Efficacy and safety of temperature-sensitive acellular dermal matrix in prevention of postoperative adhesion after thyroidectomy: A randomized, multicenter, double-blind, non-inferiority study.

INTRODUCTION: MegaShield® is a newly developed temperature-sensitive anti-adhesive containing micronized acellular dermal matrix. The aim of this study was to investigate the efficacy and safety of MegaShield® compared with Guardix-SG® in the prevention of adhesions in patients undergoing bilateral total thyroidectomy.
METHOD: We conducted a multicenter trial between October 2018 and March 2020 in patients undergoing total thyroidectomy. The patients were randomly assigned to either the MegaShield® group or the Guardix-SG® group. The primary outcome was the esophageal movement using marshmallow six weeks after the surgery and the secondary outcome was the assessed adhesion score. The safety assessment was also evaluated.
RESULTS: The study included 70 patients each in the MegaShield® and control (Guardix-SG®) groups. Baseline clinical characteristics, the mean score of marshmallow esophagography, and the sum of adhesion scores were not statistically different between the two groups. Inferiority test demonstrated that the efficacy of MegaShield® is not inferior to that of Guardix-SG®. There were no device-related complications in both groups.
CONCLUSION: The efficacy and safety of MegaShield® were not inferior than those of Guardix-SG®. MegaShield® demonstrated the potential of ADM as a potential future anti-adhesive agent. TRIAL REGISTRATION: The name of trial registry CRIS (Clinical Research Information Service) https://cris.nih.go.kr/cris/index.jsp. (The full trial protocol can be accessed) Registration number: KCT0003204.

Introduction

Postoperative adhesion refers to the formation of fibrous bands between the traumatized tissues after surgery. It is a natural phenomenon due to the proliferation and regeneration of the tissue cells which occurs after a surgical procedure. The severity of postoperative adhesions can vary, resulting in numerous complications. Sometimes, it can be hazardous and even life-threatening, leading to malfunction of organs which require re-intervention.

Surgical intervention for thyroid gland is inevitable when a patient has uncontrolled hyperthyroidism or thyroid cancer, the most common endocrine cancer worldwide [1]. Although postoperative adhesions after thyroid surgery are not life-threatening, many patients experience life-long discomfort such as pain, stiffness, hyperesthesia, paresthesia, swallowing difficulty, and in some severe cases, choking sensation. Attempts to prevent postoperative adhesion formation and associated complications include minimizing the dissection during surgery; administering anti-inflammatory agents, fibrinolytic agents, anticoagulants, and other hormonal drugs; and applying anti-adhesive agents around the surgical field [2-8].

Although complete recovery of postoperative adhesion is not possible, various anti-adhesion barriers (AABs) that are currently available are known to be helpful in reducing the extent of postoperative adhesion. AABs are divided into three categories according to their viscosity: film/membrane, solution, and gel [9-11]. Each AAB presents different disadvantages: film/membrane type shows foreign body reaction, application difficulties, and adhesion formation at the suture site; solution type may cause inaccurate application; and the gel type shows short maintenance time due to rapid dissolvement and discharge [12-14]. Nowadays, temperature-sensitive anti-adhesives using poloxamer [15], which has sol-gel transition at body temperature, has been developed, of which Guardix-SG® is the most common example.

Similarly, acellular dermal matrix (ADM) has been studied for its usefulness as an anti-adhesive agent [16-18]. ADM is derived from donated human skin supplied by the US tissue banks following the guidelines of the American Association. ADM contains various substances such as collagen and elastin, and acts as a biologic platform for re-epithelialization, neovascularization, and fibroblast infiltration while not inducing an immune response [16, 19, 20]. ADM has been applied in various surgical fields such as abdominal wall repair [21], dural repair [22], pelvic floor reconstruction [23], and bladder wall repair [24] for minimizing postoperative adhesion and facilitating wound healing and soft tissue augmentation.

In this context, we developed a new anti-adhesive agent which includes micronized ADM with sol-gel transition properties for effective application. The resulting product is MegaShield® (L&C BIO, Seongnam, Korea), consisting of ADM, poloxamer 407, sodium hyaluronate (HA), and 1,4-butanediol diglycidyl ether (1,4-BDDE). Our previous study demonstrated that MegaShield® is effective as an anti-adhesive in a rat model [17]. In this study, we demonstrate the efficacy and safety of MegaShield® in a human model. We hypothesized that the efficacy and safety of MegaShield® are not inferior to those of Guardix-SG®, and the aim of this study was to investigate our hypothesis using a randomized, multicenter, double-blind, non-inferiority study design.

Materials and methods

Patients

Between October 4, 2018 and March 26, 2020, patients who underwent total thyroidectomy were enrolled and followed up in this study. This multicenter trial was conducted by three surgeons in three different hospitals. Patients with the following were excluded: < 20 years old or ≥ 70 years old, liver or kidney function abnormality, previous history of lymphatic or hematologic disease, diabetes mellitus, immune system suppression, autoimmune disorder, severe systemic disorder, anticoagulative medication intake, chemotherapy for other cancer, or expectation of other following surgery. The study protocol was approved by each Institutional Review Board of Severance hospital (IRB Approval No. 1-2018-0034), Kangbuk Samsung Hospital (IRB Approval No. KBSMC 2018-05-028), and Seoul St. Mary’s Hospital (IRB Approval No. KC18DDDT0299), Korea. No changes to methods after trial commencement are implemented.

Products used in this study

Both MegaShield® and Guardix-SG® are temperature-sensitive anti-adhesive agents which can transform from solution to gel form at body temperature. MegaShield® comprises ADM, poloxamer 407, HA, and 1,4-BDDE. Guardix-SG® does not contain ADM and comprises poloxamer, sodium alginate, and calcium chloride. The patients received 5 g (5 mL) of MegaShield® or Guardix-SG® according to their assigned group.

