Evaluation of the Effect of the Microscopic Glass Surface Protonation on the Hard Tissue Thin Section Preparation

Round 1

Reviewer 1 Report

1. The contents of the purpose of this study described in the introduction are insufficient. These authors need to clarify the hypotheses for the study of which theories are to be proved through physical/mechanical experiments.
2. As mentioned in the introduction, when the glass was etched with hydrofluoric acid, did each group (epoxy, bone, titanium) show different patterns? What was the difference between the surface results of nitric acid etching and hydrofluoric acid etching?
3. It is difficult to read because there are so many mistakes in the grammar and typos, as well as the expressions of symbols and numbers.
4. Line 197. The Smoluchowski coagulation equation related to the results of this study is also recommended in the text.
5. Where is the experimental method described for the results mentioned in Figure 9? And where is Figure 10, and what experiment is Figure 11 also from? Quite confusing.
6. Have you measured the surface roughness and hardness before and after the nitric acid etching treatment? If these two experiments were conducted, were there any significant differences between the groups?

Author Response

Dear Reviewer 1,

Thank you very much the Reviewer for the helpful suggestions. The valuable comments are appreciated and we have made the changes according to the suggestions. Below we present the responses to the questions one-by-one. Comments and questions are marked in red, while answers to them are marked in blue:

 

1. “The contents of the purpose of this study described in the introduction are insufficient. These authors need to clarify the hypotheses for the study of which theories are to be proved through physical/mechanical experiments.”

Thank you for your comments. The aims are added to the last part of the introduction. The copy of the aim of this study section missed at the template using. The following aims are added to the text:

“The primary aim of this study was to evaluate the protonation effect of the microscopic glass surface between the frosted glass slide and the histological section content (epoxy embedding material, bone and titanium) using 10-MDP containing bond material with comparing of measured data of shear bond strength (SBS), contact angle, X-ray photoelectron spectra, zeta potential on the protonated and unprotonated glass. The secondary aim was to compare the efficacy of 10-MDP containing bond material to metallography used thermoplastic adhesive with SBS data comparison. The final aim was to improve the microscopic histological examination by safely thinning of the undecalcified, titanium-containing thin bone section during the thinning processes of the section. “

 

2. “As mentioned in the introduction, when the glass was etched with hydrofluoric acid, did each group (epoxy, bone, titanium) show different patterns? What was the difference between the surface results of nitric acid etching and hydrofluoric acid etching?”

In the introduction part it was showed that Goldman et al. used mineral acid, hydrofluoric acid, and silane primer on histologic slide. (DOI: 10.1002/sca.4950210106) They used 20% hydrofluoric acid for several minutes and then is was treated with light-curing adhesive. Their aim was to present a rapid preparation of specimens that may be examined both light microscopy (LM) and Scanning Electron Microscopy (SEM) in multiple imaging sessions. They wrote that the correlative LM and SEM techniques are not directly applicable to their applications using ground bone section. They focused on a rapid section preparation process to eliminate re-preparation steps. Therefore they did not analyse the surface before and after acid etching and not investigate the bond strength between glass slide and components of section (embedding materials, bone and titanium). The reached final thickness of Goldman’s section was 100 µm ± 5 µm. In the introduction part we emphasized that above mentioned section thickness is far from an optimal value for histological analysis. The expected, optimal section thickness is one cell thick (under 10 µm) for high-quality microscopy analysis after staining. 

 

3. “It is difficult to read because there are so many mistakes in the grammar and typos, as well as the expressions of symbols and numbers”

We tried to improve the English and correct the grammar mistakes and typos. We used symbols for encoding of the sample.

 

4. “Line 197. The Smoluchowski coagulation equation related to the results of this study is also recommended in the text.”

Thank you for your comments. We put it into the 3.2. Results of zeta potential subsection by the following:

“The surface charging of the glass surface by protonation can be described by zeta potential measurements. Zeta potential was calculated using the Smoluchowski approximation. The measured pH and calculated zeta potential data are shown in Table 2.”

and into the discussion part by the following:

“The zeta potential measurements, which were calculated by Smoluchowski coagulation equation, revealed that the glass surface becomes positively charged after nitric acid etching (Table 2).”

 

5. Where is the experimental method described for the results mentioned in Figure 9? And where is Figure 10, and what experiment is Figure 11 also from? Quite confusing.

The figures are in the discussion part because we want to show the effectiveness of our method through examples. They are not organically related to the experiments in which our aim was to investigate the usability of protonation and 10-MDP molecule.  

