Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells
and Morio Tonogi 1,4Round 1
Reviewer 1 Report
Current Issues in Molecular Biology
Manuscript ID: cimb-1357020
Article: Effect of azithromycin on mineralized nodule formation in MC3T3-E1 cells.
Authors : Kengo Kato, Manami Ozaki, Kumiko Nakai, Maki Nagasaki, Junya Nakajima, Ryosuke Koshi, Hideki Tanaka, Takayuki Kawato and Morio Tonogi
In this work authors studied effect of antibiotic azithromycin on osteoblast-like MC3T3-E1 cells. Authors measured alkaline phosphatase activity, cell proliferation, type I collagen, bone sialoprotein, osteocalcin and osteopontin mRNA, as well as mineralized nodule formation after azithromycin treatment. They found that azithromycin, at highest concentration used (10 mg/ml) suppressed mineralized nodule formation, while lower concentrations of azithromycin were without effect on osteogenic function in MC3T3-E1 cells. This work is original and involves azithromycin, an antibiotic that is of common use. However, the main claim of the manuscript lays on alizarin red staining that was not quantified and that is difficult to analyze because of high (maybe maximum) staining of control MC3T3-E1 cells. Additional experiments are needed.
Major comments :
- MC3T3-E1 cells have high alkaline phosphatase (ALP) activity in the resting state and thus might be not the best model to test ALP and mineralized nodule formation. In the figure 3 there is a strong staining of control cells. Thus, in the Figure 3, a positive control must be used, showing that in these culture conditions, ALP and mineralized nodule formation can further increase. Experiments must be evaluated not only after 10 days but also at days 3, 5 and 7. Quantification of alizarin red staining must be done.
Minor comments :
- Which subclone of MC3T3-E1 cells was used? This information should be added to Materials and Methods.
- Line 65, 66 : “in osteoblast” should be deleted.
Author Response
Response to the comments from Reviewer 1
We wish to express our gratitude for the positive and important comments. We have responded to the reviewer’s comments point-by-point as follows.
- The reviewer made the following comments “MC3T3-E1 cells have high alkaline phosphatase (ALP) activity in the resting state and thus might be not the best model to test ALP and mineralized nodule formation. In the figure 3 there is a strong staining of control cells. Thus, in the Figure 3, a positive control must be used, showing that in these culture conditions, ALP and mineralized nodule formation can further increase. Experiments must be evaluated not only after 10 days but also at days 3, 5 and 7. Quantification of alizarin red staining must be done.” In this study, MC3T3-E1 cells were cultured in the medium containing 50 mM β-glycerophosphate and 50 µg/mL ascorbic acid (described as osteogenic supplements in the revised manuscript) and were evaluated the formation of the calcified nodule. Moreover, the previous study (Cheung W.M et. al.) reported that dimethyl sulfoxide (DMSO) used as a vehicle for azithromycin in this study induced osteogenic function in MC3T3-E1 cells. The concentration of DMSO (0.1%) as a vehicle in this study was lower than the concentration that induced osteogenic function in this previous study, however our pilot study indicated that 0.1 DMSO induced Runx2 expression in MC3T3-E1 (dta not shown). Therefore, we added 0.1% DIMSO, the final concentration in each medium containing azithromycin, into the control medium. We explained these conditions in the revised manuscript (lines 165-171). We agreed with the reviewer`s suggestion to add alizarine red evaluation of days 3, 5, and 7 of culture, but we could not finish the culture within the assigned time. Instead of these additional experiments, we added the results that clarified the effects of osteogenic supplements and 0.1% DMSO on forming a calcified nodule in MC3T3-E1 (Figure 3 in the revised manuscript). We also added the quantification results of Alizarine red staining (Figure 4 in the revised manuscript). We explained the method for quantification and the results in the revised manuscript (lines 171-176). We hope these data help to dissolve the reviewer’s concern.
- The reviewer made the following comments: “Which subclone of MC3T3-E1 cells was used? This information should be added to Materials and Methods. Line 65, 66 : “in osteoblast” should be deleted.” According the reviewer’s instruction, we confirmed the datasheet of MC3T3-E1 cells used in this study and added the information in the revised manuscript (lines 74-75). We also deleted “in osteoblasts” in the revised manuscript (lines 64).
