Safety and efficacy of CO2 dot matrix laser combined with platelet‐rich plasma on depressed scar after acne vulgaris and influencing factors of its repair effect: A retrospective analysis

Acne vulgaris (AV) is the most pervasive inflammatory disease of hair follicle and sebaceous gland in the dermatology department, and depressed scar (DS) is the most common and serious sequela of AV.


| INTRODUC TI ON
Acne vulgaris (AV) is the most pervasive inflammatory disease of hair follicles and sebaceous gland in the dermatology department. The cheek, forehead, and jaw are all areas with a high incidence, and in severe cases it can even involve all parts of the whole body. 1 With an incidence rate of 40%-55%, AV is common among teenagers aged 10-18. 2 The incidence of AV is considered to be primarily related to hormones and faster sebum endocrine. 3 In recent years, as society develops, AV has shown an obvious increase in incidence. 4 As a skin inflammatory disease, AV can usually subside itself with the increase of the patients' age. 5 However, for patients with serious developed AV, AV can cause bacterial infection of skin pores, skin nodules, and cysts, which seriously compromises their normal life and communication. 6 At this time, they need targeted medical treatment. At the current stage, clinical treatment of AV has achieved relatively stable results, and the disease disappears completely in approximately 95% of patients after treatment. 7 Depressed scars (DS) left after AV treatment have become the most pervasive and serious sequela of AV treatment in clinical practice. 8 According to statistics, more than 50% of patients with moderate to severe AV suffer from different degrees of DS after treatment. 9 DS is caused by a dermal tissue defect, and it can lead to permanent skin depression due to the lack of collagen and elastin that will not reproduce. 10 DS has no effect on the physiological function of the human body, but it seriously disrupts the psychological state of patients. One survey shows that patients with DS usually suffer from a strong sense of inferiority in social life and have a sense of fear and resistance to communication with others. 11 Among them, approximately 6%-10% of patients suffer from different degrees of depression due to DS. 12 Therefore, DS has exerted a severe psychological impact on patients, and its treatment is the key to restoring patients' self-confidence.
Currently, the clinical treatment methods for DS include chemical exfoliation, grinding, radio frequency, tissue filling, dot matrix laser, microneedles, and surgery. 13 With different effects, each method has its own advantages and disadvantages. 14 Among them, the CO 2 dot matrix laser is a frequently used method for clinical treatment of DS, and its effect has been recognized in many studies. 15,16 The CO 2 dot matrix laser vaporizes deep skin tissue by surface heating and activates collagen proliferation, thus promoting skin repair such as tissue repair and collagen recombination. 17 However, its efficacy mainly depends on the quality of the laser instrument. Some common CO 2 dot matrix laser instruments may have some problems such as insufficient laser penetration ability, uneven energy distribution, and excessive thermal coagulation, which is likely to give rise to adverse reactions such as thermal damage and pigmentation to patients' skin tissues. 18 Therefore, improving the efficacy of CO 2 dot matrix laser while ensuring the economic benefits of patients has become a hot and difficult issue in clinical research.
Platelet-rich plasma (PRP), as a platelet concentrate, is not only one of the treatment schemes for skin injuries such as DS, burns, and ulcers, but also has been found as a strong assistant for laser treatment of DS in recent years. 19,20 For example, one study by Gentile et al. 21 revealed that laser combined with PRP can induce hair regeneration in patients with androgenic alopecia. For CO 2 dot matrix laser, the combination use of RPR has also been verified to rectify its limitations and improve its efficacy on patients with DS. 22 However, the current research on the combination of the two is not sufficient, and the related research is mainly limited to the preliminary observation of DS. In order to further improve the DS treatment plan, this study conducted a more in-depth and comprehensive experiment and evaluation of CO 2 dot matrix laser combined with PRP and acquired more effective experimental results for clinical reference.

