Hair Strengthening Evaluation of Anisotropic Osmolite Solutions (Inositol + Arginine): Cross-Talk between Dermal Papilla Fibroblast and Keratinocytes of the Outer Root Sheath Using a µHair Follicle 3D Model

Round 1

Reviewer 1 Report

The authors present the results of two separate studies examining a physiochemical approach to the impact of anisotropic osmolyte solutions on hair fibres and in vitro organoid cultures. This reviewer cannot understand the rationale behind the examination of these two tissues under the 'osmoprotection' effects if the materials. Perhaps the authors could present a more explicit rationale as to why these very different tissues - one a fully keratinised hair fibre and the other a living tissue organoid (100% saturated) - might respond in a favourable way to the materials tested. In other words, the study would benefit from a clear hypothesis.

The authors should consider and reference the evidence that dermal papilla cells interacting with hair ORS cells is a suitable model. The authors could discuss the limitations of their 3D cell model as an explanation of why they chose K6 as a marker of epithelial (not trichocyte) differentiation. The differentiation seen is more reminiscent perhaps of the outer root sheath or even a hyperproliferative epidermis where K6 is a useful differentiation marker.

Related to this the authors should avoid making statements about the effect of materials under test on hair growth (line 272, 284) and should not refer to these models as 'proto-hairs' as there is no 'hair' - no trichogenic activity is presented, thus speculation should be avoided.

The term 'ex vivo' is usually used to describe living tissues removed for ex vivo use. The authors do not state the origins of hair samples; for example, if the hair swatches were from a commercial supplier, I feel the term 'ex vivo' is inappropriate and should be amended.

In the anisotropic modelling, it was not clear whether the selected materials had been achieved through the screening of many materials to arrive at arginine and inositol? The last sentence of 'experimental design' does not adequately propose why the same materials/mixtures might show effects in an in vitro test and in the dead hair fibre?

In the consumer tests, the state of the hair was described as breakable damaged and weak; was this consumer perception? if so please state this.

The term electronic microscope may be an incorrect translation; it should be a scanning electron microscope throughout.

In the pull test - it is perhaps more correct now to describe hairs pulled as exogen hairs? telogen refers to the state of the follicle, not the hair fibre.

Can the authors please justify the use of cyclosporin as a hair growth benchmark? Recent data published by Hawkshaw et al 2015/2018 should be read and referenced.

I feel the data on the hair fibre and the anisotropic modelling to arrive at selected materials for hair benefit is interesting, however, that the same models can explain benefits also in a living cell model requires much more justification. The authors might consider separating the hair study and the cell study into two separate manuscripts.

Minor points

line 258/9 - consistent use of time points (days vs hours)

line 277 - define a biological duplicate; ideally, this would be two independent samples, not a replicate section from the same sample.

line 286 - figure legend is incorrect

Figure 6 showing COLIV - the localisation of staining could be better verified with a second stain such as with the K6. Please comment on the outer edge location of COLIV?

Line 339 - can the authors justify that a decrease in the hair pull test values of telogen corresponds to an increase in 'hair strength'. The latter is normally tested using an instrument. The authors could propose why they got this effect?

Graphs should show error bars and statistical comparisons should be marked on all graphs

Line 357/8. The statement that treatment is the same as control essentially means there is no effect of treatment?

359/360 and 364. The authors draw too much from the data to claim hair strength and growth stabilisation.

Author Response

1. The authors present the results of two separate studies examining a physiochemical approach to the impact of anisotropic osmolyte solutions on hair fibres and in vitro organoid cultures. This reviewer cannot understand the rationale behind the examination of these two tissues under the 'osmoprotection' effects if the materials. Perhaps the authors could present a more explicit rationale as to why these very different tissues - one a fully keratinised hair fibre and the other a living tissue organoid (100% saturated) - might respond in a favourable way to the materials tested. In other words, the study would benefit from a clear hypothesis.

Sorry but we do not understand the question/comment

 

2. The authors should consider and reference the evidence that dermal papilla cells interacting with hair ORS cells is a suitable model. The authors could discuss the limitations of their 3D cell model as an explanation of why they chose K6 as a marker of epithelial (not trichocyte) differentiation. The differentiation seen is more reminiscent perhaps of the outer root sheath or even a hyperproliferative epidermis where K6 is a useful differentiation marker.

