Ms. Ref. No.: AANAT1606 Title: Topographical Variations in Articular Cartilage and Subchondral Bone of the Normal Rat Knee are Age-Related Annals of Anatomy

Received Feb 05, 2014

Decision 1 Feb 21, 2014

Revision 1 received Apr 01, 2014

Decision 2 Apr 16, 2014

Revision 2 received Apr 24, 2014

Accepted Apr 28, 2014

1. Decision letter

Dear Dr. Hamann,

The reviewers have commented on your above paper. They indicated that it is not

acceptable for publication in its present form.

However, if you feel that you can suitably address the reviewers' comments (included

below), I invite you to revise and resubmit your manuscript.

The comments provided by the reviewers very often considerably help to improve and

strengthen a paper. In addition, the authors' response letter accompanying a revised

version often contains important information which may be regarded as an added

value to the final version of the manuscript. Therefore, Editors decided that the non-

confidential comments of the reviewers and the non-confidential authors' responses

will be published as online supplementary material together with the final version of

an eventually accepted article.

Please carefully address the issues raised in the comments.

If you are submitting a revised manuscript, please also:

a) outline each change made (point by point) as raised in the reviewer comments

AND/OR

b) provide a suitable rebuttal to each reviewer comment not addressed

I look forward to receiving your revised manuscript.

Yours sincerely,

Friedrich Paulsen

Editor-in-Chief

Annals of Anatomy

Reviewers' comments:

Managing Editor: Dear authors,

the manuscript has been reviewed by 3 experts, and at least two of them feel the

article meeds a substantial revision before being acceptable for publication.

Please address their concerns by answering each of their comments in itemized form,

preferably by

- providing a comment to each of the suggestions

- and by outlining what was done in the text to address this concern by an "author

action item" below each comment.

Reviewer #1: This manuscript deals with a highly interesting topic lying in the scope

of Annals of Anatomy.

It nicely presents topographical variations of all major components of the

osteochondral unit in dependency of the state of maturation. For studies dealing with

changes in OA in rat models this study provides essential data for comparing the

results.

The authors investigate both morphological and structural parameters as well as

material properties. The methods used are appropriate. Statistical analysis is correct.

The study design is well documented, the results are clearly written. The conclusions

and interpretations in the discussion are logical and put into a greater context

concerning biomechanical implications. I therefore recommend to accept this paper

for publication after some minor revisions.

- I suggest to shorten the discussion.

- Figure 1: Instead of this simple scheme I propose to take a real picture of the tibia or

a 3D reconstructed picture of Mikro-CT and overlay the measuring points.

- Line 40: … while a high porosity of subchondral bone plate (should be added)

- Line 91: animals and as well (and should be omitted)

Reviewer #2: The manuscript describes the use of µCT technique and biomechanical

testing for investigations of the topography of the respective joint components at 2

time points: The presented study is considered to be very helpful and an important

basis for knee joint models in rats. Some comments and additional recommendations:

1) Please correct numbering of Figures. In the manuscript Fig.3 refers to aggregate

modulus, but Fig.3 shows C.Th. Figure 4 and 5 are messed up, too. Reference to Fig.2

in line 252 describes what is shown in Fig.3. Figure 5A is not mentioned in the

manuscript, but the differences clearly visible there should be discussed.

2) In line 263 the authors refer to Fig.3 showing no differences in aggregate moduli

between lateral and medial compartment (Fig.3 is C.Th. as said). However, there is no

figure at all that shows stress curves of cartilage, but of rubber sheets only (Fig.2). I

understood that rubber sheet curves are methodological standard curves. They are not

necessarily needed in the manuscript. I recommend replacing them by or add curves

showing differences between 7 and 13 week old cartilage as described in line 263/264.

3) Fig.1 shows locations of cartilage measurement. It appears as if different distances

to the plateau boundary were chosen for lateral and medial compartments. If so, why?

And how were those exactly defined?

4) The authors have chosen to investigate 7 and 13 week old rats. What was the

rational to address these time points? Please discuss. It is not surprising that the

cartilage and subchondral bone plate have different properties between pre-mature

and mature rats. I encourage the authors to compare their finings with later time

points in following experiments.

5) In line 111 the authors mention that they have stored the samples at -20°C, but no

storage time and storage procedure is given (e.g. if all samples were frozen over the

same period). It is well known that freezing, but as well fixation methods will

influence humidity, density and biomechanical properties of tissues like cartilage or

menisci. Please add information about standardization of storage duration and discuss

potential limitations of cartilage measurements due to storage conditions. It is

important to exclude that age differences between 7 and 13 week samples in

aggregate measurement are not storage artifacts, e.g. because 7 week samples have

been frozen over 6 weeks until the 13 week animals were euthanized, while 13 week

samples were scanned freshly.

