Redox signaling induces laminin receptor ribosomal protein-SA expression to improve cell adhesion following radiofrequency glow discharge treatments

Current biomaterials effectively replace biological structures but are limited by infections and long-term material failures. This study examined the molecular mechanisms of radio frequency glow discharge treatments (RFGDT) in mediating the disinfection of biomaterial surfaces and concurrently promoting cell attachment and proliferation. Dental biomaterials were subjected to RFGDT, and viability of oral microbial species, namely Streptococcus mutants (SM), Streptococcus gordonii (SG), Moraxella catarrhalis (MC), and Porphyromonas gingivalis (PG), were assessed. Cell attachment and survival of a pre-odontoblast cell line, MDPC-23, was examined. Finally, mechanistic investigations into redox generation and biological signaling were investigated. Based on their compositions, dental biomaterials induced reactive oxygen species (ROS) following dose-dependent RFGDT. Reduced microbial viability was evident following RFGDT in the catalase-negative (SM and SG) species more prominently than catalase-positive (MC and PG) species. Cell adhesion assays noted improved MDPC-23 attachment and survival. Pretreatments with N-acetylcysteine (NAC) and catalase abrogated these responses. Immunoassays noted redox-induced downstream expression of a laminin receptor, Ribosomal Protein SA, following RFGDT. Thus, RFGDT-induced redox mediates antimicrobial and improves cell responses such as adhesion and proliferation. These observations together provide a mechanistic rationale for the clinical utility of RFGDT with dental biomaterials for regenerative clinical applications.


Results
Redox generation at biomaterial interfaces following RFGDT. The plasma generated by RFGDT affects the surface chemistry of the biomaterials based on their composition and the presence of exogenous gases. As the dental pulp is exposed to various restorative biomaterials such as glass ionomer cement, polymers, and metals, we examined the generation of redox species following RFGDT with a chemiluminescence luminol assay 31 . RFGDT generated a significant increase in ROS from glass (Fig. 1a) and platinum surfaces (Fig. 1b), while minimal expression was noted on a polymeric (Polymethylmethacrylate, PMMA) surface (Fig. 1c). RFGDT induced a dose-dependent increase in redox generation with optimal noted at 180 s (Fig. 1d). As a prior study utilized various surface treatments such as detergent, alcohol, or silane, we noted that only RFGDT generates redox (Fig. 1e) 27 . These results show that while multiple agents can disinfect biomaterial interfaces, induction RFGDT-induced redox could persistently prevent microbial colonization while favorably modulating host cell interactions.

Microbial colonization is inhibited by RFGDT-induced redox.
To examine the disinfection potential of RFGDT, we first examined the endogenous antioxidant by incubating individual microbe with hydrogen peroxide. We noted that Streptococcus gordonni and Streptococcus mutants, which are catalase-negative, failed to generate effervescence (Fig. 2a). In contrast, Moraxella catarrhalis and Porphyromonas gingivalis, which are catalase-positive, generated profuse effervescence indicating the active ROS neutralization capabilities. Glass coverslips subjected to RFGDT were seeded with S. mutant and demonstrated reduced viability with the livedead assay (Fig. 2b). Consistent with this observation, colony-forming units (CFUs) of S. mutants (Fig. 2c) and S. gordonni (Fig. 2d) were significantly reduced following RFGDT. The reduction in microbial survival could be prevented by pre-incubation with ROS scavengers N-acetyl cysteine (NAC) and catalase. Consistent with these observations, no differences in CFUs were observed with M. catarrhalis (Fig. 2e), and P. gingivalis (Fig. 2f) seeded on glass coverslips following RFGDT. These results demonstrate RFGDT-induced redox reduces viability and proliferation of susceptible (catalase-negative) microbes, while anaerobic, catalase-positive species were resistant.

