Enriched Graphene Oxide-Polypropylene Suture Threads Buttons Modulate the Inflammatory Pathway Induced by Escherichia coli Lipopolysaccharide

Graphene oxide (GO), derived from graphene, has remarkable chemical–physical properties such as stability, strength, and thermal or electric conductivity and additionally shows antibacterial and anti-inflammatory properties. The present study aimed to evaluate the anti-inflammatory effects of polypropylene suture threads buttons (PPSTBs), enriched with two different concentrations of GO, in the modulation of the inflammatory pathway TLR4/MyD 88/NFκB p65/NLRP3 induced by the Escherichia coli (E. coli) lipopolysaccharide (LPS-E). The gene and the protein expression of inflammatory markers were evaluated in an in vitro model of primary human gingival fibroblasts (hGFs) by real-time PCR, western blotting, and immunofluorescence analysis. Both GO concentrations used in the polypropylene suture threads buttons-GO constructs (PPSTBs-GO) decreased the expression of inflammatory markers in hGFs treated with LPS-E. The hGFs morphology and adhesion on the PPSTBs-GO constructs were also visualized by inverted light microscopy, scanning electron microscopy (SEM), and real-time PCR. Together, these results suggest that enriched PPSTBs-GO modulates the inflammatory process through TLR4/MyD 88/NFκB p65/NLRP3 pathway.


Introduction
Graphene, a two-dimensional (2D) nano-structure containing sp 2 carbon atoms, is a building block of several carbon-based materials, including graphite, bucky balls, and carbon nanotubes [1,2]. Graphene was discovered in 2004, and it appeared as a promising nanomaterial due to its catalytic, optical, and electrical properties as well as remarkable physical properties such as a large specific surface area and mechanical strength [3]. In the medical and biological fields, the usefulness of graphene and its derivatives is due to their ability to improve the biocompatibility of various materials already used in tissue engineering [4]. For example, the high aspect ratio, planar structure, flexibility, and hybridization of carbon atoms of graphene help to increase some material properties such as stability [5], the TLR4 pathway activation is involved in the production of pro-inflammatory cytokines as interleukin-6 (IL-6) and interleukine-8 (IL-8), which was also observed in gingival fibroblasts stimulated with E. coli LPS (LPS-E) [36]. Moreover, LPS-E induced a higher expression of inducible nitric oxide (iNOS), IL-6, and monocyte chemotactic protein-1  in an in vitro model of gingival fibroblast cells stimulated with E. coli rather than gingival fibroblasts stimulated with P. gingivalis LPS [34].
Based on this knowledge, the purpose of the current work was to analyze the biological effects of PPSTBs enriched with two different concentrations of GO in an in vitro model of primary human gingival fibroblasts (hGFs) to evaluate the potential protective role of PPSTBs functionalized with GO in the inflammatory process through modulation of the TLR4/MyD88/NFκB p65/NLRP3 pathway. bloodstream [31,32].
The endotoxin of E. coli, in contact with the cells, causes the release of pro-inflammatory cytokines after the activation of the Toll-like receptor (TLR) 2 and TLR4, thus stimulating an immune-inflammatory response [33,34]. The major part of Gram-negative bacteria is recognized to induce the production of pro-inflammatory cytokines principally through TLR4 and nuclear factor-κB (NF-κB) pathways [35]. As reported by Pansani T.N. et al., the TLR4 pathway activation is involved in the production of pro-inflammatory cytokines as interleukin-6 (IL-6) and interleukine-8 (IL-8), which was also observed in gingival fibroblasts stimulated with E. coli LPS (LPS-E) [36]. Moreover, LPS-E induced a higher expression of inducible nitric oxide (iNOS), IL-6, and monocyte chemotactic protein-1 (MCP-1) in an in vitro model of gingival fibroblast cells stimulated with E. coli rather than gingival fibroblasts stimulated with P. gingivalis LPS [34].
Based on this knowledge, the purpose of the current work was to analyze the biological effects of PPSTBs enriched with two different concentrations of GO in an in vitro model of primary human gingival fibroblasts (hGFs) to evaluate the potential protective role of PPSTBs functionalized with GO in the inflammatory process through modulation of the TLR4/MyD88/NFκB p65/NLRP3 pathway.

