Inhibitory effects and mechanisms of low-temperature plasma on hypertrophic scar

Hypertrophic scars (HSs) result from an excessive healing response to deep skin trauma, characterized by the excessive proliferation of fibroblasts and deposition of an extracellular matrix. Clinical presentation typically involves a raised, thickened lesion with hardness and is often accompanied by symptoms such as itching, swelling, and pain,^1–3 which can significantly impact patients’ physical and mental well-being. Currently, surgical resection is the preferred treatment for HSs despite the risk of recurrent pathological scars. Alternative therapies, including laser therapy, pressure therapy, and cryotherapy, have not produced satisfactory outcomes. Additionally, the prevailing therapeutic medications are mainly hormonal and can cause various adverse effects.⁴ Therefore, new methods of treating scarring have been explored, one of which is the low-temperature plasma (LTP) technology.

Plasma is a versatile source of active ingredients, encompassing ultraviolet (UV) radiation; charged particles, such as electrons and both positive and negative ions; as well as chemically active particles, such as ROS and RNS. LTP has been found to disrupt bacteria cell walls and induce DNA damage, resulting in inactivation of pathogens. Proper dosing of plasma treatment promotes the migration and proliferation of cells, induces relevant cytokines, increases inflammatory factors, enhances angiogenesis, and improves wound healing. However, administering higher doses of treatment may result in cytolethal effects. Due to its unique properties, LTP has been utilized in various fields, such as infections, wound healing, blood coagulation, stomatology, and cancer. Plasma medicine, an emerging and interdisciplinary field, has already developed as a new innovative approach for biomedical and clinical applications.^5–7 

In recent years, the efficacy of plasmas in the treatment of scarring has been found through several clinical studies. Through experimentation with cells, some scholars have proposed the opposite effects of LTP on cell migration and collagen production in keloid and normal fibroblasts, which determined that the inhibitory mechanism of low-temperature plasmas on keloid is mediated by regulation of signal pathways. This study revealed that LTP suppressed keloid cell migration via down-regulation of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription-3 (STAT3) and reduced collagen production via suppressing transforming growth factor-β (TGF-β). These findings implied that LTP has a therapeutic potential to intervene in keloids.⁸ On this basis, a study established an acute rat wound model and treated the wounds with low-temperature plasma jets. It showed that the scar width for the LTP group was not only smaller but also had superior re-epithelialization than the control group. Meanwhile, LTP reduced the TGF β1/Smad2/Smad3 signal pathway and regulated the levels of α-SMA and type I collagen in scar tissue, again suggesting a correlation between LTP and scar formation.⁹ 

However, there are limited experimental studies on the application of low-temperature plasmas to inhibit hypertrophic scar (HS) formation. Therefore, we conducted a study to investigate the efficacy of LTP in preventing HS formation in a rabbit ear scar animal model.^10–12 Based on histological observation and quantitative analysis of immunohistochemistry, we have concluded that the effects of plasma on HS tissue are similar to those of the positive drug, salvianolic acid B (SAB). Our findings provide a novel perspective on the treatment of hypertrophic scars.

The study analyzed the surface morphology of scars on rabbit ears at specific intervals after modeling and treatment administration. Measurements with a vernier caliper determined the thickness of scars and adjacent normal ear tissue. The difference between the two measurements was calculated to determine the trend in scar proliferation over time.

As shown in Fig. 1, the surface of the scar in the model group remained red and swollen for six weeks, with a protrusion in the middle. After six weeks of treatment, the scar in the positive drug group became less red and swollen, with a gradual decrease in the height of the protrusion and a softer texture compared to the model group. After undergoing LTP treatment, there was a noticeable improvement in the redness and swelling of the scar surface, and the protrusion was less apparent. The scar also became softer, indicating that LTP effectively hinders scar proliferation.

As shown in Fig. 2, both SAB gel and LTP exhibited significant inhibitory effects on the expression of TGF-β1 (P < 0.05) and Smad3 (P < 0.0001), suggesting that the inhibitory effect of low-temperature plasmas on hyperplastic scars was related to the TGF-β/Smad signaling pathway.

