Biocompatible 3D-printed tendon/ligament scaffolds based on polylactic acid/graphite nanoplatelet composites

Citation: Silva, M.; Gomes, S.; Correia, C.; Peixoto, D.; Vinhas, A.; Rodrigues, M.T.; Gomes, M.E.; Covas, J.A.; Paiva, M.C.; Alves, N.M. Biocompatible 3D-Printed Tendon/ Ligament Scaffolds Based on Polylactic Acid/Graphite Nanoplatelet Composites. Nanomaterials 2023,13, 2518. https://doi.org/10.3390/nano13182518 Academic Editors: Kinga Pielichowska, Anna Morawska-Chochol and Patrycja Domalik-Pyzik Received: 28 July 2023 Revised: 29 August 2023 Accepted: 4 September 2023 Published: 8 September 2023 Copyright: © 2023 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/). nanomaterials Article Biocompatible 3D-Printed Tendon/Ligament Scaffolds Based on Polylactic Acid/Graphite Nanoplatelet Composites Magda Silva 1,2,3 , Susana Gomes 3, Cátia Correia 1,2, Daniela Peixoto 1,2 , Adriana Vinhas 1,2 , Márcia T. Rodrigues 1,2 , Manuela E. Gomes 1,2 , JoséA. Covas 3, Maria C. Paiva 3and Natália M. Alves 1,2,* 1 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark, 4805-017 Guimarães, Portugal; [email protected] (M.S.); [email protected] (C.C.); [email protected] (D.P.); [email protected] (A.V.); [email protected] (M.T.R.); [email protected] (M.E.G.) 2ICVS/3B’s, Associate PT Government Laboratory, 4710-057 Braga/4805-017 Guimarães, Portugal 3Department of Polymer Engineering, Institute for Polymers and Composites, University of Minho, 4800-058 Guimarães, Portugal; [email protected] (S.G.); [email protected] (J.A.C.); [email protected] (M.C.P.) *Correspondence: [email protected] Abstract: Three-dimensional (3D) printing technology has become a popular tool to produce complex structures. It has great potential in the regenerative medicine field to produce customizable and reproducible scaffolds with high control of dimensions and porosity. This study was focused on the investigation of new biocompatible and biodegradable 3D-printed scaffolds with suitable mechanical properties to assist tendon and ligament regeneration. Polylactic acid (PLA) scaffolds were reinforced with 0.5 wt.% of functionalized graphite nanoplatelets decorated with silver nanoparticles ((f-EG)+Ag). The functionalization of graphene was carried out to strengthen the interface with the polymer. (f-EG)+Ag exhibited antibacterial properties against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), an important feature for the healing process and prevention of bacterial infections. The scaffolds’ structure, biodegradation, and mechanical properties were assessed to confirm their suitability for tendon and ligamentregeneration. All scaffolds exhibited surface nanoroughness created during printing, which was increased by the filler presence. The wet state dynamic mechanical analysis proved that the incorporation of reinforcement led to an increase in the storage modulus, compared with neat PLA. The cytotoxicity assays using L929 fibroblasts showed that the scaffolds were biocompatible. The PLA+[(f-EG)+Ag] scaffolds were also loaded with human tendon-derived cells and showed their capability to maintain the tenogenic commitment with an increase in the gene expression of specific tendon/ligament-related markers. The results demonstrate the potential application of these new 3D-printed nanocomposite scaffolds for tendon and ligament regeneration . Keywords: 3D printing; functionalized graphite nanoplatelets; PLA; composites; ligaments; tendons 1. Introduction In recent years, 3D printing has emerged as tool for the production of complex and personalized products, built layer by layer, and with the advantages of low cost and easy operation [ 1 , 2 ]. Three-dimensional printing finds great potential applications in the medical field, in particular in tissue engineering (TE), to produce scaffolds with complex and reproducible geometries, allowing excellent control of porosity and pore size, which is not possible with traditional manufacturing processes. It also provides the possibility to create customized, patient-specific scaffolds [ 2 – 5 ]. Fused deposition modeling (FDM) (also known as fused filament fabrication (FFF), is the most commonly used material extrusion 3D printing method, presenting several advantages compared with other techniques [ 5 ]. It is reliable and cheap and does not require solvents [6]. Nanomaterials 2023,13, 2518. https://doi.org/10.3390/nano13182518 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2023,13, 2518 2 of 23 Tendon/ligament injuries are some of the most prevailing health problems that affect the adult population worldwide [ 7 ]. Various materials and strategies have been suggested to find a scaffold that can help the regeneration of these tissues, restoring its function when it is severely injured [ 8 ]. It has been reported that highly porous scaffolds with interconnected pores are essential for nutrient and oxygen diffusion, waste removal, and cell proliferation [ 3 , 9 ]. In addition, the degradation rate of the implant should match the rate of the new tissue formation, allowing it to receive the appropriate level of mechanical load from the scaffold [9,10]. PLA is routinely used for medical applications such as sutures or orthopedic fixation devices [ 6 ], being slowly degraded and fully reabsorbed [ 11 ]. It is made from bio-based monomers that are obtained from corn or cellulose [ 6 ] with easy modulation of its physical and biochemical features by blending with different nanofillers [ 11 ]. The mixing or in situ modification with toughening agents, flame retardants, and anti-UV agents have been used to address the unfavorable inherent qualities of PLA (i.e., poor toughness, inflammability, and UV aging) and achieve high-performance PLA composites. However, the addition of these additives may increase the economic cost or even reduce the PLA mechanical properties due their agglomeration [ 12 ]. A new bio-based porphyrins approach was suggested by Yang and co-workers [ 12 ] to improve PLA’s overall multifunctional performance. The incorporation of only 3 wt.