Randomization and blinding

The patients were randomly assigned to either the MegaShield® group or the Guardix-SG® group after the anesthetic effect subsided, and the randomization was performed at a 1:1 ratio through a permuted block randomization method with a fixed block size of four. The randomization was stratified by center and performed with SAS ver. 9.4 (SAS Institute, NC, USA) software by a professional statistician of the Clinical Trials Center Yonsei University Health System. Patient identification codes were assigned only to patients who were judged to meet the inclusion/exclusion criteria among those who needed total thyroidectomy. In addition, patients were given a randomization code (3 digits) in order according to the randomization table and assigned to the corresponding application group. The randomization code and patient identification code were used as the patient number during the clinical trial. During the study, the surgeons who performed the surgeries were not blinded as the packaging of the two devices were different. Meanwhile, patients and evaluator were blinded. The clinical assessor was an independent evaluator who did not participate in the surgery; therefore, it was not possible for him/her to know which investigational product had been applied to the patient. Since he/she contacted the patient only during evaluation, blinding was maintained. Since the assigned investigational product was applied in the patient while under general anesthesia during surgery, he/she could not know which investigational product had been applied, even after the surgery.

Surgical procedure

Standard bilateral total thyroidectomy was performed in all patients. Patients underwent open thyroidectomy with central lymph node dissection by a single surgeon (K.-H.N. in Yonsei college of Medicine, J.S.B in Seoul St. Mary’s Hospital, and J.S.Y in Kangbuk Samsung Hospital) in each of the centers. After total thyroidectomy, 2 mL of MegaShield® or Guardix-SG® was applied between the space of tracheal wall and strap muscle according to their group. After closing the midline of the strap muscles, 3 mL of the identical antiadhesive agent was applied between the overlying subcutaneous fat layer and strap muscle fascia.

Data collection and interventions

All data were prospectively collected by each of the three clinical assessors in each institution. Clinical visits were conducted for 30 days at 0 day before the surgery (visit 1), day of the surgery (visit 2), postoperative week 1 (visit 3), and week 6 (visit 4). During visit 1, the baseline demographic characteristics and clinical and laboratory assessments were thoroughly recorded after obtaining written informed consent. During visit 2, the investigational device (MegaShield® or Guardix-SG®) was applied on the surgical field. Participants were followed up twice (visits 3 and 4) after device application. Clinical and laboratory variables and adverse events were evaluated during all visits.

Outcome measures

Patient demographic findings including age, sex, and body mass index were documented. The primary outcome was the esophageal movement using marshmallow [25] at visit 4. With this evaluation, the affected esophageal motility due to postoperative adhesion was checked. Esophageal motility was classified as normal and scored 3 if the esophageal transit time of the bolus marshmallow in the prone position occurred within 30 seconds. The specified score classification of the marshmallow esophagography is presented in Table 1. The mean scores of both groups were compared. The secondary outcome was the assessed adhesion score at visits 1, 3, and 4, presenting the questionnaire survey to both the patients and the clinical assessors. The questionnaire included subjective discomforts in swallowing, cosmetic changes in cervical skin wrinkles answered by the patients, and objective adhesion severity evaluated by the clinical assessors. The evaluation criteria are presented in Table 2. Each criterion was assessed using a score from 0 to 10, in which a higher score represented a more severe adhesion. The sum of all scores and the change in scores from the screening day (visit 1) after surgery were compared between the two groups. The safety outcomes included adverse events and changes in laboratory findings, vital signs, and physical examination. All data on these outcomes were collected by the clinical assessors. No changes to trial outcomes after trial commencement are implemented.

Table 1

Classification of marshmallow esophagography.

Score	Definition	Transition time of marshmallow
3	Normal	Transition within 30 seconds in supine position
2	Mild	Transition after 30 seconds in supine position and within 30 seconds in erect position
1	Moderate	Cessation in supine position and transition after 30 seconds in erect position
0	Severe	Cessation in both supine and erect positions

Table 2

Adhesion score questionnaire.

Evaluation criteria
Subjective discomforts of patients	1. Is there difficulty in swallowing saliva?
	2. Is there difficulty in swallowing water?
	3. Is there difficulty in swallowing solid food?
	4. Do wrinkles around cervical area look abnormal?
Objective evaluation of clinical assessor	5. Do wrinkles around cervical area look symmetrical and natural with natural position?
	6. Do wrinkles around cervical area look symmetrical and natural with neck extension?
	7. Is there inflammatory reaction or scar formation?

Statistical analysis

The sample size was calculated to perform the non-inferiority test [26]. We supposed that the esophageal movement score (marshmallow esophagography score) of the test group (MegaShield) was not lower than that of the control group (Guardix-SG®) after thyroidectomy. Group sample sizes of 63 each achieved 80% power to detect non-inferiority if the lower limit of the one-sided 97.5% confidence interval (CI) of the mean difference was less than -0.1 (non-inferiority margin) using two-sample unpaired t-test. The non-inferiority margin was calculated based on a previous study [27]. In the study, the difference in scores between comparator (Guardix-SG®) and placebo was 2.93–2.73 = 0.2; therefore, assuming that, the margin is 0.2/2 = 0.1. Hence, we assumed the standard deviation of 0.2, non-inferiority margin of -0.1, and equal mean difference between the two groups. After accounting for an overall dropout rate of 10%, a total of 140 patients were required. The analysis sets consisted of intent-to-treat (ITT), modified intent-to-treat (mITT), and per protocol (PP). The ITT set included any patient who was treated at least once in the assigned group, while the mITT set consisted of any patient who belonged to the ITT set and was followed up for the first evaluation for estimating the effectiveness of the medical device of the assigned group. The PP set included any patient who belonged to the ITT and mITT set and completed the trial without any major protocol violations. The efficacy analysis was performed primarily on the mITT population, and concomitantly on the PP population. A safety analysis was performed on the ITT set. For primary outcome, 97.5% CI was established with unpaired t-test. For secondary outcome and safety analysis, chi-squared test or Fisher’s exact test was applied for categorical data, and unpaired t-test or Wilcoxon signed rank test was used depending on whether the normality assumption was satisfied (Shapiro-Wilk test) for continuous data. The significance level of each statistical analysis was set at p<0.05. SAS ver. 9.4 was used for statistical computation.

