Quantitative Near-Infrared Imaging of Platelets in Platelet-Rich Fibrin (PRF) Matrices: Comparative Analysis of Bio-PRF, Leukocyte-Rich PRF, Advanced-PRF and Concentrated Growth Factors
, Tetsuhiro Tsujino 1, Taisuke Watanabe 1, Kazushige Isobe 1, Yutaka Kitamura 1, Atsushi Sato 1, Sadahiro Yamaguchi 1, Hajime Okudera 1, Kazuhiro Okuda 2 and Tomoyuki Kawase 3,*Round 1
Reviewer 1 Report
The manuscript entitled: “Quantitative near-infrared imaging of platelets in platelet-rich fibrin (PRF) matrices: comparative analysis of bio-PRF, leukocyte-rich PRF, advanced-PRF, and concentrated growth factors", by Aizawa and collaborators describes a non-invasive methodology based on near-infrared (NIR) imaging technology to visualize platelet distribution in PRF. The authors compared four types of PRF matrices, bio-PRF, advanced-PRF (A-PRF), leukocyte-rich PRF (L-PRF), and CGF.
The work described is a continuation of previously published research by the authors. It is interesting and the results obtained are reasonably discussed. The article is globally well written. The references are adequate and a considerable number is based in recent published works. The abstract is adequate. As a result, in my opinion it deserves publication in International Journal of Molecular Science.
Author Response
The manuscript entitled: “Quantitative near-infrared imaging of platelets in platelet-rich fibrin (PRF) matrices: comparative analysis of bio-PRF, leukocyte-rich PRF, advanced-PRF, and concentrated growth factors", by Aizawa and collaborators describes a non-invasive methodology based on near-infrared (NIR) imaging technology to visualize platelet distribution in PRF. The authors compared four types of PRF matrices, bio-PRF, advanced-PRF (A-PRF), leukocyte-rich PRF (L-PRF), and CGF.
The work described is a continuation of previously published research by the authors. It is interesting and the results obtained are reasonably discussed. The article is globally well written. The references are adequate and a considerable number is based in recent published works. The abstract is adequate. As a result, in my opinion it deserves publication in International Journal of Molecular Science.
Response: Thank you very much for your evaluation.
Reviewer 2 Report
In this manuscript Aizawa et al evaluate near-infrared imaging to quantify platelets in four different types of platelet rich fibrin (PRF) depending on variability of commercial sources. They found no major difference in platelet distribution in three major portions of the sample. They concluded that gravity was not a major factor in distribution of platelet in PRF. They proposed centrifugal force and distance from surface had a major impact on distribution.
Specific Points:
- Authors need to write form of CGF.
- Is there any specific reason CD41 was used for the study? Can author comment on using other CDs (e.g. CD42, 61) for this study?
- Page 2 line 65: It is advised that authors reduce the NIR imaging region to the one that is used clinically (750 – 900). NIRII imaging (1000-1500 nm) is still being developed and is not a norm. Imaging above 1500 nm is rare.
- Figure 1. X axis is hard to read.
- Figure 3. Any particular reason for omitting CGF?
- Page 7 Line 143: full form of PRP?
- Can authors comment on the sensitivity of the NIR dye to detect platelets?
- Page 12: Please delete Line 323 – 334
- Page 13: second sentence on line 346
Author Response
Specific Points:
Authors need to write form of CGF.
Response: We have added the full name of CGF in the abstract.
Is there any specific reason CD41 was used for the study? Can author comment on using other CDs (e.g. CD42, 61) for this study?
Response: We do not have any reasons to exclude CD42 or CD61. To confirm the validity of platelet imaging using the anti-CD41 antibody, we think that we should test the other antibodies against CD42 and CD61.
The main reason we used the monoclonal anti-CD41 antibody (BioLegend) in this study was that this antibody produced better results than the polyclonal anti-CD41 antibodies purchased from GeneTex and Santa Cruz Biotechnology in the preliminary experiments and in a previous study (Yamaguchi et al. Front Bioeng Biotechnol. 8:600;2020). In addition, we previously used both monoclonal and polyclonal antibodies against CD41 to identify platelets and acquire data (Biologicals 40, 323-9, 2012; Int J Implant Dent 1, 31, 2015; Biomedicines 5, 57, 2017; Micron 113, 1-9, 2018; Front Bioeng Biotechnol 6, 4, 2018; J Invest Clin Dent 10, e12458, 2019; J Funct Biomater 10, 43, 2019; Front Bioeng Biotechnol 8, 600, 2020). Therefore, to maintain data compatibility, we adopted CD41 as a pan platelet maker in this study.
Page 2 line 65: It is advised that authors reduce the NIR imaging region to the one that is used clinically (750 – 900). NIRII imaging (1000-1500 nm) is still being developed and is not a norm. Imaging above 1500 nm is rare.
Response: We have only described the electromagnetic definition of NIR wavelength range in the text. With respect to clinical imaging, as you indicated, NIR with shorter wavelengths are used. Thus, we have added some information concerning the wavelength range for clinical use in the revised manuscript.
Figure 1. X axis is hard to read.
Response: We have replaced the small labels with large ones for better visibility.
Figure 3. Any particular reason for omitting CGF?
Response: Both CGF and L-PRF were prepared using centrifuges equipped with fixed-angle rotors. The angulation of both rotors was 33°. In addition, both CGF and L-PRF showed similar fluorescence intensities and sizes (lengths) (Figure 2). In fact, comparisons of three regions did not show statistically significant differences in fluorescence intensity in bioPRF, A-PRF, and L-PRF. Therefore, we decided to omit CGF. Instead, CGF was included in the immunocytochemical data shown in Figure 4, to highlight the striking differences in platelet distribution in comparison with A-PRF.
Page 7 Line 143: full form of PRP?
Response: We have added the full name of PRP in the manuscript.
Can authors comment on the sensitivity of the NIR dye to detect platelets?
Response: Concerning the transmittance of the 800 nm NIR, we presented the data and discussed the feasibility in the previous article (Yamaguchi et al. Front Bioeng Biotechnol. 8:600;2020). The fluorescence intensity of the 800 nm wavelength was reduced by an average of approximately 20% by the compressed PRF membrane; however, reproducible data could be obtained. This information is described in Lines 299-301 of the revised manuscript.
At the same time, we should consider the specificity of the monoclonal antibody and its possible non-specific binding to fibrin fibers. Concerning the specificity, as this antibody has been sold for a long time, we think that its specificity and affinity have been established.
Our previous scientific findings obtained from PPP-derived fibrin membranes demonstrated that the signal-to-noise ratio in glass tubes was reduced significantly from 2.0 to less than 1.5. Thus, the possibility that the monoclonal antibody could, to some extent, bind to fibrin fibers cannot be ruled out. However, because of its specific binding at greater levels, we concluded that non-specific binding did not significantly interfere with the quantification.
Page 12: Please delete Line 323 – 334
Response: Thank you for the advice. We forgot to delete the sentences described in the template. We have now deleted those sentences.
Page 13: second sentence on line 346
Response: Thank you again. We have now deleted those sentences.
