**5. Conclusions**

In summary, at least five observations were highlighted: (1) There was a significant difference between non-operated and operated bone tissues. With immunostaining, in the operated bone tissue we observed mostly M2-like macrophages with smaller quantities of M1-like macrophages. No macrophages were found in the non-operated bone tissue. (2) The localization of the macrophages was limited to the wounded area. (3) The operated tissue showed a strong IL-1β and IL-6 cytokine expression as measured by RT-qPCR detection. (4) In addition, the in situ HCR method has been proved useful as it allows selectively exploring the RNA expressions of the cytokines and macrophage markers in the cells of the wounded bone tissue. With this technique, it was possible to identify the in vivo M2b-like macrophages in the wounded bone tissue. (5) It was pointed out that microspectroscopical analysis is very important in macrophage characterization because it can differentiate between fluorescence and autofluorescence.

Finally, this work highlighted the limits of immunostaining and the interest in in situ HCR method providing valuable information for in vivo macrophage characterization. This technique is an important approach for analyzing the biological mechanism of bone regeneration and offers a new perspective in the field of regenerative medicine.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/biomedicines10061385/s1. Figure S1: Identification of M1-like and M2-like macrophages in the rat femurs; (A–D) immunolabeling with anti-Iba1 and CD206 antibodies of the operated femurs; (B–D) expanded view: high magnification image of the area within the red rectangle in image A; anti-Iba1 (Alexa488, turquoise fluorescence), labeling the M1 and M2 macrophages; anti-CD206 (Alexa568, red fluorescence) labeling the M2 macrophages and satellite cells; nuclear staining with DAPI (blue fluorescence); thin arrow: M1-like macrophages; thick arrow: M2-like macrophages. Figure S2: Identification of M2-like macrophages and satellite cells in the rat femurs: (A) immunolabeling with anti-CD163 and CD206 antibodies of the operated femurs; (B) negative control; anti-CD163 (Alexa488, turquoise fluorescence), labeling the M2-like macrophages; anti-CD206 (Alexa568, red fluorescence) labeling the M2-like macrophages and satellite cells; nuclear staining with DAPI (blue fluorescence); thin arrow: M2-like macrophages; thick arrow: satellite cells. Figure S3: Identification of macrophages M1-like and M2-like in the femurs of the rat; (A) negative control of Figure 4B; (B) negative control of Figure 4C; (C) negative control of Figure 4D; (D) negative control of Figure 4E; (E) negative control of Figure 4F; (F) negative control of Figure 4G; anti-CD68 (Alexa488, turquoise fluorescence) labeling the M1-like and M2-like macrophages.; anti-CD206 (Alexa568, red fluorescence) labeling the M2-like macrophages and satellite cells; nuclear staining with DAPI (blue fluorescence). Figure S4: In situ hybridization in the non-operated femur of the rat; (A) expression of β-actin mRNA (positive control); (B) negative control; (C) CD68 mRNA; (D) IL-1β mRNA; (E) CD163 mRNA; (F) IL-6 mRNA; (G) expanded view: high magnification image of the area within the red rectangle in image E; (H) expanded view: high magnification image of the area within the red rectangle in image F. Table S1: Oligos used for RT-qPCR: FW (forward primer); RV (reverse primer). Table S2. Oligos are used for in situ hybridization experiments: T3 (T3 promoter); T7 (T7 promoter); FW (forward primer); and RV (reverse primer).

**Author Contributions:** K.N. and A.-L.F. designed the experiments; C.C. performed the cryo-coupe; K.N. and E.S. performed the immunofluorescence and in situ hybridization; J.B. performed the spectrum analysis; M.O. helped with image preparation; K.N., X.H. and A.-L.F. designed the analysis approaches; X.H. and A.-L.F. devised and conducted the analyses; M.D. and J.-M.C. provided femurs of the rat samples; K.N., X.H. and A.-L.F. wrote the manuscript with input from all the authors. All authors discussed the results. All authors have read and agreed to the published version of the manuscript.

**Funding:** Work was supported by the <sup>D</sup>élégation Générale de l'Armement (DGA, Ministry of French Army) (PDH2-NRBC-4-NR-4306 and PDH-SAN-1-217/206).

**Institutional Review Board Statement:** This study was approved by the French Army Animal Ethics Committee (N◦2011/22.1). All rats were treated in compliance with the European legislation (dir 2010/63/EU) implemented into French law (decree 2013-118) regulating animal experimentation.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are very grateful to Zsolt Kelemen for his helpful advice and critical reviewing our manuscript. We are also grateful to Xavier Butigieg for helping with image preparations.

**Conflicts of Interest:** The authors declare no conflict of interest.