RFGDT improves cell adhesion and expansion.
Cell adhesion to the substrate is crucial for attachment and subsequent proliferation. Several biomaterials, such as dental cements and composites, include glass or metals that directly contact dental pulp cells. Therefore, we next examined the effects of RFGDT on a mammalian pred-odontoblast cell line, MDPC-23. Glass coverslips were subjected to detergent, alcohol, silane (ODS), or RFGDT prior to seeding. We examined both weak and strong cell adhesions by assessing basal and centrifugal force-induced adherent and floating cell populations at 5 and 24 h after cell seeding. We observed a maximal number of adherent cells on RFGD treatment while most cells on ODS and alcohol-treated surfaces were nonadherent (Fig. 3a, b). Time course analysis after 5 and 24 h with the adhesion strength assays further validated this observation with silane and alcohol-treated surfaces, demonstrating most non-adherent cells in contrast to RFGDT that offered an optimal biomaterial interface for predominantly strong cell adhesion (Fig. 3c- www.nature.com/scientificreports/ pronounced increase in non-adherent cells in the alcohol-treated group at 5 h could be attributed to a persistent residual agent reducing cell viability. Finally, incubation with either N-acetyl cysteine or catalase was noted to specifically reduce RFGDT-improved cell attachment (Fig. 3f). These results suggest that the improved cell adhesion following RFGDT is mediated via redox-mediated signaling.
Improved cell adhesion is mediated via RFGDT-generated redox-mediated RPSA expression. Cell adhesion involves concerted actions of multiple cell membrane and cytoplasmic molecules capable of dynamic assembly of an attachment complex [31][32][33] . Among these various mediators, laminin receptors on the cell surface have been shown to play a critical role in mediating matrix interactions 37 . Elucidation of these adhesive complexes has also outlined several upstream signaling mediators that outline the role of a laminin receptor, Ribosomal Protein SA (RPSA), induced by redox signaling 34,35 . Hence, to examine the effects of redox signaling on cell adhesion, MDPC-23 cells were treated with various concentrations of hydrogen peroxide that demonstrated increased RPSA and phospho-FAK 397 expression ( Fig. 4a-c). Next, we examined if the RFGDT-improved cell adhesion could be mediated via a similar mechanism. We observed a robust increase in phospho-FAK 397 and RPSA expression following RFGDT by immunoblotting (Fig. 4d, e) and immunofluorescence (Fig. 4f, g). These responses were abrogated by pretreatment of the biomaterial surface with silane or by adding NAC, indicating these are redox-mediated. Finally, we inquired if the improved cell attachment via the enhanced laminin receptor following RFGDT would affect cell survival and proliferation. We observed a significant improvement (n = 3, p < 0.05) in RGFDT group compared to all other groups (Fig. 4h). These results suggest improved cell adhesion following RFGDT could be mediated via induced RPSA expression.