Results

Patient characteristics

The patients were enrolled between October 4, 2018 and February 11, 2020, and followed up until March 26, 2020. The patient enrollment is depicted in the flow diagram as shown in Fig 1. Among the 156 recruited patients, 16 were excluded due to screening failure and 140 were randomly assigned to either the MegaShield® group (n = 70) or the Guardix-SG® group (n = 70) and analyzed as the ITT set. There were 70 patients from Severance hospital, 44 from Kangbuk Samsung hospital, and 26 from Seoul St. Mary’s Hospital (S1 Table). No deviation occurred in the randomization process. One patient in the MegaShield® group was excluded due to loss of follow-up. One patient in the Guardix-SG® group was excluded due to the patient’s request after surgery. During the first efficacy evaluation, the marshmallow esophagography, 11 patients (7 patients in MegaShield® group and 4 in Guardix-SG® group) who swallowed saliva multiple times within 30 seconds or did not swallow saliva even after 30 seconds were considered as protocol deviation. Thus, mITT analysis was conducted on 127 patients (62 in MegaShield® group and 65 in Guardix-SG® group). After marshmallow esophagography, one patient in MegaShield® group was found to have consumed aspirin and was excluded from the study. Hence, PP set included 126 patients (61 in MegaShield® group and 65 in Guardix-SG® group). There were no significant differences in baseline clinical characteristics between the two groups (Table 3).

Fig 1

Disposition of patients.

Table 3

Demographic and baseline characteristics (ITT set).

	MegaShield (Test device)	Guardix-SG (Control device)
	(N = 62)	(N = 65)
Investigation center (n)
Severance	35	35
KangBuk Samsung	19	22
Seoul St. Mary’s Hospital	8	8
Age (years)
Mean	48.1	49.2
SD	11.2	10.8
Median	50.5	50.0
Range	(24.0–68.0)	(24.0–67.0)
Weight (kg)
Mean	66.7	65.7
SD	13.9	14.5
Median	64.9	64.0
Range	(40.0–100.3)	(44.0–118.0)
Height (cm)
Mean	163.0	162.4
SD	7.9	8.5
Median	161.0	161.0
Range	(147.0–184.0)	(145.0–186.0)
BMI (kg/m 2 )
Mean	25.0	24.7
SD	4.0	3.9
Median	24.4	24.3
Range	(15.6–34.3)	(18.4–36.4)
SBP (mmHg)
Mean	125.5	126.5
SD	10.8	13.3
Median	126.0	130.0
Range	(97.0–146.0)	(99.0–150.0)
DBP (mmHg)
Mean	77.3	78.8
SD	8.0	9.4
Median	79.0	80.0
Range	(54.0–100.0)	(59.0–97.0)
PR (beats/min)
Mean	81.5	78.9
SD	10.2	10.7
Median	82.0	77.0
Range	(54.0–105.0)	(56.0–102.0)
Sex (n [%])
Male	14 (22.6)	16 (24.6)
Female	48 (77.4)	49 (75.4)
Smoking (n [%])
Yes	1 (1.6)	1 (1.5)
No	61 (98.4)	64 (98.5)
Drinking (n [%])
Yes	10 (16.1)	7 (10.8)
No	52 (83.9)	58 (89.2)
Any medical history (n [%])
Yes	46 (74.2)	45 (69.2)
No	16 (25.8)	20 (30.8)

Efficacy results

As the primary efficacy outcome, the outcomes of the marshmallow esophagography were compared at visit 4 (Table 4). The mean score was 2.8 ± 0.6 in MegaShield® group and 2.6 ± 0.8 in Guardix-SG® group in mITT (p = 0.16) and PP set (p = 0.17) analyses. The 97.5% one-sided CI of the difference was -0.08–Inf., and the calculated lower limit of the one-sided CI exceeded the prespecified noninferiority margin of -0.1 demonstrating the noninferiority of MegaShield® on mITT set. Similarly, the 97.5% one-sided CI of the difference was -0.07–Inf., and demonstrated the noninferiority of MegaShield® on PP set.

Table 4

Summary of marshmallow esophagography results at six weeks after thyroid gland surgery, visit 4 (day 42±7).

	mITT set			PP set
	MegaShield (Test device) (N = 62)	Guardix-SG (Control device) (N = 65)	Mean Difference (97.5% one-sided CI)	MegaShield (Test device) (N = 61)	Guardix-SG (Control device) (N = 65)	Mean Difference (97.5% one-sided CI)
Marshmallow esophagography
Mean	2.8	2.6	0.16 (-0.08–Inf.)	2.8	2.6	0.17 (-0.07–Inf.)
SD	0.6	0.8		0.6	0.8
SE	0.1	0.1		0.1	0.1
Median	3.0	3.0		3.0	3.0
IQR	0.0	0.0		0.0	0.0
Range	(0.0–3.0)	(0.0–3.0)		(0.0–3.0)	(0.0–3.0)
Score frequency
Normal (3)	51 (82.3)	49 (75.4)		51 (83.6)	49 (75.4)
Mild (2)	9 (14.5)	11 (16.9)		8 (13.1)	11 (16.9)
Moderate (1)	1 (1.6)	1 (1.5)		1 (1.6)	1 (1.5)
Severe (0)	1 (1.6)	4 (6.2)		1 (1.6)	4 (6.2)

SD: standard deviation; SE: standard error; range: (Minimum–Maximum).