% of vanillin-based porphyrin (VPR) not only enhanced the anti-UV and flame-retardant properties but also enabled significant toughening of the PLA/VPR composites as well as improvements of elongation at break and impact strength. This strategy greatly increases the versatility of PLA composites [ 12 ]. Formulations based on PLA have been widely proposed for tendon/ligament regeneration [ 13 , 14 ], but there are very few studies about the use of material extrusion 3D printing for this application [ 2 , 15 , 16 ], being mainly focused on screw-like scaffolds to help tendon–bone healing after anterior cruciate ligament (ACL) reconstruction [ 2 , 16 ]. This ligament holds a commonly accepted relevance because of the huge number of related injuries and reconstructive surgeries [ 17 ]. There has been an increased interest in the use of graphene-based materials such as graphite nanoplatelets (GNPs) and carbon nanotubes (CNTs) for different tissue engineering applications such as cardiac, neural, and tendon/ligament regeneration, mainly focusing their reinforcing effect on polymer composites [ 7 , 17 ]. For instance, Belaid et al. [ 6 ] produced 3D-printed scaffolds based on PLA–graphene oxide (GO). Tensile testing demonstrated a 30% increase in the Young’s modulus with the incorporation of 0.3 wt.% GO. Composite scaffolds also promoted bone cell attachment, proliferation, and differentiation [ 6 ]. In fact, the nanoscale dimension of graphene matches the cell surface receptors and extracellular matrix (ECM) nanotopography, promoting the adhesion and proliferation of various types of cells. In addition, graphene-based scaffolds may also exhibit additional functional properties such as enhanced electrical conductivity, which may be beneficial for the cellular growth and stimulation of the healing process [ 7 , 11 ]. However, each application involving the addition of graphene in the human body should always be carefully investigated because it was observed to have a size-, shape-, and concentration-time-dependent cytotoxicity [ 7 , 11 ]. It is commonly accepted that using small loadings of graphene materials such as graphene nanoplatelets (composed of single-layer and few-layer graphene) can be effective in the reinforcement of polymeric matrices such as PLA and do not present cytotoxicity [ 6 , 11 , 17 , 18 ]. When the polymer matrix is a biomaterial, the possibility of a toxic effect of the fillers is even diminished [ 19 ]. Moreover, the functionalization of graphene is of great importance for the scaffold success. The presence of functional groups may enable stronger interactions with the matrix, thus improving simultaneously the reinforcing ability and biocompatibility [7,17]. An effective strategy to reduce the risk of infection is to confer antibacterial properties to the implant [ 20 ]. Silver nanoparticles (AgNPs) have been extensively combined with inorganic materials and biopolymers to produce 3D-printed scaffolds with antibacterial activity [21,22]. Nanomaterials 2023,13, 2518 3 of 23 The objective of this work was the production of 3D-printed scaffolds with controllable dimensions and good mechanical properties to help tendon and ligament regeneration, as well as their in vitro investigation. We functionalized the GNPs via a 1,3 dipolar cycloaddition reaction (DCA) of an azomethine ylide, which bonds pyrrolidine groups onto the graphene surface to form amide bonds with the ester groups of PLA under the composite processing conditions [ 23 , 24 ]. Silver nanoparticles were also produced by reducing silver nitrate (AgNO 3 ) in dimethylformamide (DMF) and used to decorate the functionalized graphene [ 25 ]. A synergetic effect of silver and graphene properties may also occur, such as reported by Kumar S. et al. [ 26 ], who produced sheets of reduced GO decorated with silver with improved electrical and antibacterial properties. Composite filaments based on medical-grade PLA containing a low content (0.5 wt.%) of functionalized and Ag-decorated few-layer graphene were produced and applied to form 3D-printed parts, obtaining porous and reproducible scaffolds. The antibacterial efficiency against Gram-positive and Gram-negative bacteria was confirmed. The mechanical performance and biodegradation of 3D-printed scaffolds were evaluated under physiological conditions, as well as their biocompatibility using L929 cells. The scaffolds were loaded with human tendon-derived stem cells, to investigate the tenogenic commitment and to analyze the gene expression of specific tendon/ligament-related markers. The results are promising and will hopefully widen the application of 3D-printed devices for tissue engineering and demonstrate their potential use in tendon and ligament healing and regeneration. 2. Materials and Methods 2.1. Materials Medical-grade PLA pellets (PURASORB ® PL10) were purchased from Corbion, Gorinchem, The Netherlands. The PLA exhibited an inherent viscosity in the range 0.9–1.2 dL/g and melting point between 170 and 200 ◦ C. Micrograf HC11, a micronized graphite with a purity of 99.5%, equivalent diameter of approximately 10 µ m, and few tens of nanometers of thickness, will be referred, throughout the text, as EG and was purchased from Nacional de Grafite Lda, Itapecerica, MG, Brazil. EG was functionalized (f-EG) by the DCA reaction as described previously [ 23 ] using a functionalization time of 3 h at 250 ◦ C. The decoration of f-EG with silver nanoparticles [(f-EG)+Ag] was achieved by the reduction reaction of silver ions (Ag + ). The detailed procedures and characterization were previously described [23]. 