Discussion
Biomaterial interfaces represent a major frontier for improving functional integration and determining clinical therapeutic outcomes 36 . Significant progress in biomaterial composition and design has evolved from simple biocompatible, inert interfaces to existing bioactive surfaces. More recent biomaterial systems focus on smart systems capable of sense-and-respond to biological scenarios with directed responses. A major functional characteristic of these biomaterial interfaces is to promote desirable biological responses while actively dissuading detrimental ones selectively 3 . Some of the latter responses are directed at reducing microbial (biofilm) burden to www.nature.com/scientificreports/ prevent protracted inflammation or host immune responses 37,38 . This facilitates routine tissue healing responses and may enable regenerative clinical outcomes. This study utilized common oral microbes and cells from the dental pulp to examine the effects of RFGDT on biomaterials used routinely in clinical dentistry. Pulpal inflammation due to caries results in irreversible necrosis, necessitating Endodontic intervention and coronal restorations. Various pulp capping agents such as calcium hydroxide, mineral trioxide aggregate (MTA), Emdogain, or glass ionomer cement provide cues to promote tissue healing (osteodentin). However, they do not directly address residual infection that leads to protracted inflammation. Direct plasma surface modification strategies generate antibacterial properties via modulating surface topography or chemistry 39 . Besides serving as an alternate disinfection technique, its effects on improving biocompatible interfaces have been well documented. This prominent dual-action of RFGDT noted in this study, namely antimicrobial disinfection and promoting cell adhesion and expansion, is particularly attractive for clinical use (Fig. 5) 40,41 . The antimicrobial actions of RFGDT predominantly targeting the catalase-negative microbes appear to be directly relevant to healthy clinical scenarios where aerobic bacteria predominate. The (a) Four bacterial species were subjected to hydrogen peroxide, and effervescence was assessed; (b) bacterial viability test with TTC assay was performed with S. mutans following RFGDT. Colony-forming assays were performed with four bacterial species following RFGDT namely (c) Streptococcus mutants, (d) Streptococcus gordonni, (e) Moraxella catarrhalis (f) Porphyromonas gingivalis. In some cases, incubation with N-Acetyl cysteine or catalase was performed, followed by RFGDT. n = 3, *p < 0.05. RFGDT: Radiofrequency glow discharge treatment; NAC: N Acetylcysteine; CFUs: Colony-forming units. www.nature.com/scientificreports/  www.nature.com/scientificreports/  www.nature.com/scientificreports/ reduced efficacy on catalase-negative microbes was evident in this study could be clinically addressed with additional rinses or medications prior to material placement. This study noted that the generation of redox species from biomaterial surfaces was based on their chemical composition. While both glass and platinum could induce redox, the polymeric (PMMA) substrate demonstrated minimal induction. This appears to reflect the ionization potential of the predominant elemental (Glass: Silica 786 kJ/mol and Platinum 867 kJ/mol) constituents that are representative of dental biomaterials such as reinforced cement and metal implants. Glass or metal reinforced cements and composites are routinely used as dental restorative materials. A prior study noted that Dicor glass-restorations luted with resin composites improved clinical survival than other biomaterials 42 . However, unlike glass and these two biomaterials that intimately interact with cells, the polymeric material (PMMA: 6857 kJ/mol) is predominantly used to fabricate external prosthetic devices where the disinfection non-adhesive characteristics are of most significance. Future investigations could investgate the cellular responses to PMMA including funcationalization approaches to improve cellular interface. Nonetheless, direct composite materials such as pulp dressings would appear unsuitable for the RFGDT technique.
We observed an optimal treatment time of three minutes for disinfection and improved cell adhesion with RFGDT for glass surfaces under vacuum. Besides the biomaterial composition and treatment time, other variables determining these interfacial responses include gaseous composition (such as helium or argon) within the chamber, energy density, and the presence of other trace agents 43,44 . Attention to these parameters can enable the development of future biomaterial-specific RFGDT protocols. It is also important to emphasize that the RFGDT was performed on inanimate biomaterial surfaces, before cell seeding, without direct exposure to microbes or cells. Direct treatments with non-thermal plasma on mammalian cells result in DNA damage and loss of viability 45 . This essentially implies that direct clinical RFGDT on human tissues for disinfection or improving cellular responses is an untenable strategy. Nonetheless, surface treatments of biomaterial devices could prime them for optimal tissue interactions such as osseointegration of metallic implants, glass ionomer, or metalreinforced composites as dental filling materials. The latter application was a primary motivation in our choice of the pre-odontoblast cell line, MDPC-23. Interestingly, we have observed differential redox sensitivity among cells of discrete lineage in other studies 46 . Future studies could examine the selectivity of RFGDT on complex multipartite biomaterial systems with specific cell types such as osteoblasts (osseointegration), keratinocytes (wound healing), fibroblasts (matrix production), or endothelial cells (angiogenesis) for directed clinical outcomes. This study observed RFGDT-generated ROS detected using chemiluminescence following oxidation of luminol 31 . The amounts and discrete ROS have critical roles in cell survival, migration, and apoptosis 47,48 . Redox is a physiological regulator of intracellular signaling cascades, especially tyrosine kinase-mediated growth factors, has gained recent attention due to its ability to reversibly oxidize redox-sensitive targets 49 . These reversible redox modifications of cysteine-residues in proteins, also termed thiol switches, are regulated by oxidoreductase and thiol-isomerases 50 . Their primary function involves neutralizing excessive redox in the biological system that can lead to deleterious damage. Other targets of redox-generated signaling include a broad array of cytoskeletal components, signaling intermediates, and transcriptional regulators.
A significant finding in this study is that RFGDT induced redox promotes expression of the laminin receptor, Ribosomal Protein SA (RPSA), that contributes to improved cell adhesion and survival. Laminins are high molecular weight extracellular glycoproteins that constitute the major non-collagenous component of basal lamina in cells 51,52 . Their multidomain proteins are encoded by eleven human genes characterized by heterodimeric α, β and γ chains that constitute 16 different isoforms. Their key contributions to cellular functions such as adhesion, migration, and differentiation are well described. There have been recent insights into the roles of specific Laminins to selectively drive cellular enrichment and lineage specification that has potent implications for tissue healing and regeneration 53 . Besides the prototypical members, there are many proteins with laminin-like www.nature.com/scientificreports/ domains that include RPSA, LamR, 37LRP, and LBP/p40, among others 54 . RPSA is a ribosomal protein with several pathophysiological functions in protein translation, cell adhesion, tumor cell invasion, viral cell entry, and small molecule sensing 55,56 . The amino acid sequence is evolutionarily conserved with over 98%c homology in all mammals and structural similarities with the prokaryotic gene, RPS2 54 . It would be interesting to examine the functional significance of RFGDT induced RPS2 in the microbial species in future studies. RPSA has been noted to be involved in cap-dependent eukaryotic translation, especially in transformed and viral infected cells. However, upstream induction of RPSA signaling remains to be fully elucidated. A few prior reports have noted that RPSA is induced by redox-generated signaling, suggesting it is a redox-inducible target gene 34,35 . Consistent with these reports, this study outlines the ability of RFGDT induced redox to increase RPSA expression. The increased expression of this cell adhesion receptor would contribute to improved attachment and survival of the MDPC-23 cells. Membrane clustering of these laminin receptors has been noted to induce tyrosine 397 phosphorylation of FAK, as was also noted in this study. Both FAK and Src activation at these membrane adhesion complexes further drive cellular functions such as cell spreading, migration, proliferation, and prevention of apoptosis. Thus, the use of RFGDT appears to evoke discrete redox-generated biological responses that can be harnessed for both disinfection and cellular functions such as cell attachment, expansion, and differentiation to aid tissue regeneration.
In conclusion, this study demonstrates the ability of a non-invasive, directed energy tool, RFGDT, to be capable of concurrent antimicrobial disinfection and improved directed cellular responses via redox-signaling. This showcases RFGDT as a simple, cost-effective, and convenient bench-top tool for clinical application to improve tissue healing and regenerative outcomes.

