Drug-Releasing Gelatin Coating Reinforced with Calcium Titanate Formed on Ti–6Al–4V Alloy Designed for Osteoporosis Bone Repair
, Seine A. Shintani 1, Akihiro Sudo 2 and Tomiharu Matsushita 1Round 1
Reviewer 1 Report
The authors developed Ti-6Al-4V alloy with a composite layer of minodronate-containing gelatin and calcium deficient calcium titanate and demonstrated its characteristics. I am interested to find out more on several issues:
- Why normality was tested using Kolmogorov-Smirnov test not Shapiro-Wilk test? Which test (Dunnet or Steel's) was used for comparing scratch resistance in the end? Did the author use one-way ANOVA first before running Dunnet? Apart from comparing to CT, were other pairs being compared?
- Please indicate the actual p-value in Table 3.
- Why cross-linking caused the disappearance of apatite on gelatin with phoshate but not calcium as observed in Figure 6?
- Can the authors suggest future studies to prove the usefulness of the materials for orthopedic applications: osteocompatibility assay, in vivo fracture-fixation model etc.
Author Response
Thank you very much for your useful comments. Please find the attached file. The copy of the text is posted bellow.
Comment ; The authors developed Ti-6Al-4V alloy with a composite layer of minodronate-containing gelatin and calcium deficient calcium titanate and demonstrated its characteristics. I am interested to find out more on several issues:
- Why normality was tested using Kolmogorov-Smirnov test not Shapiro-Wilk test? Which test (Dunnet or Steel's) was used for comparing scratch resistance in the end? Did the author use one-way ANOVA first before running Dunnet? Apart from comparing to CT, were other pairs being compared?
Reply: We changed the normality test method from Kolmogorov-Smirnov test to Shapiro-Wilk test, considering the relative small N number of the scratch test. As a normality was not rejected, Dunnett's test, which does not include ANOVA in its procedure, was adopted to compare the control group with the other groups. The comparison between the pairs other than CT was not performed in this study.
Because of this, the explanation of 2.5 statistical analysis was changed to the following:
Page (4, 155-159).
"Statistical tests were performed using the statistical software tool (R language; version 3.6.1, The R Foundation, Vienna, Austria). The normality of the scratch resistance measurement results was tested by the Shapiro-Wilk test. As a result, normality was not rejected for all data. The comparison between the control group and the other groups was tested by the multi-group test (Dunnett's test). The significance level for all statistical analyzes was p <0.05."
- Please indicate the actual p-value in Table 3.
Reply: We have added the actual p-value in the table 3.
- Why cross-linking caused the disappearance of apatite on gelatin with phoshate but not calcium as observed in Figure 6?
Reply: The decreased apatite formation on the cross-linked gelatin with phosphate is probably due to the suppression of phosphate ion release as explained in line 354-357 in our original manuscript. Although the release of calcium ions should be also suppressed, the cross-linked gelatin with calcium almost maintained its apatite formation. This could be explained as the calcium ions can effectively induce apatite formation by increasing ionic-activity product of apatite, Ca10(PO)4(OH)2, than phosphate ions.
This explanation was added in Discussion in our revised manuscript.
- Can the authors suggest future studies to prove the usefulness of the materials for orthopedic applications: osteocompatibility assay, in vivo fracture-fixation model etc.
Reply: Thank you very much for your valuable comment. Further in vivo studies using animal model of osteoporosis are needed to prove the usefulness of the developed material for therapeutic treatment for osteoporosis bone.
This explanation was added in Discussion in our revised manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear authors,
Please consider the comments below.
L106 - What is JIS8803? Include reference. Include the speeds used. Include the shear rate used.
Topic 2.2 - Include the parameters analyzed. Include the time performed for each run. Insert the number of replicates
Topic 2.3.1 - Include the current used
Topic 2.3.4 - Include the ratio of sample and solvents used
L138 Which Ca ion? Specify
L127 - Include the reference of JIS R 3255 method
l142 - correct the numbers in chemical formulae as subscript
2.5 topic - Include the software used.
3.1 - How does it apply to your project? I mean, discuss the role of the viscosity in the formulations (is the viscosity increase considered an advantage? Include references/expand the discussion here)
Table1/Table 2/Table3 - Do not insert table as figure. Also, include the statistical difference within the treatments and explain
Figure 8 - It is difficult to identify the captions here. Correct it, please.