CI: confidence interval for the mean difference (test device—control device) based on unpaired t-test.

Predefined non-inferiority margin (-δ): -0.1.

The secondary outcome, adhesion scores, was assessed at visits 1, 3, and 4 using questionnaire survey in both the patients and the clinical assessors. Shapiro-wilk test (p<0.001) and Wilcoxon test were applied because the sum of the adhesion scores were not normally distributed. In mITT and PP sets, there were no significant differences between the sum of adhesion scores in every visit between the MegaShield® group and the Guardix-SG® group (Table 5).

Table 5

Summary of total adhesion scores by postoperative time.

Postoperative time	MegaShield (Test device)					Guardix-SG (Control device)					bp-value	cp-value based on WMWodds
	N	Mean	SD	Median	ap-value	N	Mean	SD	Median	ap-value
mITT set
Screening	62	8.82	4.02	6.00		65	9.02	4.40	6.00		0.816	0.7955
Visit 3 (Day 7±3)	62	13.10	7.82	8.00		65	13.74	7.84	9.00		0.135	0.6423
Change from screening	62	4.27	4.48	2.00	< 0.001	65	4.72	4.46	3.00	< 0.001	0.166	0.5698
Visit 4 (Day 42±7)	62	11.47	5.30	9.00		65	11.89	5.38	9.00		0.612	0.6523
Change from screening	62	2.65	3.14	2.00	< 0.001	65	2.88	3.42	2.00	< 0.001	0.704	0.6893
PP set
Screening	61	8.85	4.05	6.00		65	9.02	4.40	6.00		0.784	0.8278
Visit 3 (Day 7±3)	61	13.20	7.84	8.00		65	13.74	7.84	9.00		0.165	0.6970
Change from screening	61	4.34	4.48	2.00	< 0.001	65	4.72	4.46	3.00	< 0.001	0.212	0.6327
Visit 4 (Day 42±7)	61	11.54	5.32	9.00		65	11.89	5.38	9.00		0.685	0.7111
Change from screening	61	2.69	3.14	2.00	< 0.001	65	2.88	3.42	2.00	< 0.001	0.801	0.7464

SD: standard deviation.

a p-value for the comparison of change from screening in each device group by Wilcoxon signed-rank test at each time point.

b p-value for the comparison between two device groups by Wilcoxon rank sum test at each time point.

c p-value for testing WMWodds equal 1(Divine et al., 2018) at each time point.

Safety results

We compared the incidence of postoperative complications and adverse events with the ITT set analysis. Table 6 shows the summary of the adverse events. There were no significant differences in rates of any adverse event or serious adverse events between the two groups. Although adverse events occurred in 35 (50.0%) patients with 82 cases in MegaShield® group and 38 (54.3%) patients with 91 cases in Guardix-SG® group, most of the cases belonged to mild adverse events. Moderate adverse events occurred in one patient in MegaShield® group who had aspartate aminotransferase (AST) and alanine aminotransferase (ALT) elevation counted as two adverse event cases, in which the elevated levels recovered. In Guardix-SG® group, moderate adverse event occurred in one patient who had postoperative shoulder pain. Four serious adverse events, which resulted in prolonged hospital stay due to the exacerbation of the general condition of the patients, occurred in two patients of the MegaShield® group. One patient had postoperative fever (38.1–38.4°C) and the other patient had postoperative fever (38.1°C) and AST and ALT elevation. None of these adverse events were related to the use of anti-adhesive agents. Other adverse events included neck pain, hypothyroidism, hypoparathyroidism, and hypocalcemia. All these adverse events were not related to the usage of the anti-adhesive agents. Table 7 presents the summary of adverse events by organ system classification.

Table 6

Summary of adverse events (ITT set).

	MegaShield	Guardix-SG	Chi-square (P-value)
	(Test device)	(Control device)
	(N = 70)	(N = 70)
Patients with any adverse event	35 (50.0) [82]	38 (54.3) [91]	0.11 (0.735)
Patients with serious adverse events	2 (2.9) [4]	-	0.02 (0.181) a
Severity
Mild	35 (50.0) [80]	37 (52.9) [90]
Moderate	1 (1.4) [2]	1 (1.4) [1]
Severe	-	-
ID relationship
Definitely related	-	-
Probably related	-	-
Possibly related	-	-
Probably not related	-	1 (1.4) [2]
Not related	35 (50.0) [82]	38 (54.3) [89]
Unknown	-	-
Outcome
Recovered/Resolved	35 (50.0) [74]	37 (52.9) [79]
Recovering/Resolving	-	1 (1.4) [1]
Not recovered/Not resolved	8 (11.4) [8]	10 (14.3) [11]
Recovered/Resolved with sequelae	-	-
Fetal	-	-
Unknown	-	-

N (%) [] = number of patients reporting at least one adverse event in that category (percentage of patients among total N) [number of adverse-event occurrences].

Table 7

Summary of adverse events by organ system classification (ITT set).

