Abstract
Background/purpose
Medication–related osteonecrosis of the jaw (MRONJ), a serious adverse effect of antiresorptive agents, remains difficult to manage. Our previous studies showed that statins, widely prescribed for dyslipidemia, promote healing in MRONJ–like lesions, however, the mechanisms underlying their therapeutic effects remain unclear. This study investigated the efficacy of the therapeutic effect of fluvastatin (FS) in a rat MRONJ–like model, with particular attention to macrophage polarization.
Materials and methods
Four–week–old female rats were allocated to three groups. Two groups received zoledronic acid (ZOL) and dexamethasone (DEX) for two weeks prior to extraction of maxillary first molars. After extraction, one group was treated with a single local injection of FS near the extraction site, whereas the other received saline. A control group underwent identical procedures, but received saline instead of ZOL, DEX, and FS. All animals were evaluated two weeks after treatment.
Results
The control (saline) group showed complete socket healing. In contrast, ZOL/DEX–treated rats exhibited incomplete epithelial closure and exposed necrotic bone. However, local FS administration in ZOL/DEX–treated rats extensively promoted healing, resulting in smaller epithelial gaps and reduced bone exposure, and decreased areas of necrotic bone. Total macrophage numbers were significantly higher in both experimental groups compared with the control group. The ZOL/DEX group showed a predominance of pro–inflammatory M1 macrophages, whereas the FS–treated group exhibited relatively increased proportions of tissue–reparative M2 macrophages.
Conclusion
Local FS administration enhances bone healing of MRONJ–like lesions by promoting macrophage polarization toward M2 phenotype.
Keywords: Bisphosphonate—associated osteonecrosis, Bone remodeling, Fluvastatin, Macrophages, Wound healing
Introduction
Medication–related osteonecrosis of the jaw (MRONJ) is a severe adverse effect of antiresorptive agents such as bisphosphonates (BP).1 It is characterized by persistent exposed bone and impaired soft/hard tissue healing, leading to chronic inflammation and infection. The proposed mechanisms of MRONJ pathogenesis include suppression of bone remodeling and excessive inhibition of osteoclastic activity by antiresorptive drugs,2 oral bacterial infection,3 and inhibition of angiogenesis;4 however, the precise mechanisms remain unclear. Currently available treatments, such as irrigation, 5 antibiotic administration,6,7 and sequestrectomy,8 are only palliative, and there remains a pressing need for an established curative therapy. We and other groups have been focused on statins, which are widely used for dyslipidemia but also exhibit pleiotropic effects, including osteogenic,9,10 epithelial healing,11 anti–inflammatory,12 and angiogenic activities.13,14 Our previous study demonstrated that fluvastatin (FS) administration promoted healing of MRONJ–like lesions in rats.15 However, the underlying mechanisms have not yet been fully elucidated.
Macrophages (Mφ) play a central role in innate immunity and the maintenance of tissue homeostasis.16 Mφ exist in several subtypes. M1Mφ produce proinflammatory cytokines and induce inflammation,17,18 whereas M2Mφ secrete anti–inflammatory cytokines and promote tissue repair.19,20 Previous studies have reported that a decreased M2/M1 ratio–characterized by an increase in M1 and reduction in M2Mφ–is associated with MRONJ–like lesions in BP–treated mice and patients.21 Conversely, statins have been reported to promote M2Mφ polarization and facilitate tissue healing.22 Based on these findings, we hypothesized that local administration of FS to MRONJ–like lesions–- where inflammation is enhanced due to M1Mφ predominance induced by BP–could shift polarization toward M2Mφ, thereby promoting tissue healing.
Therefore, this study aimed to investigate the therapeutic effects of FS on MRONJ–like lesions by observing Mφ polarization and evaluating its contribution to the healing process.
Materials and methods
Rat model
This study used female Wistar rats (four weeks old, weighing 55–70 g; n = 7 per group, three groups in total). All experiments were conducted in accordance with the ARRIVE guidelines for reporting animal research.23 The experimental protocol was approved by the Institutional Animal Care and Use Committee of Kyushu University (approval no. A24-048-0).