The hematoxylin-eosin staining results of HS tissue sections for each group are depicted in Fig. 3. A significant increase in the fibroblast count was observed in the model group compared to the blank group. Additionally, the collagen fiber density was higher, forming swirls and nodules, with a large amount of vascular proliferation observed in-between.

Conversely, the positive drug group showed a notable reduction in the fibroblast count and collagen density in comparison to the control group. Although the organization of collagen was still somewhat anomalous compared to the healthy tissue, it was relatively loose compared to the model group. In the LTP group, collagen in the nearby uppermost layer was more compact and irregularly structured, while the amount of fibroblasts in deeper tissue was higher, but their placement was more relaxed and regular.

The Masson staining results for each group’s HS tissue sections are displayed in Fig. 4. The model group exhibited significant microvascular hyperplasia and collagen accumulation in the dermis. Collagen fibers situated near the shallow surface displayed disordered arrangements, often appearing in a vortex pattern, while those near the deep layer were relatively regular.

In contrast, the positive drug group displayed a reduced proportion of blue staining in the area, along with a more consistent pattern of collagen fibers aligned almost parallel to the epidermis. Furthermore, collagen fibers located toward the middle and deeper segments of the dermis exhibited a loosely arranged configuration. Conversely, the LTP group evidenced collagen fibers that were relatively disordered in the vicinity of the surface, while gaps appeared between fibers located nearer the deeper layer, which were relatively evenly arranged.

According to Table I and Fig. 5, it can be seen that the density of fibroblasts in the model group was significantly increased (P < 0.0001) compared with the blank group. Remarkably, both SAB gel and LTP treatments resulted in a statistically significant decrease (P < 0.05) in fibroblast density.

The results are shown in Table II and Fig. 6. Compared with the model group, the HI values in the sections of the positive drug group and the plasma group were significantly reduced, while the results were statistically different (P < 0.05).

The immunohistochemical sections were photographed under a microscope with a magnification of 100×. A positive expression was determined based on the presence of brownish-yellow particles or streaks within the cytoplasm. The results are shown in Fig. 7. The immunohistochemistry pictures of each field of view were grayed out using Image J software with an 8-bit conversion. The threshold value for optical density was adjusted and calibrated in each picture to determine the percentage of immunohistochemistry-positive expression area. The resulting data were then analyzed statistically.

The percentages of the positive expression area for Col I and α-SMA are presented in Table III and Fig. 8. In comparison to the model group, a significant difference (P < 0.0001) was observed in the positive expression of Col I and α-SMA in the blank group, positive drug group, and plasma group. Notably, the results of the positive drug group and the LTP group did not significantly differ from those of the blank group. These findings provide evidence supporting the down-regulatory effect of LTP on the expression of Col I and α-SMA, ultimately contributing to the inhibition of hypertrophic scars.

Previous studies^13,14 found the inhibitory potential of salvianolic acid B (SAB) on hypertrophic scars (HSs). Therefore, SAB was selected as the positive control drug in this animal experiment. Notably, SAB represents the principal water-soluble constituent in Salvia miltiorrhiza, a traditional Chinese medicine used for promoting blood circulation and removing blood stasis. Furthermore, SAB has demonstrated significant therapeutic efficacy in treating fibrotic diseases of the heart, lungs, and kidneys. It has been discovered that the antifibrotic mechanism of SAB is related to several signaling pathways, such as TGF/Smads. It can down-regulate the expression of key cellular components, such as type I collagen (Col I), matrix metalloproteinase-2 (MMP-2), and α-smooth muscle actin (α-SMA).¹ This study used SAB as a reference to visually evaluate the impact of LTP on the scar tissue.

In this experiment, a rabbit ear model was established to evaluate the morphology of scarred surfaces after six weeks of treatment with SAB and LTP. The findings revealed that both treatments had comparable efficacy for scar inhibition. Furthermore, the scar size of the treated groups was smaller compared to the control group, and redness and swelling were significantly reduced. Moreover, the hypertrophic index values were also decreased. These results suggest that LTP can effectively prevent scar proliferation. To clarify the underlying mechanism, immunohistochemistry quantitative analysis and staining approaches to detect and estimate collagen levels were performed for further analysis of the effect of LTP on the inhibiting of scar formation.

