Split Face Randomized, Evaluator Blinded Study on the Effect of Multiple Subcisions on Rolling Acne Scars

doi:10.21203/rs.3.rs-4344878/v1

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Split Face Randomized, Evaluator Blinded Study on the Effect of Multiple Subcisions on Rolling Acne Scars

https://doi.org/10.21203/rs.3.rs-4344878/v1

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Journal Publication

published 08 Jun, 2024

Read the published version in Archives of Dermatological Research →

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Acne scarring results from a common inflammatory condition present in many people. These scars can have an impact on quality of life by influencing self-esteem and social acceptance. Current acne scarring treatments, such as chemical peels and laser treatments, often have limited success due to their time-consuming nature and the variability of acne scar types. The subcision technique has shown promise for the treatment of rolling acne scars. There are few studies to date that examine the effects of multiple subcision treatments on rolling acne scars. We evaluated whether the use of multiple subcision treatments improved the appearance of rolling acne scars compared to no treatment. Five patients with rolling acne scars on both sides of their face who met inclusion and exclusion criteria were enrolled in the study. One side of the face was randomized to receive treatment, while the contralateral side of the face received no treatment. Subjects underwent five sequential subcision treatments, spaced 4 weeks apart, with two follow-up visits at weeks 20 and 36 from the 1st treatment. Photographs were taken before and after the initial treatment visit and at each subsequent visit. Acne scar appearance was evaluated by the subject, a blinded live rater, and two double-blinded dermatologist raters. Evaluations of treatment outcomes involved overall acne scar appearance on a 5-point scale, acne scar improvement on a percentage scale, a modified quantitative global scarring grading system, and potential treatment side effects. There was a greater decrease in global scarring scores in the multiple subcision side compared to the control side. There was a greater difference in the average acne scar appearance scores between the Week 36 follow-up visit and baseline for the multiple subcision side compared to that of the control side. Fifty percent of patients reported being more satisfied with the treatment side compared to the control side in regard to overall improvement. The study results suggest that multiple subcision treatments may improve the appearance of rolling acne scars compared to no treatment.

multiple subcisions

rolling acne scars

split face

randomized controlled trial

Acne scarring occurs as a result of a common inflammatory condition that affects many people. As much as 80% of people between the ages of 11 to 30 years old have active acne, and 1.7 per 1000 individuals experience acne scarring.¹ Acne scarring can range from minute scars to immense scars impacting social acceptance and self-esteem. Current acne scarring treatments include chemical peels, dermabrasions, laser treatments, and surgical treatments such as punch excision, scar excision, and undermined scar technique^1–6. Due to the variability of acne scar types and the time-consuming nature of these treatments, many of these modalities often have limited success^7,8,9.

The subcision technique has shown promise in the treatment of rolling acne scars. Rolling acne scars occur from fibrous bands tethered to subcutaneous tissue as a result of inflamed cystic lesions and defective healing, resulting in a depressed appearance. Subcision, a method reported by Dr. Oreintreich in 1995, utilizes hollow-bore needles to break the fibrous bands adjacent to the depressed scar to allow for an elevated appearance.¹⁰ A study by Alam et al. (2005) found that one treatment of subcision resulted in ninety percent of the treated patients reporting an improvement in their appearance with only transient side effects.¹¹

To date, there are few studies offering a direct evaluation of multiple subcision treatments on the improvement of acne scars.

The primary objective of this study was to determine whether the use of multiple subcision treatments would improve the appearance of rolling acne scars compared to no treatment. We hypothesized that multiple subcision treatments would improve the appearance of rolling acne scars compared to no treatment.

Study Design and Study Population

This split-face, evaluator-blinded, randomized control trial studied whether multiple subcision treatments would improve the appearance of rolling acne scars. The study took place from October 2014 to July 2016. A total of 5 patients who met inclusion and exclusion criteria were enrolled in the study and 4 patients completed the study per protocol. Inclusion criteria for subjects included being 18 to 65 years old, having bilateral rolling acne scars on each side of their face, being in good health, and having the willingness and the ability to understand and provide informed consent for the use of their tissue and communicate with the investigator. Exclusion criteria included being under 18 years of age, being pregnant or lactating, being unable to understand the protocol or to give informed consent, having mental illness, recent Accutane use in the past 6 months, being prone to hypertrophic and keloidal scarring, subject reports of any blood diseases (HIV, Hepatitis, etc), and subject reports of history of Herpes (oral or genital).

