Non-thermal plasma accelerates the healing process of peripheral nerve crush injury in rats

Choi H., Kim H., Lee J., Joo J.

Background/Objectives: As non-thermal atmospheric pressure plasma (NTP) is known to have advantages in application in the medical field, we consider its applicability to periodontitis, a representative chronic inflammatory disease. The purpose of this study was to evaluate the effect of NTP in inhibiting the progression of periodontitis in a rat model when additionally used in scaling and root planing (SRP). Methods: To induce experimental periodontitis in 20 rats, ligatures were placed in the maxillary second molar and lipopolysaccharide from Porphyromonas gingivalis was injected around the teeth. Then, NTP treatment was performed for 2 or 5 min, together with scaling and root planing (SRP). To evaluate alveolar bone loss, micro-computed tomography (micro-CT) analysis and hematoxylin–eosin (H-E) staining were performed. Tartrate-resistant acid phosphatase (TRAP) analysis was performed to compare the number of osteoclasts, while immunohistochemistry (IHC) analysis was performed to determine the expression levels of receptor activator of nuclear factor-𝜅B ligand (RANKL) and osteoprotegerin (OPG). Enzyme-linked immunosorbent assay (ELISA) analysis was performed for the detection of cytokines (TNF-α, IL-1β, and IL-10) in tissues and sera. Results: When SRP was combined with NTP, alveolar bone loss was decreased, the number of osteoclasts and RANKL expression were decreased, OPG expression was increased, and pro-inflammatory cytokine (TNF-α and IL-1β) levels were significantly decreased. Compared with the NTP treatment for 2 min, when treated for 5 min, less alveolar bone loss, fewer osteoclasts, a lower RANKL expression level, and a higher OPG expression level were observed. Conclusions: This study evaluated the adjunctive treatment effect of NTP in periodontitis-induced rats. Based on the results of this study, we suggest that supplemental NTP treatment may be a good option for non-surgical periodontal treatment; however, further studies are needed to elucidate the mechanism through which NTP suppresses periodontal inflammation.

Gebril S.M., Lashein F.E., Khalaf M., AbuAmra E.E., El-Hossary F.M.

Betancourt-Ángeles M., López-Callejas R., Berrones-Stringel G., Jaramillo-Martínez C., Navarro-Luna B., Rodríguez-Méndez B.G., Mercado-Cabrera A., Valencia-Alvarado R.

Anastomotic leaks remain a significant challenge in intestinal surgery, often leading to severe complications. This study investigated a novel approach to enhance anastomotic healing and reduce the risk of leaks by combining traditional suturing and stapling techniques with non-thermal atmospheric pressure plasma (NTAPP) application. NTAPP, a cold atmospheric plasma generated through the ionization of ambient air, has been shown to possess antimicrobial, hemostatic, and wound-healing properties. NTAPP promotes sterilization, coagulation, and tissue regeneration by generating reactive oxygen and nitrogen species, potentially strengthening anastomotic union. This pilot study evaluated the efficacy of NTAPP in three patients undergoing intestinal anastomosis. Following the standard surgical procedure, NTAPP was applied directly to the anastomotic site. Postoperative outcomes were monitored for six months, including anastomotic leaks and healing rates. Preliminary results demonstrated promising outcomes. All three patients exhibited successful sealing of the anastomosis, with no evidence of leakage during the follow-up period, providing reassurance and confidence in the potential of sutures, staples, and NTAPP. These findings suggest that NTAPP can significantly improve the safety and efficacy of intestinal surgeries by reducing the incidence of anastomotic leaks. While further research with a larger sample is necessary to confirm these initial findings, the results of this study provide a strong foundation for exploring the potential of NTAPP as a valuable adjunct to conventional surgical techniques for preventing anastomotic leaks. This innovative approach could reduce postoperative complications, improve patient outcomes, and enhance the overall quality of care in intestinal surgery.

Bozkurt A.S., Yılmaz Ş.G., Kaplan D.S., Bal R.