Numerous studies have demonstrated that transforming growth factor-beta (TGF-β) plays a critical role in the formation of HS. TGF-β is a versatile cytokine that governs cell proliferation, differentiation, and extracellular connective tissue biosynthesis. It is involved in all wound healing processes and predominantly operates through distinctive signal pathways, such as TGF-β/Smads. Of these, TGF-β1 is the most potent profibrotic factor and the cytokine is most strongly linked to the development of HS. Smads, an intracellular kinase substrate of the TGF-β1 receptor, transmits the signal for TGF-β1 interaction with the receptor from the cytoplasm to the nucleus. This process regulates the initiation of fibroblast proliferation and division, resulting in the synthesis and over-deposition of collagen in the extracellular matrix. Consequently, fibrosis of the traumatized tissue occurs, leading to the formation of HS.^15–17 Therefore, the TGF-β1/Smad pathway is considered as one of the most important signaling pathways in scar formation. The study’s serological results demonstrate that the low-temperature plasma has the capacity to decrease the expression levels of TGF-β1 and Smad3 in HS. Furthermore, Masson’s trichrome staining confirms the regular alignment of the collagen fibers in both the SAB and LTP groups, in comparison to the model group. Meanwhile, immunohistochemical results showed that type I collagen was downregulated after LTP treatment. Furthermore, the results of the HE staining indicate a decrease in fibroblast density following treatment with either SAB gel or LTP. These observations suggest that plasmas may have the capability to impede collagen deposition and fibroblast proliferation in HSs via the TGF-β/Smad signaling pathway, ultimately enhancing skin scar appearance.

The cytoskeletal protein α-SMA is a defining component of the contractile function of muscle fibroblasts. In the formation of HS, fibroblasts undergo activation and differentiation into myofibroblasts, leading to an overproduction of α-SMA and consequent excessive tissue spasm and secretion of an extracellular matrix, which stands as an integral indicator of the degree of fibrosis in HSs.^18–20 It was concluded that immunohistochemistry only revealed a positive α-SMA expression in blood vessels in normal tissues, with almost no expression in fibroblasts. However, α-SMA was found extensively expressed in fibroblasts in nodular areas of the HS tissue. These findings are consistent with the α-SMA immunohistochemistry results obtained from the model group.²¹ In this investigation, the immunohistochemical results of the SAB gel and LTP groups revealed a decrease in the region of the α-SMA positive expression in relation to the model group. This suggests that both SAB and LTP may be effective in treating HSs by suppressing the expression of α-SMA.

Fibroblasts secrete MMP-2, which selectively degrades collagen fibers.^22,23 Our study measured MMP-2 levels in rabbit serum and found a significant increase in the LTP group compared to the model group. This increase can be attributed to reactive oxygen species (ROS), the active component of a plasma that mediates MMP-2 secretion and activation at high concentrations. These results suggest that elevated levels of MMP-2 in the LTP group can be a result of ROS-mediated activation.²⁴ 

Moreover, in this experiment, a combination treatment group was established, consisting of SAB gel and LTP. However, there was no significant variation in the expression levels of each cytokine measured in the serum analysis compared to the other two individual treatment groups. Possible mechanisms to justify the plasma-mediated enhancement of drug penetration have been proposed. Several studies suggest that plasma treatment may decrease the E-cadherin expression in keratinocytes, leading to the creation of temporary gaps between epidermal cells, known as “pore formation.” This process potentially allows drugs to bypass the keratin barrier and penetrate the cells.²⁵ During the experiment, SAB was utilized as the positive drug. SAB is a water-soluble drug consisting of small molecules. The permeation performance improvement on the drug due to the “porogenic” effect of plasma is not significant. Therefore, the performance of the combined treatment group is not remarkably different from that of the individual treatment group. Further studies are required to investigate if plasmas can enhance the transdermal permeability of macromolecular water-soluble drugs to confirm this presumption.

In conclusion, this study found that LTP could exert similar efficacy as SAB in inhibiting hypertrophic scars. It may improve skin scarring by inhibiting collagen deposition and fibroblast proliferation in HSs through the TGF-β/Smad signaling pathway. Nevertheless, the particular regulatory pathways of LTP for cytokines and α-SMA remain an open question that necessitates further reflection and exploration. Although this study is limited, it yields meaningful findings. It suggests that new therapeutic targets may be unearthed by exploring wound healing and scar proliferation from cellular and molecular perspectives. This study may further provide a theoretical basis for the application of plasmas in the field of scar treatment.