2.2. Antimicrobial Potential of Functionalized Graphite The antibacterial properties of different graphite nanoplatelets (EG, f-EG, and [(fEG)+Ag]) were evaluated directly against microorganisms of clinical relevance, namely, Gram-positive Staphylococcus aureus (S. aureus) (ATCC 25923) and Gram-negative Escherichia coli (E.coli) (ATCC 25922). First, E. coli and S. aureus strains were cultured on Mueller Hinton Broth (MHB) at 37 ◦ C and 60 rpm, and the microbial suspension was adjusted to 1.0 ×106CFU/mL . Then, different EGs were sterilized using 1 h of UV lights and dispersed in a sterilized MHB medium to obtain a concentration of 2% w/v. The different EG and EG derivatives suspensions were sonicated 1 h before the assay, and then several dilutions were prepared for the tests (1.00, 0.50, 0.25, 0.10, 0.050, and 0.025% w/v). In a 96-well plate, 50 µ L of the bacterial suspension was added to 50 µ L of the different concentrations of EGs suspensions. The plate was incubated at 37 ◦ C for 24 h. Then, aliquots from each well (10 µ L) were added onto the surface of nutrient Mueller Hinton agar (MHA) and incubated at 37 ◦ C for 24 h. The minimal bactericidal concentration (MBC) was determined as the lowest concentration that showed no bacterial growth on the agar plate. Several controls were used: a bacterial suspension without EGs, a bacterial suspension with Kanamycine (5% w/v), and different EGs suspensions without a bacterial suspension. All assays were performed in triplicate. The obtained results allowed the selection of the percentage of EGs for composites’ preparation. Nanomaterials 2023,13, 2518 4 of 23 2.3. Filaments Production and Characterization According to the literature, low concentrations of GNPs did not present in vitro cytotoxicity and may be incorporated safely in PLA to improve aspects relevant for biomedical applications, such as mechanical properties [ 18 , 27 ]. Based on this consideration and the antibacterial results, we fixed the filler content at 0.5 wt.%. To ensure a good dispersion of the reinforcements in the polymer, a pre-mixing of PLA pellets with 0.5 wt.% of EG, f-EG, and (f-EG)+Ag was prepared by manual mixing. The PLA and PLA composite filaments were obtained by using melt extrusion on a co-rotating twin-screw extruder (Microlab Rondol, Nancy, France) equipped with intermeshing screws containing three mixing zones, using a screw speed of 43 rpm and a temperature profile 135/185/ ≃ 160 ◦ C (feed/barrel/die). The extrudate diameter was controlled using two pulling rolls, distanced approximately 25 and 60 cm from the shaping die, respectively. The extrusion process followed the procedure described before [ 23 ] under nitrogen atmosphere, as recommended by the polymer manufacturer, to minimize polymer degradation. The detailed processing conditions for each material are presented in Table S1, where pulling roll 1 was located next to the extruder die and allowed the production of filaments with approximately 1.75 ±0.25 mm , suitable for FDM. Scanning electron microscopy and energy dispersive spectroscopy (SEM/EDS) were carried out on a FEI Nova 200 FEG-SEM/EDS (FEI Europe Company, Hillsboro, OR, USA) to analyze the coating of the PLA pellets with EG and EG derivatives and to observe the cross-sections of the 3D printing filaments produced. The melt flow index (MFI) of all filaments was measured at 190 ◦ C using a load of 2.16 kg on MFI equipment from Daventest (Welwyn Garden City, UK). High-definition Kelvin force microscopy (HD-KFM) was used to assess the surface electric potential of the PLA+0.5[(f-EG)+Ag] filament, using a Nano-Observer AFM microscope, CSInstruments (Les Ulis, France). The measurements were carried out on longitudinal sections of composite filaments (10 × 10 µ m), using a 1V AC signal, at 53 KHz applied to the surface. 2.4. Scaffolds Production and Characterization The 3D-printed scaffolds were designed using the Ultimaker Cura (version 4.4, Ultimaker, Geldermalsen, The Netherlands) software and printed horizontally using an Ender-3 3D Printer from Creality (London, UK). The printing parameters are given in Table 1. The scaffolds exhibited a cylindrical shape with a full length of approximately 32 mm and a diameter of 9 mm, which were comparable to the dimensions of the native ACL. Smaller specimens with an approximate diameter of 9 mm and 4 mm of thickness were also produced for further testing. Table 1. Printing settings used for printing PLA and PLA composite scaffolds. Printing Parameters Nozzle diameter 0.4 mm Nozzle temperature 190 ◦C Bed temperature 80 ◦C Printing speed 45 mm.s−1 Layer height 0.15 mm Infill distance 0.8 mm Infill density 50% Infill pattern Lines Infill lines direction 0◦; 90◦ Support contact angle 5◦ 2.4.1. Physical and Morphological Analysis of Scaffolds The scaffolds’ morphology as well as the pore size and distribution were analyzed using a Leica DMS1000 digital microscope (Wetzlar, Germany). The average pore size for Nanomaterials 2023,13, 2518 5 of 23 each scaffold was obtained by running Image J software (version 1.52, National Institutes of Health, Bethesda, MA, USA) on the digital microscopy images. Perpendicular lines at 15 pores from one border of the pore toward the other were measured by the software, at the top, front, and lateral views of the scaffold. Each measurement was taken using the green channel and with improved contrast. The porosity of the as-prepared 3D scaffolds was determined using the liquid displacement method similar to that reported by Guan et al. [ 28 ] and Zhang and Ma [ 29 ]. Ethanol was chosen as a displacement liquid because it could permeate through the porous scaffolds and did not induce swelling or shrinking of the material. Each scaffold was immersed in a cylinder containing a known volume of ethanol (V1). The sample was kept soaked in ethanol for 5 min. Then, the ethanol was pressed to force air from the scaffold and to penetrate and fill the pores. The total volume of ethanol and the ethanol-impregnated scaffold was recorded as V2. The ethanol-impregnated scaffold was removed from the cylinder, and the residual ethanol volume was recorded as V3. The porosity of the scaffold (%) was given by Porosity (%)=V1−V3 V2−V3×100. (1) The average of three measurements was taken for each sample. To confirm a homogenous dispersion of EG and EG derivatives on the PLA, the 3D-printed scaffolds were cryo-fractured, and the scaffold cross-sections were sputter-coated with gold and observed by using SEM/EDS on a FEI Nova 200 FEG-SEM/EDS (FEI Europe Company, Hillsboro, OR, USA). The topography and roughness of the PLA and PLA+[(f-EG)+Ag] scaffolds were determined at the outer layer of the measured scaffolds. The measurements were performed by using a Nano-Observer AFM, CSInstruments (Les Ulis, France), and the operation mode was oscillating, with an amplitude of 5V and automatic frequency around 60 KHz. AFM topography images with dimension (10 × 10) µ m 2 were obtained. The root mean square (RMS) surface roughness was calculated by using the statistical tool in the Gwyddion software. This represented the standard deviation of the distribution of surface heights, and it was more sensitive than the arithmetic average height (Ra) to large deviation from the mean line. At least three measurements were performed for each type of scaffold. 