Participants underwent five sequential subcision treatments, each spaced four weeks apart. One side of the face was randomized to receive multiple subcision treatments, while the contralateral side received no treatment.

Digital photographs were taken in standard front and lateral views before the subcision and immediately after the procedure on the initial visit. Sociodemographic information, including age, race, gender, ethnicity, and Fitzpatrick phototype, for each subject was collected on the initial visit. The Fitzpatrick phototype was collected as part of the sociodemographics information to provide objective data for skin color. The Fitzpatrick Scale is a standardized 6-point scale describing the pigmentation of skin, with lighter skin types comprising Fitzpatrick phototypes I-IV, brown pigmented skin comprising Fitzpatrick phototype V, and black pigmented skin comprising Fitzpatrick phototype VI.¹²

Additional photographs were taken at Weeks 4, 8, 12, 16, 20, and 36 from the initial visit. Standard assessments in acne scar appearance were made by the subject and one live blinded investigator at baseline. Assessments were also made by these individuals at Weeks 4, 8, 12, 16, 20, and 36 (compared to baseline). In addition, two double-blinded investigators made assessments utilizing photographs at Weeks 4, 8, 12, 16, 20, and 36 (compared to baseline).

Before subcision, the scar site was cleansed with chlorhexidine antibacterial solution and marked with a water-soluble marker. Subcutaneous anesthetic (1% lidocaine with epinephrine 1:100,000) was administered via a 30-gauge needle. At the start of the procedure, a Sharpoint ®"The Surgeon's Edge" 20 gauge Microsurgical (1.4 mm), straight vitrectomy knife (Ref 71-2001)¹³ was inserted subdermally and slowly advanced parallel to the dermis. Rapid advancement and retraction of the needle under the scarred area in a lancing motion were performed to abrade the underside of the dermis, followed by side to side sweeping motions attempting to break any fibrous attachments to the deeper tissues. The subcised area was covered with Vaseline and a light gauze dressing. Treatments two through five were administered in the same manner as the initial procedures on weeks 4, 8, 12, and 16 after the initial visit.

Subjects returned for follow-up visits 20 weeks and 36 weeks after Day 1. Front and lateral view photographs were taken at these visits. Subjects were asked to rate which side they were more content with in terms of overall scar improvement. They were also asked to evaluate their scars using a percent improvement scale. Live ratings were performed by a blinded rater comparing the right and left sides of the face.

Recruitment Methods

The investigator recruited patients who presented to the Department of Dermatology at Northwestern University and met the inclusion and exclusion criteria. Patients were recruited via in-network Northwestern referrals. Written announcements regarding study participation were placed inside Northwestern Memorial Hospital, Chicago Transit Authority Vehicles, the Northwestern University Feinberg School of Medicine, around Chicago stores with permission, and on the internet. Potential participants who demonstrated interest by contacting the investigators using the contact information provided on the written announcements were provided additional details regarding the study, including the study procedure and its potential risks, and were screened for study eligibility using the inclusion and exclusion criteria. Participants who met all required criteria were then scheduled for the first study visit per their availability. Follow-up phone calls were made to potential participants who were unable to answer the initial telephone call or schedule the first study visit during the initial call. This study was approved by the Institutional Review Board at Northwestern University and registered on ClinicalTrials.gov (NCT02216864).

Primary Outcome Measures

Outcomes of the procedures were assessed by the subject, one blinded live rater, and two double-blinded photograph raters. The blinded live rater rated the right and left sides of the face in terms of overall appearance on a percentage scale and calculated global scarring scores per the modified quantitative global scarring grading system (Table 1). In addition, two double-blinded dermatologist raters were asked to examine photographs of the subjects comparing the photographs at Weeks 4, 8, 12, 16, 20, and 36 to those of baseline, using the same evaluations as above. The subjects were asked to complete a scar survey assessment scale to evaluate their scars at 20 and 36 weeks from Day 1.

