Topical Retinol Restores Type I Collagen Production in Photoaged Forearm Skin within Four Weeks

First Round of Evaluation

Round 1: Reviewer 1 Report and Author Response

Report: This is a very important contribution in the field of cosmeceuticals. Recommended for acceptance.

Response: We thank the reviewer for his/her evaluation.

Round 1: Reviewer 2 Report and Author Response

Report: This clinical trial shows that a relatively short term topical application with 0.4% retinol under occlusion may increase the collagen I levels to those of young subjects. The data are convincing and confirm the interest of topical vitamin A in cosmetic formulations.

Below are some minor points to address before publication:

1.         Introduction, page 1, line 31: the percentage of collagen I in the dry skin appears very high (90%); some authors indicate that this percentage corresponds to the total skin collagen content.
Please check.

2.         Introduction, page 1, line 54: this sentence may be confusing. ROL is actually vitamin A and not a vitamin A analogue. It would be better to re-write this sentence for more clarity. It may be indicated that RA is the endogenous active form of vitamin A.

3.         Materials and Methods, page 2, lines 85–87: Why applying ROL and its vehicle to the same arm (even at a one-inch distance)? It would have been better to use the other arm for the vehicle in order to ensure no contamination of the vehicle treated area by ROL.

4.         Materials and Methods, page 3, line 120: How was erythema assessed? Indicate the procedure in the Materials and Methods section.

5.         All-trans retinal (RAL) is the natural intermediate between ROL and RA. Is there a reason to use topical RA or ROL instead of RAL? This can be added to the Discussion section.

Response: We thank the reviewer's constructive comments. We have revised the manuscript based on the Reviewer’s comments. Point-by-point responses are provided below.

Introduction, page 1, line 31: the percentage of collagen I in the dry skin appears very high (90%); some authors indicate that this percentage corresponds to the total skin collagen content. Please check.

We changed line 31 to “Collagen fibrils, which comprise the bulk of the dermis, provide structural integrity and resilience to the skin. Collagen fibrils are primarily composed of type I collagen (COL1).”

Introduction, page 1, line 54: this sentence may be confusing. ROL is actually vitamin A and not a vitamin A analogue. It would be better to re-write this sentence for more clarity. It may be indicated that RA is the endogenous active form of vitamin A.

We changed line 54 to “Vitamin A (all-trans-retinol, ROL) can be converted to retinal (RAL), which is further metabolized to all-trans-retinoic acid (RA) in epidermal keratinocytes. RA is the endogenous active form of ROL and RAL. RA exerts biological function via binding to nuclear receptors and thus regulating gene expression.”

Materials and Methods, page 2, lines 85–87: Why applying ROL and its vehicle to the same arm (even at a one-inch distance)? It would have been better to use the other arm for the vehicle in order to ensure no contamination of the vehicle treated area by ROL.

We added the following sentences to Materials and Methods. “Photo-damage between the left and right forearm in the same person may differ. To avoid this potential variation, treatments were applied on the
same arm.”

Materials and Methods, page 3, line 120: How was erythema assessed? Indicate the procedure in the Materials and Methods section.

We added the following sentences to Materials and Methods. “Skin erythema was assessed by objective measurement using a chromameter (Minolta CR200; Minolta, Osaka, Japan) and subjective visual evaluation by an experienced investigator. Results obtained by both methods were similar. The erythema level measured by chromameter was shown in the supplemental data.“

All-trans retinal (RAL) is the natural intermediate between ROL and RA. Is there a reason to use topica RA or ROL instead of RAL? This can be added to the Discussion section.

We added the following sentences to the Discussion: “We investigated ROL rather than RA and RAL because pharmaceutical grade ROL was readily available at the time when we performed this study.”

Round 1: Reviewer 3 Report and Author Response

Report: The manuscript is well written and the study described is well designed. Few questions for authors are as follows:

·           Have the authors studied the effect of application of control moisturizing lotion alone (without ROL) on collagen production in skin? Since both controls used in this study- aged and young have received the moisturizing lotion treatment, it is difficult to differentiate the effect of vehicle alone on collagen production.

Response: We added the following sentence to the Materials and Methods. “The control moisturizing lotion alone does not affect COL1 synthesis or induce histologically noticeable changes when used in various treatment regimens (authors’ unpublished observations).”

·           The authors describe in the results section that occlusion enhances response to ROL. Do the authors have any comments on effect of occlusion on control (vehicle only) skin samples?

Response: We added the following sentence to the first paragraph of the results section. ”The expression of CRABPII did not differ between occlusion versus no occlusion on vehicle-treated skin, indicating that occlusion per se does not stimulate the action of ROL.”

·           Results: Page 3-lines 133–136 and Page 4-lines 143–146, I would recommend authors to use the same units to compare between groups, either % difference or xx fold difference.

Response: We changed to use fold changes to describe differences.

·           Can authors provide additional discussion on why ROL treatment for 1 day per week for
4 weeks was better as compared to continuous ROL treatment?

Response: It is not clear to us what “continuous ROL treatment” and “better” in the comment refers to. We assume that the comment refers to the data that a single ROL treatment under 4-day or 7-day continuous occlusion did not reproducibly enhance COL1 synthesis, whereas once daily occlusion of ROL for 4 weeks did. Unfortunately, the mechanism by which ROL enhances COL1 production is not known, which does not allow us to provide an evidence-based discussion on why one ROL regimen is superior in enhancing COL1 production than another regimen.

Second Round of Evaluation

Round 2: Reviewer 2 Report

Report: The authors gave satisfactory answers to the questions asked by the referees, and changed the text accordingly. I have no more comments.

Round 2: Reviewer 3 Report

Report: Thank you for the response to the review comments.