2.4.2. Mechanical/Viscoelastic Properties The dynamical mechanical analysis (DMA) was carried out to evaluate the mechanical performance and viscoelastic properties of the scaffolds subjected to cyclic loading and immersed in physiologic fluids. The scaffolds were previously soaked overnight in a PBS solution at 37 ◦ C. The DMA analysis was performed using TRITEC2000B equipment (Triton Technology, Grantham, UK) in the compressive mode. The DMA spectra were obtained at the same temperature, applying cycles of increasing frequency from 0.1 to 16 Hz. At least three samples were tested for each composition and scaffold type. 2.4.3. Biodegradation The degradation rates of different scaffolds (9 mm of diameter and 4 mm of thickness) were evaluated in vitro by measuring their initial weight and soaking them into a phosphate buffered saline (PBS, pH = 7.4, Sigma-Aldrich, Saint Louis, MO, USA) at 37 ◦ C. The PBS solution was changed every 3 days. At predefined periods (15 days, 6 weeks, and 12 weeks) the samples were removed from the solution, washed with distilled water to remove the excess salts, and dried at 37 ◦ C for 2 days. The mass loss was calculated using the following equation: Weight loss (%)=Mi−Mf Mi×100 (2) where M i and M f are the weights of the scaffolds before and after degradation, respectively. Three replicates per composition were analyzed, and the results are presented as an averaged value ±standard deviation. Nanomaterials 2023,13, 2518 6 of 23 Specimens of PLA and PLA+0.5EG/f-EG/[(f-EG)+Ag] were also observed by SEM and compared with images of non-degraded samples, to identify the surface erosion after 12 weeks of hydrolytic degradation. To evaluate the changes in the mechanical properties after the complete degradation period, the scaffolds (n= 3 or 4 per scaffold composition per time point) were immersed in 1 mL of PBS (pH = 7.4) at 37 ◦ C overnight and tested by dynamical mechanical analysis, using the method described above. 2.4.4. Biological Assays—L929 Cell Line Cell seeding. For the in vitro cell studies, L929 mouse fibroblast-like cells (NCTC clone 929, ATCC ® CCL-1 ™ , acquired from ATCC ® (Manassas, VA, USA), passage P26) were cultured in Dulbecco’s modified minimum essential medium (low glucose DMEM, Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Paisley, UK) and 1% of an Antibiotic-Antimycotic (A/A) solution (Life Technologies, Paisley, UK), in 150 cm 2 tissue culture flasks. The cells were maintained in a humidified air atmosphere containing 5% CO 2 at 37 ◦ C to grow, and the medium was replaced every 3 days until a 90% confluence was reached. Then, the cells were washed with Dulbecco’s phosphate buffered saline (DPBS, Life technologies, Carlsbad, CA, USA) and detached with 5 mL of trypLE ™ express solution (Life technologies, Paisley, UK) for 5 min at 37 ◦ C. An amount of 10 mL of culture medium was added to inactivate the trypLE ™ . The cells were centrifuged at 300 rcf for 5 min, and the obtained pellet was resuspended in the culture medium. Before the cell seeding, the scaffolds (diameter = 9 mm, thickness = 4 mm) were sterilized by immersion in 70% ethanol (v/v) for 1 h and by exposition to UV light, for 30 min on both sides. The sterile scaffolds were placed in a 24-wells suspension culture plate, and 200 µ L of a cell suspension in DMEM culture medium (2 × 10 5 ) was added to each well. The samples were then incubated at 37 ◦ C in a humidified air atmosphere of 5% CO 2 . After 4 h of seeding, fresh culture medium was added to each well until reaching 1 mL of volume. The seeding procedure was also applied on tissue culture polystyrene (TCPS, Sarstedt, Singapore) to be used as a positive control. Live/Dead staining. The viability of the L929 cells was evaluated by Calcein AM (ThermoFisher Scientific, Bleiswijk, The Netherlands) and Propidium Iodide (PI) (ThermoFisher Scientific, Bleiswijk, The Netherlands) staining. Before staining, at each time point (1, 3, 7, and 14 days of culture), the culture medium was removed, and each sample was immersed with 1 mL of DMEM medium supplemented with 2 µ g Calcein AM and 1µg PI, for 30 min, in the dark. After that, the samples were washed with PBS and analyzed using an inverted confocal microscope with incubation (TCS SP8, Leica, Germany). All experiments were performed in triplicate. SEM. The attachment and morphology of L929 cells were analyzed by SEM. The scaffolds were removed from the wells after 1, 3, 7, and 14 days of culture; washed with PBS; and then fixed with 2.5% glutaraldehyde (Sigma-Aldrich, Saint Louis, MO, USA) for 2 h . After dehydration in a graded series of ethanol (50%, 70%, 80%, 90%, and 100%) and thermostatic drying, the scaffolds were gold sputtered for analysis. DAPI-Phalloidin staining. The morphology and cytoskeletal organization of the cells was visualized by fluorescent microscopy after staining with phalloidin tetramethylrhodamine and 4 0 ,6-diami-dino-2-phenylindole (DAPI, Sigma-Aldrich, Saint Louis, MO, USA). After 1, 3, 7, and 14 days of culture, the medium was removed and washed with PBS fixed by using neutral buffered formalin (10%, ThermoFisher Scientific, Waltham, MA, USA) for 30 min. Then, the fixed samples were permeabilized using Triton X-100 (0.2% v/vin PBS, Sigma-Aldrich, Saint Louis, MO, USA) for 5 min and immersed in bovine serum albumin (BSA, 3% w/vin PBS, Sigma-Aldrich, Saint Louis, MO, USA) for 30 min. The seeded scaffolds were stained with DAPI (1:1000 in PBS, pH = 7.4) for 5 min and rhodamine-phalloidin (1:100 in PBS, pH = 7.4, Sigma Aldrich, Saint Louis, MO, USA) for Nanomaterials 2023,13, 2518 7 of 23 30 min . The samples were extensively washed with PBS and analyzed using an AiryScan 2 confocal microscope (LSM 980, Zeiss, Germany). Alamar blue. The metabolic activity of the cells was determined by using the Alamar blue method for 1, 3, 7, and 14 days of culture. After each time point, the culture medium was removed, and a fresh medium supplemented with 20% of Alamar blue reagent (BioRad, Hercules, CA, USA) was added to the cultured scaffolds. The samples were incubated in the dark, for 4 h, at 37 ◦ C, in a humidified air atmosphere of 5% CO 2 . Following this, 100 µL of each solution was transferred to a 96-well black plate to measure the fluorescence at the 590 nm emission wavelength and the 530 nm excitation wavelength using a microplate reader (BIO-TEK Instruments, Winooski, VT, USA). 