Skin treatment with non-thermal plasma modulates the immune system through miR-223-3p and its target genes.

Non-thermal plasma, a partially ionized gas, holds significant potential for clinical applications, including wound-healing support, oral therapies, and anti-tumour treatments. While its applications showed promising outcomes, the underlying molecular mechanisms remain incompletely understood. We thus apply non-thermal plasma to mouse auricular skin and conducted non-coding RNA sequencing, as well as single-cell blood sequencing. In a time-series analysis (five timepoints spanning 2 hours), we compare the expression of microRNAs in the plasma-treated left ears to the unexposed right ears of the same mice as well as to the ears of unexposed control mice. Our findings indicate specific effects in the treated ears for a set of five miRNAs: mmu-miR-144-5p, mmu-miR-144-3p, mmu-miR-142a-5p, mmu-miR-223-3p, and mmu-miR-451a. Interestingly, mmu-miR-223-3p also exhibits an increase over time in the right non-treated ear of the exposed mice, suggesting systemic effects. Notably, this miRNA, along with mmu-miR-142a-5p and mmu-miR-144-3p, regulates genes and pathways associated with wound healing and tissue regeneration (namely ErbB, FoxO, Hippo, and PI3K-Akt signalling). This co-regulation is particularly remarkable considering the significant seed dissimilarities among the miRNAs. Finally, single-cell sequencing of PBMCs reveals the downregulation of 12 from 15 target genes in B-cells, Cd4+ and Cd8+ T-cells. Collectively, our data provide evidence for a systemic effect of non-thermal plasma.

Human coronavirus 229E inactivation on porous and nonporous materials using dielectric barrier discharge (DBD) plasma.

Viruses can spread through contaminated aerosols and contaminated surface materials, and effective disinfection techniques are essential for virus inactivation. Nonthermal plasma-generated reactive oxygen and nitrogen species can effectively inactivate the coronavirus. We aim to interpret the coronavirus inactivation level and mechanism of surface interaction with materials with and without dielectric barrier discharge (DBD) plasma treatment. Nonthermal plasma, particularly surface-type DBD plasma, can inactivate human coronavirus 229E (HCoV-229E) on porous (paper, wood, mask) and nonporous (plastic, stainless steel, glass, Cu) materials. Virus inactivation was analyzed using a 50% tissue culture infectivity dose (TCID50) using cell line, flow cytometry, and immunofluorescence. Surfaces contaminated with HCoV-229E were treated at different time intervals (0-5 h) with and without plasma exposure (natural decay in ambient air conditions). HCoV-229E persistence conformed to the following order: plastic > cover glass > stainless steel > mask > wood > paper > Cu with and without plasma exposure. HCoV-229E was more stable in plastic, cover glass, and stainless steel in 5 h, and the viable virus titer gradually decreased from its initial log10 order of 6.892 to 1.72, 1.53, and 1.32 TCID50/mL, respectively, under plasma exposure. No virus was observed in Cu after treatment for 5 h. The use of airflow, ambient nitrogen, and argon did not promote virus inactivation. Flow cytometry and immunofluorescence analysis demonstrated a low expression level of spike protein (fluorescence intensity) during plasma treatment and in E and M genes expression compared with the virus control.

Overcoming antibiotic resistance: non-thermal plasma and antibiotics combination inhibits important pathogens.

Antibiotic resistance (ATBR) is increasing every year as the overuse of antibiotics (ATBs) and the lack of newly emerging antimicrobial agents lead to an efficient pathogen escape from ATBs action. This trend is alarming and the World Health Organization warned in 2021 that ATBR could become the leading cause of death worldwide by 2050. The development of novel ATBs is not fast enough considering the situation, and alternative strategies are therefore urgently required. One such alternative may be the use of non-thermal plasma (NTP), a well-established antimicrobial agent actively used in a growing number of medical fields. Despite its efficiency, NTP alone is not always sufficient to completely eliminate pathogens. However, NTP combined with ATBs is more potent and evidence has been emerging over the last few years proving this is a robust and highly effective strategy to fight resistant pathogens. This minireview summarizes experimental research addressing the potential of the NTP-ATBs combination, particularly for inhibiting planktonic and biofilm growth and treating infections in mouse models caused by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa. The published studies highlight this combination as a promising solution to emerging ATBR, and further research is therefore highly desirable.