An MRONJ–like rat model was established based on previously described methods (Fig. 1A).15,24 Zoledronic acid (Zometa; Novartis Pharma, Tokyo, Japan) was administered subcutaneously at a dose of 66 μg/kg (0.165 mL/100 g body weight), and dexamethasone (Decadron; Aspen Japan, Tokyo, Japan) was administered at 5 mg/kg (0.15 mL/100 g body weight) three times per week. Two weeks after the start of drug administration, the maxillary first molars were extracted. Two weeks after tooth extraction, when MRONJ–like lesions had developed, fluvastatin (FS, Fujifilm Wako Chemicals, Osaka, Japan) was locally injected near the lesion site at a dose of 10 mg/kg (BP + FS group, n = 7). The FS concentration was determined according to our previous study.15 A group receiving saline instead of FS served as the BP group (n = 7). In addition, a saline group (n = 7) received the same volume of saline subcutaneously three times per week instead of zoledronic acid and dexamethasone, followed by local saline injection near the extraction socket. All rats were randomly assigned to the groups. Evaluations were performed two weeks after FS or saline administration.
Figure 1.
Timeline of the experiment and measurement methods for histological evaluation.
(A) The schedule of bisphosphonates (BP), fluvastatin (FS) and saline administration is illustrated. (B) Measurement method for epithelial gap distance (a) and exposed necrotic bone length (b) on hematoxylin and eosin (HE)–stained sections. (C) Measurement method for necrotic bone area (defined as osteocyte–lacking lacunae) on HE–stained sections.
Micro–computed tomography analysis
Maxillae were scanned by micro–computed tomography (μ– CT) at a voxel resolution of 15 μm and a tube voltage of 90 kV. Bone morphometric analysis was performed with TRI/3D–Bon software (Ratoc System Engineering, Tokyo, Japan) to determine the bone volume fraction (bone volume/tissue volume: BV/TV) in the extraction socket.
Tissue preparation
After μ–CT scanning, specimens were decalcified in 20 % ethylenediaminetetraacetic acid (EDTA; Dojindo Laboratories, Kumamoto, Japan), dehydrated with 99 % ethanol (Mitsubishi Chemical, Tokyo, Japan), cleared in xylene (Nacalai Tesque, Kyoto, Japan), and embedded in paraffin. Observations were performed using a BZ–X810 microscope (Keyence, Osaka, Japan).
Hematoxylin and eosin staining
Sections were stained with hematoxylin and eosin (HE), followed by the observation with light microscopy. The parameters measured included epithelial gap distance, exposed necrotic bone length, and necrotic bone area (Fig. 1B and C). The necrotic bone area was defined as the region containing empty lacunae and measured using BX–Z Analyzer software (Keyence).
Tartrate–resistant acid phosphatase staining
Tartrate–resistant acid phosphatase (TRAP) staining was performed to evaluate osteoclast–like cells around the extraction sockets. TRAP/ALP Stain Kit (Fujifilm Wako Chemicals) was used according to the manufacturer’s instructions. After washing with distilled water, nuclei were lightly counterstained, followed by dehydration, clearing, and mounting with Entellan® new (Merck, Darmstadt, Germany). Multinucleated TRAP–positive cells located on the bone surface were identified as osteoclast–like cells.
Immunofluorescence staining
Immunofluorescence staining was performed to evaluate the distribution and polarization of Mφ around the extraction sockets. Based on previous studies,25 CD68, CD38, and CD163 were used as markers for total Mφ, M1Mφ, and M2Mφ, respectively. NG2 was used as a pericyte marker because NG2–positive pericytes express multiple immune cell markers, including CD68. Therefore, in this study, CD68+NG2− cells within and around the extraction socket were identified as Mφ. For double staining of Mφ and pericytes, deparaffinized sections were incubated with a cocktail of primary antibodies–mouse monoclonal anti–CD68 antibody (ab31630; Abcam, Cambridge, UK) at 1:100 and rabbit monoclonal anti–NG2 antibody (clone 1L2, ZooMAb® ZRB5320; Sigma Aldrich, St. Louis, MO, USA) at 1:50–at 4 °C for 12 h. After washing, sections were incubated with Alexa Fluor 546 donkey anti–mouse IgG (A10036; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 488 goat anti–rabbit IgG (A11008; Invitrogen). For M1/M2 phenotype analysis, antigen–retrieved sections were incubated with mouse monoclonal anti–CD38 antibody (sc–374650; Santa Cruz Biotechnology, Dallas, TX, USA) for M1 (1:50) and anti–CD163 antibody (sc–58965; Santa Cruz Biotechnology) for M2 (1:50) at 4 °C for 12 h, followed by FITC–conjugated goat anti–mouse IgG (H&L) (ab6785; Abcam) at 1:200 for 1 h at room temperature. Finally, sections were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (H–1200; Vector Laboratories, Burlingame, CA, USA).