2.4.5. Biological Assays—Human Tendon-Derived Cells Human tendon-derived cells (hTDCs) — Isolation and Culture. The hTDCs were isolated from tendon surplus samples under established protocols with Hospital da Prelada (Porto, Portugal). The samples were provided with the informed consent of the patients, and the procedures were reviewed and approved by the Hospital Ethics Committee (P.I. N º . 005/2019). The hTDCs were isolated and cultured as previously described [ 30 – 33 ]. First, the tissue explants were dropwise rinsed in a sterile solution of PBS. The excess of PBS was eliminated using a filtration system for 50 mL tubes (Falcon, Shawnee, USA). The tissue samples were mechanically minced and placed into a 50 mL tube with an enzymatic solution consisting of collagenase (0.1%, Sigma-Aldrich, C6885, Saint Louis, MO, USA), 2 M CaCl 2 (1:1000, VWR, Darmstadt, Germany), and 1% BSA (Sigma-Aldrich, Saint Louis, MO, USA), followed by a 1 h incubation at 37 ◦ C under agitation. The digested tissue was centrifuged three times at 290 g for 5 min. The pellet of hTDCs was then expanded in a complete culture medium consisting of a medium essential alpha ( α -MEM, Invitrogen, Life Technologies Limited, Paisley, UK) supplemented with 10% FBS (Life Technologies) and 1% A/A in humidified 5% CO2atmosphere and used at passage 1–3. Cell culture on PLA and PLA+0.5[(f-EG)+Ag] scaffolds. The hTDCs were seeded at a density of 1.2 × 10 5 cells per scaffold and cultured in α -MEM medium for 7 and 14 days in humidified 5% CO 2 atmosphere. Two experimental conditions were considered in which the hTDCs were seeded on (i) PLA scaffolds or (ii) PLA+0.5[(f-EG)+Ag] scaffolds to investigate the potential of these scaffolds for tendon/ligament applications. The hTDCs’ response was investigated by assessing tendon-associated markers at the gene and protein levels. RNA Extraction and Real-Time RT-qPCR. The total ribonucleic acid (RNA) was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions and was quantified using a Nanodrop ® ND-1000 spectrophotometer (ThermoFisher Scientific, Wilmington, NC, USA) at 260/280 nm. Complementary DNA was synthesized from 1 µ g of RNA of each sample using a qScript TM cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, MD, USA) according to the manufacturer’s protocol using a Mastercycler ® Realplex (Eppendorf, Hamburg, Germany). The transcripts quantification presented in Table S2 was carried out via quantitative polymerase chain reaction (qPCR) using the PerfeCTA SYBR Green FastMix Kit (Quanta Biosciences, Gaithersburg, MD, USA) according to the kit instructions in a Real-Time Mastercycler ep realplex thermocycler (Eppendorf, Hamburg, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as reference genes. The relative expression level was calculated using the 2−∆∆Ct method for each target gene [34]. Immunofluorescence of tendon-related markers in 3D scaffolds. The hTDCs cultured on the 3D scaffolds were fixed with 10% (v/v) neutral buffered formalin (ThermoFisher Scientific, Waltham, MA, USA) and permeabilized with 0.025% (v/v) Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) in PBS for 10 min. Afterward, the samples were washed three times with PBS, blocked with Normal Horse Serum (RTU Vectastin Kit, PK-7200, Vector, Burlingame, CA, USA), and incubated overnight at 4 ◦ C with antibodies against Tenomodulin (TNMD, Rabbit anti-human, ab81328, 1:100, Abcam, Cambridge, UK), Scleraxis (SCX, Rabbit polyclonal anti-SCX, ab58655, 1:100, Abcam, Cambridge, UK), and Nanomaterials 2023,13, 2518 8 of 23 Collagen type I (COL1, Rabbit polyclonal anti-COL1, 47972, 1:100, Novus Biologicals TM , ThermoFisher Scientific, Waltham, MA, USA). Subsequently, the samples were washed in PBS and incubated with anti-rabbit Alexa Fluor 488-fluorescent secondary antibody or anti-rabbit Alexa Fluor 594 antibodies (ThermoFisher Scientific, Waltham, MA, USA) for 1 h at RT. All antibodies were diluted in 0.1% BSA/PBS. The samples were rinsed with PBS and stained with 4,6-Diamidino-2-phenyindole, dilactate (DAPI, 5 mg/mL, D9564, SigmaAldrich, Saint Louis, MO, USA) for 10 min. The immunolabeled samples were observed by confocal laser scanning microscopy (CLSM, TCS SP8, Leica, Wetzlar, Germany). 2.5. Statistical Analysis The presented data were expressed as the mean ± standard deviation (SD) of at least three replicates, except for RT-PCR analysis, which was expressed as the mean ± standard error of the mean (SEM) of two independent experiments (n= 2). The error bars presented in the graphs denote the SD. The statistical analysis was performed using the GraphPad Prism6 software from Windows. The statistical significance was evaluated by one-way ANOVA after performing the Shapiro–Wilk test for normal distribution and by two-away ANOVA followed by Bonferroni post hoc test multiple comparison tests for RT-PCR. A difference was considered significant with a confidence interval of 95% for different degrees of confidence, p< 0.05 (*), p< 0.01 (**), p< 0.001 (***), and p< 0.0001 (****). 