Article Development and Characterization of a Polycaprolactone/Graphene Oxide Scaffold for Meniscus Cartilage Regeneration Using 3D Bioprinting Melike Nur Özder 1, Aslihan Yelkenci 2, Mine Kucak 3 , Aylin Altinbay 4 , Cem Bülent Ustündag 1,5 and Fatih Ciftci 6,7,* 1 Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yıldız Technical University, Istanbul 34210, Turkey; mnozder@gmail.com (M.N.Ö.); cbustun@yildiz.edu.tr (C.B.U.) 2 Department of Pediatric Dentistry, Faculty of Dentistry, University of Health Sciences, Istanbul 34668, Turkey; aslihanzihni@gmail.com 3 Department of Molecular Biology and Genetics, Yildiz Technical University, Istanbul 34210, Turkey; minekucak@gmail.com 4 Department of Metallurgical and Material Engineering, Faculty of Chemical and Metallurgical Engineering, Yildiz Technical University, Istanbul 34210, Turkey; aylin.altinbay@yildiz.edu.tr 5 Health Biotechnology Joint Research and Application Center of Excellence, Istanbul 34210, Turkey 6 Department of Biomedical Engineering, Fatih Sultan Mehmet Vakıf University, Istanbul 34015, Turkey 7 Department of Technology Transfer Office, Fatih Sultan Mehmet Vakıf University, Istanbul 34015, Turkey * Correspondence: faciftcii@gmail.com or fciftci@fsm.edu.tr Highlights • GO increased the storage modulus of scaffolds from 36.1 Pa to 97.1 Pa. • Yield shear stress enhanced from 97.2 Pa to 507.1 Pa with GO. • Optimal mechanical properties achieved with 1% GO: modulus 614 MPa and strength 46.3 MPa. • GO incorporation increased the melting temperature to 60.78 ◦C and glass transition Academic Editors: Athina to 31.14 ◦C. Angelopoulou and Dimitrios • Roughened scaffold surface improved cell adhesion and cellular distribution con- A. Lamprou firmed by DAPI staining. Received: 30 December 2024 • Antibacterial zones increased against E. coli (26.21 mm) and S. aureus (15.38 mm). Revised: 3 March 2025 • Rheological results showed shear-thinning viscosity improvement up to 89.3 Pa·s with GO. Accepted: 4 March 2025 • Elongation at break improved to 10.4% with 5% GO addition. Published: 7 March 2025 • GO scaffolds enhanced cell viability to over 100% at 1:8 concentration. Citation: Özder, M.N.; Yelkenci, A.; • PCL/GO scaffolds successfully mimicked native meniscus properties with biofunc- Kucak, M.; Altinbay, A.; Ustündag, C.B.; Ciftci, F. Development and tional and mechanical advantages. Characterization of a Polycaprolactone/Graphene Oxide Abstract: Background/Objectives: Meniscus injuries represent a critical challenge in ortho- Scaffold for Meniscus Cartilage pedic medicine due to the limited self-healing capacity of the tissue. This study presents the Regeneration Using 3D Bioprinting. development and characterization of polycaprolactone/graphene oxide (PCL/GO) scaffolds Pharmaceutics 2025, 17, 346. https://doi.org/10.3390/ fabricated using 3D bioprinting technology for meniscus cartilage regeneration. Methods: pharmaceutics17030346 GO was incorporated at varying concentrations (1%, 3%, 5% w/w) to enhance the bioactivity, mechanical, thermal, and rheological properties of PCL scaffolds. Results: Rheological analy- Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. ses revealed that GO significantly improved the storage modulus (G’) from 36.1 Pa to 97.1 Pa This article is an open access article and the yield shear stress from 97.2 Pa to 507.1 Pa, demonstrating enhanced elasticity and distributed under the terms and flow resistance. Mechanical testing showed that scaffolds with 1% GO achieved an optimal conditions of the Creative Commons balance, with an elastic modulus of 614 MPa and ultimate tensile strength of 46.3 MPa, closely Attribution (CC BY) license mimicking the native meniscus’s mechanical behavior. FTIR analysis confirmed the success- (https://creativecommons.org/ ful integration of GO into the PCL matrix without disrupting its chemical integrity, while licenses/by/4.0/). Pharmaceutics 2025, 17, 346 https://doi.org/10.3390/pharmaceutics17030346 Pharmaceutics 2025, 17, 346 2 of 21 DSC analysis indicated improved thermal stability, with increases in melting temperatures. SEM analysis demonstrated a roughened surface morphology conducive to cellular adhesion and proliferation. Fluorescence microscopy using DAPI staining revealed enhanced cell attachment and regular nuclear distribution on PCL/GO scaffolds, particularly at lower GO concentrations. Antibacterial assays exhibited larger inhibition zones against E. coli and S. aureus, while cytotoxicity tests confirmed the biocompatibility of the PCL/GO scaffolds with fibroblast cells. Conclusions: This study highlights the potential of PCL/GO 3D-printed scaffolds as biofunctional platforms for meniscus tissue engineering, combining favorable mechanical, rheological, biological, and antibacterial properties. Keywords: 3D bioprinting; graphene oxide; meniscus scaffolds; cartilage; PCL 1. Introduction Meniscus injuries, commonly arising from trauma or degenerative conditions, present a significant challenge in orthopedic medicine due to the tissue’s limited healing capac- ity [1,2]. The meniscus plays a crucial role in joint stability, load distribution, and shock absorption in the knee, and damage to this tissue often leads to progressive joint deteriora- tion and osteoarthritis. Traditional treatments, such as partial meniscectomy or meniscal repair, offer limited long-term success, especially in patients with extensive damage [3]. Consequently, the field of regenerative medicine has explored novel approaches to restore meniscal function, with tissue engineering emerging as a promising solution [4–7]. 