PPSTBs, PPSTBs-GO 5 μg/mL, and PPSTBs-GO 10 μg/mL Characterization
In Figure 1, AFM topographical micrographs as well as DMT modulus channels of bare and GO-enriched PPSTBs composites, were reported. By using the Peak Force QNM mode, Young's elastic modulus for the three samples was obtained. Mean values of Young's modulus of 4.22 ± 1.49 GPa and 4.39 ± 0.99 GPa were recorded for the PPSTBs and PPSTBs-GO 5 μg/mL samples, respectively, whereas Young's elastic modulus of 8.40 ± 1.39 GPa was obtained for PPSTBs-GO 10 μg/mL samples. The diffraction pattern of PPSTBs and PPSTBs-GO, reported in Figure S3 in the Supplementary Materials, showed typical diffraction peaks of PP in the 2θ range comprised between 10 • and 30 • . These peaks were related to the crystalline phase of the isotactic PP (i-PP) located at 14 • , 17 • , 18.5 • , 21 • , and 22 • corresponding to the indexed planes of the monoclinic crystals of the α-form of i-PP (110), (040), (130), (111), and (131) + (041), and to the trigonal crystals of the β-form at 16 • and 21 • corresponding to the indexed reflections of (300) and (301), respectively [37]. The absence of peaks connected to GO ((001) peak that typically appears between 9-12 • 2θ) in the diffraction patterns of PPSTBs-GO 5 µg/mL and PPSTBs-GO 10 µg/mL indicated that the nanocomposites did not possess layered GO. The addition of GO, even at the highest investigated concentration, did not significantly alter the diffraction pattern of PP. The only difference in the diffraction pattern upon enrichment with GO was the disappearance of the β phase, likely due to a different cooling rate in the crystallization region or nucleation of β crystallites [38].

Cell Viability Assay
MTS assay was performed on hGFs, hGFs + PPSTBs, hGFs + PPSTBs-GO 5 µg/mL, and hGFs + PPSTBs-GO 10 µg/mL cultured with or without LPS-E at 24, 48, and 72 h ( Figure 2). Cell viability was significantly increased in the samples with PPSTBs-GO 5 µg/mL and PPSTBs-GO 10 µg/mL compared to PPSTBs and CTRL samples. The cell metabolic activity increased in hGFs with PPSTBs functionalized with GO with or without the LPS-E treatment.
Supplementary Materials, showed typical diffraction peaks of PP in the 2ϴ range comprised between 10° and 30°. These peaks were related to the crystalline phase of the isotactic PP (i-PP) located at 14°, 17°, 18.5°, 21°, and 22° corresponding to the indexed planes of the monoclinic crystals of the α-form of i-PP (110), (040), (130), (111), and (131) + (041), and to the trigonal crystals of the β-form at 16° and 21° corresponding to the indexed reflections of (300) and (301), respectively [37]. The absence of peaks connected to GO ((001) peak that typically appears between 9-12° 2ϴ) in the diffraction patterns of PPSTBs-GO 5 μg/mL and PPSTBs-GO 10 μg/mL indicated that the nanocomposites did not possess layered GO. The addition of GO, even at the highest investigated concentration, did not significantly alter the diffraction pattern of PP. The only difference in the diffraction pattern upon enrichment with GO was the disappearance of the β phase, likely due to a different cooling rate in the crystallization region or nucleation of β crystallites [38].

Cell Viability Assay
MTS assay was performed on hGFs, hGFs + PPSTBs, hGFs + PPSTBs-GO 5 μg/mL, and hGFs + PPSTBs-GO 10 μg/mL cultured with or without LPS-E at 24, 48, and 72 h ( Figure 2). Cell viability was significantly increased in the samples with PPSTBs-GO 5 μg/mL and PPSTBs-GO 10 μg/mL compared to PPSTBs and CTRL samples. The cell metabolic activity increased in hGFs with PPSTBs functionalized with GO with or without the LPS-E treatment.

hGFs Morphological Analysis
After 24 h of LPS-E treatment, the morphology of hGFs alone or cultured with PPSTBs, PPSTBs-GO 5 μg/mL, and PPSTBs-GO 10 μg/mL were observed using an inverted light microscope and SEM. No morphological differences have been observed among all the experimental conditions at the inverted light microscope (Figure 3(A1-D2)).

hGFs Morphological Analysis
After 24 h of LPS-E treatment, the morphology of hGFs alone or cultured with PPSTBs, PPSTBs-GO 5 µg/mL, and PPSTBs-GO 10 µg/mL were observed using an inverted light microscope and SEM. No morphological differences have been observed among all the experimental conditions at the inverted light microscope ( Figure 3A1-D2). The SEM images showed that hGFs adhere equally on PPSTBs, PPSTBs-GO 5 μg/mL, and PPSTBs-GO 10 μg/mL both in the presence or in the absence of LPS-E treatment ( Figure 4). The SEM images showed that hGFs adhere equally on PPSTBs, PPSTBs-GO 5 µg/mL, and PPSTBs-GO 10 µg/mL both in the presence or in the absence of LPS-E treatment ( Figure 4).

