Effectiveness of fractional carbon dioxide laser combined with botulinum toxin type A in a rabbit ear model with the underlying mechanism

Hypertrophic scar (HS) is a common disease in plastic and cosmetic surgery, with limited treatment options, and is a challenge for clinicians.


| INTRODUC TI ON
A hypertrophic scar (HS) is a pathological scar that occurs after burn injury, trauma, or inflammation. 1 After dermal tissues are damaged, abnormal accumulation and remodeling of the extracellular matrix (ECM) due to fibroblast proliferation increase inflammatory factors, and collagen deposition predisposes to the formation of HS. 2 There are numerous clinical treatments available, such as surgical excision, compression therapy, laser, steroid injection therapy, and radiotherapy. 3 However, the efficacy of these treatment options is sometimes unsatisfactory; therefore, the treatment of HS remains a challenge.
Recently, various studies have reported significant progress in applying lasers to treat HS. The fractional carbon dioxide (CO 2 ) laser can improve HS using selective photothermolysis by creating microscopic holes that replace collagen fibers. 4 Fractional CO 2 laser treatment results in a neat alignment of collagen fibers with a reduced, soft, and flat scar. 5 Clinical experimental studies reported that fractional CO 2 laser irradiation reduced the production of aberrant collagen fiber bundles and improved the outward appearance of HS, as assessed by the Vancouver Scar Scale and Patient and Observer Scar Assessment Scale. 6 Compared with traditional methods such as surgical excision and injectable drugs, laser therapy is noninvasive and more convenient. Researchers have concluded that fractional CO 2 laser is a safe and effective treatment option for HS. 7 Botulinum toxin type A (BTXA) is a potent neurotoxin derived from Clostridium botulinum, consisting of heavy and light chains bound to a disulfide bond. 8 BTXA can inhibit acetylcholine release, fusion, and docking by acting on presynaptic neurons. This effect suppresses the contraction of transverse muscle, resulting in muscle paralysis. 9 With >30 years of clinical use, BTXA has gradually been used in various disease areas owing to its long duration of action and mild side effects. 8 BTXA has been reported to induce fibroblast apoptosis and inhibit the expression of α-smooth muscle actin (α-SMA) and myosin II to suppress HS formation, effectively reducing scar contracture. 10 Furthermore, some scholars have reported that BTXA reduces HS by inhibiting the transforming growth factor-β1 (TGF-β1)/Smad, ERK, and JNK pathways. 8,11 BTXA's duration of action generally lasts approximately 6 months, and it is commonly injected around surgical incisions to prevent HS formation in clinical settings. 12 For decades, scholars have actively investigated the underlying mechanisms of HS and conducted various studies to find a reliable treatment method. The present study explored the efficacy and potential mechanism of fractional CO 2 laser combined with BTXA in treating HS to provide a theoretical basis for clinical treatment.

| Induction of HS model in rabbit ears
The 24 New Zealand white rabbits were housed in separate cages under standard conditions. After 1 week of acclimatization, pentobarbitone (5%, 1 mL/kg) was administered intravenously through the ear margin, and the ventral side of the ear was sterilized using 0.1% iodophor. According to the method proposed by Le and Wu,13 six circular incisions of 1.0 cm diameter, each spaced >1.0 cm apart, were made on the ventral side of each ear using a perforator without invading the large blood vessels. The subcutaneous tissue and cartilage membrane were peeled away ( Figure 1A), followed by hemostasis, sterilization, and bandaging to protect the wound. After 2 days, the gauze was removed to observe the damage and to remove secretions.

| Experimental grouping and processing
Four weeks postoperatively, the 24 New Zealand white rabbits were randomly divided into control (n = 6), laser (n = 6), BTXA injection (n = 6), and combined (n = 6) groups. The laser and combined groups were administered a fractional CO 2 laser treatment (Deep Fx: Energy: 15 mJ, Frequency: 300 Hz, Shape: 2, Size: 5, Pulse: 1, and Density: 5%) for each HS, and fractional CO 2 laser treatment was repeated once after 2 weeks to make the sample fully exposed. Next

| H&E staining
Hypertrophic scars were cut intact and fixed in 4% paraformaldehyde for 24 h. The specimens were dehydrated, routinely paraffinembedded, sectioned into 3-mm-thick sections, and left overnight at 60°C. The sections were subjected to conventional dewaxing and hydration, hematoxylin staining for 5 min, rinsing with tap water, HCl-ethanol fractionation for 30 s, eosin staining for 2 min, rinsing with tap water again, gradient concentration, ethanol dehydration, xylene removal, and neutral balsam placement. Light microscopy (Eclipse 80i Microscope; Nikon Corporation) was used to obtain images at 200× magnification. The data were recorded and analyzed using ImageJ software (National Institutes of Health).