3D bioprinting has become a transformative tool in tissue engineering due to its precision in constructing complex structures tailored to individual anatomical require- ments [8–10]. By precisely depositing biomaterials and cells layer by layer, 3D bioprinting enables the creation of tissue scaffolds that mimic the natural architecture of the menis- cus [11–14]. This approach not only supports cell growth and differentiation but also allows for customization of mechanical properties to match those of native tissue. Recent studies have focused on the use of various biomaterials to create such scaffolds, with a particular interest in polymers and nanomaterials that offer structural integrity, biocompatibility, and functionality [11,12,15,16]. Polycaprolactone (PCL), a widely used biopolymer, is notable for its biodegradability and mechanical strength, making it suitable for fabricating scaffolds that require long-term stability [14]. However, PCL alone may not provide the necessary bioactivity for effective meniscal tissue regeneration [17,18]. To address this limitation, graphene oxide (GO) has been introduced as an additive to enhance cell attachment, proliferation, and differentiation [19]. GO’s unique properties, including its large surface area, high mechanical strength, and ease of functionalization, make it an ideal candidate for composite scaffold materials in meniscal applications. GO’s conductive nature also supports the transmission of bioelectrical signals, which can further promote cellular activities beneficial for tissue regeneration [20–23]. In this study, we propose a novel composite scaffold combining GO with PCL, specifi- cally engineered for meniscal regeneration via 3D bioprinting. This scaffold is designed to not only mimic the mechanical properties of the native meniscus but also foster a con- ducive environment for cellular activity and tissue growth. The introduction of GO into the PCL matrix aims to create a biofunctional scaffold that addresses the limitations of current meniscal repair materials, potentially advancing the treatment options available for meniscal injuries. This study investigates the synthesis, characterization, and bioactivity of the PCL/GO scaffold, aiming to provide insights into its potential application in meniscal tissue engineering. Pharmaceutics 2025, 17, 346 3 of 21 2. Materials and Methods 2.1. Preparation of GO and PCL/GO In this study, GO was synthesized by Hummers’ method [24]. The chemicals used for synthesis such as Graphite flake (mesh size 300), Sulfuric acid (H2SO4), potassium permanganate (KMnO4, 99.9%), phosphoric acid (H3PO4), and hydrogen peroxide (H2O2 30%) were purchased from Merck and Sigma Aldrich (Darmstadt, Germany). In a typical synthesis method, 360 mL concentrated H2SO4, and 40 mL concentrated H2PO4 were added to the 1 mL beaker with a large stir bar. The beaker was stirred in an oil bath at 40–45 ◦C at 200 RPM. Graphite (3 g) was gradually added to the solution. Then, 18 g KMnO4 was slowly added, and a deep green color was observed. The mixture was stirred for 16 h at between 40 and 45 ◦C. Then, the mixture and 400 g of ice were mixed in a 2 L beaker. To the mixture, 3 mL of 30 wt% H2O2 was added, which changed the color of the mix to yellow. The mixture was poured into the 50 mL centrifuge tubes. Then, the mix in the centrifuge tubes is centrifuged at 3000 RPM for 45 min, and the liquid in the tubes. After the first centrifuge, the mixture was sequentially washed once with distilled water and three times with concentrated HCl (10 wt%) at 3000 RPM for 45 min. Finally, they were washed thrice with ethanol at 3000 RPM for 15 min [25]. To prepare the Poly(ε-caprolactone) PCL solutions, a solvent mixture consisting of acetic acid and formic acid in a 9:1 volume ratio was utilized. The solvents used were 100% anhydrous acetic acid (ISOLAB, Eschau, Germany) and 98–100% formic acid (EMSURE ACS, Reag., Darmstadt, Germany), ensuring high purity. The solvent mixture was prepared by mixing 9 mL of acetic acid with 1 mL of formic acid at room temperature under ambient conditions. PCL with an average molecular weight of 80,000 g/mol (Sigma-Aldrich) was added to the prepared solvent mixture in varying mass concentrations of 5%, 10%, and 20% (w/v). The PCL-solvent mixtures were allowed to stir at room temperature for 24 h to ensure complete dissolution of the polymer. This approach ensured the homogeneity of the resulting solutions used in subsequent analyses. GO was added to the PCL solutions at 1%, 3%, and 5% (w/w), relative to the PCL content (Table 1). The GO was gradually introduced into the PCL solutions under continuous stirring to ensure uniform dispersion. The mixtures were stirred for an additional 6 h at room temperature, followed by ultrasonication for 1 h to prevent agglomeration of the GO particles and achieve a homogeneous polymer matrix. The resulting PCL/GO solutions were stored under ambient conditions and used for subsequent analyses or fabrication processes. Table 1. Concentrations of PCL and PCL/GO solutions. Weight Difference (%) Solutions PCL GO GO GO(wt%) (wt%) (wt%) (wt%) PCL 20%wt 20 - - - PCL/GO (1%wt) 20 (1%wt) - - PCL/GO (3%wt) 20 - (3%wt) - PCL/GO (5%wt) 20 - - (5%wt) 2.2. Fabrication of 3D Bioprinted Scaffolds Using PCL/GO Solutions The 3D printing process was carried out to produce four different scaffold compositions at 50%, 60%, 70%, and 80% infill rates. The 3D-printed scaffolds were fabricated by using a modified GBA 3D printer (Biomaterials and Nanomaterials Laboratory (BİORGİNE Labs), Department of Biomedical Engineering, Fatih Sultan Mehmet Vakıf University, İstanbul, Turkey), Department of Biomedical Engineering, Fatih Sultan Mehmet Vakıf University), Pharmaceutics 2025, 17, 346 4 of 21 which utilized a fused deposition modeling (FDM) system. It included computer-aided design (CAD) technology using a heatable build plate. A digital syringe pump was connected to a 3D printer to control the flow rate of solutions feeding into a syringe with a 0.5 mm nozzle diameter. We fabricated 3D-printed scaffolds using conditions in build plate temperature of 38 ◦C, a flow rate of 0.2 mL/h, and 0.03 mm distance between the needle and platform. 