This experiment utilized the plasma cosmetic instrument, created by the GBA National Institute for Nanotechnology Innovation. The device was powered by a DC power supply that produced an output of 5 kV after circuit transformation and was connected to a high-voltage electrode. By maintaining a 0.8 mm gap between the ceramic sheet and the skin surface, the low-temperature plasma could be safely and stably generated. During the experiment, the “plasma” mode with an output peak voltage of 4.5 V and a rated power of 3 W was utilized (Fig. 9). Table IV presents the specific discharge parameter details. This intensity is harmless to animal tissues, and during treatment, the head of the cosmetic instrument was placed close to the scarred surface.

This experiment utilized eight standard New Zealand rabbits, equally split by sex, with weights ranging from 2.0 to 2.5 kg [provided by Suibei Experimental Animal Farm, Baiyun District, Guangzhou City, Guangdong Province. Production License number: SCXK (Guangdong) 2020-0050]. The trial rabbits were housed separately in cages and bred in the standard animal facilities situated within the animal experimentation center of Guangdong Pharmaceutical University. Unrestricted access to food and water was provided. The animal facility maintained a 12-h light-dark cycle, controlling environmental conditions with a temperature of (24 ± 1) °C and a relative humidity of (50 ± 5)%. In addition, the breeding environment was kept hygienic. The animal experiments strictly complied with the regulations of the Laboratory Animal Ethics Committee of Guangdong Pharmaceutical University.

The equipment, chemicals, and medications employed in the study are comprehensively outlined in Tables V and VI.

Weigh 1 g of carbomer 940 precisely and add it to 10 ml of distilled water. Stir the mixture gently and let it swell overnight in a refrigerator at 4 °C. Precisely weigh 40 mg of SAB raw material and dissolve it in distilled water, and then, make up the volume to 10 ml. Thoroughly mix the SAB solution with the completely swollen carbomer 940 to obtain the SAB gel, which served as a positive drug in this experiment.

Select healthy New Zealand rabbits with well-developed bilateral rabbit ears that are free from any deformities. Each rabbit must be housed separately and allowed to acclimate to the experimental conditions for at least 72 h, with a 12-h fasting period before modeling. Intraperitoneal injection of a 20% urethane saline solution (5 ml/kg) was required to anesthetize the rabbits. After the anesthesia, careful disinfection of the ventral surface of the ear was carried out using 75% medical alcohol. Blood vessels should be avoided at all times. Two circular incisions, each with a diameter of 10 mm, were made on each ear of the rabbits resulting in a total of four wounds per rabbit, with a distance of over 1 cm between each incision. Normal saline was injected into the center of the circular incision on the cartilage membrane until the skin was raised. Make a circular incision on the skin using ophthalmic scissors. Carefully separate and remove the skin and underlying subcutaneous tissue, making sure to preserve the cartilage. Extract the cartilage membrane using a surgical knife. Once the wound is created, apply pressure with a cotton ball to prevent bleeding. Afterward, apply a suitable amount of tetracycline eye ointment to the wound to prevent the possibility of infection. For three consecutive days following the procedure, continue to apply the ointment. The wound was left uncovered and did not require any bandaging. It is important to clean the secretions from the wound on a daily basis.^10–12 

The formation of scar tissue on rabbit ears was observed beginning on the first day of the modeling process. A representative image of the scar model can be seen in Fig. 10. Figure 10(a) demonstrates the condition of the scar surface immediately after modeling, while Fig. 10(b) indicates that the wound gradually dried by the third day post-modeling. On the seventh day, Fig. 10(c) displays the condition of the scar surface where granulation tissue proliferation was evident, and the formation of crusts began. On the ninth day, Fig. 10(d) exhibits the scar’s surface with formed crusts. Finally, on the 12th day, Fig. 10(e) reveals the trend of crusts beginning to fall off. Figure 10(f) shows that the majority of scar surfaces shed their crusts 15 days following modeling, while Fig. 10(g) observes scar formation after 28 days. The tissue surface was observed to be smooth, higher than its surrounding normal tissue, hard in texture, and reddish.