3. Results and Discussion 3.1. Antimicrobial Potential The potential antimicrobial activity of EG and EG derivatives against E. coli and S. aureus was tested, and the results obtained after 24 h are presented in Table 2. Soft agar plates incubated with the suspensions of bacteria and different concentrations of EG and EG derivatives observed after 24 h, as well as positive and negative controls, are shown in Figure S1. Table 2. Presence (+) or absence ( − ) of E. coli and S. aureus at concentrations (0.025–1%) of different EGs. Concentration (%) E. coli S. aureus EG 1 + + 0.5 + + 0.25 + + 0.1 + + 0.05 + + 0.025 + + f-EG 1 + + 0.5 + + 0.25 + + 0.1 + + 0.05 + + 0.025 + + (f-EG)+Ag 1− − 0.5 − − 0.25 − − 0.1 −+ 0.05 + + 0.025 + + Nanomaterials 2023,13, 2518 9 of 23 Bacterial colonies of both E. coli and S. aureus were found in the presence of EG and f-EG, at different concentrations. However, no bacterial growth of S. aureus or E. coli was observed for 0.25 up to 1% of (f-EG)+Ag. The MBC of (f-EG)+Ag was 0.25% for S. aureus, while for E. coli it was only 0.1%. These results provided evidence that the presence of silver nanoparticles conferred antimicrobial properties against these two bacteria strains. This is in agreement with the literature that reports the antibacterial efficacy of silver-containing materials such as PLA fibrous membranes for tendon repair [ 35 ] and even composite mats of PLA-GO with antibacterial effects against E. coli and S. aureus [ 36 ]. Although the mechanism of the antibacterial action of AgNPs is not fully understood, it is suggested that silver ions could interact with bacterial cells in several ways [ 20 ], such as disturbing the permeability of the cells wall or even penetrating them, causing damage and changing the microbial DNA and proteins [20]. 3.2. Filaments’ Production and Characterization A pre-coating of the PLA pellets with EG and EG derivatives was applied before the filament extrusion process. Figure S2 shows a uniform distribution of the EG flakes onto the surface of the PLA pellets. The filament cross-sections were observed by using SEM (Figure S3a–d) and revealed a homogenous dispersion of the fillers in the PLA matrix. The MFI of the filaments was measured to evaluate their potential printability. The MFI values were similar for the PLA and composite filaments, ranging from 24 to 26 g/10 min, which were adequate for use in 3D printing (Figure S3e) [37]. The incorporation of conductive particles in a polymer matrix reduces the electrical resistivity of the latter and allows electronic transport/mobility. Considering the envisaged application, it may have a positive effect on the cellular adhesion and growth [ 38 , 39 ]. In addition, the rate of wound healing in vivo is closely correlated with changes in the electrical current generated from the wound site [ 40 , 41 ]. We used high-definition Kelvin force microscopy to obtain a map of the variation in the surface potential of the PLA+0.5[(f-EG)+Ag] filaments (Figure 1). Nanomaterials2023,13,xFORPEERREVIEW10of25    Figure1.Mapsofsurfacepotential(V)obtainedfromHDKFMpotentialforcompositescontaining PLA+0.5[(f-EG)+Ag]. EGandEGderivativesareconductiveparticles(redregions)andthusshowa contrastingsurfacepotentialrelativetothepolymermatrix(darkblueregions),presenting highrelativepotentialvalues(10V)[42].Achargeoutflowfromthegrapheneflakescan beseenaroundtheseparticlessinceEGshavelowelectronaffinity,asobservedinother works[42]. 3.3.Scaffolds’Characterization 3.3.1.PhysicalandMorphologicalAnalysis The3D-printingprocesswaseasytosetupandfasttocarryout,taking7mintoprint ascaffoldwithdimensionsequivalenttothoseoftheaverageACL.Thisprocessyielded reproduciblesampleswithhighstructuralhomogeneityandcontrolledgeometry, indicatingthattheprintingparameterscouldbeeasilyadjustedaccordingtotheshape andlocationoftheinjuredtissue[43].Figure2illustratesthe3D-printedPLA(a1–a4)and PLA+0.5[(f-EG)+Ag]scaffolds(b1–b4),atthetop,front,andlateralviews,atincreasing magnificationsoftheirporousmorphology. Figure 1. Maps of surface potential (V) obtained from HDKFM potential for composites containing PLA+0.5[(f-EG)+Ag]. EG and EG derivatives are conductive particles (red regions) and thus show a contrasting surface potential relative to the polymer matrix (dark blue regions), presenting high relative potential values (10 V) [ 42 ]. A charge outflow from the graphene flakes can be seen around these particles since EGs have low electron affinity, as observed in other works [ 42 ]. Nanomaterials 2023,13, 2518 16 of 23 for all studied conditions. The proliferation was even more pronounced than on the control, with the cells covering large areas of the scaffolds’ surfaces. The nanoroughness caused by FDM and by the presence of nanoparticles increased the surface area, and this was shown to positively influence the attachment, migration, and orientation of various cell lines including fibroblasts, which play a critical role in the healing process [11,27]. Nanomaterials2023,13,xFORPEERREVIEW17of25   8a,b).TheL929morphologyandcytoskeletonorganizationwereinvestigatedbyusing SEM(FigureS7)andaDAPI-phalloidintest(Figure8c).  Figure8.Invitrocellcultureonthescaffolds.(a)MetabolicactivityofL929cellsdeterminedby Alamarbluecellviabilityassay.DatanormalizedtoPLA(100%metabolicactivity).Significant Figure 8. In vitro cell culture on the scaffolds. ( a ) Metabolic activity of L929 cells determined by Alamar blue cell viability assay. Data normalized to PLA (100% metabolic activity). Significant differences, effect of the material: p< 0.05 (*). ( b ) Representative fluorescent images of live (green)/dead (red) cells seeded on the scaffolds. ( c ) Fluorescence image of L929 cells seeded on 3D-printed scaffolds and TCPS. Cells were immunostained for F-actin with phalloidin (red), and cell nuclei were stained with DAPI (blue). Nanomaterials 2023,13, 2518 17 of 23 Human tendon-derived cells in PLA and PLA+0.5[(f-EG)+Ag] scaffolds—Expression of tendon-related markers. Due to the similarities among tissues and to the expression of common markers such as TNMD and SCX [ 31 , 67 ], engineering tendons and ligaments have been pursued with common strategies. In this work, the potential of PLA+0.5[(fEG)+Ag] scaffolds to support the tenogenic/ligamentogenic phenotype was assessed in hTDCs through gene expression analysis (Figure 9). The resident cell populations including stem/progenitor cells subsets have an epigenetic commitment to respond to tendon and ligament-specific requirements and a natural role in the renewal and maintenance and of tissue composition and properties, with impact on the healing process and its outcomes [31,67] . These cells exhibit