GO-Enriched PPSTBs Influence Protein Expression Evidenced by CLSM and Western Blot Analyses
The immunofluorescence images reported the expression levels of TLR4/MyD88/NFκB p65/NLRP3 in hGFs untreated cells, in hGFs cultured with PPSTBs, in hGFs cultured with PPSTBs enriched with GO at 5 μg/mL, in hGFs cultured with PPSTBs enriched with GO at 10 μg/mL, in hGFs stimulated with LPS-E, in hGFs cultured with PPSTBs and stimulated with LPS-E, in hGFs cultured with PPSTBs enriched with GO at 5 μg/mL and stimulated with LPS-E and in hGFs cultured with PPSTBs enriched with GO at 10 μg/mL and stimulated with LPS-E. The results showed that the TLR4/MyD88/NFκB p65/NLRP3 pathway was expressed significantly in hGFs treated with LPS-E alone or in hGFs cultured with PPSTBs and LPS-E for 24 h compared to the untreated cells. Moreover, TLR4/MyD88/NFκB p65/NLRP3 level expression was less expressed in the cells cultured with PPSTBs enriched with GO and LPS-E compared to hGFs treated with LPS-E alone or in hGFs cultured with PPSTBs and LPS-E. The hGFs cultured with PPSTBs enriched with GO at 10 μg/mL had a comparable level of expression of TLR4/MyD88/NFκB p65/NLRP3 with respect to the CTRL sample group ( Figures  5-8). The results obtained by Western blot were comparable to those obtained by confocal immunofluorescence (Figure 9).

GO-Enriched PPSTBs Influence Protein Expression Evidenced by CLSM and Western Blot Analyses
The immunofluorescence images reported the expression levels of TLR4/MyD88/NFκB p65/NLRP3 in hGFs untreated cells, in hGFs cultured with PPSTBs, in hGFs cultured with PPSTBs enriched with GO at 5 µg/mL, in hGFs cultured with PPSTBs enriched with GO at 10 µg/mL, in hGFs stimulated with LPS-E, in hGFs cultured with PPSTBs and stimulated with LPS-E, in hGFs cultured with PPSTBs enriched with GO at 5 µg/mL and stimulated with LPS-E and in hGFs cultured with PPSTBs enriched with GO at 10 µg/mL and stimulated with LPS-E. The results showed that the TLR4/MyD88/NFκB p65/NLRP3 pathway was expressed significantly in hGFs treated with LPS-E alone or in hGFs cultured with PP-STBs and LPS-E for 24 h compared to the untreated cells. Moreover, TLR4/MyD88/NFκB p65/NLRP3 level expression was less expressed in the cells cultured with PPSTBs enriched with GO and LPS-E compared to hGFs treated with LPS-E alone or in hGFs cultured with PPSTBs and LPS-E. The hGFs cultured with PPSTBs enriched with GO at 10 µg/mL had a comparable level of expression of TLR4/MyD88/NFκB p65/NLRP3 with respect to the CTRL sample group (Figures 5-8). The results obtained by Western blot were comparable to those obtained by confocal immunofluorescence (Figure 9).

Genes Expression
Histogram showed the gene expression of TLR4/MYD88/RELA/NLRP3 and FN1/VIM/VCL/PTK2/ITGA5/ITGA1 evaluated by Real-Time PCR (Figures 10 and 11). The hGFs treated with LPS-E reported a significantly higher gene expression of TLR4/MYD88/RELA and NLRP3 compared to the untreated cells. Moreover, hGFs cultured with PPSTBs enriched with GO at 5 μg/mL and 10 μg/mL and stimulated with LPS-E evidenced a remarkably lower gene expression compared to hGFs stimulated with LPS-E confirming the qualitative results obtained by CLSM observations and Western blot analysis ( Figures  5-9).