Table 1

Modified Quantitative Global Scarring Grading System
(Grade) Type	Number of lesions on left side of face:	Number of lesions on right side of face
(A) Milder scarring
• Macular erythematous or pigmented
• Mildly atrophic dish-like
(B) Moderate scarring
• Moderately atrophic dish-like
• Punched out with shallow bases small scars (< 5 mm)
• Shallow but broad atrophic areas
(C) Severe scarring
• Punched out with deep but normal bases, small scars (< 5 mm)
• Punched out with deep abnormal bases, small scars (< 5 mm)
• Linear or troughed dermal scarring
• Deep, broad atrophic areas

The primary outcome measure was the change in blinded rater assessment of acne scar appearance score on a 5-point scale (0 = no acne scarring; 1 = trace acne scarring, 2 = mild, 3 = moderate, 4 = severe) at Day 1, 20 weeks, and 36 weeks compared to baseline.

Secondary Outcome Measures

Any induration, edema, and bruising were noted at Day 1, Weeks 4, 8, 12, 16, 20, and 36. The occurrence and extent of any adverse events (AE) during the entire duration of the study were noted as well. This included if the subject contacted the investigators on days that were not visit days.

Statistical Methods

Assuming a standard deviation of 1.1, a sample size of 19 patients gives 80% power to detect a 1-unit difference in mean acne scarring between treatments. This calculation assumes α = 0.05 and a two-sided test. As the sample size was below 19 patients, analysis of the 5-point acne scar appearance scores using the Wilcoxon Signed-Rank test and analysis of the scores of the percent improvement scale and the Quantitative Global Scarring Grading System using paired t-tests were not performed. Descriptive statistics evaluating the mean and standard deviation were performed for these outcomes to compare the treatment and control sides.

Five subjects were enrolled in the study and four subjects completed the study per the protocol, with the mean age and age range of patients being 41.4 years (20–54). One subject withdrew from the study due to swelling and lack of perceived improvement with treatments. Patient demographics are reported in Table 2.

Table 2

Patient Demographics
Subject	Age	Gender	Race	Ethnicity	Fitzpatrick Phototype
1	30	Male	White/Caucasian	Not Hispanic or Latino	III
2	20	Female	White/Caucasian	Not Hispanic or Latino	II
3	54	Female	White/Caucasian	Not Hispanic or Latino	II
4	49	Female	African American	Not Hispanic or Latino	V
5	54	Female	White/Caucasian	Not Hispanic or Latino	III

The average measurement and standard deviation for the 5-Point Acne Scar Appearance score for the treatment side and control side at Weeks 0, 4, 8, 12, 16, 20, and 36 are presented in Table 3 and Fig. 1, with lower scores indicating a better appearance. Across all rater types, there was greater difference in the average Acne Scar Appearance scores between the Week 36 follow-up visit and baseline for the multiple subcision side compared to that of the control side. The difference in 5-Point Acne Scar Appearance score between the Week 36 follow-up visit and baseline as rated by the subject, live blinded investigator, and double-blinded investigators evaluating photographs were, for the multiple subcision treatment side, 1.54, 1.65, and 1.85, respectively, and were, for the control side, 0.02, -0.3, and − 0.3, respectively.

Table 3

Summary of Average 5-Point Acne Scar Appearance Scores Across Time (lower scores indicate a better appearance)
Rater	Treatment	Score, Mean (SD)
		Visit
		Week 0	Week 4	Week 8	Week 12	Week 16	Week 20	Week 36	Difference Baseline vs Week 36
Subject	Multiple Subcision	3.27 (0.44)	2.98 (0.71)	2.76 (0.43)	2.26 (0.42)	1.68 (0.53)	1.54 (0.64)	1.73 (0.65)	1.54
Subject	Control	2.35 (1.11)	2.50 (0.87)	2.66 (1.17)	2.38 (1.13)	2.36 (1.25)	2.41 (1.25)	2.33 (1.16)	0.02
Live	Multiple Subcision	2.40 (0.89)	2.20 (0.84)	1.60 (1.14)	1.00 (0.71)	1.00 (1.00)	0.75 (0.50)	0.75 (0.50)	1.65
Live	Control	2.20 (1.10)	2.00 (0.71)	2.40 (1.14)	2.20 (0.84)	3.00 (1.00)	2.25 (0.96)	2.50 (1.29)	-0.3
Photoraters	Multiple Subcision	2.60 (1.14)	1.80 (1.10)	1.40 (1.14)	1.00 (0.71)	1.00 (1.00)	0.75 (0.50)	0.75 (0.50)	1.85
Photoraters	Control	2.20 (1.10)	2.20 (0.84)	2.20 (0.84)	2.20 (0.84)	2.67 (0.58)	2.25 (0.96)	2.50 (1.29)	-0.3