3. Physical Properties and Characterizations 3.1. Rheological Characterization All rheology was performed on an Anton Parr MCR 302 (Graz, Austria) rheometer equipped with a parallel plate configuration (50 mm or 8 mm diameter) at 37 ◦C, keeping the normal force constant at 0 N. During the optimization, in a typical rheological test for gelation kinetics (i.e., evolution of the storage modulus (G′) and loss modulus (G) as a function of time; time sweep), hydrogels were spread on the bottom plate at 37 ◦C. The top plate was immediately lowered to a plate separation of 0.5 mm, and the measurement was started. A frequency of 5 Hz and a strain of 1% were applied to minimize interference with the gelation process and to keep the measurement within the linear viscoelastic region. The normal force was also kept constant at 0 N. The gel point was determined by the crossover between G‘ and G′. 3.2. Mechanical Properties A tissue analyzer (Stable Micro Systems, Godalming, UK) was used to evaluate the mechanical properties, including tensile strength and elongation at break, of the designed 3D-printed materials. Composite materials were prepared as cylindrical films with a diameter of 6 mm and a height of 100 mm for compressive tests and as films measuring 5 cm in length, 3 cm in width, and 3 mm in thickness for tensile tests. The composite materials were tested at a 10 mm/min speed up to a maximum strain of 80%. Each sample was measured in quadruplicate. The compressive modulus and Young’s modulus of the bioactive composite materials were calculated using the slope of the initial linear section of the strain-stress curves corresponding to 0–25% strain. To assess the flexibility and recoverability of the composite materials, cyclic compressive tests were performed for four consecutive compressive cycles up to 70% strain, following the same rate. Each sample was tested in four repetitions [26]. 3.3. Uniaxial Tensile Tests All uniaxial tensile tests were conducted using an M100-1CT Testometric (Guangdong, China) machine equipped with a 1 kN load cell. Specimens (n = 8–10) were tested either immediately after preparation (i.e., following overnight gelation) or after immersion in culture medium for 1, 7, 14, and 21 days. A preload force of 0.1 N was applied, and each test was performed at a compression rate of 5 mm/min. Each sample was subjected to 98% tension to determine the final compressive stress and strain. For cyclic compression tests, each sample was subjected to 30% tension at a rate of 5 mm/min for both loading and unloading phases. Data were analyzed using Wintest version 4.55.0 analysis software. 3.4. Morphological Properties The morphological properties of the samples were analyzed using a scanning electron microscope (Hitachi SU3500 T2, Graz, Austria). The SEM was operated in secondary electron mode at 5 kV, and further image analysis was conducted at a voltage of 10 kV. We ensured conductivity was applied to all samples, as polymers inherently lack this property. Pharmaceutics 2025, 17, 346 5 of 21 3.5. FTIR Analysis FTIR spectroscopy was conducted to analyze the functional groups present in the produced composite materials. The characterization studies were performed using a Bruker Alpha FTIR (Karlsruhe, Germany) device. The spectral range from 4000 to 400 cm−1 was utilized to identify and analyze the functional groups within the samples. 3.6. DSC Analysis Differential scanning calorimetric (DSC) analysis was performed with a Diamond DSC (Shelton, CT, USA). The sample (~5 mg) was placed in an aluminum container and sealed and heated above the melting temperature (Tm) (60 ◦C) of PCL at 10 ◦C min−1. 3.7. In Vitro Swelling and Degradation Test The swelling and degradation behaviors of 3D composite scaffolds were investigated for PCL, PCL/GO (1%), PCL/GO (3%), and PCL/GO (5%) scaffolds with 3 mg samples. Swelling behavior was investigated at 0, 1, 2, 4, 8, 12, 16, 20, 24, 48, 72, and 96 min and calculated according to Equation (1). The degradation behavior was investigated for 5, 10, 15, 30, 45, and 60 min and calculated and plotted according to Equation (1). W − W Swelling rate (SR w) = d × 100 (1) Wd The values of wet and dry weight were indicated by Ww and Wd, respectively. W − W Degradation rate DR w( ) = d × 100 (2) Wd For the swelling ability test of 3D tissue scaffolds, the scaffold samples are first weighed in the dry state, and the dry weight (Wd) is obtained. They are then immersed in phosphate buffer saline (PBS) at a constant temperature of 37 ◦C for a fixed time interval. Afterwards, excess water is removed with tissue paper, and the wet weight (Ww) is evaluated. The mass swelling ratio (SR) is calculated using Equation (1). For the frostbite test of 3D tissue scaffolds, the scaffold samples are first weighed dry, and the dry weight (Wd) is obtained. They are then immersed in PBS at a constant temperature of 37 ◦C for a fixed time interval, and after drying at room temperature for 24 h, the wet weight (Ww) is weighed. The mass degradation rate (DR) is calculated using Equation (2). 