In vivo study on the repair of rat Achilles tendon injury treated with non-thermal atmospheric-pressure helium microplasma jet.

Non-thermal atmospheric-pressure plasma (NTAPP) has been widely studied for clinical applications, e.g., disinfection, wound healing, cancer therapy, hemostasis, and bone regeneration. It is being revealed that the physical and chemical actions of plasma have enabled these clinical applications. Based on our previous report regarding plasma-stimulated bone regeneration, this study focused on Achilles tendon repair by NTAPP. This is the first study to reveal that exposure to NTAPP can accelerate Achilles tendon repair using a well-established Achilles tendon injury rat model. Histological evaluation using the Stoll's and histological scores showed a significant improvement at 2 and 4 weeks, with type I collagen content being substantial at the early time point of 2 weeks post-surgery. Notably, the replacement of type III collagen with type I collagen occurred more frequently in the plasma-treated groups at the early stage of repair. Tensile strength test results showed that the maximum breaking strength in the plasma-treated group at two weeks was significantly higher than that in the untreated group. Overall, our results indicate that a single event of NTAPP treatment during the surgery can contribute to an early recovery of an injured tendon.

Modeling the growth of Aspergillus brasiliensis affected by a nonthermal plasma.

AIM: The main objective of the study was to develop and validate a model for the growth of Aspergillus brasiliensis on surfaces, specifically on agar culture medium. An additional aim was to determine conditions for complete growth inhibition of this micromycete using two different nonthermal plasma (NTP) sources. METHODS AND RESULTS: The developed model uses two key parameters, namely the growth rate and growth delay, which depend on the cultivation temperature and the amount of inoculum. These parameters well describe the growth of A. brasiliensis and the effect of NTP on it. For complete fungus inactivation, a single 10-minute exposure to a diffuse coplanar surface barrier discharge was sufficient, while a point-to-ring corona discharge required several repeated 10-minute exposures at 24-h intervals. CONCLUSIONS: The article presents a model for simulating the surface growth of A. brasiliensis and evaluates the effectiveness of two NTP sources in deactivating fungi on agar media.

Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro.

Epidermal melanin synthesis determines an individual's skin color. In humans, melanin is formed by melanocytes within the epidermis. The process of melanin synthesis strongly depends on a range of cellular factors, including the fine-tuned interplay with reactive oxygen species (ROS). In this context, a role of cold atmospheric plasma (CAP) on melanin synthesis was proposed due to its tunable ROS generation. Herein, the argon-driven plasma jet kINPen® MED was employed, and its impact on melanin synthesis was evaluated by comparison with known stimulants such as the phosphodiesterase inhibitor IBMX and UV radiation. Different available model systems were employed, and the melanin content of both cultured human melanocytes (in vitro) and full-thickness human skin biopsies (in situ) were analyzed. A histochemical method detected melanin in skin tissue. Cellular melanin was measured by NIR autofluorescence using flow cytometry, and a highly sensitive HPLC-MS method was applied, which enabled the differentiation of eu- and pheomelanin by their degradation products. The melanin content in full-thickness human skin biopsies increased after repeated CAP exposure, while there were only minor effects in cultured melanocytes compared to UV radiation and IBMX treatment. Based on these findings, CAP does not appear to be a useful option for treating skin pigmentation disorders. On the other hand, the risk of hyperpigmentation as an adverse effect of CAP application for wound healing or other dermatological diseases seems to be neglectable.

The Synthesis and Analysis of the Cytotoxicity of Al2O3-Supported Silver Nanoparticles Prepared by the Plasma Chemical Process Initiated by Pulsed MW Radiation in the Al2O3-Ag Powder Mixtures.

An original plasma chemical process initiated by microwave discharge in a mixture of metal and dielectric powders was applied to prepare specific materials, which consisted of microsized spherical particles of aluminum oxide covered with silver nanoparticles. The prepared materials are highly uniform in shape, size distribution, and composition. Their cytotoxicity was investigated using the human cell lines MCF7, HEK293T, A549, and VA-13 and the bacterial strains E. coli JW5503 (ΔtolC) and E. coli K12. Their cytotoxicity was found not to exceed the cytotoxicity of the starting materials. Thus, the prepared materials can be considered highly promising for catalysis and biotechnology applications.

Antimicrobial efficacy of direct air gas soft jet plasma for the in vitro reduction of oral bacterial biofilms.

