Plasma‐activated medium triggers immunomodulation and autophagic activity for periodontal regeneration

Abstract Periodontitis is an infection‐induced inflammation, evidenced by an increase in inflammatory macrophage infiltration. Recent research has highlighted the role of plasma‐activated medium (PAM) as a regulator of the innate immune system, where macrophages are the main effector cells. This study therefore aims to investigate the immunomodulatory effects of PAM on macrophages and its potential applications for periodontitis management. PAM was generated using an argon jet and applied to culture macrophages. Proinflammatory macrophage markers were significantly reduced after PAM stimulation, and this was correlated with the activation of autophagy via the Akt signaling pathway. Further investigations on the proregenerative effects of PAM‐treated macrophages on periodontal ligament cells (PDLCs) revealed a significant increase in the expression of osteogeneis/cementogenesis‐associated markers as well as mineralization nodule formation. Our findings suggest that PAM is an excellent candidate for periodontal therapeutic applications.


| INTRODUCTION
As an essential component of the innate immune system, macrophages protect against harmful pathogenic microorganisms and regulate immune responses. 1 This is accomplished by both the classically activated M1 subtype and the alternatively activated M2 subtype. 1,2 The proinflammatory functions of M1 macrophages include phagocytosing pathogens or foreign particles, presenting antigens to other immune cells, and secreting inflammatory cytokines to trigger the inflammation. 3 However, M1 macrophages would cause chronic inflammation and damage the tissues if they continuously released cytotoxic substances into the microenvironment. 4 Anti-inflammatory cytokines produced by M2 macrophages, on the other hand, would encourage tissue regeneration. 1 Periodontitis, a common bacterial infection-induced chronic inflammation, often results in tooth loss leading to major aesthetic and functional issues if left untreated. 5 Excessive M1 macrophages and uncontrolled M1 macrophagemediated cytokine secretion are the significant factors that cause the destruction of periodontium, including periodontal ligament (PDL), cementum and alveolar bone. 4 Therefore, restoring the balance between M1 and M2 macrophages is essential for periodontitis patients to reduce chronic inflammation and enhance tissue regeneration.
Plasma is an important source of charged particles, free radicals, electric fields, and metastable species. 6 Cold atmospheric plasma (CAP) has shown great promise in treating conditions such as cancer, skin infections, and blood clotting. [7][8][9][10][11] In clinical research of oral cancers and infections, CAP has been applied orally without safety concerns, [12][13][14] suggesting that CAP is safe to be used as a therapeutic option. Since it is challenging to deliver CAP directly to the target in vivo, CAP activity is often converted to liquid, providing a versatile method for therapeutic application. 10,11 One plasma-activated liquid that exhibits therapeutic properties and has recently been explored, is plasma-activated medium (PAM). 10,15 PAM produced from CAPactivated medium is cytotoxic to cancer cells but has no discernible impact on normal cells. 10 One notable advantage of PAM compared to CAP is its ability to access oral cavities and penetrate deep tissues which CAP cannot. Additionally, PAM is considered safer for cell treatment as it does not retain components such as ultraviolet rays and electromagnetic fields. Moreover, the effective treatment time for PAM treatment (hour level) is longer than that for CAP maintenance (minute level). Therefore, to promote the practical application of cold plasma technology in clinical settings, PAM is utilized as a versatile CAP method in this study.
Our previous studies [15][16][17] demonstrated that PAM is distinct from individual ROS/RNS compound solutions. Through secondary reactions, PAM can continuously generate nanosecond and microsecond short-lived free radicals through secondary reactions, such as hydroxyl radicals (ÁOH), singlet oxygen ( 1 O 2 ), and peroxynitrite anions (ONOO À ), when applied to biological tissues. PAM can also act as an inducer in the innate immune system, according to previous studies. 18,19 However, more research is needed to determine whether PAM has the potential to promote periodontal regeneration by modulating macrophage responses. This project therefore investigated the immunomodulatory effect of PAM on macrophages and its application as a therapeutic approach for periodontitis.