3.8. Antibacterial Analysis The pathogens used were obtained from Fırat University, Faculty of Veterinary Medicine (Elazığ, Turkey). Bacterial studies were carried out at the BİORGİNE Laboratory, Department of Biomedical Engineering, Fatih Sutan Mehmet Vakıf University. The pathogens used for antibacterial assays consisted of Gram-negative bacteria Escherichia coli (ATCC 25922, Manas- sas, VA, USA) and Gram-positive bacteria Staphylococcus aureus (ATCC 25923, Manassas, VA, USA). A disk diffusion test was performed to determine the antibacterial activity of the 3D meniscus scaffolds. E. coli and S. aureus suspensions were collected from 18 h nutrient broth cultures, adjusted to 0.5 McFarland standard turbidity (1.5 × 108 CFU/mL) and diluted to the desired bacterial density (1:10). Mueller–Hinton agar plates were inoculated with 0.1 mL of bacterial suspension (1.5 × 106 CFU/mL). The 3D meniscus scaffolds were sliced into x (3), y (2), z (1) mm thickness and placed in bacteria-coated Petri dishes after UV sterilization for 2 h. The plates were incubated at 37 ◦C for 24 h, and the zones of inhibition around the disks were measured with a digital micrometer. Four separate groups were prepared to select the optimum 3D meniscus scaffolds, and the tests were performed three times. Pharmaceutics 2025, 17, 346 6 of 21 3.9. Cytotoxicity In vitro cell viability was assessed using the indirect MTT test. L929 mouse fibroblast cell line was used for the biological characterization of the 3D meniscus scaffolds. Before the experiment, the 3D meniscus scaffolds were cut into pieces of 1 cm × 1 cm, UV-C sterilized for 20 min on both sides, and incubated in sterile tubes with the complete medium (DMEM-low glucose, FBS (10%, v/v), and penicillin-streptomycin (1%, v/v)) at 37 ◦C for 24 h. L929 cells were seeded in a 96-well plate at a density of 1 × 104 cells/mL per well and incubated at 37 ◦C with 5% CO2 for 24 h. Sterile scaffolds were incubated in the medium for 24 h, and scaffold extracts were obtained. After 24 h of incubation, L929 cells were treated with different concentrations of scaffold extracts at the ratios of (1:1), (1:2), (1:4), and (1:8). Different concentrations of scaffold extracts were prepared by diluting with the complete medium. After treatment, 10 µL of an MTT (Gold Biotechnology®, St Louis, MO, USA) solution was added to each well, followed by incubation for three hours. The wells were then aspirated, and 100 µL of DMSO was added to each to dissolve formazan crystals, followed by incubation for 30 min at room temperature. Absorbance was measured at 570 nm using a microplate reader (Biotek, Dallas, TX, USA). Optical density was measured with a multilayer reader at 450 nm, and cell viability was calculated using the following Equation (3): (Optical density (OD) of treated cells) Viability (%) = × 100 (3) OD of control cells L929 cells were treated with scaffold extracts at specified concentrations for 24 h and then washed with PBS. A DAPI staining solution was added and incubated in the dark for 5 min. After removing the dye solution, the cells were washed 2–3 times with PBS and observed under a fluorescence microscope. 3.10. Cell Adhesion This study investigated the adhesion and growth of cells on membranes. A mouse fibroblast cell line L929 was grown in a DMEM medium supplemented with 10% FBS, 1% L-glutamine, 1% penicillin, and streptomycin in a humidified incubator with 5% CO2 and 100% relative humidity at 37 ◦C. The 3D scaffold samples were cut into 1 cm × 1 cm pieces. After UV-C sterilization, they were placed in a 24-well medium, and L929 cells were seeded at a concentration of 5 × 104 in 1 mL of the medium per sample. The scaffold samples were incubated at 37 ◦C for 24 h and 48 h to allow cells to adhere to the scaffold samples. After 48 h of incubation, the growth medium was discarded, and the cells were fixed with 2.5% glutaraldehyde. The fixed cells were washed with PBS and then dehydrated with ethanol (30%, 50%, 70%, 90%, and 100%). Visualization of cell adhesion in the samples was performed by scanning electron microscopy [27]. 3.11. Statistical Analysis All the statistical analyses of the data were performed through ANOVA using the GraphPad Prism version 8 software (GraphPad Software Inc., San Diego, CA, USA). The values were given as means ± standard deviation (SD), and the statistical differences were analyzed by t-tests. In all cases, p < 0.05 and p < 0.0001 were considered statistically significant. 4. Results and Discussion 4.1. Rheological Analysis The rheological parameters of the prepared hydrogels such as storage modulus (G′), loss modulus (G′′), Shear-Thinning Viscosity, or tan δ (damping factor) are shown in Table 2. Pharmaceutics 2025, 17, 346 7 of 21 Table 2. Rheological characteristics of the PCL and PCL/GO hydrogels at room temperature with 1% strain at 1 Hz. G′ (Pa) G′′ (Pa) Shear-Thinning Viscosity Shear Rate Yield Shear Stress(Pa·s) (s−1) (Pa) PCL 36.1 ± 2.13 8.7± 0.19 19.1 ± 1.22 8.7 97.2 ± 8.09 PCL/GO (1%wt) 48.5 ± 1.12 14.6 ± 1.33 39.5 ± 0.13 14.1 308.4 ± 4.17 PCL/GO (3%wt) 59.1 ± 0.25 23.7 ± 2.11 50.9 ± 4.04 26.3 385.2 ± 2.09 PCL/GO (5%wt) 97.1 ± 0.08 34.4 ± 1.04 89.3 ± 1.17 49.8 507.1 ± 3.63 While the G′ value of the PCL hydrogel was 36.1 Pa, this value increased significantly as the GO content increased. In the PCL/GO hydrogel containing 5% GO, the G′ value reached about 97.1 Pa. This indicated that GO greatly strengthened the elastic structure of the material [28,29]. On the other hand, the value of G′′ increased similarly. It increased from 8.7 Pa for PCL to 34.4 Pa for 5% GO. This increase revealed that GO improved the viscoelastic properties and increased the shear resistance. The viscosity increased significantly with the increase in GO concentration [30–32]. The shear-thinning viscosity of PCL was measured as 19.1 Pa.s, and the shear-thinning viscosity of 5% GO was measured as 89.3 Pa.s. This indicates that GO increases the resistance to flow by integrating into the matrix. The shear rate of the PCL hydrogel was 8.7 s−1, while it was measured as 49.8 s−1 in 5% GO. Yield shear stress of the PCL hydrogel was 97.2 Pa, while it was observed as 507.1 Pa in 5% GO. This proved that GO makes the structure more durable. 