Author Response
Dear Reviewers
Thank you for your kind review comments. Our reply to your comments and revised manuscript are attached herewith. We edited our manuscript using Track Changes function and highlighted revised positions so that they are clearly visible.
Comment ;Please consider the comments below.
L106 - What is JIS8803? Include reference. Include the speeds used. Include the shear rate used.
Reply: JIS8803 is one of Japanese Industrial Standards that describes methods for viscosity measurement of liquid. The spindle speeds (and corresponding shear rates) were optimized for each gelatin solution sample between 11.52 and 576 1/s (corresponding shear rates were 3 to 150 rpm), and four spindle speeds were examined for each sample.
These information and analyzed parameters, time performed for each run, and number of replicates were added in 2.2 Viscosity of gelatin solution, as following.
“Viscosity of gelatin solution (5-20%) with or without additives as a function of gelatin concentration was measured by Brookfield viscometer (LVDVNextCP;.Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA) according to JIS8803 (Japanese Industrial Standards Z8803:2011, Methods for viscosity measurement of liquid). The spindle speeds (and corresponding shear rates) were optimized for each sample between 11.52 and 576 1/s (corresponding shear rates were 3 to 150 rpm), and four different spindle speeds were examined for each sample instead of increasing the number of replicates. The measurements were performed using 2 ml of samples at 60 °C, and the viscosity (mPa·s) of the sample obtained for each run after 30 sec holding time was used for analysis.”
Topic 2.2 - Include the parameters analyzed. Include the time performed for each run. Insert the number of replicates
Reply: Thank you for your comment. We included these information in section 2.2. Please see our reply to your first comment.
Topic 2.3.1 - Include the current used
Reply: The information of electric current used in SEM/EDX analysis was added.
Topic 2.3.4 - Include the ratio of sample and solvents used
Reply: The information of solvent/sample ratio was added.
L138 Which Ca ion? Specify
Reply: The Ca ions are originated from CaCl2 and thus considered as Ca2+ ions. These information was added in our revised manuscript.
L127 - Include the reference of JIS R 3255 method
Reply: The information of JIS R 3255 was added.
l142 - correct the numbers in chemical formulae as subscript
Reply: Thank you for your comment. We corrected the numbers in chemical formulae.
2.5 topic - Include the software used.
Reply: For statistical analysis, we used the R language, which is a programming language for statistical analysis of open source software supported by The R Foundation, Vienna, Austria. This information was added to our revised manuscript.
3.1 - How does it apply to your project? I mean, discuss the role of the viscosity in the formulations (is the viscosity increase considered an advantage? Include references/expand the discussion here)
Reply: The viscosity of the gelatin solution strongly affects the thickness of the gelatin layer after the dip coating as seen in Figure 2. Its coating thickness increased with increase of gelatin concentration as a result of increased viscosity of gelatin solution. Optimum thickness exists since thinner coating cannot cover all the sample surface while thicker coating can be easily damaged. In this study, we determined 10% gelatin, thus corresponding viscosity of 15.1 mPa·s, is optimum since the thickness of gelatin coating became comparative to that of cd-CT layer.
These discussions are already written in the Discussion section, and we would like to keep their position since they seem to be not suitable in Results section. In order to clarify the relation between the viscosity and coating thickness of gelatin, the following explanation was added.
“Therefore, we determined the 10% gelatin, thus corresponding viscosity of 15.1 mPa·s, as an optimized condition.”
Table1/Table 2/Table3 - Do not insert table as figure. Also, include the statistical difference within the treatments and explain
Reply: The tables were inserted as original table format. The statistical difference with actual p-value was included and explained in the main text.
Figure 8 - It is difficult to identify the captions here. Correct it, please.
Reply: We are sorry for the difficulty. We corrected the captions.
Reviewer 3 Report
Totally, the research has been well designed. Although, there are some points to review.
Line 90: please mention the bloom of used gelatin in your study.
Line 104: please add the gelatin concentration for viscosity determination in material and method section.
Author Response
Dear Reviewers
Thank you for your kind review comments. Our reply to your comments and revised manuscript are attached herewith. We edited our manuscript using Track Changes function and highlighted revised positions so that they are clearly visible.
Comment ; Totally, the research has been well designed. Although, there are some points to review.
Reply: Thank you very much for your valuable comment.
Line 90: please mention the bloom of used gelatin in your study.
Reply: The bloom of used gelatin was 316g. This information was added in our revised manuscript.
Line 104: please add the gelatin concentration for viscosity determination in material and method section.
Reply: The gelatin concentration was added in the material and method section.