System Organ Class	MegaShield (Test device)	Guardix-SG (Control device)
	(N = 70)	(N = 70)
Total	35 (50.0) [82]	38 (54.3) [91]
Musculoskeletal and connective tissue disorders	35 (50.0) [35]	35 (50.0) [36]
Neck pain	35 (50.0) [35]	34 (48.6) [35]
Musculoskeletal pain	-	1 (1.4) [1]
Endocrine disorders	12 (17.1) [15]	13 (18.6) [16]
Hypothyroidism	8 (11.4) [8]	9 (12.9) [9]
Hypoparathyroidism	6 (8.6) [6]	7 (10.0) [7]
Hyperthyroidism	1 (1.4) [1]	-
Metabolism and nutrition disorders	11 (15.7) [11]	12 (17.1) [12]
Hypocalcemia	11 (15.7) [11]	12 (17.1) [12]
Gastrointestinal disorders	3 (4.3) [4]	9 (12.9) [10]
Nausea	3 (4.3) [3]	5 (7.1) [5]
Constipation	1 (1.4) [1]	4 (5.7) [4]
Dysphagia	-	1 (1.4) [1]
Investigations	3 (4.3) [6]	5 (7.1) [9]
Aspartate aminotransferase increased	2 (2.9) [3]	5 (7.1) [5]
Alanine aminotransferase increased	2 (2.9) [2]	4 (5.7) [4]
Blood creatine increased	1 (1.4) [1]	-
Injury, poisoning and procedural complications	6 (8.6) [6]	4 (5.7) [4]
Post procedural swelling	4 (5.7) [4]	3 (4.3) [3]
Post procedural edema	1 (1.4) [1]	1 (1.4) [1]
Post procedural hematoma	1 (1.4) [1]	-
General disorders and administration site conditions	4 (5.7) [4]	1 (1.4) [1]
Pyrexia	4 (5.7) [4]	1 (1.4) [1]
Infections and infestations	1 (1.4) [1]	1 (1.4) [1]
Tonsillitis	1 (1.4) [1]	1 (1.4) [1]
Nervous system disorders	-	1 (1.4) [1]
Hypoesthesia	-	1 (1.4) [1]
Vascular disorders	-	1 (1.4) [1]
Hypertension	-	1 (1.4) [1]

N (%) [] = number of patients reporting at least one adverse event in that category (percentage of patients among total N) [number of adverse-event occurrences].

Discussion

This study was conducted through a randomized, controlled, double-blinded, multicenter, non-inferiority trial in order to evaluate the efficacy and safety of MegaShield®, a newly developed temperature-sensitive anti-adhesive agent. The efficacy and safety of ADM in prevention of adhesion formation after thyroid surgery was demonstrated by Kang et al., using ADM membrane (MegaDerm; L&C BIO, Seongnam, Korea). Although the study reported favorable results, the application of membrane-type anti-adhesive agent was considered more difficult than that of sol or gel forms, and adhesion formation at the suture site was of concern. Hence, we developed MegaShield®, a combination of temperature-sensitive sol-to-gel barrier and physical barrier with micronized ADM. In the control group, a commercially available temperature-sensitive anti-adhesive agent, Guardix-SG®, which was evaluated to be effective and safe in various surgical fields [28-30], but did not contain ADM was used.

In this study, the primary outcome was esophageal movement using marshmallow, which was objective, and the secondary outcome was the adhesion score, which was subjective. Although it is difficult to calculate the amount of adhesion, the phenotype of adhesion which is represented by dysfunction or certain symptoms of the patients can be evaluated by several measurements, and esophageal movement using marshmallow can be one of the choices. This is known to be useful as one of the good functional methods for detecting esophageal dysmobility and swallowing difficulty, which is triggered by the abnormal pharyngeal and upper esophageal muscle movement induced by post-thyroidectomy adhesion [31, 32].

The MegaShield® and Guardix-SG® groups did not show statistically significant difference in each criterion. In non-inferiority study, both ITT and PP group results are important [33], and we confirmed that they yield the same results. In addition, the trend of the adhesion score was noticeable. The adhesion score is a parameter to evaluate discomforts from postoperative adhesion such as swallowing difficulty, skin wrinkle, and scar formation, in which a higher score implies a more severe adhesion. In both MegaShield® and Guardix-SG® groups, the adhesion scores at visit 3 scored the highest and showed a decrease at visit 4. According to previous studies [34, 35], although scar remodeling process persists for more than two months, most of the significant scar changes can develop within two to three months after the surgery. However, preventive management for postoperative adhesion such as pulse dye laser or application of a silicone gel sheet were available after the 6-week period, and it was ethically difficult to deny the patients of the chance of additional treatments.

In our previous study [19], a histological analysis revealed that the deposition of collagen and elastin fibers increased at six months compared to that at one month after ADM implantation. Moreover, microvessel density increased at three and six months compared to that at one month after ADM implantation. These results suggest that slower and controlled extracellular matrix (ECM) remodeling lead to long-term structural integrity and increased durability. Although this trial focused on the acute phase of scar changes after open thyroidectomy, these findings suggest that the efficacy of MegaShield® which includes micronized ADM can be increased during long-term follow up.

In the safety assessment, there were no device-related complications. There were two patients who had serious adverse events in the MegaShield® group. Both of these patients had high fever (>38°C) without symptoms, in which one patient showed moderate increase in AST/ALT levels. Postoperative fever or AST/ALT elevation can be induced by analgesic agents used during the surgery, and we could not find direct connection between the application of the device and the adverse events. In the Guardix-SG® group, five cases of AST elevation and four cases of ALT elevation were counted as adverse events, in which none of them were device-related.

