**1. Introduction**

Macrophages have an essential role both in osteoblast-mediated bone formation [1] and in osteoclast development [2,3], but the detailed function of these cells is not ye<sup>t</sup> fully understood. In addition, the cytokines and other soluble factors secreted by macrophages can induce bone formation in vitro and in vivo [1,4–8].

For a long time, in vitro cultures were used to study the phenotypic characterization of macrophages as a model to control the extracellular environment [9–11]. In vitro macrophages can be classified into two families: (i) M1 macrophages and (ii) M2 macrophages. The M1 family expresses pro-inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-23 (IL-23), and interleukin-28 (IL-28) mediating inflammation. The M2 family expresses anti-inflammatory cytokines, such as interleukin-10 (IL-10), interleukin-1β receptor antagonist (IL-1RA), transforming

**Citation:** Nikovics, K.; Durand, M.; Castellarin, C.; Burger, J.; Sicherre, E.; Collombet, J.-M.; Oger, M.; Holy, X.; Favier, A.-L. Macrophages Characterization in an Injured Bone Tissue. *Biomedicines* **2022**, *10*, 1385. https://doi.org/10.3390/ biomedicines10061385

Academic Editor: Chikafumi Chiba

Received: 25 May 2022 Accepted: 8 June 2022 Published: 11 June 2022

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growth factor-beta (TGF-β) and proangiogenic cytokines (vascular endothelial growth factor (VEGF) that resolve inflammation and also modulate the extracellular matrix (ECM). In vitro studies have shown that the M2 family characterization is more complex and can be subdivided into four subtypes: M2a, M2b, M2c, and M2d [12–16]. Each subtype expresses a distinctive panel of cytokines and plays a different role in tissue regeneration (Figure 1) [17–23]. Surprisingly, M2b and M2d macrophages also express pro-inflammatory cytokines including TNF, IL-1β, IL-6, or IL-12 [16,24]. This division is not perfect because specific (nontypical) macrophages do not belong to either group but play an important immunoregulatory role. An in vitro approach does not allow the study of other immune cells and their respective secreted cytokines normally present at the site of the regeneration. Indeed, the host response in vivo is more complex highlighting the difficulty to deduce in vivo results from in vitro observations. The knowledge of macrophage phenotypes under in vivo conditions is still poorly understood and further investigations are essential, especially in our case, to study macrophage involvement during bone regeneration. Accordingly, in vivo macrophages are named M1-like and M2-like macrophages or resolving macrophages (Figure 1) [23,25–30].

**Figure 1.** Macrophage polarization subtypes; different cytokine expressions and functions of the macrophage populations in vitro and in vivo.

An additional category of macrophages called "Tissue-resident macrophages" is thought to participate in bone repair. Contained in almost all tissues, they are called 'osteomacs' when localized in the bone [1,2,31,32].

Biomaterial-based therapy is a useful method to improve bone regeneration; however, its underlying repair mechanism is not ye<sup>t</sup> elucidated [33–40]. About 30 years ago, the French surgeon Alain-Charles Masquelet developed a new technique to repair bone defects called the Masquelet induced membrane [41]. The surgeon places a fixator along

the bone and completes its missing parts with a biomaterial, an inert polymer that has been engineered for interacting with biological systems, usually polymethylmethacrylate (PMMA). Later on, a membrane called the Masquelet induced membrane will be generated all around the biomaterial as an immune reaction against a foreign body. It is essential for further bone regeneration [41–43]. In the literature, biomaterials are described to induce the appearance of the macrophages at the bone injury site [1,7,31,32,44–46].

One approach to characterize macrophages is the identification of their expressed cytokines. Different techniques, such as Northern blot, qPCR, microarray, flow cytometry analysis, and next-generation deep sequencing methods can be used. These methods only provide a result from a mixture of different cell types. In addition, measuring the expression level of the cytokines is not sufficient; the localization of the cytokine-expressing cells in the tissue should also be determined [47].

To localize the cytokine-expressing cells in the tissue, different methods are available: (i) expression of reporter constructs, but the limitation of this technique is the requirement of a transgenic animal; (ii) classical immunostaining techniques using antibodies against markers to detect specific proteins of the macrophages. However, since cytokines are generally secreted, it is difficult to determine exactly which cells produce this protein or peptide [48]. This technique can only distinguish between M1-like and M2-like macrophages. However, it is not appropriate for identifying M2-like subtypes, as there is currently no cell surface marker available to distinguish between the different subtypes; and (iii) in situ hybridization is one of the most convincing methods to identify the cytokine-expressing cells because it is based on messenger RNA (mRNA) detection of the targeted genes [47].

In the present study, the cryosections of non-decalcified rat femur surrounded by muscle were investigated by immunostaining, in situ hybridization, and in situ HCR to identify phenotypes of macrophages involved in bone regeneration. In these challenging conditions, expression of CD68, CD163, IL-1β, IL-6 and β-actin genes was successfully detected resulting for the first time in the identification and localization of M2b-like macrophages in vivo in the bone of rats during bone regeneration.

### **2. Materials and Methods**

### *2.1. Rat Animal Model*

All experiments were approved by the IRBA Institutional Animal Care and Use Committee (protocol 65 DEF\_IGSSA\_SP). Surgeries were carried out in an accredited animal facility. Eight-week-old (200 g average weight) male Sprague Dawley rats (Charles River Laboratories, Freiburg, Germany) were housed individually in cages in a temperature and light-controlled environment with food and provided water ad libitum. Before collecting femurs with muscles, animals were euthanized at 12 weeks old with an overdose of sodium pentobarbital (150 mg/kg) administrated intraperitoneally.

### *2.2. Embedding and Cryosectioning of the Entire Femur (Bone and Muscle Together) of the Rat*

Embedding and cryosectioning methods were performed as described in [49]. RNasefree instruments, materials, and buffers were used to collect bone samples. After euthanasia of the rat, the whole femur was cleaned rapidly, and a part of the muscles around the bone was kept. The femur was placed at the bottom of the embedding mold and covered with cryomoun<sup>t</sup> medium (CM) (00890-EX, HistoLab, Askim, Norway). Samples were snap-frozen with 2-methyl butane cooled in liquid nitrogen to obtain a block.

### *2.3. Histological Staining*

Histological staining was performed as described in [49]. Hematoxylin and phloxin (HP) staining was performed as follows: the sections were incubated in several successive baths: 40 s in a hemalum (11,487, Merck, Darmstadt, Germany) buffer (0.2 g hemalum, 5 g aluminum potassium sulfate in 100 mL distilled water), 3 min in water, 30 s in a phloxin (15,926, Merck, Darmstadt, Germany) buffer (0.5 g phloxin in 100 mL distilled water), 1 min in water, 2 min in 70% ethanol, 30 s in 95% ethanol, 1 min in 100% ethanol, and 1 min in 100% ethanol. In the end, nuclei were colored in blue and cytoplasm in pink.