clonogenicity, self-renewal, and multipotency, as well as a high expression of scleraxis (SCX), tenomodulin (TNMD), and collagen type I (COL1) [31]. Nanomaterials2023,13,xFORPEERREVIEW19of25    Figure9.AssessmentofthegeneticexpressionoftenogenicmarkersofhTDCsin3Dscaffoldsafter 7and14daysofculture.Relativegeneexpressionof(a)SCX,(b)TNMD,and(c)COL1.Symbols denotestatisticaldifferences:*forp<0.05,**forp<0.01,***forp<0.001,and****forp<0.0001. Cellsculturedonbothtypesofscaffoldsshowedincreasedexpressionof tendon/ligamentmarkers,namely,SCX,TNMD,andCOL1,duringtheexperimental setup.SCX,TNMD,andCOL1evidencedanupregulationfromday7today14, suggestingthatthe3D-printedscaffoldsassistedthemaintenanceofthehTDCs’ phenotype[68].Inaddition,thegeneexpressionlevelsofSCX,TNMD,andCOL1were significantlyincreasedbythepresenceof(f-EG)+Ag,suggestingthatthecomposite scaffoldssupportedthetenogenicphenotype[30].Inaccordance,theexpressionsofSCX andCOL1weresignificantlyincreasedinhTDCsculturedonthecomposites(SCX:atday 7,p<0.05,andatday14,p<0.01,andCOL1:atday7,p<0.05,andatday14,p<0.0001), relativetohTDCsonthePLAscaffolds.ThelevelsofTNMDshowedasignificantincrease incompositescaffoldswhencomparedwithPLA,ateachandeverytimepoint(p< 0.0001).Wefurtherinvestigatedthelocationsofthetendon-relatedproteinsinthe scaffolds,whichwereanalyzedbyimmunofluorescence(Figure10a,b).SCX,TNMD,and COL1wereobservedovertimeincellsculturedonbothtypesofscaffolds.Thecellswere homogenouslydistributed,evidencingadensecolonizationofthescaffolds’structures. Moreover,seededcellsexhibitafusiformmorphologyandatendencytoorganize themselvesintoparallelalignmentonthesurfaceofthescaffolds,whichistypically observedintendoncellsinnativetissues.Theobtainedresultssupportthat3D-printed scaffoldsencouragethecolonizationofhTDCsandtheexpressionofgenesandproteins associatedtothetenogenic/ligamentogenicphenotypeand,therefore,holdthepotential tosustainhealingstrategiesaimingtoregeneratetendonsandligaments.Similarresults werefoundforothergraphene-basedpolymerscaffolds[69,70].Theseareencouraging resultsforfurtherinvivoexperiments. Figure 9. Assessment of the genetic expression of tenogenic markers of hTDCs in 3D scaffolds after 7 and 14 days of culture. Relative gene expression of ( a )SCX, ( b )TNMD, and ( c )COL1. Symbols denote statistical differences: * for p< 0.05, ** for p< 0.01, *** for p< 0.001, and **** for p< 0.0001. Cells cultured on both types of scaffolds showed increased expression of tendon/ ligament markers, namely, SCX,TNMD, and COL1, during the experimental setup. SCX, TNMD, and COL1 evidenced an upregulation from day 7 to day 14, suggesting that the 3D-printed scaffolds assisted the maintenance of the hTDCs’ phenotype [ 68 ]. In addition, the gene expression levels of SCX, TNMD, and COL1 were significantly increased by the presence of (f-EG)+Ag, suggesting that the composite scaffolds supported the tenogenic phenotype [ 30 ]. In accordance, the expressions of SCX and COL1 were significantly increased in hTDCs cultured on the composites (SCX: at day 7, p< 0.05, and at day 14, p< 0.01 , and COL1: at day 7, p< 0.05, and at day 14, p< 0.0001), relative to hTDCs on the PLA scaffolds. The levels of TNMD showed a significant increase in composite scaffolds when compared with PLA, at each and every time point (p< 0.0001). We further investigated the locations of the tendon-related proteins in the scaffolds, which were analyzed by immunofluorescence (Figure 10a,b). SCX, TNMD, and COL1 were observed over time in cells cultured on both types of scaffolds. The cells were homogenously distributed, evidencing a Nanomaterials 2023,13, 2518 18 of 23 dense colonization of the scaffolds’ structures. Moreover, seeded cells exhibit a fusiform morphology and a tendency to organize themselves into parallel alignment on the surface of the scaffolds, which is typically observed in tendon cells in native tissues. The obtained results support that 3D-printed scaffolds encourage the colonization of hTDCs and the expression of genes and proteins associated to the tenogenic/ligamentogenic phenotype and, therefore, hold the potential to sustain healing strategies aiming to regenerate tendons and ligaments. Similar results were found for other graphene-based polymer scaffolds [ 69 , 70 ]. These are encouraging results for further in vivo experiments. Nanomaterials2023,13,xFORPEERREVIEW20of25    Figure10.Immune-locationof(a)COL1(red),SCX(red),andTNMD(green)inhTDCs-laden scaffoldsafter7and14daysofculture(20×,scalebar250µm).NucleiwerestainedwithDAPI(blue). (b)A3Dimagereconstructionofthez-stackscollectedfromthehTDCs-ladenPLA+[0.5(f-EG)+Ag] scaffold,evidencingscaffoldstructureanddistributionofTNMD(green)andSCX(red)produced byhTDCs(nuclei,blue). The3D-printedscaffolddevelopedcouldbefurtherexploredbothasanacellularand cell-ladenscaffold.AsapromisingtendonandligamentTEproduct,theinclusionofcells asacomponentintroducesrisks,andforthatreasontheselectionforanacellularscaffold asmedicaldeviceshouldbelesstime-consumingandfacelessregulatoryscrutiny.In EuropeanUnion(EU),medicaldevicesarestrictlyregulatedbothbynationalcompetent authoritiesandbytheEuropeanMedicinesAgency(EMA),whileintheUSAtheextensive regulatoryrequirementsaredefinedbytheU.S.FoodandDrugAdministration(FDA). Figure 10. Immune-location of ( a ) COL1 (red), SCX (red), and TNMD (green) in hTDCs-laden scaffolds after 7 and 14 days of culture (20 × , scale bar 250 µ m). Nuclei were stained with DAPI (blue). ( b ) A 3D image reconstruction of the z-stacks collected from the hTDCs-laden PLA+[0.5(f-EG)+Ag] scaffold, evidencing scaffold structure and distribution of TNMD (green) and SCX (red) produced by hTDCs (nuclei, blue). The 3D-printed scaffold developed could be further explored both as an acellular and cell-laden scaffold. As a promising tendon and ligament TE product, the inclusion of cells as a component introduces risks, and for that reason the selection for an acellular scaffold as medical device should be less time-consuming and face less regulatory scrutiny. In Nanomaterials 2023,13, 2518 19 of 23 European Union (EU), medical devices are strictly regulated both by national competent authorities and by the European Medicines Agency (EMA), while in the USA the extensive regulatory requirements are defined by the U.S. Food and Drug Administration (FDA). 