| Masson's trichrome staining
Masson's trichrome staining was performed to observe the arrangement of collagen fibers. Briefly, the tissue wax block sections were dewaxed, rinsed, and stained. Collagen fibers were stained blue, whereas keratin and muscle fibers were stained red. The cytoplasm and nucleus were stained light pink and dark brown, respectively. 14 Tissues were observed under a microscope (Olympus) and photographed. Images were captured at 200× magnification. The ratio of the collagen deposition area was recorded and analyzed using ImageJ software.

| Immunohistochemistry
TGF-β1, α-SMA, COL-I, and COL-III expressions were studied using immunohistochemistry. Sections were rehydrated in xylene and graded in alcohol. Endogenous peroxidase was blocked with 3% hydrogen peroxide and then soaked in a citric acid buffer to recover and repair the antigen. After incubation with primary and secondary antibodies, the sections were stained with Mayer's hematoxylin (MHS32, Sigma) and DAB color-developing solution (DA1010, Solarbio) at room temperature. A Zeiss optical microscope (Stemi 305) was used to obtain images at 200× magnification. The average optical density (AOD) of antigen-positive cells was recorded and analyzed using the ImageJ software.

| Western blotting
Hypertrophic scars in rabbit ears were collected to extract proteins. Protein concentrations were measured using an Enhanced Bicinchoninic Acid Assay Protein Assay Kit (P0012S, Beyotime).
The samples were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Gels were transferred after electrophoresis, and the membranes were blocked with a protein-free fast-blocking solution for 30 min and then incubated with primary antibodies overnight at 4°C. The Bands on the membranes were detected using ImageQuant (LAS 500) after incubation with the secondary antibodies for 1 h at room temperature, and protein bands were quantified by measuring the gray value of the signal intensity using Image Pro-Plus v6.0 (Media Cybernetics).

| Statistical analysis
SPSS software (version 20.0; SPSS Inc.) was used to analyze the data, presented as mean ± standard deviation (SD). Tukey-Kramer and analysis of variance tests using GraphPad Prism 8.0.2 Software (Version X, USA) were used for multiple comparisons and determining p-values among the four groups. Statistical significance was set at *p < 0.05; **p < 0.01; ***p < 0.001.

F I G U R E 1
The gross observation of rabbit ear wounds after molding and the formation of HS in each group after 9 weeks. Fractional CO 2 laser irradiation combined with BTXA injection improved HS. (A) HS was reduced in the fractional CO 2 laser and BTXA groups compared to the control group. The combined group had the best treatment effect; HS almost disappeared and closed to normal skin. (B) Scar thickness is the distance from the highest point of the scar to the cartilage membrane. Taking HS samples, measure each group's scar thickness. The data were shown as mean ± standard errors of the means and were compared by one-way ANOVA, followed by the Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. CO 2 , fractional carbon dioxide laser.

| Fractional CO 2 laser combined with BTXA injection inhibited the expression of pro-fibrotic factors and collagen-related proteins
TGF-β1 is a crucial pro-fibrosis cytokine, and its high expression is one of the main factors leading to HS formation. 17  Notably, the combination therapy results were better (Figure 2A,B; ***p < 0.001).
Immunohistochemistry was performed in the present study to determine the expression of pro-fibrotic and collagen-related proteins ( Figure 2C). Consistent with the Western blot analysis results, TGF-β1, α-SMA, and COL-I expression levels were significantly lower in the scar tissues of each treatment group ( Figure 2D; ***p < 0.001).