Genes Expression
Histogram showed the gene expression of TLR4/MYD88/RELA/NLRP3 and FN1/ VIM/VCL/PTK2/ITGA5/ITGA1 evaluated by Real-Time PCR (Figures 10 and 11). The hGFs treated with LPS-E reported a significantly higher gene expression of TLR4/MYD88/ RELA and NLRP3 compared to the untreated cells. Moreover, hGFs cultured with PPSTBs enriched with GO at 5 µg/mL and 10 µg/mL and stimulated with LPS-E evidenced a remarkably lower gene expression compared to hGFs stimulated with LPS-E confirming the qualitative results obtained by CLSM observations and Western blot analysis (Figures 5-9).

Discussion
GO plays a pivotal role in the biological and medical field, as well as in tissue repair, due to its ability to enhance cell adhesion, proliferation, and differentiation. In addition, GO possesses anti-inflammatory and antibacterial properties. As reported by Radunovic et al., many biomaterials, such as titanium disks and collagen membranes functionalized with GO, showed a reduced bacterial biofilm formation when compared with non-functionalized biomaterials [15]. AFM was used to characterize the surface mor- Figure 11. (a-f) Histograms of RT-PCR showed the mRNA levels of FNT1, VIM, VCL, PTK2, ITGA5, and ITG1b in untreated cells (CTRL), in hGFs cultured with PPSTBs, in hGFs cultured with PPSTBs enriched with GO at 5 µg/mL, in hGFs cultured with PPSTBs enriched with GO at 10 µg/mL, in hGFs stimulated with LPS-E, in hGFs cultured with PPSTBs and stimulated with LPS-E, in hGFs cultured with PPSTBs enriched with GO at 5 µg/mL and stimulated with LPS-E and in hGFs cultured with PPSTBs enriched with GO at 10 µg/mL and stimulated with LPS-E. * p < 0.05; ** p < 0.01; *** p < 0.001.