4.2. Mechanical Analysis In the mechanical test results for 3D meniscus scaffolds, as the PCL/GO ratio increases (1%, 3%, and 5%), a decrease in the elastic modulus value was observed as 614.1, 592.1, and 588 MPa, respectively (Table 3). The GO additive reduces the elastic modulus, making the material slightly more flexible. The high surface area and flexibility of GO may have slightly reduced the material’s stiffness. However, this may be advantageous for flexible and load-bearing structures such as the meniscus. The PCL/GO (1%) 3D meniscus scaffold showed a slightly higher ultimate stress of 46.3 MPa than pure PCL. However, for PCL/GO (3%), this value was observed as 43 MPa. For PCL/GO (5%), it increased again and reached 46 MPa. It was observed that GO increased the strength at low ratios (1%) but lost its optimal effect at 3% and stabilized again at 5% (Figure 1). When GO was added, the elongation at break increased to 9.2% (PCL), 9.9% (GO 1%), 9.3% (GO 3%), and 10.4% (GO 5%), respectively, compared to pure PCL. Table 3. Elastic modulus, ultimate stress and strain at break of 3D PCL and PCL/GO meniscus scaffolds. E modulus (MPa) Ultimate Stress (MPa) Strain at Break (%) PCL 635.3 45.3 9.2 PCL/GO (1%) 614.1 46.3 9.9 PCL/GO (3%) 592.1 43 9.3 PCL/GO (5%) 588 46 10.4 Pharmaceutics 2025, 17, x FOR PEER REVIEW 8 of 21 Table 3. Elastic modulus, ultimate stress and strain at break of 3D PCL and PCL/GO meniscus scaf- folds. E modulus (MPa) Ultimate Stress (MPa) Strain at Break (%) PCL 635.3 45.3 9.2 PCL/GO (1%) 614.1 46.3 9.9 PCL/GO (3%) 592.1 43 9.3 PCL/GO (5%) 588 46 10.4 In similar studies, 3D artificial meniscus-mimicking PCL-agarose (Ag)-gelatin meth- acrylate (GelMA) hydrogels were designed. Compressive moduli of PCL, PCL-Ag, and PCL-GelMA were 11.5 ± 1.1, 8.4 ± 2.2, and 10.0 ± 1.9 MPa; tensile moduli of the constructs were 30.8 ± 6.6, 27.4 ± 3.8, and 28.1 ± 6.9 MPa. It was observed that the addition of Ag and GELMA hydrogels to the PCL hydrogel and its 3D design caused a decrease in its me- chanical properties [33]. In similar studies, 3D scaffolds were designed from PCL-biomi- Pharmaceutics 2025, 17, 346 metic coating of chitosan/ECM hydrogel for use in meniscal injuries. The compressi8veof 21 modulus of 3D PCL scaffolds was measured as 5.65 MPa [14]. Fiigure 1.. SSttrreessss––ssttrraaiinn ccuurrvveess oof f33DD-p-prirnintetded PPCCLL anandd PCPCL/LG/OG Omemneisnciussc usscasffcaoflfdosl dwsitwh ivtharvyainrygi nGgOG O ccooncceenttrraattiioonss ((11%, ,33%%, ,aanndd 55%%).) . 4.3. STEhMe A5n%alGysOis additive reached the highest elongation capacity of the material. The increAascecoirnditnhge teolo mngoarptihoonloagticthale abnraelaykseosf oGf Othea tPtChLe 5sc%affloevlde,l tshheo pwoerdest h(paotrteh seizmesa t~e1r ial mbemca)m weemreo orebseelravsetidc aans da irtesgduelfaorr manadt iohnomcaopgaecnietyouins csrterauscetdu.reR e(Fgiagrudrien g2Am)e. cThhaen iucnailfroersmu lts, sPuCrLfa/cGe mOo(r1p%h)ooloffgeyr sreaflbeacltas nthcee dhipgrho wfiloerrkeagbailridtyin agndth ceoenltarsotlilcabmleo dpruolpues,rtsiterse onfg pthu,rae nPdCeLl.o n- Igna ttihoen .PTChLi/sGrOat i(o1%m)a sycabffeoildde, asllifgohrta iprprelgicualtairointys wwahse roebmseervchedan oicna lthper ospurefratciees waritehc GritOic al, dsuocphinags (Fthigeumree 2nBi)s.c Tuhs.is Tmhaey ciumrpvreosvde ebmiooconmstrpaatteibtihlietyi nanfldu ecnelcl eadohf etshieonG. OThceo pnotreen sttrounc-the teularest (ipcomreo sdiuzelu ~s2, mulmtim) ias treegsturleasrs, ,bauntd a smtroarien partotnhoeubnrceeadk hoeftethroegsecnaefiftoyl diss o, bhsigerhvleigdh itnin thget he wmaelclhs.a Tnhicisa lchpaenrfgoer mina tnhcee susurfiatacbe lme faoyr smligehntilsyc uinsctriesassuee menecghinaeneicrainl gstraepnpgltihca wtiohnilse. creating In similar studies, 3D artificial meniscus-mimicking PCL-agarose (Ag)-gelatin methacrylate (GelMA) hydrogels were designed. Compressive moduli of PCL, PCL-Ag, and PCL-GelMA were 11.5 ± 1.1, 8.4 ± 2.2, and 10.0 ± 1.9 MPa; tensile moduli of the constructs were 30.8 ± 6.6, 27.4 ± 3.8, and 28.1 ± 6.9 MPa. It was observed that the addition of Ag and GELMA hydrogels to the PCL hydrogel and its 3D design caused a decrease in its mechanical properties [33]. In similar studies, 3D scaffolds were designed from PCL-biomimetic coating of chitosan/ECM hydrogel for use in meniscal injuries. The compressive modulus of 3D PCL scaffolds was measured as 5.65 MPa [14]. 4.3. SEM Analysis According to morphological analyses of the PCL scaffold, the pores (pore sizes ~1 mm) were observed as a regular and homogeneous structure (Figure 2A). The uniform surface morphology reflects the high workability and controllable properties of pure PCL. In the PCL/GO (1%) scaffold, slight irregularity was observed on the surface with GO doping (Figure 2B). This may improve biocompatibility and cell adhesion. The pore structure (pore size ~2 mm) is regular, but a more pronounced heterogeneity is observed in the walls. This change in the surface may slightly increase mechanical strength while creating a more favorable environment for biological integration. The PCL/GO (3%) scaffold showed more pronounced Pharmaceutics 2025, 17, x FOR PEER REVIEW 9 of 21 Pharmaceutics 2025, 17, 346 9 of 21 a more favorable environment for biological integration. The PCL/GO (3%) scaffold microstructural irsrheogwuleadr imtieosreo pnrtohneousunrcfeadc emwicirtohstarnucitnucrraela isrirneguGlaOritcioesn toenn tth(eF siguurfraec2e Cw)i.tTh haen increasing connections betwGeOen cothnetepnot r(eFsigauprep e2aCr).s Thhaerp ceornannecdticolnesa rbeert,wweehnic thhem paoyrepsr oapvpideearf ashvaorrpaebrl eand clearer, effects on mechawnihcaiclhl omaady bperaorviindge. faTvhoerapbolere effsiezcetss orenm maeicnheadniccoanl lsotaadn tbaetaraibnog.u Tth1e mpomre, sbiuzets remained the shape of the pcoonrestsagnat iante adbaousth 1a rmpemr,a bnudt mthoe rsehdapiset ionfc tthgee opmoreetsr yg.aTinheids caa snhaimrpperro