| Data about patients
A total of 81 patients with DS at the rear of AV in our hospital were selected as the research objects from December 2018 to October 2019 for retrospective analysis. Among them, 39 patients received CO 2 fractional laser combined with PRP, which belonged to the observation group (OG); the other 42 patients received CO 2 fractional laser, which were the control group (CG). This study was approved by the Ethics Committee of our hospital. Informed consent was obtained from all patients to be included in the study.

| Inclusion and exclusion criteria
The inclusion criteria: Patients ≥ 18 years old, patients with a course of DS ≥1 year, patients with DS area ≥ 3 cm 2 , patients with detailed case data, patients without a major medical history, patients whose routine physical examination results were normal. After admission, he received CO 2 fractional laser therapy or CO 2 combined PRP therapy. The exclusion criteria: patients with comorbid photosensitive diseases, patients with scar constitution, patients who had received surgery, injection filling or other laser treatment within half a year before admission, patients with metal supports buried in their bodies, pregnant patients, lactating patients, patients who received sunlight exposure within 1 month before admission, and those allergic to laser or drugs used in this study.

| PRP preparation
The PRP preparation were performed referring to the previous study. 23 Before treatment, 40 ml of venous blood was drawn from fasting humans using vacuum blood collection tube with sodium citrate (Sanli).
In order to separate serum and plasma, the blood samples were collected and centrifuged at 1400 g for 15 min at 4°C using a Allegra 64R High-speed refrigerated centrifuge (Backman). The supernatant was discarded and the yellowish-brown portion at the bottom was the PRP.

| Treatment method
Each patient in the two groups was anesthetized with lidocaine gel for 1 h before laser treatment, and then was treated by CO 2 dot matrix laser according to the patient's scar type, area, and soft tissue. CHX-100H CO 2 laser medical equipment (Wuhan Chuangxin Photoelectron Company) was used for treatment, and the parameters were set as follows: dot-matrix scanning mode, coverage rate of 10%-25%, wavelength of 10 600 nm, spot spacing of 1-2 mm, and energy of 42-60 mJ. During the treatment, the treatment was immediately adopted once the patient showed uniform punctate or flaky bleeding. After treatment, each patient was given ice compress for 30 min, during which water should be avoided in the treatment area, and nursing measures such as moisturizing and protection against sun were strictly implemented. Each patient was treated three times in total at an interval of 1 month. After laser treatment, for each patient in the OG, the treatment area (thickness: 0.5 mm) was evenly applied with a mixed solution of autologous PRP and 10% calcium gluconate (1: 10). After solidification, the treatment area was repeatedly applied with PRP, and ice compress was carried out after 30 min. After treatment, the patient was notified by telephone every 6 months to return to the hospital to review the recovery of AV and scar.

| Efficacy assessment
The efficacy was assessed with the scar repair area as the evaluation criteria. Cure: The patient's appearance returned to normal and the repaired area was larger than 90%; markedly effective: The skin in the affected area was similar to normal skin and the repaired area was <90% but larger than 60%; effective: The appearance of the affected area was improved to some extent, and the repaired area was <60% but >30%. Ineffective: None of the above criteria were met. The total effective rate = (The number of cured patients + the number of markedly effectively treated patients + the number of effectively treated patients)/the total number of patients × 100%.

| Outcome measures
Clinical efficacy: The clinical efficacy was assessed according to the criteria in 2.5. Safety: The adverse reactions of patients during treatment were recorded and the incidence of adverse reactions was calculated.
Healing time: The scar scabbing time and decrustation time of the two groups after treatment were recorded. Scar repair: The length, width, and thickness of the scar in each patient were measured before and after treatment. The face of each patient was photographed with the same camera under the same light source and the same angle, and the relative gray level of the DS area in the patient and the surrounding normal skin was analyzed using the gray value analysis tool in PS software.
The gray level value = (gray level of normal skin -gray level of skin with DS)/gray level of normal skin × 100%. The Vancouver scar scale (VSS) was also adopted for evaluation of the scar status of each patient. 24 Pain and comfort: Before and after treatment, the pain of each patient was evaluated with the Numerical Rating Scale (NRS), and a higher score indicated more severe pain. The Kolcaba's general comfort questionnaire (GCQ) was adopted for evaluation of the comfort of each patient, and a higher score indicated higher comfort. Skin condition: Before and after treatment, a VISIA digital skin analyzer was adopted to shoot and analyze the facial skin texture and skin pores of each patient, and the VISIA score was positively associated with the skin condition. Questionnaire (Acne-QOL) was adopted to investigate the life quality of each patient, which covered symptoms, emotion, self-perception, and social function. A higher acne-QOL score indicated a better quality of life. And record the recurrence of AV and scar during the prognosis of the patient and calculate the recurrence rate. Recurrence rate = number of recurrence cases/total number × 100%.