Discussion
GO plays a pivotal role in the biological and medical field, as well as in tissue repair, due to its ability to enhance cell adhesion, proliferation, and differentiation. In addition, GO possesses anti-inflammatory and antibacterial properties. As reported by Radunovic et al., many biomaterials, such as titanium disks and collagen membranes functionalized with GO, showed a reduced bacterial biofilm formation when compared with non-functionalized biomaterials [15]. AFM was used to characterize the surface morphology of the samples. AFM height images of PPSTBs-GO 5 µg/mL ( Figure 1E) and PPSTBs-GO 10 µg/mL ( Figure 1H) samples showed a less uniform morphology compared to the PPSTBs sample ( Figure 1B). Indeed, the dispersion of GO in PP comprises the establishment of new interactions between PP and GO that require the breaking of PP intermolecular interactions. This rearrangement implies a complete reorganization of PP molecules around GO sheets and may alter the apparent morphology of the PPSTBs-GO compared to that of pure PPSTBs. Nevertheless, no relevant differences in surface roughness were observed on GO-enriched samples in comparison with PPSTBs without GO.
As far as the stiffness is concerned, the obtained Young's elastic modulus values demonstrate that the addition of GO at a concentration of 5 µg/mL did not influence the stiffness characteristics of the starting material. On the contrary, the increase in the elastic modulus of PP composites in the presence of 10 µg/mL was well-defined, and it can be attributed to stress transfer from the polymer matrix to well-dispersed strong GO sheets, enhancing the mechanical properties of the material.
Similarly, XRD analysis does not evidence the presence of aggregated/layered GO, confirming the good dispersion of the GO in the PP matrix. The disappearance of the β phase in PPSTBs enriched GO agrees with the different mechanical properties observed by AFM, at least for the highest investigated concentration of GO [38].
In the present work, the biological effects of PPSTBs with GO in an in vitro model of hGFs were evaluated in the inflammatory process through modulation of the TLR4/MyD88/ NFκB p65/NLRP3 pathway. Toll-like receptors family (TLRs) are receptors present on cell surfaces or in internal compartments such as ERs, endosomes, and lysosomes. These are formed by an ectodomain responsible for the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecules patterns (DAMPs) by a transmembrane domain and a cytoplasmic domain Toll/IL-1 receptor (TIR), which intervenes in the activation of downstream signaling [39]. LPS binds and activates TLR4 through the formation of a complex composed of LPS binding protein (LBP) and accessory proteins CD14 and MD2 [40]. In turn, the activated TLR4 binds myeloid differentiation factor 88 (MYD88), which activates the intracellular signaling cascade that ends with the phosphorylation of the inhibitors serine residues of the transcription regulator nuclear factor kappa B (NFκB) [41,42]. The activated form of NFκB is translocated from the cytoplasm to the nucleus, where it binds specific DNA elements and regulates the transcription of target genes resulting in increased IL-18, IL-6, IL-1β, tumor necrosis factor-α (TNF-α), and MCP-1 [43,44].
Based on the literature, the stimulation with LPS is responsible for the activation of the TLR4/NFκB pathway, which is involved in the increment of NOD-Like Receptor Protein 3 (NLRP3), a component of the NOD-like receptors that form the inflammasome complex [45,46]. The inflammasome is a group of intracellular protein complexes that assemble in response to PAMPs or DAMPs and induces the inflammatory reaction through the activation of caspase 1 [45]. Moreover, it has been demonstrated that LPS induces intracellular ROS and promotes the differentiation of M1 macrophages, which are key effector cells for the elimination of pathogens, virally infected, and cancer cells [47].
Our in vitro data suggest that both hGFs cells alone and cultured with PPSTBs show an increment in the expression of the inflammatory mediators TLR4/Myd88/NFκB p65/NLRP3 when treated with LPS-E. Instead, hGFs cells cultured with PPSTBs enriched with 5 µg/mL or 10 µg/mL of GO showed a significant reduction of TLR4/Myd88/NFκB p65/NLRP3 level expression. The reduction of these inflammatory mediators observed in hGFs cultured with PPSTBs enriched with both concentrations of GO showed that GO could be responsible for modulating the inflammatory process. The results are particularly relevant because the concentration of GO in these PPSTBs is very low (<0.1%).
Moreover, to further support the data obtained, the gene expression of the principal markers involved in cell adhesion, such as Fibronectin, Vimentin, Vinculin, Focal Adhesion Kinase (FAK), and Integrin α5β1, was also investigated.
Cell adhesion to extracellular matrix (ECM) proteins is essential for regenerative processes and for maintaining tissue homeostasis as well as for wound healing processes [48]. Cell adhesion is a fundamental biological event that defines cell and tissue morphogenesis by intervening in the modulation of cell differentiation, cycle, and survival. The adhesion proteins, the main players of this event, are membrane receptors that allow the cells to arrange themselves three-dimensionally to form the tissue and allow their interaction with the surrounding environment.
The affinity of the cells for the biomaterial substrate depends on the ECM molecules and represents a key factor for the development of the biomaterial [49]. In our study, an increase in the gene expression of the principal markers involved in cell adhesion processes was detected in hGFs untreated and treated with LPS-E and cultured with PPSTBs-GO 5 µg/mL and PPSTBs-GO 10 µg/mL. In detail, a significant increase of FNT1/VIM/VCL/PTK2/ITGA5 and ITG1b transcribing, respectively, for Fibronectin, Vimentin, Vinculin, Focal Adhesion Kinase (FAK), and Integrin α5β1 underlines that GO added to PPSTBs promotes cell-to-cell interactions and cell interactions with the surrounding environment.
Fibronectin is an ECM protein involved in cell adhesion, spreading, migration, proliferation, and apoptosis [50]. Its interaction with the heterodimeric cell surface glycoprotein regulates the mechanical anchoring and the formation of focal cell-cell and cell-material adhesion contacts [51]. Specifically, Integrin α5β1 is reported to be highly expressed in human fibroblasts, promoting their motility and survival [52,53], as well as in hGFs [54]. The interaction of Fibronectin with Integrins determines a receptor conformational change and its consequent activation resulting in a mechanical coupling to the ligand. Successively, the receptors form an adhesion complex containing structural proteins, such as Vinculin, and signaling molecules, such as FAK involved in the association with cytoskeletal actin and cell anchoring as well as in sending signals relating to ECM [55,56]. Vimentin, known as one of the principal proteins of cell intermediate filaments, is reported to enhance integrin α5β1 binding fibronectin and to improve cell-cell interactions through its association with hemidesmosomes and desmosomes [57,58].
Despite the limitations of the present in vitro study, relevant and positive outcomes have been obtained. Taken together, these findings highlight the anti-inflammatory effects and the capacity of GO to improve cell adhesion abilities, which may play an important role in the early stage of wound healing. Understanding the mechanisms of the release of ECM components and their regulation is essential for developing novel strategies in the field of tissue engineering and regenerative medicine.
The biological effects of GO, evidenced by our data, could result in better and faster healing of the tissues treated with suture thread enriched with GO. Consequently, the potential use in the clinical setting of these sutures enriched with GO could reduce hospitalization times of treated patients and limit, thank also to the demonstrated antibiofilm activity [9], the use of postoperative antibiotic therapies.