Materials and methods
Cell culture. Mouse dental pulp cells (MDPC-23, kind gift from Drs. Jacques Nor and Tatiana Botero, Universithy at Michigan) were maintained in DMEM (Glutamax, high-glucose) supplemented with 10% Fetal Bovine Serum (certified grade) and 100 units mL −1 Penicillin and 100 µg mL −1 Streptomycin (all from Thermo-Scientific, USA). Cells were grown at 37 °C in a humidified chamber with 5% CO 2 .
Sample preparation and sterilization. Cover glass (18 mm, 0.12-0.17 mm thickness, Matsunami micro cover glass) were first cleaned with 10% detergent (Sparkleen, Fisher Scientific, USA) and sonicated (Aristocrat Ultrasonic, Healthco, USA) for 10 min, followed by three washes in distilled water and allowed to dry vertically in a holder. Following this, some covers slips were soaked in 95% Ethanol (Sigma-Aldrich, USA) for 10 min, while another set was soaked in 2% n-octadecyl trichlorosilane (ODS) for 10 min. Finally, another set of coverslips was subjected to RFGDT, as described below. Except for the RFGDT, all samples were sterilized under UV light or 15 min before cell seeding.

Radiofrequency glow discharge treatments (RFGDT).
A partial air-vacuum RFGD device (PDC-32G, Harrick Scientific, USA) was used as per the manufacturer's instructions. The biomaterials (glass coverslips, platinum sheets, or PMMA discs) were placed within the chamber and treated for various times as indicated in individual studies. Biomaterial specimens were used immediately for biochemical, microbiological, or cell adhesion/survival studies.
AlamarBlue assay. MDPC-23 cells were seeded at a cell density of 1 × 10 5 cells mL −1 on the treated glass surfaces described above placed in a 12 well dish. Cell viability and proliferation were assessed with the Alamar-Blue assay as per the manufacturer's instructions. Briefly, following 24 h of cell incubation, 10% (v/v) of Alamar-Blue reagent (Thermo Fisher Scientific, USA) was added to complete media and incubated for 2 h. Replicates of conditioned media from each well were transferred to black wells, and fluorescence was assessed using a plate reader (Victor3, Perkin Elmer, USA).
Cell adhesion assay. MDPC-23 cells were seeded at a cell density of 1 × 10 5 cells mL −1 on the treated glass surfaces described above placed in a 12 well dish. Cell adhesion was assessed after 5 h of incubation as described previously. As equivalent cells were seeded on all substrate conditions and no proliferation is expected within 5 h, the non-adherent, floating cells (higher, more reliable global numbers) in each condition were counted rather than imaging multiple high-power fields (lower, less reliable local numbers) per well. Media from each well was collected, stained with Trypan Blue (0.4% w/v, Sigma-Aldrich, USA), and assessed for floating (nonseeded) cells. After replacing the media, plates were subjected to centrifugation (Eppendorf, USA) at 2400 rpm (weak adhesions) and 4000 rpm (strong adhesions) for 6 min, and the media was collected to assess cell numbers. Images were also captured of these wells before and after spinning to document the number and morphology of the adherent cell populations. In some studies, Catalase (10 uM), N-acetyl cysteine (1 mM), or hydrogen peroxide (all from Sigma-Aldrich, USA) were included in the media prior to cell seeding of the glass surfaces to assess the role of RFGDT-induced ROS in mediating cell adhesion.

