*2.5. RT-qPCR*

Frozen femur samples were homogenized in liquid nitrogen. Total RNA was then isolated using an RNeasy Fibrous Tissue mini kit (HB-0485, Qiagen, Courtaboeuf, France) according to the m anufacturer's recommendations. RNA extracts were recovered with 20 μL RNase-free water. Reverse transcription (RT) was performed with oligo-dT primers following the instructions of the Sensiscript transcription kit (205211, Qiagen, Courtaboeuf, France); cDNA synthesis was carried out for 1 h at 37 ◦C using 50 ng of RNA with 10 μM oligod(T) primers, RNase inhibitor (2 IU), and Sensiscript reverse transcriptase.

Real-time qPCR was carried out in a 20 μL final volume using LC480 SybrGreen I Mastermix (Roche Applied Science, Mannheim, Germany) using 0.25 μL of cDNA. Oligos designed for RT-qPCR experiments are listed in Table S1.

### *2.6. In Situ DIG Hybridization*

In situ hybridization methods were performed as described in [49]. Oligos designed for in situ hybridization experiments are listed in Table S2.

Digoxigenin (DIG)-labeled cRNA probes were used for in situ hybridization. Briefly, the tissues were fixed in 4% ( *w*/*v*) in PBS/paraformaldehyde (PBS, *w*/*o* Ca and Mg, GAUPBS0001, Eurobio, Les Ulis, France; PFA, P6148, Sigma, Lezennes, France) for 30 min and then treated with 100% methanol for 15 min and air-dried. The sections were prehybridized for 2 h at 45 ◦C in a pre-hybridization buffer (50% formamide (GHYFOR0402, Eurobio, Les Ulis, France), 0.5× sodium ch loride citrate (SSC) (GHYSSC007, Eurobio, Les Ulis, France) buffer, 50 μg mL −1 heparin (H3393, Sigma, Lezennes, France), 100 μg mL−<sup>1</sup> transfer RNA, and 0.1% (*v*/*v*) Tween 20 (822184, Merck, Darmstadt, Germany). Finally, the sections were incubated overnight at 45 ◦C with the RNA probes (2 μL probe in 200 μL hybridization buffer (50% formamide, 100 μg mL−<sup>1</sup> transfer RNA, 7.5% (*v*/*v*) Tween 20, 8.5% NaCl, 20% dextran sulfate (GHYDEX000T, Eurobio, Les Ulis, France), and 2.5× Denhardt's Solution (50× stock, D2532, Sigma, Lezennes, France)), which were previously denaturized for 2 min at 80 ◦C in the hybridization buffer. Non-specific hybrids were dissociated with the following washes: 30 min in 0.1× SSC + 0.5% SDS at 45 ◦C, 2 h in 2× SSC + 50% formamide at 45 ◦C, 5 min in NTE (0.5 M NaCl, 10 mM Tris pH 8, 1 mM EDTA) at 45 ◦C,

30 min in NTE + 10 mg mL−<sup>1</sup> Rnase A (10109169001, Roche, Boulogne-Billancourt, France) at 37 ◦C, 1 h in 2× SSC + 50% formamide at 45 ◦C, 2 min in 0.1× SSC at 45 ◦C, and finally 15 min in PBS at RT.

Immunodetection of the DIG-labeled probes was performed using an anti-DIG antibody coupled to alkaline phosphatase as described by the manufacturer (11093274910, Roche, Boulogne-Billancourt, France). Afterward, the sections were incubated for 1–2 days in a buffer containing 337 μL BCIP (5-Bromo-4-chloro-3-indolyl-phosphate) and 225 μL NBT (Nitroblue tetrazolium chloride) in a 50 mL solution (100 mM Tris pH 9.5, 100 mM NaCl and 50 mM MgCl2) until a blue precipitate adhering to the sections were formed. The DIG sections were observed with an epifluorescence microscope DM6000 (Leica, Schönwaldeglien, Germany) equipped with monochrome and color digital cameras, while the HCR sections were observed with a confocal microscope (LSM700, Zeiss, Dresden, Germany).

### *2.7. In Situ HCR Hybridization*

In situ hybridization methods were performed as described in [49]. Oligos designed for in situ hybridization experiments are listed in Table S2.

The HCR protocol of Choi and colleagues (2014, 2016, 2018, 2020) was performed with some modifications as described below to enhance mRNA localization in the femur of the rat [50–53].

The sections were pre-hybridized for 10 min at RT in a hybridization buffer (50% formamide, 5× SSC, 9 mM citric acid pH 6, 50 μg mL−<sup>1</sup> heparin, 1× Denhardt's Solution, 0.1% (*v*/*v*) Tween 20 and 10% dextran-sulfate). Previously, the hybridization probes (2 pmol per slide) were denatured for 2 min at 80 ◦C. Finally, the sections were incubated in a hybridization buffer together with probes overnight at 45 ◦C. Nonspecific hybrids were dissociated with the following washes: 30 min in 0.1× SSC + 0.5% SDS at 45 ◦C, followed by 2 h in 2× SSC + 50% formamide at 45 ◦C, then 2 min in 0.1× SSC at 45 ◦C, and finally 15 min in PBS at RT.

Sections were first incubated for 2 h at RT with an amplification buffer (5× SSC, 0.1% (*v*/*v*) Tween 20, 10% dextran-sulfate, and 100 μg mL−<sup>1</sup> salmon sperm ADN) and subsequently for 12 to 16 h with the DNA hairpins marked with a fluorophore (Alexa Fluor488) (diluted in amplification buffer as previously described). The hairpins were previously heated at 95 ◦C for 90 s and cooled to RT for 30 min. The sections were then washed 2× 30 min in 5× SSCT (5× SSC and 1% (*v*/*v*) Tween 20) and 5 min with 5× SSC without Tween at RT.

### *2.8. Microspectroscopical Analysis*

For spectroscopic analysis, an LSM 780 (Carl Zeiss, Jena, Germany) confocal microscope was used to acquire a lambda stack of 10 nm wavebands between 425 nm and 625 nm. Then, Zen Black software (Carl Zeiss, Jena, Germany) built-in plugin was used to perform linear unmixing using the automatic component extraction algorithm. It was possible to extract Alexa 488 spectrum, and the second spectrum was considered the sum of all other sources of endogenous fluorescence (autofluorescence).

### *2.9. Quantification of HIS by ImageJ*

To quantify the percentage of the marked region on in situ hybridization slides, each slide was acquired with the Hamamatsu Nanozoomer S60 at 20x (Tokyo, Japan). On the virtual slide, three regions of interest (ROI) were drawn in the immediate neighborhood of the biomaterial; three others were done away from it (internal negative control). Each zone was analyzed using the Fiji software. The ratio between the surface of the marked region and the surface of the entire ROI was computed after color deconvolution and thresholding (using the MaxEntropy algorithm).

Statistical analyses were performed with a *t*-test.