avnedt hmeore distinct structure stabilitygeaonmdetthrye.f Tavhiosr caabnle imenpvroirvoen tmhee nsttrfuocrtuthree satarrbailnitgye amnedn tthoef fcaevlolsra. bTlhe een5v%iroGnOment for the doping created siganrriafincagnemt ierrnetg oufl acreiltlys.o Tnhteh e5%su rGfaOc ed(oFpiginugr ec2reDa)t.eTdh sisigmniafiycafanvt oirrraebglyulaafrfietcyt othne the surface (Figure 2D). This may favorably affect the attachment and proliferation of cells to the scaf- attachment and proliferation of cells to the scaffold. However, excessive surface modifications fold. However, excessive surface modifications may cause weaknesses in mechanical may cause weaknstersesnegsthin. Pmoerec hsaizneisc ailncsrtreeansegdth t.o Paorroeunsidz e2s minmcr,e aansedd irtroegaurolaurnitdies2 imn mth,ea pnodre walls in- irregularities in tchreeapsoedre. Twhael ldseinnscitrye aosfe md.icTroh-e andde nnsaintyosocfalme sictrruoc-taunreds noann tohsec saulerfastcreu mctauyr egsive positive on the surface mareysguilvtse rpegoasritdivinegr beisoullotgsirceagl apredrfinorgmbainocloeg. Aicsa lthpee rGfoOr mraatinoc ien.cAresatshese iGn O3Dr amtieoniscus scaf- increases in 3D mfeonldis dcuessigscna, fafo nldotdiceesaibglne, cahnanogtiec eianb sluercfhacaen gmeoirnphsuorlofagcye omccourrpsh. oTlhoigs ybeoncecfiutrss .cell attach- This benefits cell matteancth, pmreonlitf,eprartoiolinfe, raantdio bni,oalnogdicbailo ilnotgeigcraaltiinotne g[3r4a]t.i on [34]. Figure 2. SEM imaFgiegsuirlleu 2s.t SraEtMe tihmeasguersf iallcuesmtraotrep thhoe lsougryfaacne dmsotrrpuhcotulorgeyo afn(dA s)tPruCcLtu, r(eB o) fP (CAL) /PGCLO, ((B1%) P)C, L/GO (1%), (C) PCL/GO (3%),(aCn) dPC(DL/)GPOC L(3/%G),O an(d5% (D) )3 DPCmL/eGnOis c(5u%s )s c3aDf fmolednsi.scus scaffolds. 4.4. DSC Analysis4.4. DSC Analysis The peak temperTahtue rpeesaokf tPemCLp,erPaCtuLr/esG oOf P(1C%L), ,PPCCLL/G/GOO (1(%3)%, P),CaLn/dGPOC (L3%/G), Oan(d5 %PC) L3/DGO (5%) 3D scaffolds were 58s.c4a6ff, o6l0d.0s 3w, e6r1e.6 578,.4a6n, d606.013.6, 761◦.C67,,r aensdp e6c1t.i6v7e °lyC,( rFeisgpuerceti3v)e.lyT h(Fisigsumrea 3ll).i nTchries assmeall increase in peak temperatiunr epmeaaky tienmdpiceartaetuthrea tmthaey inincdoircpaoter atthioatn tohfeG iOncoinrptoortahteioPnC oLf mGaOtr iixntsoli gthhetl yPCL matrix improves the thersmligahl tsltya bimiliptyr.oTvhese tphree sthenercme aolf sgtraabpilhiteyn. eTohxei dperessheonwcee odf tghraatpthheenme oelxtiidneg spheoawked that the was slightly enlamrgeeldtinagn dpehaikg whears. sTlihgihstlsyu gengleasrtgsetdh aant dG hOigmheary. Tinhfilsu seungcgeetshtse tchrayt sGtaOll imzaatyio innfluence the behavior of the pocrlyysmtaelrliaznatdiopno bseshibalvyioinr corfe athsee tphoelyomrdeerr aonfdt hpeoscsriybsltya lilnicnreeastsreu tchteu roer.dIetrc oafn tbhee crystalline structure. It can be observed from the DSC curves that GO changes the melting tempera- observed from the DSC curves that GO changes the melting temperature and thermal behavior ture and thermal behavior of the PCL matrix. The addition of GO generally improves the of the PCL matrixth. eTrhmeaald sdtaibtiiolintyo [f3G5,O36]g. enerally improves the thermal stability [35,36]. PPhahramrmacaecueutitciscs2 2002255, ,1 177, ,3 x4 6FOR PEER REVIEW 10 1o0f o2f12 1 FFiigguurree 33.. DDiiffffeerreennttiaialls sccaannnnininggc aclaolorirmimetertyryc ucruvrevseos fo3fD 3-Dp-rpinritnedtePdC PLCaLn danPdC PLC/LG/OGOm emneisnciuscsussc asfcfaofl-ds wfoiltdhsv warityhin vgarGyOingco GnOce cnotrnacteionntrsat(i1o%n,s 3(%1%, a, n3%d ,5 a%n)d. 5%). 4.5. FTIR Analysis 4.5. FTIR Analysis In the spectrum (Figure 4A), a broad peak belonging to hydroxyl (OH) groups was In the spectrum (Figure 4A), a broad− 1peak belonging to hydroxyl (OH) groups was oobbsseerrvveedd iinn tthhee rarannggee ofo 3f530500–03–230200 c0mcm−1 [37,3[387]., 3T8h].e iTnhteensinittye ninscitryeaisnec rinea tsheisi nregthioisn riengdiio-n icnadteicsa tthese tchoenctroinbturtibiount ioofn OofHO gHrogurposu pins tihnet hsetrsutcrtuucrteu roef oGfOG.O T.hTish iisnidnidcaictaeste tshteh esuscuccecsessfsuflu l iinnccoorrppoorat −1 ratiioonn ooff GGOO inintoto ththe ePCPCL Lmmataritxri. xIn. tIhnet h29e5209–5208–5208 c5m0−c1 mranger a[3n9g]e, s[y3m9]m, seytmricm aentdri c aansydmasmyemtrmice strtircetscthreintcgh ivnigbrvaitbiorantsi oonfs tohfet hcehacrhaacrtaecrtisetriics tmicemtheythleynlee n(eC(HCH) 2g)rgoruopusp osfo Pf CPCL L2 ssccaaffffoolldd( (FFiigguurree4 4BB))w weererec lceleaarlrylyd deteetcetcetded. D. Desepspitieteth tehea daddidtiiotinono foGf OGOin idni dffieffreenretnpte prceerncetangte-s, naogessi,g nnoif isciagnntifipcoasnitti opnoscihtiaonng cehwanagseo wbsaesr vobedseirnvetdhe inb athned sbainndths iisnr ethgiiso nre.gHioonw. Hevoewr,esvliegrh, t isnlcigrehats ienscirneathseesi nint etnhsei tiyntoefntshietyb oafn tdhsea braenodbss earrvee odbpsearrvaleldel ptoartahlelealm too tuhnet aomf GouOn. tI nofP GCOL/. IGnO (P1C%L) /(GFiOgu (r1e%4)C ()F,iPgCurLe/ 4GCO), (P3C%L) /(GFiOgu (r3e%4)D (F),iagnudreP 4CDL)/, GanOd (P5C%L) /(GFiOgu (r5e%4)E ()Fciogmurpeo 4sEit)e cs,oCmH- 2 gproosuitpess,b CecHam germouoprse bperocamminee mnto. rTeh pis2 rosumgigneesntts. tThhaitsb seusigdgeesstths ethinatt ebreascitdioenss thofe GinOtewraicthtioPnCsL , m −1ofe GthOyl ewniethg ProCuLp,s mareethmyloerneep grreoseunptsi narteh me sotrreu cptruerseenotf tinh ethpeo lsytrmuecrt.uIrne tohfe th17e 2p0o–l1y7m40erc.m In rtahneg 1e7[2400–],1a74s0tr ocnmg−1p reaankgbee l[o4n0]g,i na gsttrootnhge cpaerabko nbyello(Cng=iOn)gg troo uthpes coafrPbCoLnywl a(sCo=bOs)e rgvreodu.pTsh oefs e pPeCaLk swoafsc oarbbsoernvyeldg.r Touhepssem paeianktsa ionfe cdartbhoeinrypl ogsriotiuopnsi nmtahinetparineseedn tcheeoirf pGoOsitainodn sinh othwee pdroesn-ly semncaell oinf tGenOs iatyndch sahnogwese.dT ohnislyi nsdmicaallt eisnttehnastiGtyO chhaansgwees.a kThpihs yisnidcaiclaotresc htheamt iGcaOl i nhtaesr awcteiaokn s w −1phityhsiPcCalL o.rT chheemraincgael i1n1te5r0a–c1t2io5n0sc wmith P[4C1L] .r eTphree sraenntgse t1h1e5C0–-1O25s0tr ecmtch−1i n[4g1]v ribeprarteisoennstso fthteh e eCs-tOer sbtroentcdhsining tvhiebrPaCtiLonsst rouf ctthuer ee.stDere bsponitdest hine tchoen PtrCibLu sttirounctoufreG. OD,enspoitsei gthneif iccoannttripboustiiotino n cohf aGnOge, nwoa ssigdneitfieccatendt pinostihtiiosnr ecghiaonng,es uwgagse dsteitnegcttehda itnG thOisd roeegsionno, tsaufgfegcetsteisntge rthbaotn GdOs. dInoeths e 5n0o0t– a1ff00e0ct cm −1 ester braonngdes.