Reviewer 2 Report
The paper is interesting and well written, I only have some conserns about the trend of osteopontin, osteocalcin, BSP and collagen I trend following the treatment with 10 µg/ml azithromycin.
The authors should display these parameters possibly starting from T0 and also evaluate the statistical significance respect to this T0. However, it is also strange that in the graphs the histograms of controls does not start from 1, as first value on y axis. The authors should correct graphs.
Author Response
Response to the comments from the Reviewer 2
We wish to express our gratitude for the important comments. We have responded to the reviewer’s comments point-by-point as follows.
- The reviewer made the following comments: “The authors should display these parameters possibly starting from T0 and also evaluate the statistical significance respect to this T0. However, it is also strange that in the graphs the histograms of controls does not start from 1, as first value on y axis. The authors should correct graphs.” To address the reviewer’s comment, we determined the expression level of each gene in cells without exposing azithromycin and DMSO on the day when cells were seeded (T0). Then, the expression level of each gene was calculated and expressed as a ratio to the expression level in These results were shown in Figure 5 in the revised manuscript, but we could not start from 1, as first value on the y-axis. This was because there was a large difference between T0 and control on days 7 and 10 of culture in the presence of azithromycin and 0.1% DMSO as a vehicle. We also thought the comparison between the vehicle control and azithromycin was appropriate to evaluate the effect of azithromycin on cell proliferation, alkaline phosphatase activity, osteopontin protein secretion because alizarine red staining indicated that 0.1% DMSO upregulated the osteogenic function in MC3T3-E1. We explain them in the revised manuscript (lines 165-176, 221-228). We hope these revisions help to dissolve the reviewer’s concern.
Round 2
Reviewer 1 Report
Current Issues in Molecular Biology
Manuscript ID: cimb-1357020-v2
Article: Effect of azithromycin on mineralized nodule formation in MC3T3-E1 cells.
Authors : Kengo Kato, Manami Ozaki, Kumiko Nakai, Maki Nagasaki, Junya Nakajima, Ryosuke Koshi, Hideki Tanaka, Takayuki Kawato and Morio Tonogi
Authors did not answered the main demand of the major comment. Instead of adding positive control in the experiment with azithromycin they did additional figure showing positive control effect but no azithromycin treatment was compered in this new figure. In addition, cells cultured in medium containing osteogenic supplements in the new figure 3 stained very differently than in the figure 4 (old figure 3). Why? Experiments have to be repeated in which all controls have to be compared with azithromycin treatment. Experiments must be evaluated not only after 10 days but also at days 3, 5 and 7.
Author Response
Dear editor and reviewers,
We thank you so much for taking the time to review our manuscript and give important comments. We have responded to the comments point-by-point as follows and have made revisions according to the comments.
Response to the comments from Reviewer 1
We have responded to the reviewer’s comments point-by-point as follows.
- The reviewer made the following comments “Authors did not answered the main demand of the major comment. Instead of adding positive control in the experiment with azithromycin they did additional figure showing positive control effect but no azithromycin treatment was compered in this new figure.” We had agreed to the reviewer’s comment and immediately started the additional experiments. However, we could not finish this until the deadline for the first revision. We would like to apologize for lacking data. In the 2nd revised manuscript, we added the new data of alizarine red staining on days 3, 5, 7, 10 and 14 of culture.
- The reviewer made the following comments “In addition, cells cultured in medium containing osteogenic supplements in the new figure 3 stained very differently than in the figure 4 (old figure 3).” In the original manuscript, we presented the image of a digital camera. In the 1st revised manuscript, we used a scanner to take a picture of the same plate to improve the quality. This is why there was a difference in the image of the same plate between the original manuscript and 1st revised manuscript.
- The reviewer made the following comments “Experiments have to be repeated in which all controls have to be compared with azithromycin treatment. Experiments must be evaluated not only after 10 days but also at days 3, 5 and 7.” In the original manuscript, we had conducted alizarine red staining on day 14 of culture, but we had indicated “day 10” in the figure by mistake. We would like to apologize for this mistake. Following the reviewer’s suggestion, we conducted red staining before day 10 of culture and compared the effect of azithromycin with all control, in the 2nd revised manuscript.
Author Response File: 
Author Response.pdf
Round 3
Reviewer 1 Report
Authors answered to major comments and their manuscript is recommended for publication.