This study presents several limitations. First, there is no overall agreement on the criteria used for the adhesion score. The grading system we used is subjective and may not reflect the precise degree of adhesion. However, we also evaluated the objective assessment with the marshmallow esophagogram which can compensate the subjective grading system. Moreover, subjective symptoms of the patient can be considered as an important parameter as we can assess the effect of adhesion by the symptoms. Second, this study might require a subgroup analysis to determine if there is a difference in the results between the institutions; however, the number of cases was not enough for subgroup analysis. Third, we used Wilcoxon rank-sum test for the statistical analysis of this study, based on its widespread use to analyze non-normally distributed data, with the Shapiro-Wilk test. Although it is not entirely suitable as a median test, we applied the Wilcoxon rank-sum test to demonstrate if the two populations are equally distributed or not [36]. Fourth, this trial focused on the acute phase of scar changes after open thyroidectomy due to ethical issues. Although we did not restrict additional anti-adhesive treatment after the six-week period, an observational study for the two groups after the follow-up period may be conducted in future.

Conclusion

This randomized, controlled, double-blind, multicenter trial demonstrated that the efficacy and safety of MegaShield® was not inferior compared to that of Guardix-SG®. MegaShield® demonstrated the potential of ADM as a potential future anti-adhesive agent.

CONSORT 2010 checklist of information to include when reporting a randomised trial*.

(DOC)

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Total setting of the enrolled patients.

(DOCX)

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(PDF)

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(PDF)

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(XLSX)

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21 Sep 2021

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors reported the results of a randomized, multicenter, double-blind

Non-inferiority study to investigate the efficacy, determined by the extent of adhesion as assessed using Marshmallow esophagography 6 weeks after the surgery, of MegaShield ® compared with Guardix-SG ® in patients undergoing bilateral total thyroidectomy. They concluded, “The efficacy and safety of MegaShield ® was not inferior compared to that of Guardix-SG ®”.

In general I think the paper describes a well planned study, but the description of the result need to address the planning details much more specific. In the following. I will start with some more general comments:

• As the study aims to prove a non-inferiority hypothesis the appropriate CONSORT Checklist should be applied and the structure of the paper should follow the section/topics of the guideline. That helps by identification of the important information of this study type. Among other, the study typ as non-inferiority study should be included in the title. Please note also that ervey point in the guideline should be addressed, e.g. 3b should be commented as: No changes to methods after trial commencement are implemented. In particular this information is important as the trial register does not allow to follow the history of changes. See: Piaggio G (2012), PMID: 23268518.

• There is no appended individual patient level data file, so that reproducibility is limited.

• As most of the details, e.g. the non-inferiorty margin including justification, these information is included only on the side so that study objective is unclear most the time. This would be solved, if the CONSORT recommendation is followed strictly.

Detailed comments:

L83ff: Include the hypothesis and endpoint in the definition of the aim.

L105: Block size is missing in the description of PBR.

L105f: It is unclear whether the randomization is stratified by center.

L109: It is unclear how blinding is implemented. Please give details. I assume that treatments could not be blinded. Give details for blinding of clinical assessors.

L132: As biostatistican it is not clear to me, whether the rimary outcome in the manuskrip “extent of adhesion as assessed using Marshmallow esophagography” equals “esophageal movement using Marshmallow” or “” which is given in the protocol.

L161: Statistical tests on baseline characterisics is not. Useful in randomized trials. Delete column 4. Give numbers recruited patients in groups Per “center”.

L172f: Give more details for sample size calculation. I guess your argument is based on the z-statistic as in the protocol. Describe your arguments for the non-inferiority margin. Give arguments for the dropout rate, e.g. by referring to the literature. The ICH E9 stated to do the ITT and PP and they should yield the same results. SO you may proceed similarly by giving the two onesided 95% Cis. Actually, the method does not result in a conservative test decision and the method of Sanchez (Statistics in Medicine, 2006) may be a useful as an alternative. You just have to argue.

L183: I assume you mention statistical tests on the secondary endpoint etc. There are good arguments to avoid pretest for normality – so may be you delete that text. Second, the tests could not be associated to the parameters, so it is best to mention the test in the result section at places where it is used. Give the software used for statistical computation.

L187f: Describe the setting first. Report on enrollment time, patients per center and in particular deviations in the randomization process.

L193: I see the exclusion of 11 patients critical with regard to the ITT principle. As there are longitudinal measurements other strategies to handle missing observations may be more appropriate. In particular with regard to figure 1, where only 1 patient is withdrawn in each group.

L209f: Use the analysis population mentioned above. Skip the p-values.

- Give p-values with at least three decimal digits!

L226: Please improve the layout of the table5, so that no line break occurs in numbers. As the data are compared by Wilcoxon test, the appropriate statistic based on provision Probability should be stated (WMWodds, see Divine, 2018). On the other hand, the longitudinal measurements may be analysed by Mixed effects models, rather than splitting over time.

L288: Limitation should address and discuss potential sources of bias in the trial and success of implemented measures against bias.

L299: This is rather a result than a clinical conclusion. What does the result mean for practice?

Reviewer #2: Well designed study.

I would have liked a more detailed explanation of the marshmallow oesophagography technique. The transit time is used as a surrogate marker for adhesions. I would have liked to see some evidence or justification that an increased transit time correlates with the degree of adhesions.

The subjective scoring system to assess adhesions could be improved

**********

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Reviewer #1: No

Reviewer #2: No

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3 Nov 2021

November 4, 2021

Emily Chenette

Editor-in-Chief

PLOS ONE

Dear Editor:

Thank you for giving us the opportunity to submit a revised draft of our manuscript titled “Efficacy and Safety of Temperature-sensitive Acellular Dermal Matrix in Prevention of Postoperative Adhesion after Thyroidectomy: A Randomized, Multicenter, Double-blind, Non-inferiority study”

to PLOS ONE. We appreciate the time and effort that you and the reviewers have dedicated to providing valuable feedback regarding our manuscript. We are grateful to the reviewers for their insightful comments on our paper. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. The changes made within the manuscript have been highlighted to aid navigation.

Here are our point-by-point responses to the reviewers’ comments and concerns.