Statistical analysis
Normality was assessed using the Shapiro–Wilk test. When normality was not satisfied, the Kruskal–Wallis test was applied, followed by the Steel–Dwass method for multiple comparisons among the three groups. When normality was confirmed, one–way analysis of variance (ANOVA) was performed, followed by Tukey’s honestly significant difference (HSD) test for multiple comparisons. A two–tailed P–value < 0.05 was considered statistically significant. Statistical analyses were conducted using JMP® Student Edition 18 (SAS Institute Inc., Cary, NC, USA).
Results
Evaluation of tissue healing
μ–CT analysis revealed minimal new bone formation within the extraction sockets of the BP group, whereas newly formed bone was observed in the BP + FS group. The extraction sockets in the saline group were completely filled with new bone (Fig. 2A–C). Statistical analysis demonstrated that the bone volume fraction (BV/TV) was significantly lower in the BP group than in the BP + FS and saline groups, with no significant difference between the BP + FS and saline groups (Tukey’s HSD: BP vs. BP + FS, P < 0.05; BP vs. Saline, P < 0.01; BP + FS vs. Saline, P = 0.13) (Fig. 2G). Histological examination at 4 weeks post–extraction showed that in all rats of the BP group, epithelial continuity was disrupted, necrotic bone was exposed, connective tissue formation was insufficient, and numerous inflammatory cells were observed around the necrotic bone. Necrotic bone was mainly present at the exposed surface, but not in the apical area. In the BP + FS group, partial or complete epithelial closure of the extraction sockets was observed, along with reduced necrotic bone and fewer inflammatory cells compared to the BP group. In the saline group, complete epithelial closure and the formation of osteoid tissue were observed in the extraction sockets, with connective tissue filling the space between bone and epithelium. Neither necrotic bone nor inflammatory lesions were detected in the extraction or non–extraction sides (Fig. 2D–F). Statistical analyses showed that the epithelial gap distance was significantly shorter in the BP + FS and saline groups than in the BP group, with no significant difference between the BP + FS and saline groups (Kruskal–Wallis test with Steel–Dwass post hoc test: BP vs. BP + FS, P < 0.01; BP vs. Saline, P < 0.01; BP + FS vs. Saline, P = 0.36) (Fig. 2H). Similarly, the length of exposed necrotic bone was significantly shorter in the BP + FS and saline groups than in the BP group, with no significant difference between the BP + FS and saline groups (BP vs. BP + FS, P < 0.05; BP vs. Saline, P < 0.01; BP + FS vs. Saline, P = 0.36) (Fig. 2I). The necrotic bone area was significantly smaller in the BP + FS and saline groups than in the BP group, with no significant difference between the BP + FS and saline groups (BP vs. BP + FS, P < 0.05; BP vs. Saline, P < 0.01; BP + FS vs. Saline, P = 0.18) (Fig. 2J).
Figure 2.
Micro–computed tomography and histological evaluation.
(A–C) Representative micro–computed tomography (–CT) images of the Saline, BP, and BP + FS groups. (D–F) Representative hematoxylin and eosin (HE)–stained images of the extraction socket. Scale bars: 500 μm. (G) Quantification of bone volume fraction (bone volume/tissue volume: BV/TV). Values are expressed as mean ± SD. The significance of differences between groups was determined by one–way analysis of variance with post hoc Tukey’s honestly significant difference test. *P < 0.05; **P < 0.01. (H–J) Quantification of epithelial gap distance, exposed necrotic bone length, and necrotic bone area. Values are expressed as median (interquartile range). The significance of differences between groups was determined by the Kruskal–Wallis test with Steel–Dwass post hoc test. *P < 0.05; **P < 0.01. BP, bisphosphonates; FS, fluvastatin.
Evaluation of osteoclasts
Numerous TRAP–positive multinucleated cells were observed on the bone surface in the saline group. The BP group exhibited a significantly lower number of TRAP–positive cells compared to the saline group. The BP + FS group showed a significant increase in TRAP–positive multinucleated cells compared to the BP group, but no significant difference was found between the BP + FS and saline groups (Tukey’s HSD: BP vs. BP + FS, P < 0.01; BP vs. Saline, P < 0.01; BP + FS vs. Saline, P = 0.30) (Fig. 3A–D).
Figure 3.
Osteoclast activity.
(A–C) Representative tartrate–resistant acid phosphatase (TRAP) staining images of the Saline, BP, and BP + FS groups. Scale bars: 50 μm. (D) Quantification of TRAP–positive multinucleated cells. Values are expressed as mean ± SD. The significance of differences between groups was determined by one–way analysis of variance with post hoc Tukey’s honestly significant difference test. **P < 0.01. BP, bisphosphonates; FS, fluvastatin.