Six New Zealand rabbits were selected to establish a rabbit ear scar model. There were randomly divided into three groups of two: a model group, a positive drug group, and a low-temperature plasma group. In addition, two rabbits were selected as the blank group without any modeling. Administration of treatment for the positive drug and low-temperature plasma groups began on the 28th day after modeling. The treatment protocol involved topical application of SAB gel to scars on the positive drug group, twice daily, for a period of six weeks. Members of the low-temperature plasma group received twice-daily treatments with a Healthplas plasma cosmetic tool, lasting 1 min during the first and second weeks, 2 min during the third and fourth weeks, and 3 min during the fifth and sixth weeks. Neither the blank group nor the model group underwent treatment.

The rabbits used in the experiment were euthanized via air embolism. A full pathological tissue block, which included the cartilage layer, was excised. Normal tissue at the edges of both sides, ∼0.50 cm on each side, was preserved. The tissue was fixed in a 4% paraformaldehyde solution for more than 24 h and subsequently readied for paraffin sectioning. Hematoxylin-eosin (HE) staining^26,27 and Masson’s trichrome staining (Masson)²⁸ were performed to observe fibroblasts and collagen fibers under an optical microscope.

HE-stained sections of the HS tissues from each group were prepared and examined under a microscopic examination at 400× magnification. Ten fields of view were precisely selected, comprising the central superficial, central intermediate, and central deep regions, as well as the left and right regions of the HS tissue, with two fields of view obtained from each region. The fibroblast cells in the chosen fields of view were counted, and the fibroblast density per unit area (mm²) was calculated.

The prepared paraffin sections were deparaffinized to water, followed by antigen retrieval, endogenous peroxidase blocking, and serum blocking. The blocking solution was then removed, and rabbit primary antibodies for type I collagen and α-smooth muscle actin, diluted in PBS as per a predetermined ratio, were added to the sections. The sections were subsequently incubated overnight at a temperature of 4 °C in a humidified chamber. After completion of the secondary antibody incubation, the sections underwent DAB color development. Next, the nuclei were restained, and the sections were dehydrated and sealed.

After discontinuing medication for one day, draw ∼4 ml of venous blood from rabbit’s ear vein using a vacuum blood collection needle. Then, separate the serum and measure the levels of transforming growth factor-beta 1 (TGF-β1), Smad3, and matrix metalloproteinase-2 (MMP-2) in the rabbit serum following the instructions provided in the ELISA kit.

HE-stained sections were obtained from the HS tissues of all groups. Using a 100-fold microscope, images of the entire HS tissue were captured, and the distance from the HS surface to the cartilage surface was measured with Image J software. This measurement was recorded as A. Furthermore, the distance from the normal skin surface to the cartilage was measured and recorded as B. The hypertrophic index (HI) was calculated as A/B, as in Fig. 11.

OriginLab software (OriginLab, Massachusetts, United States) was employed for statistical analysis. Homogeneity of variance was tested using the Levene test, and a one-way ANOVA was conducted, followed by Tukey’s test to compare differences between groups. Mean ± standard deviation was used to express parameters, and significance was considered at p-values less than 0.05.

This work was supported by the National Natural Science Foundation of China (Grant Nos. 52130701, 11805075, 51977096, and 52277150) and the National Key Research and Development Program of China (Grant No. SQ2020YFE010195).

The authors have no conflicts to disclose.

New Zealand rabbits were provided by Suibei Experimental Animal Farm, Baiyun District, Guangzhou City, Guangdong Province. Production License No. SCXK (Guangdong) 2020-0050. The animal experiments involved in this study strictly abide by the management regulations of the Laboratory Animal Ethics Committee of Guangdong Pharmaceutical University. The animal experiments in this paper have been approved by the Animal Ethics Committee of Guangdong Pharmaceutical University, with Approval No. gdpulacspf2022028.

Lanlan Nie: Writing – original draft (equal); Writing – review & editing (equal). Yali Wang: Data curation (equal). Xi Chen: Data curation (equal). Xinpei Lu: Formal analysis (equal). Lu Gan: Writing – original draft (equal). Dongrong Liu: Data curation (equal). Jun Shi: Writing – original draft (equal); Writing – review & editing (equal).

The data that support the findings of this study are available from the corresponding authors upon reasonable request.