4. Conclusions and Future Work The 3D-printed scaffolds based on medical-grade PLA reinforced with 0.5 wt.% of EG, f-EG, and (f-EG)+Ag were successfully produced and characterized. EG was organically functionalized and decorated with silver nanoparticles. The aim of the organic functionalization of EG was to strengthen the interface with the polymer and to provide anchoring sites for Ag, allowing the inclusion of a small concentration of Ag. The Ag anchored on EG acted as an anti-microbial agent, as confirmed against microorganisms of clinical relevance S. aureus and E. coli, an important feature for the healing process and prevention of bacterial infections. PLA composite filaments were melt-extruded with a good filler dispersion and used for the fabrication of customized 3D porous scaffolds. Highly reproducible scaffolds were obtained with a porosity of 64–71% and a network of interconnected pores of around 400 µ m. The scaffolds’ biodegradation and mechanical properties were evaluated. All scaffolds exhibited high stability and surface nanoroughness, which was increased by the fillers’ presence. The wet state dynamic mechanical analysis proved that the addition of reinforcements led to a significant increase in the storage modulus, being mechanically adequate for tendon and ligament applications. The highest increase was observed for scaffolds with PLA+0.5[(f-EG)+Ag], which represents an increase of ≃ 55% compared with PLA scaffolds. Similar to native living tissues, the scaffolds exhibited a viscoelastic behavior. The PLA+[(f-EG)+Ag] scaffolds were non-toxic and showed capability to maintain the tenogenic commitment of human tendon-derived cells, with an increase in the gene expression of specific tendon/ligament-related markers. The results demonstrate the possibility for easy, cost-effective, and personalized 3D-printed scaffolds with great potential applications for tendon and ligament regeneration. We believe that this article presents compelling in vitro results for further in vivo experiments. Some strategies may be considered for future directions of this research, to improve the scaffolds’ overall performance and clinical applications, namely, their capability to inhibit mycobacteria [ 71 ] or even adapt and change over time [72]. Supplementary Materials: The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/nano13182518/s1: Table S1: Operating parameters used for the production of composite filaments. Table S2: Primers used for real-time quantitative RT-PCR analysis. Figure S1: Minimum bactericidal concentration of different EGs (EG, f-EG, and (f-EG)+Ag) against E.coli and S. aureus. Figure S2: SEM images of (a) PLA pellet and PLA pellet coated with 0.5 wt.% of (b) pristine EG, (c) f-EG, and (d) (f-EG)+Ag powder. The insets represent different magnifications. Figure S3: SEM images of filaments’ cross-sections: (a) PLA, (b) PLA+0.5EG, (c) PLA+0.5f-EG, and (d) PLA+0.5[(f-EG)+Ag]. (e) MFI values of filaments. Table S3: Mean pore size of the 3D-printed scaffolds. Figure S4: SEM images of the cross-section of 3D-printed scaffolds formed by (a) PLA, (b) PLA+0.5EG, (c) PLA+0.5f-EG, and (d) PLA+0.5[(f-EG)+Ag]. Figure S5: EDS of PLA+0.5[(f-EG)+Ag] scaffolds. Figure S6: SEM images of the surface of (a,a1) PLA, (b,b1) PLA+0.5EG, (c,c1) PLA+0.5f-EG, and (d,d1) PLA+0.5(f-EG)+Ag scaffolds at stage 0 and after 12 weeks of degradation, respectively. Figure S7: SEM images of the L929 cells seeded on PLA, PLA+EG, PLA+f-EG, and PLA+[(f-EG)+Ag] scaffolds, after 1, 3, 7, and 14 days. Magnifications for closer observation of L929 cells. Author Contributions: M.S., investigation, writing—original draft, and formal analysis; S.G., investigation; C.C., investigation; D.P., investigation; A.V., studies with hTDCs, conceptualization, investigation, and writing—review and editing; M.T.R., studies with hTDCs, conceptualization, and writing—review and editing; M.E.G., conceptualization and writing—review and editing; J.A.C., conceptualization and writing—review and editing; M.C.P., conceptualization and writing—review and editing; N.M.A., conceptualization and writing—review and editing. All authors have read and agreed to the published version of the manuscript. Nanomaterials 2023,13, 2518 20 of 23 Funding: This research was funded by the Portuguese Foundation for Science and Technology (FCT) through the National Funds Reference UIDB/05256/2020 and UIDP/05256/2020, the FCT, EU and European Social Fund (FSE) through the Ph.D. Grant References SFRH/BD/138244/2018, COVID/BD/153245/2023, and SFRH/BD/143209/2019, the FCT in cooperation with the Northern Portugal Regional Coordination and Development Commission (CCDR-N) through the project “TERM RES Hub—Scientific Infrastructure for Tissue Engineering and Regenerative Medicine”, reference PINFRA/22190/2016 (Norte-01-0145-FEDER-022190), and the ERC CoG MagTendon (No. 772817). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article and Supplementary Materials. Acknowledgments: IPC acknowledges the support of the FCT through the National Funds Reference UIDB/05256/2020 and UIDP/05256/2020. Silva M. acknowledges FCT for the Grant References SFRH/BD/138244/2018 and COVID/BD/153245/2023 and is thankful to Alberto Campuzano for the support provided in the 3D-printing process. Correia C. acknowledges FCT for the Grant Reference SFRH/BD/143209/2019. Silva M., Correia C., Peixoto D., Vinhas A., Rodrigues M.T., Gomes M.E. and Alves N.M. would like to acknowledge the contributions to this research from the project “TERM RES Hub—Scientific Infrastructure for Tissue Engineering and Regenerative Medicine”, reference PINFRA/22190/2016 (Norte-01-0145-FEDER-022190), funded by the FCT in cooperation with the Northern Portugal Regional Coordination and Development Commission (CCDR-N), for providing relevant lab facilities, state-of-the art equipment, and highly qualified human resources. The authors are thankful to Hospital da Prelada (Porto, Portugal) for providing tendon samples. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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