| PAM generation
A conventional commercial argon plasma jet (model kINPen 09, INP Greifswald, Germany) was used in this study to generate CAP. 20 Aliquots of 1.5 mL serum-free medium (Dulbecco's Modified Eagle Medium, DMEM; Life Technologies, USA) containing 50 U/mL penicillin and 50 μg/mL streptomycin (P/S; Life Technologies Australia Pty Ltd.) was activated by CAP for 10 min at a flow rate of 5.0 Standard Liter per Minute while being exposed to pure argon gas. The plasma jet nozzle was 10 mm away from the medium surface. Serum-free DMEM containing 50 U/mL penicillin and 50 μg/mL streptomycin was used to dilute PAM to various concentrations (5%, 10%, and 30%). Optical emission spectroscopy (OES), SpectraPro-750i monochromator (Acton Research Corporation, USA), was used to identify several reactive atoms and molecules found in PAM. 11

| Macrophage culture stimulation
The murine macrophage cell line RAW264.7 was grown in DMEM with 1% P/S, 10% fetal bovine serum (FBS) at a density of 5000 cells per cm 2 . To induce the proinflammatory M1 phenotype, 10 ng/mL lipopolysaccharide (LPS; Sigma, USA) and 100 ng/mL interferon gamma (IFN-γ; R&D, USA) were added to the culture medium. The induction was conducted at 37 C with 5% CO 2 for 12 h. The cells were then washed with phosphate buffered saline (PBS) for three times to completely remove the residue of LPS and IFN-γ. M1 or untreated macrophage cultures were supplemented with PAMactivated DMEM at a dose of 5%, 10%, or 30% (100 μL per 5000 cells) for 12 h at 37 C with 5% CO 2 . To fully eliminate the excess PAM-activated DMEM, the cells were washed three times with PBS.
Before collecting the conditioned medium, phenotype-switched macrophages underwent a 24-h starvation period. The conditioned medium was labeled as M1CM and M0CM, respectively, depending on whether the macrophages had been treated with or without LPS/IFN-γ.

| Live/dead cell viability assay
The cellular supernatants were removed after 12 h of PAM culture, and the cells were washed with PBS. Each well received 50 μL of PBS that contained 10 μg/mL propidium iodide and 10 μg/mL Hoechst 33342 for a 30-min incubation period at 37 C. Incubated cells were then scanned using an In-Cell Analyzer 6500HS (GE Healthcare, USA, 10Â objective). Propidium iodide (PI) and Hoechst 33342 staining were identified at a wavelength of 642 nm and 405 nm, respectively.
Live/Dead cell viability analysis was performed using IN Carta Image Analysis Software. 10 Cell viability is calculated as follows: cell count (PI)/cell count (Hoechst 33342) Â 100%.

| Scanning electron microscope (SEM)
LPS/IFN-γ stimulated macrophages were cultured with PAM for 12 h and then fixed with 2.5% glutaraldehyde (Sigma, G5882). The fixed samples were postfixed in 1% osmium tetroxide and then processed through dehydration by increasing ethanol concentrations (50%, 70%, 90%, and 100% vol/vol). The samples were finally gold-coated for observation of the cell morphology using SEM (FESEM, Zeiss Sigma) under 20 kv using SE2 mode.

| Osteogenic differentiation of periodontal ligament cells (PDLCs)
Human research ethics approval was obtained from the Office of Research Ethics and Integrity (OREI), Queensland University of Technology (QUT). The PDLCs used in this study were harvested from caries-free and periodontally healthy premolars extracted for orthodontic treatment purposes (n = 6). The periodontal ligament tissues were isolated from the mid-third of the tooth root surfaces, trimmed to fine pieces, and rinsed with PBS. 4 The tissue explants were transferred to a T75 flask with DMEM containing 10% FBS and 1% P/S. The culture medium was replaced twice a week and the outgrown cells were passaged after they had reached around 80% confluence. The cells from passages 2 to 4 were used in the following experiments. For osteogenic differentiation of PDLCs, the macrophage-derived conditioned medium was mixed 1:1 with osteogenic medium (DMEM supplemented with 20% FBS, 1% P/S, 20 mM β-glycerophosphate, 100 μM ascorbic acid, and 200 nM dexamethasone) to culture PDLCs for 7 days at 37 C and 5% CO 2 .

