PONE-D-21-20663Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercisePLOS ONE

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2. In your Methods, please provide the product number for the KRG extract you obtained from Korea Ginseng Corporation, and include details of any quality assessments provided.

3. Please provide details about any efforts to minimise distress during the jumping task.

4. To comply with PLOS ONE submissions requirements, please provide the method of euthanasia in the Methods section of your manuscript.

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6.Please state specifically whether the animal ethics committee specifically approved the study.

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Reviewers' comments:

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Comments to the Author

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

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Reviewer's comments on paper “Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercise” submitted to Plos One (PONE-D-21-20663).

Although some interesting results seems to be reported in this work, Authors didn't draft the manuscript well and I cannot recommend the publication of the article in its present form.

The (L24) abstract and introduction (L49-59) suggest that the aim of the study was to compare the effect of KRG with Jump-exercise in rat osteoporosis model. There is no explanation how 11 wk-old (male rats can serve as a osteoporosis model animal - no osteoporosis agent was introduced to animals in this study (ovariectomy, orchidectomy, hypophysectomy, parathyroidectomy, immobilization, or dietary manipulation). In rats, first osteoporotic changes in long bone metaphysis occurs in older animals, peak bone mass in trabeculae of femur metaphysis is reached at the age of ca. 6 months. On the other hand, the last sentences of the introduction indicate that the true aim of the study was to assess the influence of KRG as an effective strategy for improving bone strength during growth (L91) and compare this effect with well-known method of stimulation of bone formation in young adults – exercises. Therefore, I suggest the correct the manuscript by removing most of the osteoporosis-related fragments and make it cleaner that this study is focused on the young individuals during development.

There is only one dose examined, which selectin was based on a single reference data in which a different osteoporosis model (OVX) was under study. In this manner, the idea comparison of KRG (dietary supplement) with jump-exercised group is not sufficiently explained.

Limitations of the study should be extended as there are three main limitation of this study: the mentioned issue with the single-dose selection (and a single jump regime in lesser extent), the lack of the KRG+JUM group and the single time-point analysis.

Distal femoral histomorphometry was performed both using uCT and histological analysis. Were these results somehow consistent? It should be discussed.

Minor comments (selected):

L31 the information about KRG form is missing – extract? powder?

L53 the direct reference to WHO should be given (but I recommend remove all fragments related to primary osteoporosis)

L53 “bone destruction” – rephrase

L85 add information that this correspond to “rat model studies” as all references [25-27] are rat-related

L117 was it pure EtOH or phosphate-buffered EtOH?

L142 copper

L157 what was the mineral density of hydroxyapatite phantom

L173-174 I think that this information should be placed earlier, when animal management is described

L178 Correct to Villanueva osteochrome bone stain. Also was it ready-to-use kit ? If yes, please give the information of manufacturer.

L179-186 How all of these indices were calculated? what software was used ?

Statistics: As you present the results of statistical analysis as a single comparison of KGR group vs CON or vs JMP, why didn't you perform simple t-test or contrast analysis of KGR vs appropriate group instead of Tukey’s test where all three groups are examined ?

L203 What was the idea of presenting the weight of the calf muscle ? Was the increase of muscle weight expected (any references?)? Also, why tight muscle, which is more appropriate for any femur analysis than calf, was not examined? Finally, if some changes in muscle structure were expected, muscle histology also should be performed.

L301 hPTH? What is the connection of human parathyroid hormone with current study ?

Fig 2 - No A, B ... H on figure 2.

Fig 5 - more interesting than showing trabeculae would be showing calcenin labeling lines

which are barely visible in presented figures

Reviewer #2: Review of PONE-D-21-20663

Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercise

General Comments

This study examined the effects of KRG on the trabecular bone of the distal femur in young growing male Wistar rats. Trabecular architecture and dynamic histomorphometry were employed as primary measures, complementing with computational models of the extracted trabecular architecture. Only the distal femur trabecular compartment was measured, with no axial skeleton examined and no diaphyseal cortex examined. As a comparator, the jump exercise model was examined in a matched cohort of rats, demonstrating a larger positive effect on this compartment. Both models suggested a shift to increased osteoblast numbers and decreased osteoclast numbers. The typical combined therapeutic (KRG + exercise) was not examined in this study, but has likely been pursued by this group (I encourage inclusion of such data in this paper if available).

