 displayed a stiffer behavior than dry PA12 owing to the molecular interactions between water and N–H groups. On the other hand, FOMM and wFOMM presented similar results to native cartilage of older patients. This similarity could be beneficial as most cases of cartilage replacement occur in aged patients. The obtained results provide promising evidence that 3D printed parts may be part of the future of regenerative medicine. Furthermore, this study highlighted new research paths, such as designing multimaterial structures with a PA12 core and outer shell in wFOMM. Future work should include the preparation of such multimaterial structures and their in vitro characterization, which will include prokaryotic and eukaryotic cell tests. Author Contributions: Conceptualization, G.F.D., A.C.P. and A.P.P.; methodology, A.C.P. and A.P.P.; validation, A.C.P. and A.P.P.; formal analysis G.F.D. and A.C.P.; investigation, G.F.D. and A.C.P.; resources, A.P.P.; writing—original draft preparation, G.F.D. and A.C.P.; writing—review and editing, A.P.P.; visualization, A.C.P.; supervision, A.C.P. and A.P.P.; project administration, A.P.P.; funding acquisition, A.P.P. All authors have read and agreed to the published version of the manuscript. Funding: The research was developed within the scope of the project CEMMPRE, UIDB/00285/2020, financed by national funds through the FCT. 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