| Gene expression detection
Using the TRIzol ® reagent, total RNA from macrophages or PDLCs was extracted (Life Technologies Pty Ltd., Australia). The purity and quantity of RNA were determined spectrophotometrically using a NanoDrop instrument (Thermo Fisher Scientific). The cDNA was syn-  (Table 1). All experiments were performed in triplicate.

| Alizarin Red S staining
After 14 days of culture with the macrophage-derived conditioned medium, the PDLCs were fixed with 4% paraformaldehyde (PFA). The fixed cell samples were incubated with 1% Alizarin Red S (A5533, Sigma-Aldrich) for 20 min and rinsed with distilled water. The dye of each sample was extracted using 300 μL 50% acetic acid, let to sit at room temperature for 30 min, and then neutralized with 10% ammonium hydroxide (320,145, Sigma-Aldrich) with the pH set to 4.1. The solutions were centrifuged at 10,000 RPM for 10 min. Finally, 100 μL supernatant of each sample was transferred into a 96-well plate and measured at 405 nm using a BIO-RAD microplate absorbance spectrophotometer.

| Western blot
The protein of each sample was collected and suspended in 500 μL RIPA lysis buffer (R0278, Sigma) with protease inhibitor

| Statistical analysis
All data were presented as mean ± standard deviation (SD) for three independent experiments. Statistical differences between each group were determined by one-way ANOVA with Bonferroni's multiple comparison tests. A p < 0.05 was considered statistically significant.  Figure 1A). However, when the macrophages were treated with PAM at various concentrations (5%, 10%, or 30%), the number of nuclei, which was around 34,000, did not change significantly compared to the M0 subtype ( Figure 1A). Additionally, PAM at various concentrations (5%, 10%, or 30%) had no effect on the proliferation of macrophages since, in comparison to M1 macrophages without PAM treatment, the average nuclei number of PAM-treated macrophages did not vary significantly.
Cell viability was measured as cell count (PI)/cell count (Hoechst 33342) Â 100%. Our results indicate that the cell viability was not significantly impacted by LPS and IFN-γ stimulation, and the same pattern was seen in M0 or M1 macrophages cultured in PAM at various concentrations (5%, 10%, or 30%) ( Figure 1B). LPS/ IFN-γ stimulation on macrophages resulted in the extension of dendritic structure T A B L E 1 Primers used for RT-qPCR.

Target gene
Forward primer Reverse primer Figure 1Cb,Cb1), while turning to a spindle-like shape after PAM treatment (Figure 1Cc,Cc1).  Figure 2Cc3,Cd3,Ce3). . Data from five randomly selected field of view (FOV) were analyzed using the ImageJ software to determine the intensity of CD86 and CD206. The results are presented as mean ± SD (*p < 0.05, one-way ANOVA).

| PAM increased the autophagic activity in M1 macrophages
Autophagic activity is represented by the quantity of autophagosomes, which can be stained with MDC. 21 PAM-treated M1 macrophages exhibited a higher level of intercellular autophagosomes than the M1 macrophages (Figure 3Ai). This was further supported by the higher intensity detected in PAM-stimulated M1 macrophages than that in M1 macrophages (Figure 3Aii). Western blot was used to detect the intercellular protein markers of autophagic activity, ATG5, and LC3II/LC3I. The relative band intensity demonstrated that both ATG5 and LC3II/LC3I levels in PAM-treated M1 macrophages were elevated ( Figure 3B). Spautin-1 (10 μM) was added to the PAMtreated M1 macrophages to deactivate autophagy, which led to a reduction in the expression of ATG5 and LC3II/LC3I ( Figure 3C).