[ 3In9, 4th2]e, 5C0H0–21o0s0c0i lclamto−1r yravnigber a[t3i9o,n4s2]o, fCPHC2L o-sacinlldatGorOy- svpibercaiftiiconbsa nodf s wPCerLe- oabnsde rGvOed-s. pIetcwifiacs bdaentdersm winereed otbhsaetrtvheed.b Iatn wdaisn tdeentseirtmiesiniendc rtehaaste tdhein btahnids riengteionnsitwieist h ainncirnecarseeads iinng thGisO rergaitoion. wTithhi sanin idniccraetaessinthga Gt OG Oratiiso.h Tohmiso ginedniecoautessl ythdaits GpeOr sies dhoinmtohgeenPeC-L mouastrlyix dainspdetrhseedco imn pthoes iPteCsLtr mucatturriex manadin tthaein csoimtspinotseigter isttyr.uActsuarere msualitn, tFaTinIRs iatns ailnytseigsrrietyv.e Aales d tah aretstuhlet,i nFcToIRrp aonraatliyosniso rfeGveOaliendt oththate tPhCe Linsctorrupcoturaretiodnid onf oGtOle aindtoto thaes iPgCniLf icstarnutcctuhraen gdeidi n cnhoetm leicaadl btoo nad siinggnibfiuctacnatu csheadndgieff ienre cnhceems iincaplr boopnerdtiinesg sbuucth caasuisnecdre dasiffederienntecness iitny ipnrothpeebrtainesd s bseulcohn gaisn igntcoreOasHedg rionutepnss. iTtyh einse tfhine dbianngdssr ebveelaolntghiantgt htoe iOntHer agcrtoiounpss.o Tf hGeOsew fiinthdPinCgLs orecvcuerala t ttheatp thyes iinctaelrlaecvteiolnasn odf tGheOc wheitmh iPcCalLs torcuccutru aret tohfeP pChLysiiscpalr elesverevl eadnd, b tuhte tchheeimncicoarlp sotrruatcitounreo f GO improves the properties of the composite material. Pharmaceutics 2025, 17, x FOR PEER REVIEW 11 of 21 Pharmaceutics 2025, 17, 346 of PCL is preserved, but the incorporation of GO improves the properties of the compos1i1teo f 21 material. FiFgiugruere4 .4.( A(A))F FTTIIRRs sppeeccttrruummss ooff PPCCLL aanndd PPCCLL//GGOO mmeneinscisucsu sscascffaoflfdosl dwsiwthi tvharvyainrygi nGgOG cOonccoenntcrean-tra- tiotinosns( 1(1%%, ,3 3%%,, aanndd 55%%)),, ((BB) )PPCCLL 3D3D prpinrtinedte (d12( 1la2ylearys)e,r (sC),) (PCC)LP/GCOL /(1G%O) 3(1D% p)ri3nDtedp r(i1n2t leadye(r1s2),l (aDy)e rs), (DP)CPLC/GLO/G (3O%()3 3%D) p3rDinpterdin (t1e2d la(1y2erlsa)y, e(Ers) )P, C(EL)/PGCOL (/5%GO) 3(D5 %pr)in3Dtedp (r1in2 tleadye(r1s2).l ayers). 4.46..6S. Swwelellilningga anndd Weeiigghhtt Loossss Raattee TThhees wsweelllilninggb beehhaavviioorr of PCL and PCL//GGOO cocmompopsoisteitse (s1(%1%, 3,%3,% an, adn 5d%5)% is) eixsperxepssredss ed asasa af ufunncctitoionno offt tiimee ((Figure 5A).. IIniittiiaallyly,, tthhee sswweelllilningg rartaet eofo tfhteh PeCPLC sLcasffcaoflfdo lrdemreaminasi ants at lolwowererl elevveelslsc coomppaarreed tto the GO-reiinffoorrcceedd ccoommppoosistiet escsacffaofflodlsd. sT.hTish icsanca bne binetienrtperrepterdet ed asasG GOOi ninccrereaasseesst thhee watter absorptiion ccaappaaccitiyty dduuee toto itist shyhdyrdorpohpihliicl incantuartue.r Ae. sAigsniigfinciafincta nt swswelelilnlinggin incrceraeasesei siso obbsseerrvveeddi inn aallll ssamplleess,, eesspeecciialllly ffrrom 2244 minin oonnwwaardrds,s ,aanndd reraecahcehse s a plaa tpelaautealeuv leelvaetl 7a2t –7926–9m6 imn.inH. oHwoewveevr,ear,s atsh tehpe eprecrecnetnatgaegeo foGf GOOin icnrceraesaesse,s,t htheei ninccrereaasseei nint he swtheel lsinwgelrlainteg breacteo mbeecsomeosr emporroen poruonncoeudn.cTehdi. sTihnids iicnadteicsatthesa tththaet thhoem hoomgeongeeonuesoduiss dtriisbtruit-ion ofbGutOionin otfh GeOp oilny mthee rpmolaytmriexra mndattrhixe ahnydd trhoeg ehnydbroongdesn fboornmdesd fobremtwede ebnetGwOeeann GdOp oalnydm er atptroalyctmwera taettrrmacot rweaetfefre cmtiovreel yeff[e4c3t,i4v4e]l.y [43,44]. ItItw waassa alslsooo obbsseerrvveedd tthhaatt tthhee ccoommppoossitiete ssccaafffofoldlds scocnotnatianiinnign 1g%1 %anadn 3d%3 %GOG rOearcehaecdh ed aah ihgihgheerrs wsweelllilinngg rraattiioo ccoommppaarreedd ttoo tthhee ssaammpplele coconntatianiinnign g5%5% GOG.O T.hTish issusguggesgtes stthsatth iaf t if GGOOi sisu useseddi ninh higighh pprrooppoorrttiioonnss,, iitt mmaayy ppaarrttiaialllyly reredduucec ewwataetre pr epremrmeaebailbitiyli tbyy bcyaucsaiunsgi ng agglomeration in the matrix. Overall, these results reveal that GO as a hydrophilic addi- agglomeration in the matrix. Overall, these results reveal that GO as a hydrophilic additive tive can optimize the swelling properties [44–46]. can opFtiigmuirzee 5tBh eesxwamelilninesg wpreoigphetr tlioesss [a4s4 –a4 6fu].nction of time and reflects the degradation behFavigiourr eof5 tBhee sxaammpilneess. Twhee iwghetiglhots lsoasss oaf ftuhen cPtCioLn socaffftoimlde isa ngednreerafllelyc thsigthheerd tehgarna tdhaatti on beohf atvheio Gr oOf-dthoepseadm cpomlesp.oTsihtee swcaeffigohldt slo. sTshoisf tihndeiPcaCtLess tchaafft oGldOi senghenanercaesll ythhei gmheecrhtahnaincatlh at ofsttahbeiliGtyO a-nddo ptheedrmcoaml dpuorsaibteilistyca offf othldes p. oTlyhmiseirn mdiactaritxe.s Inth paatrGticOuleanr,h caonmcepsostihteesm coencthaainn-ical stianbgi l3it%y aanndd t5h%er GmOal sdhuorwab liolwityero wf tehieghpto lloysms einr mloantgr-itxe.rImn pteasrttsi.c Tuhlaisr, ccaonm bpeo esxitpelsacinoendta binyi ng 3%thea nfadct5 t%haGt GOOs phroowvidloews ae rbwareriiegrh etffleocsts, liinmliotinngg- ttherem intteersatsc.tiTonh iosf ctahne pboelyemxpelra sintreudctbuyret he fawctitthh wataGteOr apndro tvhiudse ssloawbianrgr ideorwefnf ethcte, dliemgritaidnagtitohne pirnotceersasc [t4io7n–4o9]f. tWhehepno alynmaleyrzesdtr ausc tau re wfiuthncwtioante orf atnimdet, hauns