Comments from Reviewer 1

�  Comment 1: As the study aims to prove a non-inferiority hypothesis the appropriate CONSORT Checklist should be applied and the structure of the paper should follow the section/topics of the guideline. That helps by identification of the important information of this study type. Among other, the study type as non-inferiority study should be included in the title. Please note also that ervey point in the guideline should be addressed, e.g. 3b should be commented as: No changes to methods after trial commencement are implemented. In particular this information is important as the trial register does not allow to follow the history of changes. See: Piaggio G (2012), PMID: 23268518.

Response: Thank you for your valuable comment. In this revision, we did our best to comply with the CONSORT guidelines. We have also commented, “No changes to methods after trial commencement are implemented” in the methods section (page 4, paragraph 3, line 100).

�  Comment 2

There is no appended individual patient level data file, so that reproducibility is limited.

Response: It is not clear as to what the individual patient level data imply, as the PLOS ONE guidelines do not specifically obligate it. However, we have provided the score frequency in Table 4, which made it easier to analyze the primary outcome and arrive at the same conclusion.

�  Comment 3

As most of the details, e.g. the non-inferiorty margin including justification, these information is included only on the side so that study objective is unclear most the time. This would be solved, if the CONSORT recommendation is followed strictly

Response: Thank you for your comment which made the structure of this manuscript more systematic. As we have commented above, we have tried to follow the CONSORT recommendation strictly in the revised manuscript.

�  Comment 4

L83ff: Include the hypothesis and endpoint in the definition of the aim.

Response: Thank you for your comment. Accordingly, we have described the hypothesis and outcomes (page 4, paragraph 2, line 86–88).

�  Comment 5

L105: Block size is missing in the description of PBR.

Response: Thank you for your comment. Accordingly, we have added the information on the block size. (page 5, paragraph 2, line 111).

�  Comment 6

L105f: It is unclear whether the randomization is stratified by center.

Response: According to your comment, we have clearly stated that the randomization is stratified by center. (page 5, paragraph 2, line 111).

�  Comment 7

L109: It is unclear how blinding is implemented. Please give details. I assume that treatments could not be blinded. Give details for blinding of clinical assessors.

Response: Thank you for your valuable comment. We agree that the implementation of blinding can be explained more clearly based on the study protocol. We have added this information in the manuscript. (page 5, paragraph 2, line 114–121).

�  Comment 8

L132: As biostatistican it is not clear to me, whether the primary outcome in the manuskrip “extent of adhesion as assessed using Marshmallow esophagography” equals “esophageal movement using Marshmallow” or “” which is given in the protocol.

Response: Thank you for your insightful comment. We agree that the similarity of the expressions should be resolved. We think that the “extent of adhesion as assessed using marshmallow esophagography” is what we wanted to evaluate with “esophageal movement using marshmallow”. We have changed the expression in the manuscript to “esophageal movement using marshmallow”, following the expression in the guideline. To make this more logical, we have explained it in the discussion section. (page 17, paragraph 2, line 298–304).

�  Comment 9

L161: Statistical tests on baseline characterisics is not. Useful in randomized trials. Delete column 4. Give numbers recruited patients in groups Per “center”.

Response: Thank you for your suggestion. As per your comment, we have modified Table 1.

�  Comment 10

L172f: Give more details for sample size calculation. I guess your argument is based on the z-statistic as in the protocol. Describe your arguments for the non-inferiority margin. Give arguments for the dropout rate, e.g. by referring to the literature. The ICH E9 stated to do the ITT and PP and they should yield the same results. SO you may proceed similarly by giving the two one sided 95% Cis. Actually, the method does not result in a conservative test decision and the method of Sanchez (Statistics in Medicine, 2006) may be a useful as an alternative. You just have to argue.

Response: Thank you for your suggestion. I agree that a detailed explanation of the protocol should be presented in the manuscript. We have described it in the manuscript with the arguments for non-inferiority margin (page 8, paragraph 1, lines 168–174). Regarding the ICH E9, we have commented that we obtained the same results with both ITT and PP groups with the reference that you had recommended (page 17, paragraph 3, lines 305–307).

�  Comment 11

L183: I assume you mention statistical tests on the secondary endpoint etc. There are good arguments to avoid pretest for normality – so maybe you delete that text. Second, the tests could not be associated to the parameters, so it is best to mention the test in the result section at places where it is used. Give the software used for statistical computation

Response: I appreciate your professional comment. Based on your comment, we have deleted the relevant sentence. We included the details of the software used for statistical computation (SAS ver. 9.4; SAS Institute, NC, USA) at the beginning of the paragraph (page 8, paragraph 1, line 183-184). Further, if possible, we would like to add footnotes to the tables pertaining to the correlated statistical test to add clarity to the manuscript.

�  Comment 12

L187f: Describe the setting first. Report on enrollment time, patients per center, and in particular deviations in the randomization process.

Response: I appreciate your comment. We have described the setting including enrollment time and patients per center. We have also commented that no deviations occurred during the randomization process. (page 8, paragraph 2, lines 188–189 and page 9, paragraph 1, lines 191–193).

�  Comment 13

L193: I see the exclusion of 11 patients critical with regard to the ITT principle. As there are longitudinal measurements other strategies to handle missing observations may be more appropriate. In particular with regard to figure 1, where only 1 patient is withdrawn in each group.

Response: We admit that Figure 1 may be appear confusing to the readers. Therefore, we have revised it to explain the whole patient setting. Further, the data of the patients who were included in ITT but excluded in mITT were not formally approved at the time of the first postoperative assessment (visit 3). Alternatively, there were no missing observations to substitute in mITT group.

�  Comment 14

L209f: Use the analysis population mentioned above. Skip the p-values. Give p-values with at least three decimal digits!