6) Please add in mat&meth section whether you used any filters for µCT scanning.

7) How was the VOI determined in its sagittal dimension? How were lateral and

medial compartments separated? Did the authors use standardized geometric figures

(e.g. cylindrical) or manually drawn interpolated ROIs based on anatomical markers?

Please add to mat&meth section. If the authors have analyzed the whole area shown

in Fig.5A over the described number of 100 and 125 slides, BV/TV may have been

underestimated by including proximal and distal condensed areas. Therefore, in

Fig.5A differences in BV per µm³ between lateral and medial compartment may

appear more prominent than depicted by Fig.5C. Smaller VOIs could be beneficial in

this setting.

8) Different thresholds were needed for binarization of 7 and 13 week old rats. Why?

Please add BMD analysis or grey scale indices and discuss, especially exclude

potential storage artifacts (see #5).

9) Line 378-380: Age-dependency yes, but the study is limited to 7 and 13 weeks. To

conclude articular cartilage differentiates later or slower than subchondral bone would

need more time points and longitudinal measurement. Please rephrase.

10) Please rephrase lines 295-298 "There was a significant (p<0.05) compartment

effect on the Tb.N and Tb.Sp, with higher values at the medial compared to the lateral

compartment for the Tb.N and lower values at the medial compared to the lateral

compartment for the Tb.Sp (Table 1)." Data for Tb.N and Tb.Sp in medial and lateral

compartments are equal for 7 weeks time points. The authors have described that in

the next sentence. Rephrase or delete that sentence.

11) Starting in line 389 the authors discuss why differences between lateral and

medial compartment in cartilage thickness and subchondral bone did not translate into

different compressive properties. Did cartilage thickness and equilibrium modulus not

correlate positively only in the earlier experiment from 2013 (line 396) or in this study,

too? Particularly when discussing that "cartilage mechanical quality is rather

determined by its organization and composition than its thickness" it is crucial to

make sure that changes in mechanical properties are not caused by storage conditions.

It should be added that maybe the method is not sensitive enough to measure minor

differences in equilibrium modulus between lateral and medial compartments.

12) Please reassess statistics. In line 228-230: The authors made a two-way

(compartment x age) ANOVA with repeated measures to examine the effect of

compartment and age on tibial articular cartilage and subchondral bone. 2-way

ANOVA with repeated measures is unsuitable, as different animals were used for

different time points.

Reviewer #3: The study is generally interesting in within scope for readers of Annals

of Anatomy. Methods and study design are adequate. However, the manuscript is

poorly written, with shortcomings in English language and some sloppy mistakes (e.g.

figure captions in the text). The manuscript maybe accepted after a major revision,

preferably assisted by an expert in English medical writing.

General comments:

- A hypothesis that can be tested and then supported or declined would be very

helpful. Otherwise the study could be titled exploratory.

- Please try to use abbreviations sensibly. Don't introduce abbreviations which are not

used throughout the text, and don't use them several paragraphs later than introduced,

but reintroduce then.

Specific comments:

Abstract:

- The abbreviation for micro CT (µ CT) is established but not used throughout the

abstract - please remove

Introduction:

- Page 3, lines 58-60: The verb "to arise" used in a past form ("arose") here does not

fit in this context. I think the authors want to say something like "…cartilage differs

depending on the loads it is subjected"

- Page 3, lines 64-67: Please provide a reference for this statement.

- Page 3, line 71: What is meant by "cartilage receives the primary impact"? Cartilage

is not a shock absorber, but distributes loads homogeneously to the subchondral bone.

- Page 3, line 72: I do not have the work by Layton et al at hand, but a current work of

Malekipour et al. (2013, http://dx.doi.org/10.1016/j.jmbbm.2013.05.005) might be

better suited. However, it should be stressed that neither cartilage nor subchondral

bone can effectively absorb energy from impacts.

- Page 4, lines 78-80: Maybe it could be explained what "stress raisers" mean in the

context of subchondral sclerosis.

- Page 4, line 85: Please substitute "creatures" with "models" or something similar.

- Page 4, line 87-90: Please use abbreviations only if they are used further in the text.

"MIA", "MMT" and "ACLT" seem to be used nowhere else in the text.

- Page 4, line 90-92: reads like a German to English translation with BabelFish

(http://www.babelfish.com/)

Material and methods:

- Page 5, lines 124-126: Please use abbreviations only if they are used further in the

text. "NaCl", EDTA", "HCL" and "NEM" seem to be used nowhere else in the text.