Exosomes (Exos) are candidates for functional recovery and regeneration following sciatic nerve crushed (SNC) injury due to their composition which can accelerate tissue regeneration. Therefore, mouse embryonic fibroblast-derived exosomes were evaluated for their regenerative capacity in SNC injury. In the study, 40 Balb/c males (20 ± 5 g) and two pregnant mice (for embryonic fibroblast tissue) were used and crushed injury was induced in the left sciatic nerve with an aneurysm clamp. Sciatic nerve model mice were randomly divided into 5 groups (n = 8; control, n = 8; sham, n = 8; SNC, n = 8; Mouse embryonic fibroblast exosome (mExo), n = 8; SNC + Mouse embryonic fibroblast exosome (SNC + mExo). Rotarod tests for motor functions and hot plate and von Frey tests for sensory functions were analyzed in the groups. Expression changes of exosome genes (RARRES1, NAGS, HOXA13, and MEIS1) immunohistochemical analysis of these gene proteins, and structural exosome NF-200 and S100 proteins were evaluated by confocal microscopy. Behavioral analyses showed that the damage in SNC was significant between groups on day14 and day28 (P < 0.05). In behavioral analyses, it was determined that motor functions and mechanical sensitivity lost in SNC were regained after mExo treatment. While expression of all genes was detected in MEF-derived exosomes, the high expression was MESI1 and the low expression was HOXA13. NF200, an indicator of axon number and neurofilament density, was found to decrease in SNC (P < 0.001) and increase after treatment, but not significantly. The decreased S100 protein levels in SNC and the increase detected after treatment were not significant. The expression of four mRNAs in mExos indicates that these genes may have an effect on regenerative processes after SNC injury. The regenerative process supported by tissue protein expressions demonstrates the therapeutic potential of mExo treatment.

de Moura C.E., Francelino L.E., da Silva G.R., Júnior C.A., Façanha D.A., Nunes T.L., de Paula V.V.

Cold atmospheric plasma (CAP) has been employed as a therapy against both acute and chronic skin lesions, contaminated or not, and has effects on angiogenesis and reepithelialization promoting healing. In this context, the present study aimed to evaluate the effects of a CAP jet associated with pharmacological treatment described by the 2015 AAHA/AAFP pain management guidelines and the 2022 WSAVA guidelines for the recognition, assessment, and treatment of pain, on the healing of chronic skin lesions caused by a pruritic reaction resulting from post-surgical neuropathic pain. To this end, a single CAP application was performed on a feline patient with a 6 months old recurrent contaminated cervical skin lesions along with administration of ketamine (10 µg/kg/min) following the prescription of prednisone (1 mg/kg, SID, 6 days), gabapentin (8 mg/kg, BID, 60 days) and amitriptyline (0.5 mg /kg, SID, 60 days). A single application of plasma associated with an NMDA antagonist, anti-inflammatory steroid, tricyclic antidepressant and gabapentinoid thus provided a significant improvement in the macroscopic appearance of the lesion within 10 days, and the owner reported the cessation of intense itching within the first four hours after treatment and a consequent improvement in the animal's quality of life. The medical treatment was finished almost a year since the writing of this paper, without clinical or reported recurrent signs of the condition. Therefore, we observed that single dose CAP application associated with ketamine, gabapentin, amitriptyline and prednisone leads to significant healing of chronically infected skin lesions resulting from post-surgical neuropathic pain.

de Moura C.E., Francelino L.E., Silva G.R., de Paula V.V., Nunes T.L., Façanha D.A., Júnior C.A.

Abstract Cold atmospheric plasma (CAP) has been employed as a therapy against both acute and chronic skin lesions, contaminated or not, and indirectly exerts an influence on adjacent tissues, promoting healing and other benefits, such as nervous tissue repair and differentiation. In this context, this pilot study aimed to evaluate the effects of a CAP jet on the healing of chronic skin lesions caused by a pruritic reaction resulting from post-surgical neuropathic pain. To this end, a single CAP application was performed on a feline patient with recurrent cervical skin lesions associated with specific drug therapy aiming at resolving neuropathic pain. The single plasma application led to significant macroscopic lesion appearance improvement within 10 days, and the owner reported the cessation of intense itching within the first four hours after treatment and a consequent improvement in the animal's quality of life. Therefore, CAP application leads to significant healing of chronically infected skin lesions resulting from post-surgical neuropathic pain.

Elhessy H.M., Habotta O.A., Eldesoqui M., Elsaed W.M., Soliman M.F., Sewilam H.M., Elhassan Y.H., Lashine N.H.