The figure in the discussion part helps to understand and present the problems of decalcified titanium containing thin bone section preparation. Because of the titanium content of the section it cannot be decalcified and cut with traditional microtome, therefore undecalcifying section preparation method was chosen for histological analysis. In this multistep procedure, the cut section have to grinding or polishing until we get the optimal thickness for histological examination. Among many detrimental factors, one can be incorrectly chosen polishing step. This factor can cause damage in the valuable section that can lead to loss of section. The Figure 9 present the inadequate and adequate polishing steps (time of polishing, correct or incorrect order in abrasive paper applying that can form cracks or perforation in section) and their consequences. In the Figure 9, the experimental method description of the SEM measurements are the following: these electron micrographs were taken from polished specimen with Hitachi S-4300 SEM at accelerating operating voltages of 5 and 15 kV. Before the taking of SEM picture the surface of the samples was sputter-coated with a 50 nm gold layer.     

There was a mistake in the numbering of the figures, therefore the Figure 11 correctly Figure 10 and Figure 12 is correctly Figure 11. It was corrected in the text. The corrected Figure 10. presents an example for a Haematoxylin-eosin stained section with sintered titanium implants with different thickness.

6. “Have you measured the surface roughness and hardness before and after the nitric acid etching treatment? If these two experiments were conducted, were there any significant differences between the groups?”

Jang et al. investigate the effects of chemical etching with nitric acid on glass surfaces. (DOI: 10.1116/1.1333087) In their study commercial glass slide was soaked in nitric acid with different way (sonicated in nitric acid, boiled in nitric acid) and different nitric acid concentration. Based on their AFM measurements, which was used to study surface morphology and to measure the root-mean square (rms) surface roughness, it was found that the surface morphology of acid etched glass is very similar regardless of above listed etching treatments. Surface roughness of acid etched glass is significantly rougher than that of bare glass. The rms value of bare glass was 0.58 and acid etched was around 6.

Among the different hardness measurement methods, the nanoindenter can provide more precise information about the hardness of interfaces involved in bonding. The numbers of cohesive failure mode 2 (fracture appear inside the glass in the interface) increased significantly compared to unprotonated tested group (Figure 8). If the nanoindenter can provide further help in clarifying the processes taking place at the interfaces, we will consider performing this measurement in our later work. 

Author Response File: Author Response.docx

Reviewer 2 Report

Mainly grammatical changes

replace we and us

p58 grammar

p76 remarkable needs changing

p143 replace get

p258 title too long split and add text

p277 for equation use scientific format

p301 suggest use using histogram

fig 9 as p258

p410 replace remarkable

conclusions need expanding

Author Response

Dear Reviewer 2,

Thank you very much the Reviewer for the helpful suggestions. The valuable comments are appreciated and we have made the changes according to the suggestions. Below we present the responses to the questions one-by-one. Comments and questions are marked in red, while answers to them are marked in blue:

Mainly grammatical changes

replace we and us

Thank you very much for your comments. We replaced we and us words by rewriting the sentences.

p58 grammar

In the line 58 before correction:

“However, when having metal content samples, this traditional microtome cannot be sliced the metal part. The solution of the processability in metal content cases was first examined by Gross and Strunz in 1977”

after correction:

“However, metal samples cannot be sliced with this traditional microtome. The solution of the processability in metal content cases was first examined by Gross and Strunz in 1977”

p76 remarkable needs changing

Thank you for your comments. We think that the sentence is also understandable without remarkable word.

p143 replace get

Thank you for your comments. We replace the get word with achieve word.

p258 title too long split and add text

Thank you for your suggestion.  The length of figure caption is reduced. The color coding is added to the text. 

p277 for equation use scientific format

Thank you for your suggestion. We used scientific format for shear bond strength equation. We used equation editor of Microsoft Word.

p301 suggest use using histogram

Thank you for your comments. Instead of histogram we used bar graph where significant differences among mean values can also be illustrated.

fig 9 as p258

We would like to present a sample for a typical problems at the section preparation. The SEM picture were taken from sections after polishing, not a shear bond strength tested specimens.

p410 replace remarkable

Thank you for your comments. We replace the remarkable word with increased  word with rewriting of the sentence.

Original sentence was:

Epoxy sample of Group 2 and Group 3 showed still remarkable failure modes at adhesive - interface 1 because of the nonreactive molecular motif of epoxy. 

Corrected sentence was

Epoxy sample of Group 2 and Group 3 showed increased number of failure modes at the adhesive - interface 1 because of the nonreactive molecular motif of epoxy.  

conclusions need expanding

Thank you for your suggestion. We modified the conclusion. 

Author Response File: Author Response.docx