The average percent improvement scores, comparing scar appearance at Weeks 20 and Week 36 follow-up visits to that of Week 0, as rated by the subject, live blinded investigator, and double-blinded investigators evaluating subject photographs are presented in Table 4 and Fig. 2. Across all rater types and at both time points, the average percent improvement score was greater for the multiple subcision side compared to those of the control side.

Table 4

Summary of Average Percent Improvement Scores Comparing Weeks 20 and 36 to Baseline
Rater	Treatment	Visit
Rater	Treatment	Week 20	Week 36
Subject	Multiple Subcision	63.00 (16.37)	72.25 (21.14)
Subject	Control	6.25 (12.5)	25.00 (35.36)
Live	Multiple Subcision	6.25 (2.50)	6.25 (2.50)
Live	Control	-1.25 (2.50)	-4.25 (5.06)
Photoraters	Multiple Subcision	7.50 (2.89)	8.75 (2.50)
Photoraters	Control	-2.5 (5.00)	-5.00 (5.77)

There was a greater decrease in global scarring scores in the multiple subcision side compared to those of the control side. The differences in the modified quantitative global scarring scores between the Week 36 follow-up visit and baseline as rated by the live blinded investigator and double-blinded investigators evaluating subject photographs were, for the multiple subcision side, 4.05 and 7.8, respectively, and were, for the control side, -1.3 and − 2.3, respectively (Table 5 and Fig. 3).

Table 5

Summary of Average Quantitative Global Scarring Scores Across Time
Rater	Treatment	Ave. Score, Mean (SD)
		Visit
		Week 0	Week 4	Week 8	Week 12	Week 16	Week 20	Week 36	Difference Baseline vs Week 36
Live	Multiple Subcision	7.80 (5.02)	5.6 (3.78)	6.80 (5.76)	4.00 (2.83)	5.33 (3.06)	4.00 (2.31)	3.75 (2.63)	4.05
Live	Control	9.20 (7.53)	8.80 (6.83)	8.20 (6.06)	8.80 (5.93)	10.50 (7.33)	9.75 (6.40)	10.50 (7.33)	-1.3
Photoraters	Multiple Subcision	11.80 (6.34)	7.00 (4.58)	8.80 (6.10)	6.40 (4.98)	5.67 (3.21)	4.50 (3.00)	4.00 (3.16)	7.8
Photoraters	Control	9.20 (7.53)	8.80 (7.26)	9.20 (7.36)	10.20 (7.85)	11.50 (8.43)	10..75 (7.50)	11.50 (8.43)	-2.3

At the end of the study at Week 36, fifty percent of the patients reported being happier with the multiple subcision treatment side in regard to overall improvement, while the other fifty percent of patients were happier with the control side. Reported side effects were induration, bruising, and edema, and all side effects were resolved by the end of the study at Week 36.

The results of this study suggest that multiple subcision treatments may be more effective for treating the appearance of rolling acne scars compared to no treatment, as the appearance of the acne scars treated by multiple subcisions improved when evaluated by raters. A greater percent improvement in the appearance of rolling acne scars on the multiple subcision side was noted compared to the control side. There was also a downward trend in the acne scar appearance scores and global scarring scores across all rater types involved for each measure. However, only fifty percent of patients were happier in regard to overall improvement with the multiple subcision treatment side by the end of the study. This does not align with the rest of the study results, including the acne scar evaluations performed by the subject. A possible reason for this discrepancy may be confusion due to the wording of the question and the placement of the question directly after the visual analogue scale for percentage improvement of scar appearance.