| Statistical analysis
SPSS24.0 software was used for statistical analyses of data. Data such as clinical efficacy and incidence of adverse reactions were presented by (n/%) and compared between groups using the chi-square test.
Other data were expressed as mean ± standard deviation (SD), and inter-group differences of them were analyzed by the independentsamples T test, paired t test, one-way ANOVA, and Bonferroni post hoc test. In addition, logistic regression analysis was carried out for analysis of related factors. p < 0.05 denotes a significant difference.

| Comparison of baseline data
In this study, 81 patients with DS were collected and randomized to OG group (n = 39) and CG group (n = 42). Figure 1 shows the flow diagram of the progress in this study. Patients in the OG group and CG group were not greatly different in clinical baseline data like age, gender, course and disease, scar degree, and so on (p > 0.05, Table 1).

| Comparison of clinical efficacy and incidence of adverse reactions
The OG group showed a notably higher total effective rate than the CG group (94.87% vs. 78.57%, p < 0.05, Table 2). However, there F I G U R E 1 Flow diagram of enrollment, treatment, and analysis was no significant difference in the incidence of adverse reactions between the two groups (17.95% vs.16.67%, p > 0.05, Table 3).

| Comparison of clinical recovery status
The scar scabbing time and decrustation time experienced in the OG group were (28.59 ± 9.83) h and (6.97 ± 0.99) days, respectively, both of which were shorter than those of the CG group (both p < 0.05, Figure 2). Before treatment, no difference was found in scar status between the two groups (p > 0.05), while after treatment, the scar thickness, length, width, gray level, and VSS score of both groups decreased (all p < 0.05), and the scar repair in the OG group was significantly superior to that in the CG group (p < 0.05, Figure 3).
Before treatment, the two groups were not greatly different in NRS and GCQ scores (both p > 0. 05 Figure 4). Before treatment, no difference was found between the two groups in VISIA score and visual score (both p > 0.05), while after treatment, the VISIA scores of both groups increased and visual score of them decreased (both p < 0.05). In addition, after treatment, the VISIA scores of texture and pores in the OG group were higher than those in the CG group, while the visual score were lower (p < 0.05) ( Figure 5). Before treatment, the two groups were not greatly different in SAS and SDS scores (both p > 0.05), while after it, SAS and SDS scores of both groups decreased (both p < 0.05), and the OG got lower SAS and SDS scores than the CG (both p < 0.05) ( Figure 6).

Representative photographs of clinical improvement in OG
group are shown in Figure 7.

| Comparison of satisfaction
The satisfaction toward scar repair in the OG group was 92.31%, higher than that in the CG group (p < 0.05), and the satisfaction toward treatment service in the OG group reached 100%, also notably higher than that in the CG group (p < 0.05), (Table 4).   Table 5).

| Multivariate analysis of factors affecting repair effect
The above-mentioned influential single factors were assigned (

| Comparison of prognosis
During the 2-year follow-up, 4 patients were lost to follow-up in OG group due to the loss of contact, and 10 patients were lost to follow-up in CG group, including 6 patients that loss of contanct and 4 patients that moved. Finally, 35 cases in the OG group and 32 cases in the CG group were successfully followed. The results of Acne-QOL score revealed that the symptom score, emotion score, self-cognition score, and social function score of the OG group were all higher than those of the CG group (all p < 0.05), but there is no notable difference between the two groups in the recurrence of AV and scar (both p > 0.05) (Figure 8).