Many details regarding the computational models are not included and need to be clarified in the manuscript. Some additional computational series should be run to answer the questions posed in the specific comments below.

Jump exercise had a measured effect of reducing femur length by 0.6-0.7 mm, which is a substantial amount. As an initial bound on this examination, t-tests performed without statistical penalty for JUM vs KRG and JUM vs CON demonstrate p values of 0.014 and 0.057, substantially more indicative of real difference than afforded by osteoblast numbers that are noted in presentation of results with p values > 0.5 due to variation. What occurs with jump exercise to stunt longitudinal growth? Is there damage to the physis?

The importance of ginseng processing is detailed, but the processing and verification of constituents and potency of the commercial product used in this study were not measured or reported. A test of residual KRG product should be performed prior to publication of this study.

The study would be improved with the inclusion of measurements for the axial skeleton (e.g. lumbar vertebrae) and cortical bone (e.g., femoral mid-diaphysis cortex) as is customary for bone morphometry measurements in studies of this nature. These data should be included for publication.

Specific Comments

Line 33: State the actual estimated jump height, and not the box height of 40 cm, as that seems misleading. See comment below regarding line 122.

Lines 75-77: These studies thus implicate direct action on osteoclasts. Did any of these in vivo studies perform histological measurements?

Lines 93-94: Cite references that provide data demonstrating this conclusion of ideal.

Line 109: Should “anesthetized” be “euthanized”?

Line 117: Why are no histological data reported in the abstract methods and results? They are very important.

Line 122: As described with a 40 cm tall box, how high must the rats jump from standing stretched position in order to grasp the box wall? It seems that the jump height would be 10-20 cm. Please clarify in the manuscript.

Lines 168-169: The method and algorithm implemented for assigning elastic modulus based on micro-CT derived tissue mineral density (using the Carter & Hayes relationship) must be provided. Was there any volumetric averaging of discrete values or was every voxel assigned a unique value? (unique assignment to each voxel does not seem justified)? Are your results different if one homogeneous value of elastic modulus is assigned to all regions of all models? Typically, trabecular structure dominates the variance in modulus assigned via such mineralization rules. Most importantly, what failure criterion was implemented for strength?

Lines 129-130: Measurements should have been performed to assess the true composition of ginsenosides, etc. within this commercial sample of KRG. Very frequently the true composition of such products is not as stated on the label.

Line 166: The boundary condition of uniform applied force rather than uniform displacement (Fig 1E) does not seem realistic, given the heterogeneous assignment of modulus values. That is, the lack of geometrical constraint due to extraction of trabecular bone, along with the spatial difference in elastic modulus, would seemingly allow each boundary location of applied force to deform differentially and unrealistically relative to the anatomical condition.

Line 166-168: What is the basis for selecting 2.8% as a threshold for defining failure. How do the results change if that value is perturbed upward and downward? Most importantly, what material failure criterion was applied in the definition of failure? This is a critical part of this method.

Lines 216-217: The stated differences in vBMD of 2% and 4% for jump exercise relative to control and KRG do not match the bar charts in Figure 2, which show large percentage differences similar to BV/TV (as they should). The data in figure 2 are almost certainly values of vBMD (i.e., apparent density) and report essentially the same comparative data as BV/TV. Presumably this statement regarding 2-4% pertains to the tissue-level density, which typically varies at most by a few percent. However, the manuscript makes no reference to tissue mineral density measurement throughout.

Lines 237-238: It does appear that osteoblast numbers were 25-30% higher in the experimental groups, but that measurement variation swamped the statistical comparison. It might be more insightful to the reader to provide the information in that manner.

Table 2: The histological data indicate that KRG lowered BFR/BS relative to control and jump exercise. Is this secondary to depletion of osteoclasts?

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Reviewer #2: No

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Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercise

PONE-D-21-20663R1

Dear Dr. Yong-In Ju,

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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Additional Editor Comments (optional):

Reviewers' comments:

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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

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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

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

Reviewer #1: Yes

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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: (No Response)

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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

**********

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: Dear authors,

The manuscript has been substantially improved, so my recommendation is to accept the manuscript in its present form.

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Reviewer #1: No