Although these are common used abbreviations, there is no point in using them, if not

needed further in the text.

- Page 5, line 127: The adjective "perpendicular" needs to be used as "perpendicular

to…" I think the authors would want to use "upright" or something similar.

- Page 5, line 129: Perpendicular to what?

- Page 7, line 157: 10, 15 and 20% strain are substantially more than reported

physiologically and in-vivo for human knee cartilage (5-6%). Please give a rationale

for these amounts.

- Page 7, line 158: What is meant by "equilibrium"? "Cartilage deformation

equilibrium" usually means that deformational, external forces and cartilage internal

pressure are in equilibrium, with no more deformation. As here deformation is fixed, I

do not understand what is meant by equilibrium.

- Page 8, line 177: The abbreviation "µCT" which was introduced in the abstract is

used as sub header. The sub header should be spelled out, and the abbreviation

introduced here.

- Page 8, line 182: Perpendicular is again used to describe an upright position of the

tibia. Please substitute "perpendicular" or describe perpendicular to what - ground

surface or table surface.

- Page 8, line 183: Please use abbreviations only if they are used further in the text.

"EtOH" seems to be used nowhere else in the text.

- Page 8, line 185: What was the orientation of the 2D images in relation to the

cartilage surface or the tibial axis?

- Page 10, line 233: Please spell "p-value"

- Page 10, line 233: It seems that the numerous cartilage and bone measurements are

all correlated against each other. In this case the p-value needs to be adjusted for

multiple testing via Bonferroni correction.

Results:

- Page 11, lines 247-249: Please be very careful about presenting non-significant

results as trends (difference between medial/lateral cartilage thicknesses in 13-week

old rats).

- Page 11, line 252: Probably "cartilage" is missing in "…articular compared..."

- Page 11, lines 252 and 257: The text references "figure 2", but the corresponding

figure is figure 3

- Page 11, line 263 and page 12, lines 266 and 268: The text references "figure 3", but

the corresponding figure is figure 4. The caption seems to correspond to figure 5A.

- Page 12, lines 275, 279, 283 and 285: All references to figure 4 actually mean figure

5.

- Page 13, line 295: The text references "figure 4", but the corresponding figure is

figure 5

- Page 14, line 315: Please correct Pl,Th to Pl.Th

Discussion:

- Page 15, lines 347-350: "Two-sided sword" is a German idiom, but not an English

one. Please just note the relevant facts (reference for the OA initiation is missing)

without using colorful metaphors.

- Page 15, lines 350-352: Please give references for this "widely accepted" fact.

- Page 15, line 352-354: Repetition of the sentence before, this time with a reference.

- Page 15, line 360- page 16, line 362: It is very bold to state that higher cartilage

thickness is protective for OA, without a reference it is plain speculation!

- Page 17, line 405-406: I see no evidence for the statement!

- Page 18, first paragraph: This seems to be a limitation of the study. Why is this

introduced with "In addition…"? Should be moved to the limitations.

- Page 18, line 409: Please change "posterior at the respective compartment" to

"posterior to the respective compartment"

Conclusions:

- Page 18, line 429: "initially" would be a correct adjective here, not "firstly"

- Page 18, lines 434-438: Please don't use references in the conclusions. The entire

block should be moved to the discussion section.

- The statement that subchondral bone architecture is mostly unaffected by age seems

to contradict the results, where significant differences due to age were found.

Tables and Figures:

- Fig 3, legend: Actual p-values should be noted with brackets, as "A" is unusual and

readers could evaluate the p=0.07 themselves, instead of talking about "statistical

tendencies". Exact distribution of the group sizes (n) is complicated and repetitive

between the different figure legends - please refer to the text.

- Fig 4, legend: Actual p-values should be noted with brackets, as "A" is unusual.

Exact distribution of the group sizes (n) is complicated and repetitive between the

different figure legends - please refer to the text.

- Fig 5, legend: Actual p-values should be noted with brackets, as "A" and "C" is

unusual and complicated. 2. Decision letter

Dear Dr. Hamann,

The reviewers have commented on your above paper. I would be happy to accept your

manuscript provided you incorporate the minor changes suggested by the reviewers.

The comments provided by the reviewers very often considerably help to improve and

strengthen a paper. In addition, the authors' response letter accompanying a revised

version often contains important information which may be regarded as an added

value to the final version of the manuscript. Therefore, Editors decided that the non-

confidential comments of the reviewers and the non-confidential authors' responses

will be published as online supplementary material together with the final version of

an eventually accepted article.