insclorewasiningg dtroewndn inth we edigehgtr alodsast iios nobpsreorvcesds, e[s4p7e–c4i9a]l.ly Winh tehne fiarnsat l1y0z ed asmainf.u Tnhcitsi omnayo fbtei mduee, taon thinec irneiatisailn dgistsroelnudtioin owf ethigeh wt alotesrs ciosnotabcste arrveeads ,oens ptheec ipaolllyminert he firssutrf1a0cem. Hino.weTvheirs, imn laoyngber dteustes,t tohitsh tereinndit sialolwdsi sdsowlunt iaonnd orefatchheesw aa ctoenrstcaonntt laecvtela. rTehaiss on thperopcoelsys msleorwssu drfoawcen. eHveonw mevoerre, winithlo ningcerreatseisntgs , GthOi scotrnecnedntsralotiwonss.d oInw pnaratnicdulraera, cthe s a cownesitgahntt lloesvs eolf. tThhe icsopmropcoessitse sclonwtasindionwg n5%e vGeOn ims othre lwowithesitn, icnrdeaicsaitninggG thOatc othnics esnatmraptlieo ns. In particular, the weight loss of the composite containing 5% GO is the lowest, indicating that this sample may be the most suitable option for long-term durability and stability in biomedical applications. In conclusion, these graphs show that GO-doped PCL composites increase the usability in biomedical applications by affecting both swelling and weight loss behaviors. Future studies could focus on evaluating the performance of such materials in Pharmaceutics 2025, 17, x FOR PEER REVIEW 12 of 21 may be the most suitable option for long-term durability and stability in biomedical ap- Pharmaceutics 2025, 17, 346 plications. In conclusion, these graphs show that GO-doped PCL composites increase the 12 of 21 usability in biomedical applications by affecting both swelling and weight loss behaviors. Future studies could focus on evaluating the performance of such materials in different dbiifofelorgeinctalb eionlvoigroicnaml eenntvs.i rTohnism pernotvsi.dTesh aisnp imropvoidrteasnta bnaismis pfoorr ttahne tdbeavseilsopfomretnhte odf bevioemloi-pment of bmioemtici mstreuticctusrtersu acntudr tehsea enxdpatnhseioenx pofa tnhseiior napopfltichaetiirona parpelaics.a tion areas. FFiigguurree 55.. SSwweelllilningg (A(A) a)nadn ddedgeragdraatdioanti (oBn) (bBeh) abveihorasv oiof rthseo 3fDth-per3inDte-dp rsicnatffeodldssc.a ffolds. 4.7. Antibacterials Result The PCL/GO (1%) scaffold showed a pronounced antibacterial effect on E. coli (Figure 6A). The 26.21 mm inhibitory zone was much larger than the 12.65 mm zone pro- vided by PCL alone. GO was observed to potentiate the antibacterial properties of PCL. This showed that it provides a much more effective solution against Gram-negative bacteria. This is because GO’s surface properties and load-carrying capacities can enhance antibacterial properties by acting on the bacterial cell wall. The PCL/GO (1%) combination also showed a stronger effect on S. aureus (Figure 6B), but this effect was not as pronounced as against E. coli. The inhibitory zone of 15.38 mm is larger than the PCL effect of 10.01 mm. Similar studies Pharmaceutics 2025, 17, x FOR PEER REVIEW 13 of 21 4.7. Antibacterials Result The PCL/GO (1%) scaffold showed a pronounced antibacterial effect on E. coli (Figure 6A). The 26.21 mm inhibitory zone was much larger than the 12.65 mm zone provided by PCL alone. GO was observed to potentiate the antibacterial properties of PCL. This showed that it provides a much more effective solution against Gram-negative bacteria. Pharmaceutics 2025, 17, 346 This is because GO’s surface properties and load-carrying capacities can enhance antibac- 13 of 21 terial properties by acting on the bacterial cell wall. The PCL/GO (1%) combination also showed a stronger effect on S. aureus (Figure 6B), but this effect was not as pronounced as haagvaeinssht oEw. cnolit.h TahteG inOhiibnittooryP CzoLnes coaf f1f5o.l3d8s mimm pisr olavregsert htheainr tahnet iPmCiLc reoffbeicat lopf r1o0.p0e1r mtiems.. In one stSuimdyil,aPr CstLudiinecso hrapvoer sahtoewdnw thitaht GreOd uincteod PCgrLa spchaffenoledos ximidpero(rvGesO th)ewira asnftiomuincrdobtoiael xphroibpi-t potent anerttiibeas.c Itne roianle astcutidvyi,t yPCaLg ainincostrpboortahtedG wraimth- rpeodsuictievde g(rSa.phauenreeu osx)iadne d(rGGOra) mw-ans efoguantidv etob acteria (Ee.xchoiblii)t, paontdenht iagnhtiebractoenricael natcrtaivtiitoyn asgoaifnrsGt bOo-tehn Ghraanmc-epdosbitaicvtee (rSio. asutaretuics)e affnedc tGsr[a5m0-]n. eIng-another stautidvye, bealecctetrioaa (cEti. vceoliP),C aLndsc haifgfohledr scownictehnthraetriomnas lolyf rGedOu-ecnedhagnrcaepdh beanceteorixoisdtaeti(cT reGffeOct)ss howed co[5m0]p. Ilent aeneortahderic satutidoyn, eolfeSct.raouacrteiuvse uPnCdLe srcaeffleocltdrsi cwailtsht tihmeurmlaatliloyn r,ehdiugcheldi gghratipnhgenthe eoxpiodtee ntial of co(TmrGbiOn)i nshgoewleecdt rcooamcptilveetea enrdadaicnattiiboanc otef rSi.a aluprreoups eurntdieesr feolercetrnichaal nsctiemdutliastsioune, rheiggehnliegrhat-tion [51]. Tihnegs tehefi pnodtienngtisals oufp cpoomrbtinthinegi edleeactrtohaacttitvhee anindc aonrtpiboarcatteiroianl porfoGpeOrt-ideso fpoerd enPhCanLcesdc atifsf-olds not osnulye rseugpenpeorrattsiotnis [s5u1e]. rTehgeesne efirnadtiinogns bsuupt paolsrot tmhei itdigeaat tehsatb tahcet einrciaolrpgororawtiothn, owf GhiOch-diospcerdu cial for PCL scaffolds not only supports tissue regeneration but also mitigates bacterial growth, pwrehviecnh tiisn cgruincifaelc ftoior npsreivnemntiendgi cinafleacptiponlisc ainti monesdilcikale amppelnicisactiuosnsim likpel amnetns.iscus implants. FiFgiguurree 66.. AAsssseessssmmeennt toof fanatnimtimicriocbroiabl ieaffleecftfievcetnievsesn feosrs tefsotredte 3sDte dme3nDismcues nsicsacffuosldssc avfifao aldgasrv diiasca gar disc ddififffuussiioonn mmeetthhooddss, ,rerespspecetcivtievlye,l yb,yb ryecroercdoinrdg iinnghiibnithioibni tzioonnesz.o (nAe) sE. . (cAol)i, E(B. )c oSl.i ,au(Bre)uSs .(*a*u**r etu tses(t*,* ** t test, GGrarapphhPPaadd;; tthhee ssttaattiisstticicaal lsisginginfiicfiacnacne cleevleelv welasw daestedremteinremdi anse pd