à In the present work, the selected compounds have been screened on a new 3D in vitro model based on the state of the art on hair follicle models which has been developed to be comparable to other test systems in terms of response and reproducibility (Havlockova et al 2009). We have developed a model which is morphologically representative, although simplified, and is able to respond at transcriptional and metabolic level to external stimulation by active compounds and growth factors.

For this reason, we selected CK6 as established marker for the epithelial compartment of the model since, although it is not peculiar, it is highly expressed in hair follicle and present also in similar in vitro 3D model as demonstrated by Havlockova et al 2009.

In case of more specific hair follicle cytokeratins (as K6IRS, Rogers, 2003), their expression is connected to specific hair follicle populations and could be assessed and evaluated in future experiments. In this study our interest was to obtain evidences of and the effect of active compounds on the epithelial compartment and on a keratin fundamental not only for the skin but also for hair follicle 

 

Havlickova et al. Journal of Investigative Dermatology (2009) 129, 972–983; doi:10.1038/jid.2008.315; published online 16 October 2008

Rogers et al., 2003 https://doi.org/10.1046/j.1523-1747.2003.12087.x

 

3. Related to this the authors should avoid making statements about the effect of materials under test on hair growth (line 272, 284) and should not refer to these models as 'proto-hairs' as there is no 'hair' - no trichogenic activity is presented, thus speculation should be avoided.

à Protohair has been substituted by µHF Revised

 

 

4. The term 'ex vivo' is usually used to describe living tissues removed for ex vivo use. The authors do not state the origins of hair samples; for example, if the hair swatches were from a commercial supplier, I feel the term 'ex vivo' is inappropriate and should be amended.

à More simply, the term ex vivo means that the samples to be tested have been extracted from the organism. This was reported also to underline that the used material was not synthetic

 

5. In the anisotropic modelling, it was not clear whether the selected materials had been achieved through the screening of many materials to arrive at arginine and inositol? The last sentence of 'experimental design' does not adequately propose why the same materials/mixtures might show effects in an in vitro test and in the dead hair fibre?

à The screening included Arginine, Inositol and Taurine at different possible combination

 

6. In the consumer tests, the state of the hair was described as breakable damaged and weak; was this consumer perception? if so please state this.

Revised

 

 

7. The term electronic microscope may be an incorrect translation; it should be a scanning electron microscope throughout.

Revised

 

8. In the pull test - it is perhaps more correct now to describe hairs pulled as exogen hairs? telogen refers to the state of the follicle, not the hair fibre.

Revised

 

 

9. Can the authors please justify the use of cyclosporin as a hair growth benchmark? Recent data published by Hawkshaw et al 2015/2018 should be read and referenced.

à We selected Cyclosporin A as known promoter of hair follicle both in vivo and ex vivo, since the underlyng mechanism was not completely understood. The recent studies of Hawkshaw et al 2015/2018 clarify the biological mechanisms of action of Cyclosporin which, involving the Beta-catenin/WNT pathway, in particular suppressing WNT inhibitor SFRP1, induces a prolongation of anagen phase, cell proliferation, melanin production and, as final macroscopic phenomenon hair growth in vivo and ex vivo.

Hawkshaw (2018). PLoS Biol 16(5): e2003705.

Hawkshaw (2015) Journal of Investigative Dermatology (2015) 135, 2129 – 2132;

 

10. I feel the data on the hair fibre and the anisotropic modelling to arrive at selected materials for hair benefit is interesting, however, that the same models can explain benefits also in a living cell model requires much more justification. The authors might consider separating the hair study and the cell study into two separate manuscripts.

We do not agree

 

11. line 258/9 - consistent use of time points (days vs hours)

Revised

 

 

12. line 277 - define a biological duplicate; ideally, this would be two independent samples, not a replicate section from the same sample.

à The histological analyses have been performed on section deriving from 2 µMTS with exception of sample Cyclosporin A CK6 staining (n=1) due to difficult inclusion of samples

 

13. line 286 - figure legend is incorrect Revised

 

 

 

14. Figure 6 showing COLIV - the localisation of staining could be better verified with a second stain such as with the K6. Please comment on the outer edge location of COLIV?