Response: As we have commented previously, it seems that the analysis of mITT group does not contain statistical problems. We have described this in the discussion section, which also corresponds to the answer to your comment 10.

�  Comment 15

L226: Please improve the layout of the table 5, so that no line break occurs in numbers. As the data are compared by Wilcoxon test, the appropriate statistic based on provision Probability should be stated (WMWodds, see Divine, 2018). On the other hand, the longitudinal measurements may be analysed by Mixed effects models, rather than splitting over time.

Response: We thoroughly read the article written by Divine et al. Indeed, the Wilcoxon test has a limitation as a test of medians. However, this study aimed to evaluate the difference between two groups rather than perform a median test. As we had planned to use the Wilcoxon-Mann-Whitney test for statistical analysis in this study, based on its widespread usage for analysis of non-normally distributed data, we would like to retain the statistical results. We have described this as a limitation in the discussion section (page 18, paragraph 4, lines 334– page 19, paragraph 1, line 337).

�  Comment 16

L288: Limitation should address and discuss potential sources of bias in the trial and success of implemented measures against bias

Response: We agree that we must add a comment pertaining to the potential source of bias in this study. However, as evident from the study guideline (11.3.1), we attempted to minimize the statistical biases with double-blinding and randomization generated using SAS by a statistical expert who was not directly related to the institution. For blinding, before the study was terminated, only a minimum number of personnel who are not directly related to the institution were able to access the randomization table and code. Regarding financial bias, this study was stated to be sponsored by L&C bio in the protocol; however, the study grant was finally sponsored by the Ministry of Health & Welfare as mentioned in the financial disclosure.

�  Comment 17

L299: This is rather a result than a clinical conclusion. What does the result mean for practice?

‘Response: We appreciate your comment. We have added a sentence to the conclusion to show the clinical usefulness of this study (page 19, paragraph 2, line 342–344).

Comments from Reviewer 2

�  Comment 1

Well designed study.

I would have liked a more detailed explanation of the marshmallow oesophagography technique. The transit time is used as a surrogate marker for adhesions. I would have liked to see some evidence or justification that an increased transit time correlates with the degree of adhesions.

The subjective scoring system to assess adhesions could be improved.

Response: We appreciate your comment. We agree that the association between the extent of adhesion and the transit time on marshmallow esophagography can be explained more thoroughly. Based on your comment, we have added more references and relevant explanations. (page 17, paragraph 2, lines 298–304).

In addition to the above comments, all spelling and grammatical errors have been corrected.

We look forward to hearing from you in due course regarding our submission and will be glad to respond to any further questions and comments that you may have.

Kee-Hyun Nam

Address: Department of Surgery, Yonsei University College of Medicine,

50-1, Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea

Phone: +82-2-2228-2100

Email: khnam@yuhs.ac

Submitted filename: Response to reviewers.docx

Click here for additional data file.

17 Dec 2021

2 Jun 2022

Journal Requirements

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: As per your comment, we have changed or deleted several references. The four references listed below were not retracted articles, but were published in Korean journals, which means that they were not internationally acceptable. We apologize for our disregard for proper referencing.

Reference 3 (Oh et al. Trends of anti-adhesion adjuvant-review) was deleted. The journal (biomaterials research) was originally a Korean journal and became an international journal after 2014. Unfortunately, the reference we had used was from 2013. We found that deleting this reference made no difference in the content, so we decided to delete this article from the reference list.

Reference 12 (Lee et al. The efficacy and safety of HA/CMC anti-adhesion barrier solution with varying viscosities) was changed to reference 11, which is a reference from an international journal (Leach et al, Reduction of postsurgical adhesion formation in the rabbit uterine horn model with use of hyaluronate/carboxymethylcellulose gel. Fertility and sterility. 1998;69: 415-418).

Reference 26 and 27 (Song et al. Clinical value of marshmallow esophagography in detecting esophageal dysmotility and Park et al. Significance of functional esophagogram with a marshmallow bolus) were changed to reference 25, which is an internationally acceptable reference (Ott et al. Evaluation of the esophagus with a marshmallow bolus: Clarifying the cause of dysphagia. Gastrointestinal radiology. 1991;16: 1-4

Comments from Reviewer 1

Comment 1: Thank you for considering my comments. There is only one point to be addressed in the final review. The presentation of the results of the Wilcoxon test needs providing the appropriate statistic based on provision Probability, i.e. WMWodds (see Divine, 2018).

Response: We thank the reviewer for this valuable comment regarding the appropriate reference for the latest analysis. Accordingly, the results are now presented in Table 5 (page 13). We used the SAS program provided in Divine et al., (2018). We hope that this revision is in accordance with the statistical standards of the journal.

Submitted filename: Responst to Reviewers.docx

Click here for additional data file.

5 Aug 2022

Efficacy and Safety of Temperature-sensitive Acellular Dermal Matrix in Prevention of Postoperative Adhesion after Thyroidectomy: A Randomized, Multicenter, Double-blind, Non-inferiority study

PONE-D-21-04211R2

Dear Dr. Nam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Miquel Vall-llosera Camps

Senior Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Substitute "p-value for the median comparison between two device groups by WMWodds (Divine et al., 2018) at each time point" by

p-value for testing WMWodds equal 1 (Divine et al., 2018) at each time point

Reviewer #2: The revised manuscript has addressed all the comments raised previously. Well written and researched

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

9 Sep 2022

PONE-D-21-04211R2

Efficacy and Safety of Temperature-sensitive Acellular Dermal Matrix in Prevention of Postoperative Adhesion after Thyroidectomy: A Randomized, Multicenter, Double-blind, Non-inferiority study

Dear Dr. Nam:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Staff Editor

PLOS ONE