| Effect of fractional CO 2 laser combined with BTXA injection on pathological scar injuries
To explore the efficacy of the fractional CO 2 laser combined with BTXA in the rabbit ear model, we used hematoxylin and eosin (H&E) staining to observe pathological scar injury in each treatment group.
Compared to rabbits' normal dorsal ear skin, we observed chromatin condensation and loss of cell polarity of fibroblasts in the control group, and fibroblasts were disorganized and more numerous.
Fractional CO 2 laser treatment or BTXA injection inhibited fibroblast proliferation and restored cell polarity ( Figure 3A). The effect of the fractional CO 2 laser combined with BTXA injection was significantly enhanced ( Figure 3A).
The treatment status of each group was assessed by measuring the scar elevation index (SEI). The SEI is the ratio of the distance from the highest point of a scar to the ear cartilage to the distance from the normal skin to the ear cartilage. 19,20 The SEI decreased significantly in the fractional CO 2 laser and BTXA treatment groups than in the control group; however, the effect of the combined therapy was better ( Figure 3B; ***p < 0.001).  In the present study, four groups were designed using the rabbit ear HS model, and the therapeutic effects of the fractional CO 2 laser, BTXA, and a combination of both treatments were observed. After 4 weeks of modeling, the surface of the HS in rabbit ears was rough and protruded from the normal skin, and the texture was tough.

| Fractional CO 2 laser combined with BTXA injection inhibited collagen production and deposition
After fractional CO 2 laser or BTXA treatment, the scars decreased, became soft, and were similar to normal skin. In particular, the curative effects of the combined treatment were more significant, and the appearance of scars in the combined treatment group was similar to that of normal skin. By measuring the scar thickness to further evaluate the therapeutic effect, we observed that the scar thickness decreased significantly in all treatment groups, and this trend was more evident after combined treatment.
TGF-β1 is a significant regulator of tissue repair, inflammation, and fibrosis and can activate multiple cellular pathways, such as the TGF-β1/Smad, ERK, and MAPK pathways, to participate in scar formation. 8,21 Under normal conditions, the production and degradation of the ECM is a dynamic and balanced process. However, in pathological states, due to the activation of excessive myofibroblasts, ECM secretion increases, and its degradation is reduced, leading to the formation of HS. 22 Myofibroblasts are differentiated from fibroblasts by inflammation and other stimuli. α-SMA is a marker for the transformation of fibroblasts into myofibroblasts.
Collagen COL-I and COL-III are essential indicators of collagen deposition. 18 Generally, their levels are dramatically increased in HS.
In the present study, Western blotting and immunohistochemical analysis results revealed that treatment with fractional CO 2 laser or BTXA alone reduced the expression of TGF-β1 and α-SMA and inhibited collagen deposition. However, fractional CO 2 laser irradiation combined with BTXA injection increased these effects. Abnormal proliferation of fibroblasts is the most crucial factor leading to HS formation. 23  Tension is one of the major causes of HS formation, and persistent tension in the lesion is more likely to cause HS formation. 25 The most common clinical locations for HS are in the chest, back, and joint motion areas with high tension. Because of persistent tension, HSs in these areas are more likely to recur after treatment.
BTXA acts on motor nerve endings and can induce chemodenervation by inhibiting acetylcholine binding to receptors. 9 Striated muscle will be functionally denervated for approximately 6 months after injection. BTXA has been used more frequently in recent years to treat HS, owing to its ability to reduce muscle tension. Furthermore, some studies have reported that BTXA improves the appearance of HSs and inhibits their growth by affecting the cell cycle and regulating the TGFβ signaling pathway. 8

| Study limitations
This study has some limitations. First, the potential molecular mechanism of fractional CO 2 laser treatment combined with BTXA for treating HS need to be more deeply understood. Second, there is a significant difference between rabbit ears and human skin; therefore, the rabbit ear model cannot completely represent human HS.

| CON CLUS IONS
Fractional CO 2 laser treatment combined with BTXA more effectively reduced HS by inhibiting fibroblast proliferation, decreasing TGF-β1 and α-SMA expressions, and causing collagen remodeling.
Fractional CO 2 lasers and BTXA have been used individually in the clinical treatment of HS for many years. However, both treatments are seldom used in combination. Therefore, this study provided a reliable therapeutic strategy and a theoretical basis for the clinical treatment of HS.

AUTH O R CO NTR I B UTI O N S
Jianxiang Xiong involved in study design, animal modeling, implementing the operation, data collection, data analysis, data inter-

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
Rabbits were provided from the Laboratory Animal Center of Medical College, Nanchang University. All experimental procedures in this study were approved by the Laboratory Animal Ethics Committee of the Nanchang University School of Medicine(2022CDYFYYLK10-08).
The animal experimental procedures strictly followed the guidelines of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.