à In µHF model the secretion of collagen IV seems to be due to the activity of the epithelial compartment. Further investigation by double staining CK6-COLIV have not been performed  however I agree that they could better clarify the balance between the differentiation process and the anchoring of keratinocyte envelope to the dermal papilla core. 

 

15. Line 339 - can the authors justify that a decrease in the hair pull test values of telogen corresponds to an increase in 'hair strength'. The latter is normally tested using an instrument. The authors could propose why they got this effect?

Revised

 

 

16. Graphs should show error bars and statistical comparisons should be marked on all graphs

à If the statistical comparison is not present and, in the test the word “tendential” is reported, there is not a significant difference

 

17. Line 357/8. The statement that treatment is the same as control essentially means there is no effect of treatment?

à the statement was exclusively related to the maintain of the morphological characteristics of the model and that the treatment has not determined significant morphological modification (layers differentiation and disposition) even if the expression of CK6 is increased.

 

 

18. 359/360 and 364. The authors draw too much from the data to claim hair strength and growth stabilisation. Revised

Reviewer 2 Report

This paper shows Inositol and arginine is good for hair strengthening using MM calculation, cell culture and 30 female volunteers.

 

However, the meaning of hair strength is used for Fig 1 - 3 as hair protein strength. But Fig 4-6 show the effect of inositol and arginine on hair follicle cell growth or keratin synthesis. It may not show hair strength. In Fig 7, hair loss is measured, but it does mean hair strength, it means decreased hair number in pulling test.  Therefore, please specify the aim of this paper.

 

Why did not use more accurate method of computer program like semi-empirical calculation for example Mopac software etc. As computer is getting high performance, this size molecule may be possible to calculate with semi-empirical method rather than MM.

 

Minoxidil is not used in this experiment as positive control. Why? As minoxidil is famous reagent that increase hair growth.

 

In fig 7., why did not control the brushing times. If someone seldom brush her hair, pulling test results will be smell number, but brushing many times, hair may lost during brushing, and pulling test shows small value.

Author Response

1. However, the meaning of hair strength is used for Fig 1 - 3 as hair protein strength. But Fig 4-6 show the effect of inositol and arginine on hair follicle cell growth or keratin synthesis. It may not show hair strength. In Fig 7, hair loss is measured, but it does mean hair strength, it means decreased hair number in pulling test. Therefore, please specify the aim of this paper.

à Sorry, could you specify your request? Fig. 7 is not related to the hair loss.

 

2. Why did not use more accurate method of computer program like semi-empirical calculation for example Mopac software etc. As computer is getting high performance, this size molecule may be possible to calculate with semi-empirical method rather than MM.

 In this work we performed molecular dynamics simulation of a protein and some small molecules in water solution. As the solvent is explicitly represented, each system is composed by thousands of atoms.

Considering that the time evolution of each system has been modeled (rather than a single minimum energy structure), calculating 100 ns long simulations, it is evident that a quantum mechanical description of the systems (even at the semi empirical level) is out of the possibility of current calculators, including the most powerful GPU based architectures.

The proposed classical dynamics simulations are thus the highest possible level of theory currently affordable to model the time evolution and the properties of large systems as those considered here.

 

3. Minoxidil is not used in this experiment as positive control. Why? As minoxidil is famous reagent that increase hair growth.

à We worked on possible ingredients for cosmetic products. The Minoxidil is a drug and the comparison between cosmetic ingredients and drug was not consider suitable.

 

4. In fig 7., why did not control the brushing times. If someone seldom brush her hair, pulling test results will be smell number, but brushing many times, hair may lost during brushing, and pulling test shows small value.

 Revised

Reviewer 3 Report

The manuscript is well written and it is necessary to review the english language from a native speaker

there are some mistakes in the text

Moreover no conflict of interest is written and it is a reaserch for a new product for hair damage

Author Response

1. The manuscript is well written and it is necessary to review the english language from a native speaker

Revised

 

2. There are some mistakes in the text

Revised

 

3. Moreover no conflict of interest is written and it is a reaserch for a new product for hair damage

Sorry, but what should we write?

Round 2

Reviewer 2 Report

Your response is acceptable.

Author Response

Thank you

Reviewer 3 Report

Do the authors have any conflict of interest with this paper?

you have to write if there is or not conflict of interest with this product and if there is a contribution from any cosmetic or pharmaceutical company

Author Response

Statement on conflict of interest intoduced