The aim of this study was to evaluate the antimicrobial efficacy of an air gas soft jet CAP for its potential use in removing oral biofilms, given that plasma-based technologies have emerged as promising methods in periodontology. Two types of biofilms were developed, one by Streptococcus mutans UA 159 bacterial strain and the other by a complex mixture of saliva microorganisms isolated from a patient with periodontitis. This latter biofilm was characterized via Next Generation Sequencing to determine the main bacterial phyla. The CAP source was applied at a distance of 6 mm for different time points. A statistically significant reduction of both CFU count and XTT was already detected after 60 s of CAP treatment. CLSM analysis supported CAP effectiveness in killing the microorganisms inside the biofilm and in reducing the thickness of the biofilm matrix. Cytotoxicity tests demonstrated the possible use of CAP without important side effects towards human gingival fibroblasts cell line. The current study showed that CAP treatment was able to significantly reduce preformed biofilms developed by both S. mutans and microorganisms isolated by a saliva sample. Further studies should be conducted on biofilms developed by additional saliva donors to support the potential of this innovative strategy to counteract oral pathogens responsible for periodontal diseases.

Cold Atmospheric Pressure Plasma-Activated Medium Modulates Cellular Functions of Human Mesenchymal Stem/Stromal Cells In Vitro.

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

Non-invasive Physical Plasma Modulates Macrophage Polarization: A Potential Strategy for Tumor Microenvironment Remodeling.

BACKGROUND/AIM: Non-invasive physical plasma (NIPP) has shown promise in the treatment of cancer. However, conflicting results have been reported regarding the effect of NIPP on macrophage polarization. As tumor-associated macrophages (TAMs) are essential in the regulation of cancer development, this study aimed to determine the role of NIPP treatment in macrophage polarization and tumor-microenvironment (TME) remodeling. MATERIALS AND METHODS: A portable NIPP device, Plasma Care (Terraplasma Medical, Garching, Germany), was employed as the source of NIPP. The human monocytic cell line THP-1 was adopted as the cell model for macrophage differentiation and polarization. The effects of NIPP treatment on temperature, pH value, and oxidative stress induction of the culture medium were examined to validate the feasibility of applying the NIPP device in subsequent cell treatment. The changes in morphology, viability, and proliferation of THP-1 cells after NIPP treatment were determined. The expression of M1/M2 macrophage markers was examined by real-time quantitative polymerase chain reaction. RESULTS: No significant changes were observed in temperature and pH value after NIPP treatment, while the formation of hydrogen peroxide was promoted in a time-dependent manner. Cell morphology, viability, and proliferation were not affected by up to 6 minutes of NIPP treatment. In monocytes, 6 minutes of NIPP treatment significantly increased the expression of M1 markers (TNF-α and IL-6) and suppressed the M2 marker (CD206), findings which were consistent in the monocyte-derived macrophages. Furthermore, NIPP treatment also significantly promoted M1 polarization in the monocyte-derived macrophages induced by phorbol 12-myristate 13-acetate. CONCLUSION: NIPP is a safe and robust oxidative stress inducer and showed potential in TAM regulation by promoting M1 macrophage polarization.

Enhancing high-efficiency breeding and microbial microdroplet cultivation techniques for Ganoderma lucidum.

Ganoderma lucidum is known for its bioactive compounds, such as polysaccharides and triterpenoids, which are crucial in food and medicine. However, liquid fermentation encounters challenges in terms of strain differentiation and stability. In this research, we employed atmospheric room temperature plasma mutation and a microbial microdroplet culture system to identify strains with enhanced biomass and triterpenoid production. The three mutant strains, YB05, YB09, and YB18, exhibited accelerated growth rates and antagonized the initial strain G0023 more effectively than the controls. Notably, YB18 displayed the fastest growth, with a 17.25% increase in colony radius. Shake flask cultivation demonstrated that, compared with the initial strain, YB05 and YB18 had 26.33% and 17.85% greater biomass, respectively. Moreover, the triterpenoid production of YB05 and YB18 surpassed that of the control by 32.10% and 15.72%, respectively, as confirmed by colorimetric detection. Importantly, these mutant strains remained stable for five generations. This study revealed a comprehensive screening system utilizing atmospheric pressure, room temperature plasma mutation technology and microbial droplet cultivation. This innovative approach offers a promising pathway for obtaining advantageous Ganoderma strains for liquid fermentation. The methodology of atmospheric room temperature plasma mutation and microbial microdroplet culture systems is detailed for better comprehension.