Graphene Oxide (GO)
The GO aqueous solution was obtained as a commercial sample from Graphenea (Graphenea, Donostia-San Sebastian, Spain) and already characterized by the manufacturing company in terms of exfoliation (monolayer content > 95%), size (<10 µm), and oxidation degree (Elemental analysis: carbon: 49-56%; oxygen: 41-50%). This characterization is very important because it has been proven that the above-mentioned "biological" properties of GO depend strictly on those features. Due to the good properties of this commercial sample, we decided to use this material and add it as a solid [59]. The commercial aqueous solution of 4 g/L GO was added to Ultrapure MilliQ water (electric resistance > 18.2 MΩ cm −1 ) from a Millipore Corp. model Direct-Q 3 system (Merk, Burlington, Massachusetts, US) in order to reach the concentration of 1 mg/mL, and bath ultrasonicated for 10 min (37 kHz, 180 W; Elmasonic P60H; Elma). The concentration of GO has checked spectrophotometrically at λ max 230 nm by using a Varian Cary 100 BIO UV-Vis spectrophotometer. Dimensions of GO flakes were measured by using dynamic laser light scattering (DLS) (90Plus/BI-MAS ZetaPlus multi-angle particle size analyzer; Brookhaven Instruments Corp., Holtsville, NY, US) in order to check that micrometric GO have been obtained, and ultrasonication did not reduce significantly GO flakes dimensions (see Figure S1, Supplementary Materials). In order to further characterize the commercial GO, ζ-potential measurements and Raman spectroscopy (XploRA PLUS, HORIBA, Kyoto, Japan) analyses have been performed (see Supplementary Materials). GO dispersion was divided into different aliquots and transferred at −80 • C overnight. After GO aliquots were completely frozen, the samples were placed in a freeze dryer for 48 h, generating black GO sponges. An aliquot of dried GO was redispersed in water and characterized by DLS, ζ-potential (see Supplementary Materials), and UV-Vis spectrophotometry. The UV-Vis spectrum was registered in order to observe the dispersion behavior and check the real final concentration of GO after the freeze-drying process ( Figure 12). Analogously, Raman spectroscopy analyses were performed on the dry GO sample after lyophilization (see Figure S2 in the Supplementary Materials). The amount of GO necessary for the production of 50 g PPSTBs was 5 mg GO for 5 µg/mL samples and 10 mg GO for 10 µg/mL samples. mercial aqueous solution of 4 g/L GO was added to Ultrapure MilliQ water (electric resistance > 18.2 MΩ cm −1 ) from a Millipore Corp. model Direct-Q 3 system (Merk, Burlington, Massachusetts, US) in order to reach the concentration of 1 mg/mL, and bath ultrasonicated for 10 min (37 kHz, 180 W; Elmasonic P60H; Elma). The concentration of GO has checked spectrophotometrically at λmax 230 nm by using a Varian Cary 100 BIO UV-Vis spectrophotometer. Dimensions of GO flakes were measured by using dynamic laser light scattering (DLS) (90Plus/BI-MAS ZetaPlus multi-angle particle size analyzer; Brookhaven Instruments Corp., Holtsville, NY, US) in order to check that micrometric GO have been obtained, and ultrasonication did not reduce significantly GO flakes dimensions (see Figure S1, Supplementary Materials). In order to further characterize the commercial GO, ζ-potential measurements and Raman spectroscopy (XploRA PLUS, HORIBA, Kyoto, Japan) analyses have been performed (see Supplementary Materials). GO dispersion was divided into different aliquots and transferred at −80°C overnight. After GO aliquots were completely frozen, the samples were placed in a freeze dryer for 48 h, generating black GO sponges. An aliquot of dried GO was redispersed in water and characterized by DLS, ζ-potential (see Supplementary Materials), and UV-Vis spectrophotometry. The UV-Vis spectrum was registered in order to observe the dispersion behavior and check the real final concentration of GO after the freeze-drying process (Figure 12). Analogously, Raman spectroscopy analyses were performed on the dry GO sample after lyophilization (see Figure S2 in the Supplementary Materials). The amount of GO necessary for the production of 50 g PPSTBs was 5 mg GO for 5 μg/mL samples and 10 mg GO for 10 μg/mL samples. Figure 12. (a) Photograph of GO aqueous dispersion 1 mg/mL (left) and the obtained GO sponge after freeze-drying process (right) and (b) UV-Vis spectra of GO aqueous dispersion before freeze-drying (straight line) and GO aqueous dispersion obtained by redispersion of GO sponge (dotted line). The decrease of the absorbance after lyophilization is due to the loss of material during the process.