Limitation of the study and future directions:

The major limitation of this study is that the number of enrolled subjects was relatively small. Data collection from nineteen patients would allow this study to be adequately powered for further statistical analysis. As such, adding fourteen additional subjects would increase our confidence that multiple subcision treatments are an effective treatment for rolling acne scars. The limited number of subjects enrolled was likely due to the lengthy nature of the study. The recruitment methods resulted in a large quantity of phone calls from potential subjects; however, many of them were ineligible to participate due to the exclusion and inclusion criteria and/or were unwilling to participate after learning about the minimally invasive procedure and 9-month duration of the study. An analysis of the sociodemographic information of potential subjects who decided not to participate may provide further insight into the low number of subjects for this study. This data was not collected during the pre-screening telephone conversations with these unenrolled potential subjects in accordance with the IRB-approved protocol, as these individuals did not consent to the study. However, Table 6 is a de-identified list displaying the potential subjects who contacted the investigators, if they were ultimately enrolled in the study, and, if applicable, the reason for their not being enrolled in the study. Of note, a sizable number of potential subjects (14 of 46) were unable to be unrolled as they met exclusion criteria or did not meet the inclusion criteria. Reasons for not meeting inclusion criteria included not having the appropriate type of acne scar for the study or not having bilateral facial rolling acne scars, which is required for the split-face design of the study. Additionally, 23 of the 46 potential subjects did not respond to the follow-up phone calls made by the investigators despite having expressed interest in the initial telephone conversation or telephone message. Another limitation of this study is that although a split-face study design allowed subjects to act as their own control, there may be a difference in the baseline rolling acne scar appearance on each side of the patient. Additionally, it was difficult to guarantee that the same dermatologist performed the subcision treatment at each treatment visit and for all subjects due to the high-volume nature of the clinic in which this study was performed. This may have led to a variability in treatment outcomes. Lastly, as this study was only blinded to the evaluators and no sham procedure was performed for the control, there may be a placebo effect present in the evaluations completed by the subjects.

Table 6

List of Pre-Screened Potential Subjects and Reasons for Not Enrolling (If Applicable)
Pre-Screening ID	Enrollment Status	Reason for Not Enrolling
1	Not enrolled	Did not meet inclusion criteria
2	Not enrolled	Did not respond to follow-up phone call
3	Not enrolled	Did not respond to follow-up phone call
4	Not enrolled	Did not respond to follow-up phone call
5	Not enrolled	Did not want to participate after discussion of study protocol
6	Enrolled	N/A
7	Not enrolled	Did not respond to follow-up phone call
8	Not enrolled	Did not respond to follow-up phone call
9	Not enrolled	Did not respond to follow-up phone call
10	Not enrolled	Did not want to participate after discussion of study protocol
11	Enrolled	N/A
12	Not enrolled	Did not respond to follow-up phone call
13	Not enrolled	Did not want to participate after discussion of study protocol
14	Not enrolled	Did not meet inclusion criteria
15	Not enrolled	Did not respond to follow-up phone call
16	Not enrolled	Did not respond to follow-up phone call
17	Not enrolled	Did not meet inclusion criteria
18	Not enrolled	Did not respond to follow-up phone call
19	Enrolled	N/A
20	Not enrolled	Did not respond to follow-up phone call
21	Not enrolled	Did not respond to follow-up phone call
22	Not enrolled	Did not meet inclusion criteria
23	Not enrolled	Did not respond to follow-up phone call
24	Not enrolled	Did not respond to follow-up phone call
25	Not enrolled	Did not meet inclusion criteria
26	Not enrolled	Did not want to participate after discussion of study protocol
27	Not enrolled	Did not respond to follow-up phone call
28	Not enrolled	Did not meet inclusion criteria
29	Not enrolled	Did not respond to follow-up phone call
30	Not enrolled	Did not respond to follow-up phone call
31	Enrolled	N/A
32	Not enrolled	Did not respond to follow-up phone call
33	Not enrolled	Did not meet inclusion criteria
34	Not enrolled	Did not respond to follow-up phone call
35	Not enrolled	Did not respond to follow-up phone call
36	Not enrolled	Did not respond to follow-up phone call
37	Not enrolled	Did not meet inclusion criteria
38	Enrolled	N/A
39	Not enrolled	Did not respond to follow-up phone call
40	Not enrolled	Did not meet inclusion criteria
41	Not enrolled	Did not meet inclusion criteria
42	Not enrolled	Did not meet inclusion criteria
43	Not enrolled	Did not respond to follow-up phone call
44	Not enrolled	Did not meet inclusion criteria
45	Not enrolled	Did not meet inclusion criteria
46	Not enrolled	Met exclusion criteria