Biocompatible plasma-treated liquids: A sustainable approach for decontaminating gastrointestinal-infection causing pathogens.

Nosocomial infections are a serious threat and difficult to cure due to rising antibiotic resistance in pathogens and biofilms. Direct exposure to cold atmospheric plasma (CAP) has been widely employed in numerous biological research endeavors. Nonetheless, plasma-treated liquids (PTLs) formulated with physiological solutions may offer additional benefits such as enhanced portability, and biocompatibility. Additionally, CAP-infused long-lived reactive oxygen and nitrogen species (RONS) such as nitrite (NO2-), nitrate (NO3-), and hydrogen peroxide (H2O2) can synergistically induce their antibacterial activity. Herein, we investigated those argon-plasma jet-treated liquids, including Ringer's lactate (RL), phosphate-buffered saline (PBS), and physiological saline, have significant antibacterial activity against nosocomial/gastrointestinal-causing pathogens, which might be due to ROS-mediated lipid peroxidation. Combining the conventional culture-based method with propidium iodide monoazide quantitative PCR (PMAxx™-qPCR) indicated that PTLs induce a minimal viable but non-culturable (VBNC) state and moderately affect culturable counts. Specifically, the PTL exposure resulted in pathogenicity dysfunction via controlling T3SS-related effector genes of S. enterica. Overall, this study provides insights into the effectiveness of PTLs for inducing ROS-mediated damage, controlling the virulence of diarrheagenic bacteria, and modulating homeostatic genes.

Comprehensive studies on the biological activities of human metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer cell lines, directly or combinedly treated using non-thermal plasma-based approaches.

Progressive incidence and a pessimistic survival rate of breast cancer in women worldwide remains one of the most concerning topics. Progressing research indicates a potentially high effectiveness of use cold atmospheric plasma (CAP) systems. The undoubted advantage seems its simplicity in combination with other anti-cancer modalities. Following observed trend of studies, one inventory CAP system was applied to directly treat human breast cancer cell lines and culturing in two different Plasma Activated Media (PAM) for combined utilization. Proposed CAP treatments on MCF-10 A, MCF-7, and MDA-MB-231 cell lines were studied in terms of impact on cell viability by MTT assay. Disturbances in cell motility following direct and combined CAP application were assessed by scratch test. Finally, the induction of apoptosis and necrosis was verified with annexin V and propidium iodide staining. Reactive species generated during CAP treatment were determined based on optical emission spectrometry analysis along with colorimetric methods to qualitatively assess the NO2-, NO3-, H2O2, and total ROS with free radicals concentration. The most effective approach for CAP utilization was combined treatment, leading to significant disruption in cell viability, motility and mostly apoptosis induction in breast cancer cell lines. Determined CAP dose allows for mild outcome, showing insignificant harm for the non-cancerous MCF-10 A cell line, while the highly aggressive MDA-MB-231 cell line shows the highest sensitivity on proposed CAP treatment. Direct CAP treatment seems to drive the cells into the sensitive state in which the effectiveness of PAM is boosted. Observed anti-cancer response of CAP treatment was mostly triggered by RNS (mostly NO2- ions) and ROS along with free radicals (such as H2O2, OH•, O2-•, 1O2, HO2•). The combined application of one CAP source represent a promising alternative in the development of new and effective modalities for breast cancer treatment.

Cold atmospheric plasma enhances morphological and biochemical attributes of tomato seedlings.

Cold atmospheric plasma (CAP) is a physical technology with notable effects on living organisms. In the present study, tomato seeds (Solanum lycopersicum var. Bassimo Mill.) were exposed to CAP for various time intervals, ranging from 1 to 5 min, in both continuous and intermittent periods, and were compared with a control group that received no CAP treatment. Seedlings grown from treated seeds exhibited improvements in levels of growth traits, photosynthetic pigments, and metabolite contents when compared to the control group. Seedlings from seeds treated with S04 displayed significant increases in shoot and root lengths, by 32.45% and 20.60% respectively, compared to the control group. Moreover, seedlings from seeds treated with S01 showed a 101.90% increase in total protein, whereas those treated with S02 experienced a 119.52% increase in carbohydrate content. These findings highlight the substantial improvements in growth characteristics, photosynthetic pigments, and metabolite levels in seedlings from treated seeds relative to controls. Total antioxidant capacity was boosted by CAP exposure. The activities of enzymes including superoxide dismutase, catalase, and peroxidases were stimulated by S02 and exceeded control treatment by (177.48%, 137.41%, and 103.32%), respectively. Additionally, exposure to S04 increased the levels of non-enzymatic antioxidants like flavonoids, phenolics, saponins, and tannins over the control group (38.08%, 30.10%, 117.19%, and 94.44%), respectively. Our results indicate that CAP-seed priming is an innovative and cost-effective approach to enhance the growth, bioactive components, and yield of tomato seedlings.