Please carefully address the issues raised in the comments.

If you are submitting a revised manuscript, please also:

a) outline each change made (point by point) as raised in the reviewer comments

AND/OR

b) provide a suitable rebuttal to each reviewer comment not addressed

I look forward to receiving your revised manuscript.

Yours sincerely,

Friedrich Paulsen

Editor-in-Chief

Annals of Anatomy

Reviewers' comments:

Reviewer #1: The authors have done an intensive and careful revision of their

manuskript and taken into account the suggestions of all reviewers.

I therefore recommend to accept this paper for publication.

Reviewer #2: I have great regard on the work the authors made. They have

implemented almost all comments reasonably. Though obviously a few

misunderstandings arose (see below). I support the manuscript for publication after

minor corrections.

1) I really like the new figure 1. Much better now!

2) I am really happy to read that storage conditions were kept equal for both time

points and that findings cannot be a result of different freezing durations.

3) I still think figure 2 is not necessarily needed, but description of rubber sheet

standard curves in mat&meth would be enough. However, that´s a matter of taste.

4) The quality of figure 6 B and C is low. P-values are hardly readable in pdf. The

original image has a resolution of 37 pixel/cm. That may be not enough for readable

printing?

5) To my comment #6: As said, please add whether you used any filters for

scanning. I did not ask for the algorithm of image noise filtering during reconstruction

- though, that information is worth mentioning, too.

However, again, pleased add whether you used in example aluminum or copper or

whatever material filters or no filter for scanning. Weaker filters may enhance

contrast produced by soft materials, while stronger filters lead to a more concentrated

beam and reduce beam-hardening in scans of dense tissue like bone.

To make it short: I suggest to add to mat&meth (line 206): …400 ms integration time.

A xyz filter was used. Or: No filter was used.

6) To my comment #11: The authors argument that they don´t believe the method

was not sensitive enough to find statistically significant differences in equilibrium

modulus between compartments, because they showed high reproducibility on rubber

sheets. Reproducibility on rubber is good, but actually I didn´t question the

reproducibility, but suggested to discuss whether maybe the method is not sensitive

enough to measure small differences between different locations on the tibia plateau

of rats - even though, that then very reproducible.

To make it short: I suggest to add the following to your new limitations discussion

section (481): … between the two compartments. Furthermore, the method may not

be sensitive enough and may have underestimated minor differences in biomechanics

of very thin rat cartilage at different locations.

----------------------------------------------------------------------------------------------------------------------------

Authors response letter Revision 1

Revision 2

Dear Reviewer,

we greatly acknowledge the suggestions of the reviewer. We have modified our

manuscript accordingly and changes are highlighted in the marked copy.

Yours sincerely,

Nina Hamann

Reviewer #2:

Reviewer comment 4: The quality of figure 6 B and C is low. P-values are hardly readable in pdf. The

original image has a resolution of 37 pixel/cm. That may be not enough for readable

printing?

Author response: We thank the reviewer for this comment. Accordingly, we improved

the quality/resolution of Figure 6.

Reviewer comment 5: To my comment #6: As said, please add whether you used any filters for scanning. I

did not ask for the algorithm of image noise filtering during reconstruction - though,

that information is worth mentioning, too.

However, again, pleased add whether you used in example aluminum or copper or

whatever material filters or no filter for scanning. Weaker filters may enhance

contrast produced by soft materials, while stronger filters lead to a more concentrated

beam and reduce beam-hardening in scans of dense tissue like bone.

To make it short: I suggest to add to mat&meth (line 206): …400 ms integration time.

A xyz filter was used. Or: No filter was used.

Author action:

We thank the reviewer for this comment. The correction has been made. We added

that a 0.5 mm aluminum filter was used (Line 186).

Reviewer comment 6: To my comment #11: The authors argument that they don´t believe the method was

not sensitive enough to find statistically significant differences in equilibrium

modulus between compartments, because they showed high reproducibility on rubber

sheets. Reproducibility on rubber is good, but actually I didn´t question the

reproducibility, but suggested to discuss whether maybe the method is not sensitive

enough to measure small differences between different locations on the tibia plateau

of rats - even though, that then very reproducible.

To make it short: I suggest to add the following to your new limitations discussion

section (481): … between the two compartments. Furthermore, the method may not

be sensitive enough and may have underestimated minor differences in biomechanics

of very thin rat cartilage at different locations.

Author action:

The correction has been made. The sentence stated by the reviewer has been added to

the limitations section (Lines 412-414).