Multiple subcision treatments have potential for the improvement of rolling acne scar appearance and further studies are required to evaluate its effects. Future studies could involve a larger sample size, investigate the effects of different schedules for multiple subcision treatments, and require that the same dermatologist perform all treatments. Additionally, this study could also be performed at multiple institutions to facilitate the recruitment of a greater number of subjects with rolling acne scars. To further aid in the recruitment of subjects, the study method may be modified in several aspects. For instance, the split-face design of the study required rolling acne scars to be present on both sides of the face to allow one side of the face to serve as the control. However, this caused potential subjects who were interested in the study to be ineligible. A possible solution is that the inclusion criteria could be modified so that it only requires multiple rolling acne scars on any region of the face. One of the rolling acne scars can undergo treatment, while the other can be untreated. Thus, the untreated rolling acne scar, regardless of its location on the face, can serve as the control for the rolling acne scar that underwent multiple subcision treatments. Another issue with recruitment was the potential subjects’ lack of interest in the study after learning about its long duration. It is likely that there would be a greater number of subjects if there were 2 or 3 fewer subcision treatments, as this would shorten the study duration by 2 to 3 months. With these modifications to the methods and having the study performed at multiple sites, future studies may provide more insight into the efficacy of multiple subcision treatments on rolling acne scars due to their greater sample sizes.

Funding: This study was supported by Departmental Research Funds, Department of Dermatology, Northwestern University.

Conflicts of Interest: None declared.

Rivera AE. Acne scarring: a review and current treatment modalities. J. Am. Acad. Dermatol. 2008; 59: 659-76.
Jemec GB, Jemec B. Acne: treatment of scars. Clin. Dermatol. 2004; 22: 434-8.
Goodman GJ. Management of post-acne scarring. What are the options for treatment? Am J Clin Dermatol 2000; 1: 3-17.
Jacob CI, Dover JS, Kaminer MS. Acne scarring: a classification system and review of treatment options. J. Am. Acad. Dermatol. 2001; 45: 109-17.
Goodman G. Post acne scarring: a review. J Cosmet Laser Ther 2003; 5: 77-95.
Alam M, Dover JS. Treatment of acne scarring. Skin Therapy Lett 2006; 11: 7-9.
Goodman GJ, Baron JA. The management of postacne scarring. Dermatol. Surg. 2007; 33: 1175-88.
Goodman G. Post acne scarring: a review. J Cosmet Laser Ther 2003; 5: 77-95.
Alster TS, Tanzi EL, Lazarus M. The use of fractional laser photothermolysis for the treatment of atrophic scars. Dermatol Surg 2007;33:295–9.
Orentreich, D.S. and N. Orentreich, Subcutaneous incisionless (subcision) surgery for the correction of depressed scars and wrinkles. Dermatol Surg, 1995. 21(6): p. 543-9.
Alam, M, Omura, N, Kaminer, MS. Subcision for Acne Scarring: Technique and Outcomes in 40 Patients. Dermatol Surg. 2005;31:310-317.
S. Tejiram, S.L. Kavalukas, J.W. Shupp and A. Barbul, 1 - Wound healing, In Wound Healing Biomaterials. Woodhead Publishing, 2016. P. 3-39.
Ayeni, O., W. Carey, and C. Muhn, Acne scar treatment with subcision using a 20-G cataract blade. Dermatol Surg, 2011. 37(6): p. 846-7.

No competing interests reported.

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Journal Publication

published 08 Jun, 2024

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Editorial decision: Accepted
08 May, 2024
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Reviewers agreed at journal
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You are reading this latest preprint version

Split Face Randomized, Evaluator Blinded Study on the Effect of Multiple Subcisions on Rolling Acne Scars

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Abstract

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Introduction

Hypothesis and/or Research Question

Materials and Methods

Study Design and Study Population

Recruitment Methods

Primary Outcome Measures

Secondary Outcome Measures

Statistical Methods

Results

Discussion

Limitation of the study and future directions:

Declarations

References

Additional Declarations

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