Alterations in macrophage polarization in the craniofacial and extracranial skeleton after zoledronate application and surgical interventions - an in vivo experiment

Abstract

Purpose: Medication-related osteonecrosis occurs exclusively in the jaw bones. However, the exact pathogenesis of medication-related osteonecrosis of the jaw (MRONJ) and the unique predisposition of the jaw bones have not been elucidated, making its treatment a challenge. Recent evidence indicates that macrophages might play a pivotal role in MRONJ pathogenesis. The aim of the present study was to compare the macrophage populations between the craniofacial and extracranial skeleton and to investigate the changes induced by zoledronate (Zol) application and surgical interventions.

Materials and methods: An in vivo experiment was performed. 120 wistar rats were randomized to 4 groups (G1, G2, G3, G4). G1 served as an untreated control group. G2 and G4 received Zol injections for 8 weeks. Afterwards, the right lower molar of the animals from G3 and G4 was extracted and the right tibia osteotomized followed by osteosynthesis. Tissue samples were taken from the extraction socket and the tibia fracture at fixed time points. Immunohistochemistry was conducted to determine the labeling indexes of CD68⁺ and CD163⁺ macrophages.

Results: Comparing the mandible and the tibia, we observed a significantly higher number of macrophages and a heightened pro-inflammatory environment in the mandible compared to the tibia. Tooth extraction caused an increase of the overall number of macrophages and a shift toward a more pro-inflammatory microenvironment in the mandible. Zol application amplified this effect.

Conclusion: Our results indicate fundamental immunological differences between the jaw bone and the tibia, which might be a reason for the unique predisposition for MRONJ in the jaw bones. The more pro-inflammatory environment after Zol application and tooth extraction might contribute to the pathogenesis of MRONJ. Targeting macrophages might represent an attractive strategy to prevent MRONJ and improve therapy. In addition, our results support the hypothesis of an anti-tumoral and anti-metastatic effect induced by BPs. However, further studies are needed to delineate the mechanisms and specify the contributions of the various macrophage phenotypes.

Keywords: MRONJ; bisphosphonate; macrophage; osteonecrosis; tooth extraction.

Figure 1

Photos of the surgical interventions. At a time point of 8 weeks, the right tibia was osteotomized followed by a microplate osteosynthesis in rats from group 3 and 4 (A, B). The site of the incision was closed by multilayer suturing (C, D). Additionally, the right lower molar of the animals was extracted with forceps (E, F). Surgical interventions were conducted under general anesthesia using 100 mg/kg of ketamine hydrochloride (Parke-Davis, Berlin, Germany) and 2.5 mg/kg of Xylocain (Bayer, Leverkusen, Germany) via intraperitoneal injection.

Figure 2

Study schedule. Rats from group (G) 2 and 4 were injected with zoledronate weekly for 8 weeks. After 8 weeks, surgical interventions (i.e, tooth extraction, tibia fracture followed by osteosynthesis) surgery was undertaken in rats from G3 and G4. Rats were euthanized after 8 (G1 and G2 only), 10, 12, and 16 weeks. Afterwards, tissue harvesting took place immediately. Tissue samples from the extraction socket (bone marrow and the cancellous bone of the mandible) as well as the repaired tibia fracture (bone morrow ot the tibia) were analyzed and further investigated by immunohistochemistry.

Figure 3

Representative images of CD68 immunohistochemical staining in the bone marrow (A, B) and cancellous bone of the mandible (C, D) as well as the bone marrow of the tibia (E, F).

Figure 4

Representative images of CD163 immunohistochemical staining in the bone marrow (A, B) and cancellous bone of the mandible (C, D) as well as the bone marrow of the tibia (E, F).

Figure 5

Influence of zoledronate treatment and surgical interventions on the macrophage polarization in the bone marrow and the cancellous bone of the mandible. Labeling index (LI) of (A) CD68⁺ cells, (B) CD163⁺ cells, and the (C) expression ratio of CD68 and CD163 (LI CD163/CD68) in the bone marrow of the jaw depending on the interventions. LI of (D) CD68⁺ cells, (E) CD163⁺ cells, and the (F) expression ratio of CD163 and CD68 (LI CD163/CD68) in the the cancellous bone of the tibia depending on the interventions. Rats from group (G) 1 were compared to those from G2 and G3. Rats from G4 were compared to those from G2 and G3. The box plot diagrams represent the median, interquartile range, minimum and maximum. For statistical analysis, the Mann-Whitney-U-test was used. Two-sided adjusted p values ≤0.05 were considered statistically significant. Statistically significant differences are marked with an asterisk.

Figure 6

Comparison between both sides of the jaw bone of rats treated with surgery and combinatorial treatment with surgery and zoledronate treatment regarding the macrophage polarization. Surgical interventions were conducted on the right side whereas the left jaw side served as control. Labeling index (LI) of (A) CD68⁺ cells, (B) CD163⁺ cells, and the (C) expression ratio of CD68 and CD163 (LI CD163/CD68) in the bone marrow of the mandible depending on the interventions. LI of (D) CD68⁺ cells, (E) CD163⁺ cells, and the (F) expression ratio of CD68 and CD163 (LI CD163/CD68) in the cancellous bone of the mandible depending on the interventions. The box plot diagrams represent the median, interquartile range, minimum, and maximum. For statistical analysis, the Mann-Whitney-U-test was used. Two-sided adjusted p values ≤0.05 were considered statistically significant. Statistically significant differences are marked with an asterisk.

Figure 7

Influence of zoledronate treatment and surgical interventions on the macrophage polarization in the bone marrow of the tibia. Labeling index (LI) of (A) CD68⁺ cells, (B) CD163⁺ cells, and the (C) expression ratio of CD68 and CD163 (LI CD163/CD68) in the bone marrow of the tibia depending on the interventions. Rats from group (G) 1 were compared to those from G2 and G3. Rats from G4 were compared to those from G2 and G3. The box plot diagrams represent the median, interquartile range, minimum, and maximum. For statistical analysis, the Mann-Whitney-U-test was used. Two-sided adjusted p values ≤0.05 were considered statistically significant. Statistically significant differences are marked with an asterisk.