ROS detection assay.
To assess RFGDT-generated ROS, a chemiluminescence assay with Luminol (detects both extra-and intracellular ROS) was utilized as described previously 33 . Briefly, a solution was prepared by mixing 5 mg of luminol powder with 100 mL of 1 M Sodium hydroxide (both Sigma-Aldrich, USA) and mixed gently. The freshly prepared luminol solution was added to the cells in a 12 wells plate at a ratio of 1 mL/well (v/v) immediately after RFGDT treatments at different doses, and chemiluminescence was detected at 0.5, 1, 3, and 5 min using a multiwell detector (Victor3, Perkin Elmer, USA). www.nature.com/scientificreports/ Western blotting. Glass coverslips were RFGDT treated and seeded with MDPC23 cells. After 5 h of incubation in CO 2 incubator, the attached cells on the coverslip were collected from different groups, and collected cells were re-suspended in RIPA buffer (Sigma-Aldrich, USA) with Mini Protease Inhibitor (Thermo Scientific, USA). Cells were disturbed with two cycles of sonication (QSonica, USA) for 5 s each, and lysates were centrifuged at 14,000 rpm at 4 °C for 20 min. The total protein in the lysates was quantified with the Bradford assay kit (BCA Protein Assay, Thermo Scientific Inc). Protein lysates were separated in precast mini-protein TGX stain-free gels and transferred to PVDF membranes (both Bio-Rad, USA). Blots were initially blocked with 1% BSA for 1 h and further incubated with primary antibodies for Ribosomal Protein SA (RPSA, Abcam, USA) or phospho-FAK (Cell signaling, USA) at 4 °C overnight. Following washes, blots were incubated for normalization with appropriate species-specific HRP conjugated secondary antibodies (Cell signaling, USA) or HRP-conjugated β-actin (Cell signaling, USA). Finally, the blots were developed by using chemiluminescent substrates (Thermo Scientific, USA), and images were scanned digitally using ChemiDoc MP Imaging System (Bio-Rad Laboratories, USA). Western blots protein band intensities were quantified by using ImageJ software. Multiple blot exposures are provided in supporting figures.
Immunofluorescence staining. In all groups, immunofluorescent experiments were carried out using primary laminin antibody (RPSA) (Cat # ab133645, Abcam, USA) and Alexa fluor 488 secondary antibody (Cat # 4412S, Cell signaling, USA), image were captured by using ZOE Fluorescent Cell Imager (Bio-rad, USA), where nucleus was counterstained stained with DAPI. Digital images were captured, and following thresholding, fluorescence intensity over the entire field was quantified using NIH ImageJ software as described previsouly 57,58 .

Microbial survival assay.
For the CFU assays, coverslips were inoculated with 250 uL respective bacterial culture and incubated in species-specific growth conditions for 24 h (S. gordonni, M. catarrhalis, S. mutants) or 5 days (P. gingivalis). Aliquots of bacterial culture from glass coverslips were serially diluted by tenfold dilutions and plated via the drop plate technique.
Statistical analyses. Data were analyzed using Excel (Microsoft, USA), and statistical significance was assessed using either a one-way ANOVA or t tests, where p < 0.05 was considered statistically significant. All studies were repeated at least twice, with each assay performed in minimum duplicates.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files. However, any further information or material is available from the authors upon reasonable request.