Predictive modeling of molds effective elimination by external inactivation sources.

Presented paper deals with a novel application of the (nonlinear) logistic equation to model an elimination of microscopic filaments types of fungi-molds from affected materials via different external inactivation techniques. It is shown that if the inactivation rate of the external source is greater than the maximum natural growth rate of mycelium, the mold colony becomes destroyed after a finite time. Otherwise, the mycelium may survive the external attack only at a sufficiently large initial concentration of the inoculum. Theoretically determined growth curves are compared with the experimental data for Aspergillus brasiliensis mold inactivated by using both cold atmospheric plasma (CAP) and UV-germicidal lamp. Model presented in the article may be applied also to other classes of microorganisms (e.g. bacteria).

Enhancing starch functionality through synergistic modification via sequential treatments with cold plasma and electron beam irradiation.

The impact of dual sequential modifications using radio-frequency (RF) plasma and electron beam irradiation (EBI) on starch properties was investigated and compared with single treatments within an irradiation dose range of 5-20 kGy. Regardless of sequence, dual treatments synergistically affected starch properties, increasing acidity, solubility, and paste clarity, while decreasing rheological features with increasing irradiation dose. The molecular weight distribution was also synergistically influenced. Amylopectin distribution broadened particularly below 10 kGy. Amylose narrowed its distribution across all irradiation doses. This was due to dominating EBI-induced degradation and molecular rearrangements from RF plasma. With the highest average radiation-chemical yield (G) and degradation rate constant (k) of (2.12 ± 0.14) × 10-6 mol·J-1 and (3.43 ± 0.23) × 10-4 kGy-1, respectively, upon RF plasma pre-treatment, amylose underwent random chain scission. In comparison to single treatments, dual modification caused minor alterations in spectral characteristics and crystal short-range order structure, along with increased granule aggregation and surface irregularities. The synergistic effect was dose-dependent, significant up to 10 kGy, irrespective of treatment sequence. The highest synergistic ratio was observed when RF plasma preceded irradiation, demonstrating the superior efficiency of plasma pre-treatment in combination with EBI. This synergy has the potential to lower costs and extend starch's technological uses by enhancing radiation sensitivity and reducing the irradiation dose.

Simulation and experimental study of a cold atmospheric pressure plasma and comparison of efficiency in boosting recombinant Endoglucanase II production in Pichia pastoris.

Recombinant proteins are essential in various industries, and scientists employ genetic engineering and synthetic biology to enhance the host cell's protein production capacity. Stress response pathways have been found effective in augmenting protein secretion. Cold atmospheric pressure plasma (CAP) can induce oxidative stress and enhance protein production. Previous studies have confirmed the applicability of CAP jets on Phytase and green fluorescent protein (GFP) production in Pichia pastoris hosts. This study investigates the effect of CAP treatment on another valuable recombinant protein, Endoglucanase II (EgII), integrated into the Pichia pastoris genome. The results demonstrated that plasma induction via two different ignition modes: sinusoidal alternating current (AC) and pulsed direct current (DC) for 120, 180, and 240 s has boosted protein secretion without affecting cell growth and viability. The AC-driven jet exhibited a higher percentage increase in secretion, up to 45%. Simulation of plasma function using COMSOL software provided a pattern of electron temperature (Te) and density distribution, which determine the plasma cocktail's chemistry and reactive species production. Furthermore, electron density (ne) and temperature were estimated from the recorded optical spectrum. The difference in electron properties may explain the moderately different impressions on expression capability. However, cell engineering to improve secretion often remains a trial-and-error approach, and improvements are, at least partially, specific to the protein produced.

The synergistic effect of dielectric barrier discharge plasma and phycocyanin on shelf life of Oncorhynchus mykiss rainbow fillets.