GO-Enriched PPSTBs
PPSTBs of pure PP were enriched with two different concentrations of GO. Briefly, 50 g of PPSTBs were dissolved at the temperature of 160°C and thoroughly mixed with 5 mg GO for 5 μg/mL samples and 10 mg GO for 10 μg/mL samples, respectively. As the final step, the molten product was placed in molds to create the PPSTBs (produced and furnished by Assut Europe S.p.A).

PPSTBs, PPSTBs-GO 5 μg/mL, PPSTBs-GO 10 μg/mL Characterization
The PPSTBs substrates were characterized by AFM using the MultiMode 8 AFM microscope (Bruker, Billerica, MA, USA) equipped with a Nanoscope V controller. The Peak Force Quantitative Nanomechanics (PFQNM) mode was used to map the morphology and to acquire quantitative insight into nanomechanical parameters of PPSTBs

GO-Enriched PPSTBs
PPSTBs of pure PP were enriched with two different concentrations of GO. Briefly, 50 g of PPSTBs were dissolved at the temperature of 160 • C and thoroughly mixed with 5 mg GO for 5 µg/mL samples and 10 mg GO for 10 µg/mL samples, respectively. As the final step, the molten product was placed in molds to create the PPSTBs (produced and furnished by Assut Europe S.p.A).

PPSTBs, PPSTBs-GO 5 µg/mL, PPSTBs-GO 10 µg/mL Characterization
The PPSTBs substrates were characterized by AFM using the MultiMode 8 AFM microscope (Bruker, Billerica, MA, USA) equipped with a Nanoscope V controller. The Peak Force Quantitative Nanomechanics (PFQNM) mode was used to map the morphology and to acquire quantitative insight into nanomechanical parameters of PPSTBs substrates, such as Young's elastic modulus. The PPSTBs and PPSTBs-GO 5 µg/mL samples were mapped using a precalibrated RTESPA-300-30 probe (spring constant 38.904 N/m and resonance frequency of 350.251 kHz), while for PPSTBs-GO 10 µg/mL samples, the precalibrated RTESPA-525-30 cantilever (spring constant 266.124 N/m and resonance frequency of 582.946 kHz) was chosen. The deflection sensitivity of both types of cantilevers was measured against a standard Sapphire 12-M sample, and after the calibration, images of 512 × 512 pixels were collected with scan sizes of 5 × 5 µm. To analyze the images, the Nanoscope Analysis 1.8 software was used [60]. The elastic modulus values were calculated by using the Derjaguin-Muller-Toropov (DMT) model, extracting them from each force-distance curve registered at each point of the scanned surface. XRD analysis was performed using the D2 Phaser X-ray diffractometer apparatus (Bruker, Billerica, MA, USA) with Cu Kα radiation (λ = 0.154 nm, 30 kV, 10 mA) as an X-ray source. Scattered X-ray intensities were collected over a range of scattering angle 2θ = 5 • to 50 • with a scan velocity of 0.05 2θ s −1 .

Experimental Study Design
The experimental points shown in the following study design were performed in triplicate with hGFs at passage 5. The cells were stimulated with LPS derived from E. coli

Scanning Electron Microscopy (SEM)
hGFs cells were seeded on PPSTBs, PPSTBs-GO 5 µg/mL, and PPSTBs-GO 10 µg/mL attached to the bottom of a 12-well plate with and without stimulation with LPS-E. After 24 h of culture, cells were fixed for 1 h at 4 • C in 2.5% glutaraldehyde (Electron Microscopy Sciences, EMS, Hatfield, PA, USA), in 0.1 M sodium phosphate buffer (PB), pH 7.3, rinsed three times with PB, and post-fixed for 1 h in 1% aqueous osmium tetroxide (EMS) at 4 • C. The cells were dehydrated through an ethanol series (30%, 50%, 70%, 90%, 95%, and two times 100%) followed by drying in air and carbon. Morphological analysis was carried out using a high-resolution scanning electron microscope (SEM) Regulus 8220 (Hitachi, Ltd., Tokyo, Japan) operated at 1 kV.

