**4. Conclusions**

Bone healing is very complex process. For proper regeneration, dynamic interplay between external and internal signals is essential. Cytokines of the macrophages and other cellular factors act at diverse times, and have indispensable functions during repair. In order to make progress in bone regeneration understanding, the development of new tools is essential. These tools will provide opportunities to explore in situ the spatial actors involved in inflammation and bone tissue regeneration. Indeed, the application of distinct ISH approaches can bring new comprehension to bone gene expression and tissue regeneration. Combination of cryofixation with ISH techniques is a relevant approach to study the molecular and spatial biological mechanisms of bone regeneration and provides advanced perspectives in the field of regenerative medicine to induce bone regeneration for developing new treatments.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10020484/s1. Figure S1: Workflow of cryo-sample preparation. (A) Covering mandrel with cryomoun<sup>t</sup> solution. (B) Positioning the block on the mandrel. (C) Hardening of the cryomoun<sup>t</sup> and fixation of the sample on the mandrel. (D) Trimming of the sample. (E) Positioning of the adhesive film on the surface of the block. (F) Application of the roller to improve the adhesion of the film. (G) Cutting the sample slowly and uninterruptedly by holding the lower part of the adhesive film. (H) Obtaining of 5 μm tissue section on adhesive film. (I) Positioning the adhesive film on the precoated slide. (J) Application of the roller to improve the adhesion to the slide. (K) Positioning the slide in CryoJane flash unit. (L) Triggering two flashes at 30 s intervals. (M) Removing the film with cold tool. Figure S2: Workflow to optimize the transfer of the cryo-section. (A) Block trimming to adjust to the sample surface. (B) Positioning the block on the mandrel. (C) Estimation of the adhesive film surface. (D) Cutting of the adhesive film. (E) Positioning of the adhesive film on the surface of the block. (F) Adhesion of the film on the surface of the sample. Table S1: (A) The cDNA sequence of the β-actin used for in vitro transcription. The T3 and T7 promoter sequences labeled with red. (B) Oligos used for in situ hybridization experiments. T3 (T3 promoter), T7 (T7 promoter), FW (forward primer), RV (reverse primer).

**Author Contributions:** C.C. performed cryofixation, cryosectionning of bone; K.N. and H.M. performed ISH and HCR experimentations, designed probes and produced cDNA; M.D. performed rat animal model experimentation; C.C. and K.N. collected the data; X.H., K.N. and A.-L.F. designed the work; K.N., A.-L.F. and X.H. performed data analysis, interpretation; K.N. and A.-L.F. drafted the paper; C.C., K.N., X.H., M.D., H.M., A.B. and A.-L.F. approved the final version to be published. 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) (PDH2-NRBC-4-NR-4306).

**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.

**Acknowledgments:** We are very grateful to Martine Miquel for her helpful advice and critical reviewing of our manuscript. We are thankful to Zsolt Kelemen for his invaluable help on microspectrofluorimetry. We thank the Imagery platform of INRA-Versailles for technical assistance on confocal microscopy. This work was supported by the Service de Santé des Armées and a gran<sup>t</sup> (NBC-4-NR-4306) from Direction Générale de l'Armement (DGA, Paris, France).

**Conflicts of Interest:** Authors declare no conflict of interest.