| PAM inhibited the Akt signaling pathway in macrophages
One important mechanism that controls autophagic activity is the Akt signaling pathway, the activation status of which may be determined by calculating the ratio of p-Akt/Akt. 22

| DISCUSSION
Macrophage, belonging to innate immune system, is one of the first members response to infectious pathogens, implants, and biomaterials. 26,27 Recent research has focused on macrophages because of their regulatory property function, which is essential for tissue repair. 28 Indirectly promoting tissue regeneration via stimulating macrophages is a significant concept of drug discovery, therapy design, and biomaterial synthesis. 26 were found to be enriched ( Figure S1). Reactive nitrogen species (RNS) and reactive oxygen species (ROS) were found to be the most stable and bio-reactive compounds in PAM. 10,15 High levels of intercellular ROS may force cells to experience more oxidative stress, which lowers cell viability. 29 Interestingly, even at a concentration of  (Figure 1Cb,Cb1), which resembled the feature of a typical M1 macrophage subtype. 1,3,31 Morphologic change from a star-like shape to a spindle-like shape was observed after applying PAM to M1 macrophages (Figure 1Cc,Cc1), which is normally present by the M2 subtype. 31 It can be confirmed that, rather than affecting cell viability, PAM tends to cause macrophage subtype switching.
It is generally established that exogenous stimuli such as LPS and IFN-γ can polarize macrophages towards an M1 subtype. 32 The effi-   Different plasma treatment parameters and application scenarios, as shown in Table 2, may produce opposite outcomes. However, the above results confirmed that cold plasma can be a modulator of immune cell activation and can stimulate macrophage polarization through M1/M2 differentiation under various treatment conditions. The use of cold plasma in this project to investigate macrophage polarization in an inflammatory environment is therefore of significant scientific value.
The molecular mechanism involving cold plasma and macrophages may become a hot topic of future plasma medicine research.
The process of osteogenesis requires the participation of macrophages. 45  resorption. 52,53 The increasing expression of bone homeostasisrelated cytokines also suggests the potential of applying PAM in periodontal therapy. However, bacterial infection is the primary cause of periodontitis, and its defense requires the involvement of inflammatory cytokines. 5 It is possible that PAM inhibits macrophage-derived inflammatory cytokine secretion and may have a negative effect on bacterial resistance. In fact, the overexpression of tissue-damaging inflammatory cytokines indicates that the disadvantages outweigh the anti-bacterial benefits. 4,5 Other studies have demonstrated the antibacterial property of PAM, 30 which may increase the efficacy of periodontitis treatments. Because PAM is the liquid form of CAP, it can be used as a mouthwash to deliver charged particles, free radicals, electric fields, and metastable species to macrophages in the periodontium. In addition to inflammatory cytokines, macrophage is one of the major producers of exosomes, which contain regulatory factors and play an important role in tissue regeneration. 54,55 Close correlations exist between lysosomal-fusion autophagosomes and membrane-fusion exosomes. 56 Therefore, PAM may influence the release of macrophage-derived exosomes by enhancing autophagic activity, a phenomenon worthy of further investigation. As a potential periodontal therapy candidate, the safety of applying PAM orally should be carefully investigated. There were no observable changes in gene expressions in PDLCs treated with PAM ( Figure S3). Furthermore, clinical reports that are currently available showed no damage to oral mucosa when applying CAP orally for bacterial infection and oral cancer treatments. 12,13,57 Our study has demonstrated that PAM treatment did not affect the morphology and functions of PDLCs ( Figure S3), and caused no apoptosis to macrophages ( Figure 1B), suggesting that PAM is a safe anti-inflammatory therapeutic option for periodontitis.

| CONCLUSION
Plasma activation transforms the culture medium into a microenvironment replete with ROS and RNS. The ROS/RNS boosts autophagic activity by deactivating the Akt signaling pathway, which prevents macrophages from switching towards the proinflammatory M1 subtype. In addition to having anti-inflammatory properties, PAM also promotes the production of factors associated with osteogenesis and cementogenesis, making it an excellent candidate for periodontal therapeutic applications ( Figure 6). writingreview and editing (supporting). Yin Xiao: Funding acquisition (supporting); project administration (supporting); resources (supporting); supervision (supporting); writingreview and editing