Confocal Laser Scanning Microscope (CLSM)
The hGFs were cultured in 8-well culture glass slides (Corning, Glendale, AZ, USA) at the density of 1.3 × 10 4 /well. After 24 h of treatment, the cells were fixed 1 h at room temperature with 4% of paraformaldehyde (PFA) (BioOptica, Milan, Italy) in 0.1 M in PBS (Lonza, Basel, Switzerland). After 3 washes in PBS, the cells were permeabilized with 0.1% Triton X-100 (BioOptica) in PBS for 5-6 min and blocked with 5% of non-fat milk in PBS for 1 h at RT. Successively, the primary antibodies were prepared in 2.5% non-fat milk in PBS and maintained overnight at 4 • C. The primary antibody used in this study were all purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and were used, as suggested by their datasheet, at the concentration of 1:200: TLR4 (sc-293072), anti-MyD88 (sc-74532), anti-NFκB p65 (sc-8008), anti-NLRP3 (sc-134306). The secondary antibody Alexa Fluor 568 red fluorescence-conjugated goat anti-mouse (A11031, Invitrogen, Eugene, OR, USA) has been prepared 1:200 in 2.5% non-fat milk in PBS and added 1 h at 37 • C. The cytoskeleton actin and the nuclei have been stained, respectively, with Alexa Fluor 488 phalloidin green fluorescent conjugate (A12379, Invitrogen) and TOPRO (T3605, Invitrogen), both prepared 1:200 in 2.5% non-fat milk in PBS and maintained 1 h at 37 • C. The images were acquired through Zeiss LSM800 confocal system (Carl Zeiss, Jena, Germany) [62].

RNA Isolation and Real-Time RT-PCR Analysis
TLR4, MyD88, NFκB p65, and NLRP3 mRNA expression were analyzed by Real-Time PCR. Total RNA was extracted using PureLink RNA Mini Kit (Ambion, Thermo Fisher Scientific, Milan, Italy) according to the manufacturer's instructions. Three independent biological replicates were analyzed for each sample. One microgram of total RNA was retrotranscribed using M-MLV Reverse Transcriptase (M1302 Sigma-Aldrich) to synthesize cDNA for 10 min at 70 • C, 50 min at 37 • C and 10 min at 90 • C according to the technical bulletin. Real-Time PCR was performed with Mastercycler ep real plex Real-Time PCR system (Eppendorf, Hamburg, Germany). The levels of mRNA expression of TLR4, MYD88, RELA, NLRP3, FN1, VIM, VCL, PTK2, ITGA5, ITG1B, and Beta-2 microglobulin (B2M) (endogenous marker) were evaluated in hGFs cells cultured alone, in hGFs cultured with PPSTBs, in hGFs cultured with PPSTBs enriched with GO at 5 µg/mL, in hGFs cultured with PPSTBs enriched with GO at 10 µg/mL, in hGFs stimulated with LPS-E, in hGFs cultured with PPSTBs and stimulated with LPS-E, in hGFs cultured with PPSTBs enriched with GO at 5 µg/mL and stimulated with LPS-E and in hGFs cultured with PPSTBs enriched with GO at 10 µg/mL and stimulated with LPS-E. Commercially available PrimeTime™ Predesigned qPCR Assays TLR4 (Hs.PT. 58 Table 1). Beta-2 microglobulin (B2M Hs.PT.58v.18759587, Tema Ricerca Srl) was utilized for template normalization. The amplification program included a preincubation step for cDNA denaturation (3 min at 95 • C), followed by 40 cycles consisting of a denaturation step (15 s at 95 • C) and an annealing step (1 min at 60 • C). Expression levels for each gene were performed according to the 2 −∆∆Ct method. Real-Time PCR was performed in three independent experiments.

Statistical Analysis
Statistical significance was established with GraphPad 5 (GraphPad, San Diego, CA, USA) software utilizing one-way ANOVA followed by post hoc Tukey's multiple comparisons analysis. Values of p < 0.05 were considered statistically significant.

Conclusions
The current work aimed to investigate the possible therapeutic benefit of commercial PP suture threads enriched with GO in a gingival fibroblasts cellular model. Our results showed that GO-fabricated PP suture threads modulated the inflammatory effects induced by LPS-E through TLR4/MyD88/NFκB p65/NLRP3 pathway. The biological effects of suture thread enriched with GO may represent a promising strategy that can be applied in clinical medicine.