This study aimed to evaluate the efficacy of dielectric barrier discharge treatment (DBD) combined with phycocyanin pigment (PC) in extending the shelf life of Oncorhynchus mykiss rainbow fillets stored at 4 ± 0.1 °C. Microbiological, physicochemical, sensory and antioxidant properties were assessed over an 18-day storage period. The combined DBD and PC treatment significantly inhibited total viable counts and Psychrotrophic bacteria counts compared to the rest of the samples throughout storage. While Total Volatile Nitrogen concentrations remained below international standard until day 18, they exceeded this threshold in control sample by day 9. DBD treatment notably reduced Trimethylamine levels compared to controls (p < 0.05). PC and DBD combined inhibited DPPH and ABTS radical scavenging capacities by 80% and 85%, respectively, while demonstrating heightened iron-reducing antioxidant activity compared to controls. Analysis of 24 fatty acids indicated that PC mitigated DBD's adverse effects, yielding superior outcomes compared to controls. The ratio of n-3 to n-6 fatty acids in all samples met or fell below international standard. Thus, the combined use of DBD and PC shows promise in extending fillet shelf life by over 15 days at 4 °C.

Fracture healing on non-union fracture model promoted by non-thermal atmospheric-pressure plasma.

Non-thermal atmospheric-pressure plasma (NTAPP) is attracting widespread interest for use in medical applications. The tissue repair capacity of NTAPP has been reported in various fields; however, little is known about its effect on fracture healing. Non-union or delayed union after a fracture is a clinical challenge. In this study, we aimed to investigate how NTAPP irradiation promotes fracture healing in a non-union fracture model and its underlying mechanism, in vitro and in vivo. For the in vivo study, we created normal and non-union fracture models in LEW/SsNSlc rats to investigate the effects of NTAPP. To create a fracture, a transverse osteotomy was performed in the middle of the femoral shaft. To induce the non-union fracture model, the periosteum surrounding the fracture site was cauterized after a normal fracture model was created. The normal fracture model showed no significant difference in bone healing between the control and NTAPP-treated groups. The non-union fracture model demonstrated that the NTAPP-treated group showed consistent improvement in fracture healing. Histological and biomechanical assessments confirmed the fracture healing. The in vitro study using pre-osteoblastic MC3T3-E1 cells demonstrated that NTAPP irradiation under specific conditions did not reduce cell proliferation but did enhance osteoblastic differentiation. Overall, these results suggest that NTAPP is a novel approach to the treatment of bone fractures.

How does atmospheric pressure cold helium plasma affect the biomechanical behaviour on alkali-lesioned corneas?

BACKGROUND: Melting corneal ulcers are a serious condition that affects a great number of animals and people around the world and it is characterised by a progressive weakening of the tissue leading to possible severe ophthalmic complications, such as visual impairment or blindness. This disease is routinely treated with medical therapy and keratoplasty, and recently also with alternative regenerative therapies, such as cross-linking, amniotic membrane transplant, and laser. Plasma medicine is another recent example of regenerative treatment that showed promising results in reducing the microbial load of corneal tissue together with maintaining its cellular vitality. Since the effect of helium plasma application on corneal mechanical viscoelasticity has not yet been investigated, the aim of this study is first to evaluate it on ex vivo porcine corneas for different exposition times and then to compare the results with previous data on cross-linking treatment. RESULTS: 94 ex vivo porcine corneas divided into 16 populations (healthy or injured, fresh or cultured and treated or not with plasma or cross-linking) were analysed. For each population, a biomechanical analysis was performed by uniaxial stress-relaxation tests, and a statistical analysis was carried out considering the characteristic mechanical parameters. In terms of equilibrium normalised stress, no statistically significant difference resulted when the healthy corneas were compared with lesioned plasma-treated ones, independently of treatment time, contrary to what was obtained about the cross-linking treated corneas which exhibited more intense relaxation phenomena. CONCLUSIONS: In this study, the influence of the Helium plasma treatment was observed on the viscoelasticity of porcine corneas ex vivo, by restoring in lesioned tissue a degree of relaxation similar to the one of the native tissue, even after only 2 min of application. Therefore, the obtained results suggest that plasma treatment is a promising new regenerative ophthalmic therapy for melting corneal ulcers, laying the groundwork for further studies to correlate the mechanical findings with corneal histology and ultrastructural anatomy after plasma treatment.