BackgroundThe majority of the suggested experimental modalities for peripheral nerve injury (PNI) result in varying degrees of recovery in animal models; however, there are not many reliable clinical pharmacological treatment models available. To alleviate PNI complications, research on approaches to accelerate peripheral nerve regeneration is encouraged. Cerebrolysin, dexamethasone, and ascorbic acid (vitamin C) drug models were selected in our study because of their reported curative effects of different mechanisms of action.MethodologyA total of 40 adult male albino rats were used in this study. Sciatic nerve crush injury was induced in 32 rats, which were divided equally into four groups (model, Cerebrolysin, dexamethasone, and vitamin C groups) and compared to the sham group (n = 8). The sciatic nerve sensory and motor function regeneration after crushing together with gastrocnemius muscle histopathological changes were evaluated by the sciatic function index, the hot plate test, gastrocnemius muscle mass ratio, and immune expression of S100 and apoptosis cascade (BAX, BCL2, and BAX/BCL2 ratio).ResultsSignificant improvement of the behavioral status and histopathological assessment scores occurred after the use of Cerebrolysin (as a neurotrophic factor), dexamethasone (as an anti-inflammatory), and vitamin C (as an antioxidant). Despite these seemingly concomitant, robust behavioral and pathological changes, vitamin C appeared to have the best results among the three main outcome measures. There was a positive correlation between motor and sensory improvement and also between behavioral and histopathological changes, boosting the effectiveness, and implication of the sciatic function index as a mirror for changes occurring on the tissue level.ConclusionVitamin C is a promising therapeutic in the treatment of PNI. The sciatic function index (SFI) test is a reliable accurate method for assessing sciatic nerve integrity after both partial disruption and regrowth.

Shaw P., Vanraes P., Kumar N., Bogaerts A.

Cold atmospheric plasma and nanomedicine originally emerged as individual domains, but are increasingly applied in combination with each other. Most research is performed in the context of cancer treatment, with only little focus yet on the possible synergies. Many questions remain on the potential of this promising hybrid technology, particularly regarding regenerative medicine and tissue engineering. In this perspective article, we therefore start from the fundamental mechanisms in the individual technologies, in order to envision possible synergies for wound healing and tissue recovery, as well as research strategies to discover and optimize them. Among these strategies, we demonstrate how cold plasmas and nanomaterials can enhance each other’s strengths and overcome each other’s limitations. The parallels with cancer research, biotechnology and plasma surface modification further serve as inspiration for the envisioned synergies in tissue regeneration. The discovery and optimization of synergies may also be realized based on a profound understanding of the underlying redox- and field-related biological processes. Finally, we emphasize the toxicity concerns in plasma and nanomedicine, which may be partly remediated by their combination, but also partly amplified. A widespread use of standardized protocols and materials is therefore strongly recommended, to ensure both a fast and safe clinical implementation.

Do C.B., Jaiswal M.S., Jang Y., Kim U., Kim G., Hwang D.

Among the various methods, Non Thermal Plasma (NTP) has been recently introduced and is being studied to recover the damaged nerve. In the recent years, several studies have suggested that NTP accelerates nerve cell regeneration, but the mechanism remains unknown. This study evaluated the effect of NTP on neuronal proliferation in SH-SY5Y (Human neuroblastoma cells) cells differentiated by retinoic acid (RA) and investigated the mechanism by which NTP promotes cell proliferation. We analyzed the morphology of differentiated SH-SY5Y cells, and performed western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Immunofluorescence analysis was performed in an in vivo study by categorizing Wistar A rats into three groups: non-nerve damage (Non-ND), nerve damage (ND), and nerve damage + NTP treatment (ND + NTP). The cell morphology analysis revealed that the number of cells increased and axonal elongation progressed after NTP treatment. In addition, western blots indicated that tau expression increased significantly after NTP treatment. The RT-PCR results revealed that the expression of tau, wnt3a, and β-catenin increased after NTP treatment. The in vivo immunofluorescence assay showed that NTP increased the markers for tau and S100B while regulating the over-expression of MAP2 and GAP43. NTP treatment accelerated cell proliferation and regeneration of damaged neurons in differentiated SH-SY5Y cells. These results establish the fact of NTP as a noninvasive and effective treatment for nerve injury.

Choi J., Gu H., Park K., Hwang D., Kim G.

Oral squamous cell cancer (OSCC) is the most common type of oral cancer (about 80–90% of cases) and various research is being done to cure the disease. This paper aims to verify whether treatment with no-ozone cold plasma (NCP), which is designed for safe usage of the plasma on oral cavities, in combination with gold nanoparticles conjugated with p-FAK antibody (p-FAK/GNP) can trigger the selective and instant killing of SCC-25 cells both in vitro and in vivo. When SCC25 and HaCaT cells are exposed to p-FAK/GNP+NCP, the instant cell death was observed only in SCC25 cells. Such p-FAK/GNP+NCP-mediated cell death was observed only when NCP was directly treated on SCC25 harboring p-FAK/GNP. During NCP treatment, the removal of charged particles from NCP using grounded electric mesh radically decreased the p-FAK/GNP+NCP-mediated cell death. This p-FAK/GNP+NCP-mediated selective cell death of OSCC was also observed in mice xenograft models using SCC25 cells. The mere treatment of p-FAK/GNP and NCP on the xenograft tumor slowly decreased the size of the tumor, and only about 50% of the tumor remained at the end of the experiment. On the other hand, 1 week of p-FAK/GNP+NCP treatment was enough to reduce half of the tumor size, and most of tumor tissue had vanished at the end. An analysis of isolated tissues showed that in the case of individual treatment with p-FAK/GNP or NCP, the cancer cell population was reduced due to apoptotic cell death. However, in the case of p-FAK/GNP+NCP, apoptotic cell death was unobserved, and most tissues were composed of collagen. Thus, this paper suggests the possibility of p-FAK/GNP+NCP as a new method for treating OSCC.