| DISCUSS ION
As the most familiar sequela of AV, DS is usually strongly associated with the severity, treatment, and other factors of AV. 25 27,28 However, one of the limitations of the CO 2 dot matrix laser is the high price of the precision laser, and treatment with it costs significant more. The second limitation is that for Asians with dark skin color, many times the CO 2 dot matrix laser may cause erythema and pigmentation. 29 There has been many research and analyses on the efficacy of CO 2 dot matrix laser on DS, 30,31 and it is also agreed that the shortcomings of CO 2 dot matrix laser can be effectively addressed by applying it with other therapeutic schemes in the meantime. For instance, a study by Artzi et al. 32 has revealed the remarkable efficacy of CO 2 dot matrix laser combined with antimony sodium gluconate on leishmaniasis. Mu et al. 33 have also pointed out that CO 2 dot matrix laser combined with other schemes can produce better results in the treatment of AV scars.
In other DS treatment schemes, PRP, as a high concentration platelet plasma, can be extracted from patients' autologous blood, so it has great advantages in terms of acquisition. 34 PRP can release a variety of cytokines related to skin tissue regeneration and can exert a relatively stable efficacy on all skin wounds. 35 In addition, PRP is extracted from autologous blood, so its safety is the highest among all treatment plans for DS. 36 At present, the combined use of PRP and CO 2 dot matrix laser has become a hot spot in clinical research. This study analyzed the efficacy of the combination of the two on DS from many aspects, and could provide reliable reference and suggestions for follow-up research and clinical application of the combination.
This study first evaluated the clinical efficacy and safety of CO 2 dot matrix laser combined with PRP. The results are consistent with our expectations. The OG group treated by the combination of the two showed a higher effective rate than the CG group treated by CO 2 dot matrix laser alone, and the two groups were not greatly different in the incidence of adverse reactions. The results are also consistent with those in a previous study, 37 which can support the conclusion of our study. Our study also revealed that the scar healing and repair of the OG were better than those of the CG, indicating that CO 2 dot matrix laser combined with PRP could effectively promote the wound healing of patients. Obviously, PRP plays a crucial role in wound healing. As we all know, CO 2 dot matrix laser will form micropores with specific depth on the skin surface of patients during treatment, and then will make use of the thermal effect of micropores to start deep tissue healing. 38 However, the healing is complicated, which is strongly related to inflammatory reaction, coagulation function, granulation, and epithelialization. 39 In our study, The mechanism may be as follows: After treatment of both PRP and CO 2 dot matrix laser, the established microporous tissue enters the subcutaneous tissue, which strengthens the activity of dermal cells  basic reason is that the CO 2 dot matrix laser combined with PRP can deliver a remarkable scar treatment effect. Finally, our Logistic regression analysis revealed that the course of disease, scar degree, staying up late, and repair method were all independent factors affecting the repair effect of DS, and the results were basically consistent with those of the previous studies. 43,44 However, there are some shortcomings in this study. First, in previous studies, CO 2 dot matrix laser and PRP have also been verified to have favorable efficacy on hypertrophic scars. [45][46][47] This study has only included patients with DS, so the efficacy of the combination on hypertrophic scars is not yet clear. Additionally, because of the small number of cases in this study, it cannot be excluded that the statistical calculation of some results may be accidental. Moreover, there are many clinical therapeutic schemes for DS at present, but in this study, only CO 2 dot matrix laser therapy has been used as a control, so the effect of CO 2 dot matrix laser combined with PRP may not be as significant as expected when compared with other schemes in the future. Therefore, the above are the focus and direction of our research in the future. We will continue to conduct a longer follow-up investigation on subjects in this study and carry out in vitro experiments to clarify the exact mechanism of CO 2 dot matrix laser combined with PRP in the treatment of DS, so as to obtain more comprehensive experimental results for clinical reference.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any 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
This study was approved by the Ethics Committee of Shanxi Bethune Hospital (YXLL-KY-2021-022).