Liu Z., Tong H., Li J., Wang L., Fan X., Song H., Yang M., Wang H., Jiang X., Zhou X., Yuan H., Wang Y.

A hydrogel system loaded with mesenchymal stem cell–derived exosome (MSC-Exos) is an attractive new tool for tissue regeneration. However, the effect of the stiffness of exosome-loaded hydrogels on tissue regeneration is unclear. Here, the role of exosome-loaded hydrogel stiffness, during the regeneration of injured nerves, was assessed in vivo. The results showed that the photocrosslinkable hyaluronic acid methacrylate hydrogel stiffness plays an important role in repairing nerve injury. Compared with the stiff hydrogels loaded with exosomes, soft hydrogels loaded with exosomes showed better repair of injured peripheral nerves. The soft hydrogel promoted nerve repair by quickly releasing exosomes to inhibit the infiltration of macrophages and the expression of the proinflammatory factors IL-1β and TNF-α in injured nerves. Our work revealed that exosome-loaded hydrogel stiffness plays an important role in tissue regeneration by regulating exosome release behavior and provided important clues for the clinical application of biological scaffold materials.

Park N., Yun S., Lee H., Lee H.J., Choi J., Kim G.

To apply the sterilisation effect of low-temperature plasma to the oral cavity, the issue of ozone from plasma must be addressed. In this study, a new technology for generating cold plasma with almost no ozone is developed and is named Nozone (no-ozone) Cold Plasma (NCP) technology. The antimicrobial efficacy of the NCP against four oral pathogens is tested, and its specific mechanism is elucidated. The treatment of NCP on oral pathogenic microbes on a solid medium generated a growth inhibition zone. When NCP is applied to oral pathogens in a liquid medium, the growth of microbes decreased by more than 105 colony forming units, and the bactericidal effect of NCP remained after the installation of dental tips. The bactericidal effect of NCP in the liquid medium is due to the increase in hydrogen peroxide levels in the medium. However, the bactericidal effect of NCP in the solid medium depends on the charged elements of the NCP. Furthermore, the surface bactericidal efficiency of the dental-tip-installed NCP is proportional to the pore size of the tips and inversely proportional to the length of the tips. Overall, we expect this NCP device to be widely used in dentistry in the near future.

Lee S., Jang Y., Kim U., Kim H., Ryu M., Kim G., Hwang D.

This experimental research aimed to investigate the effects of non-thermal plasma on nerve regeneration after transected nerve damage using the sciatic nerve in Wistar albino (A) rats. The experiments were performed on 27 Wistar A rats. The rats underwent surgery for right sciatic nerve exposure and were divided into three groups (each group, n = 9) according to sciatic nerve transected injury (SNTI) and non-thermal plasma application: a non-nerve damage (non-ND) group, a only nerve damage without non-thermal plasma application (ND) group, and a nerve damage with non-thermal plasma application (ND + NTP) group. Subsequent to SNTI and immediate suture, non-thermal plasma was administered three times per week for eight weeks. Evaluation for functional recovery was performed using the static sciatic index measured over the full treatment period of eight weeks. The sciatic nerve specimens were obtained after euthanasia and third day from the last non-thermal plasma application. The sciatic nerve tissues were subjected to histological analysis. Behavior analysis presented that the ND + NTP group showed improved static sciatic index compared with the nerve damage group. Histopathological findings demonstrated that the ND + NTP group had more dense Schwann cells and well-established continuity of nerve fibers, greater than the nerve damage group. Immunohistochemistry showed that the ND + NTP group had increased levels of markers for microtubule-associated protein 2 (MAP2), tau, S100 calcium-binding protein B, and neurofilament-200 and regulated the overexpression of CD68 and MAP2. These results indicated that non-thermal plasma enhanced the motor function and restored the neuronal structure by accelerating myelination and axonal regeneration. Additionally, non-thermal plasma was confirmed to have a positive effect on the recovery of SNTI in rats.