Evaluation of macrophages
In the saline group, extraction sockets showed a healed appearance with a relatively small number of Mφ distributed broadly. In the BP group, granulation tissue was abundant within the extraction socket, and numerous Mφ were detected in and around the lesion. The proportion of M1Mφ was higher than that of M2Mφ. Conversely, in the BP + FS group, extraction sockets showed a healing tendency, and numerous Mφ were observed predominantly around the socket. The proportion of M2Mφ was greater than that of M1Mφ (Fig. 4A–I). Statistical analysis revealed that the number of CD68+NG2− cells (Mφ) was significantly higher in the BP and BP + FS groups than in the saline group (Tukey’s HSD: BP vs. BP + FS, P = 0.066; BP vs. Saline, P < 0.01; BP + FS vs. Saline, P < 0.01) (Fig. 4J). The number of CD38+ cells (M1Mφ) was significantly higher in the BP group than in the BP + FS and saline groups (BP vs. BP + FS, P < 0.05; BP vs. Saline, P < 0.01; BP + FS vs. Saline, P = 0.31) (Fig. 4K). In contrast, the number of CD163+ cells (M2Mφ) was significantly higher in the BP + FS group than in the BP and saline groups (BP vs. BP + FS, P < 0.05; BP vs. Saline, P = 0.91; BP + FS vs. Saline, P < 0.05) (Fig. 4L). The M2/M1 ratio was significantly higher in the BP + FS groups and saline group than in the BP group (Tukey’s HSD: BP vs. BP + FS, P < 0.01; BP vs. Saline, P < 0.01; BP + FS vs. Saline, P = 0.30) (Fig. 4M).
Figure 4.
Macrophage distribution and polarization.
(A–C) Representative images of CD68 (red), NG2 (green), and DAPI (blue). CD68+NG2− cells were defined as macrophages (Mφ). Scale bars: 100 μm. (D–F) Representative images of M1 macrophages (M1Mφ) (CD38+). Scale bars: 100 μm. (G–I) Representative images of M2 macrophages (M2Mφ) (CD163+). Scale bars: 100 μm. (J–M) Quantification of Mφ, M1Mφ, M2Mφ, and the M2/M1 ratio. Values are expressed as mean ± SD. The significance of differences between groups was determined by one–way analysis of variance with post hoc Tukey’s honestly significant difference test. *P < 0.05; **P < 0.01. BP, bisphosphonates; FS, fluvastatin.
Discussion
In the present study, the BP + FS group showed restoration of epithelial continuity accompanied by connective tissue formation in the extraction socket compared with the BP group. Previous studies have reported that FS promotes soft tissue healing in extraction sockets by enhancing the proliferation of oral epithelial cells and fibroblasts.11 In addition, farnesyl pyrophosphate (FPP), a downstream metabolite of the mevalonate pathway, has been shown to inhibit keratinocyte migration and delay epithelial healing. 26 Furthermore, statins have been reported to upregulate mRNA expression of FGF–2 and TGF–β, which promote fibroblast proliferation and collagen production.27,28 Taken together, FS may accelerate epithelial closure by inhibiting FPP synthesis and enhance connective–tissue formation through growth–factor–mediated fibroblast activation.
In addition, the BP group showed a pronounced reduction in TRAP–positive osteoclasts, whereas the BP + FS group exhibited significant recovery of osteoclast numbers compared with the BP group. BP binds strongly to hydroxyapatite in bone and accumulates at resorptive sites, where it is internalized by osteoclasts. It inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, leading to impaired prenylation of small GTP–binding proteins and suppressing osteoclast resorptive capacity, cytoskeletal organization, and vesicular transport.34 Although statins also inhibit HMG–CoA reductase, they do not preferentially accumulate on bone surfaces like BP. Therefore, the decreased osteoclast count in the BP + FS group is unlikely to result from a direct inhibitory effect of FS, but rather from BP–induced osteoclast apoptosis. The partial recovery of osteoclast numbers in the BP + FS group may be attributed to the anti—inflammatory effects of FS and its indirect activation of osteoblasts. Statins promote osteoblast differentiation, enhance BMP—2 and VEGF expression, and reduce osteoblast apoptosis.35–37 These findings suggest that FS promotes osteoblast differentiation and increases osteoblast—derived RANKL expression, thereby facilitating the differentiation of osteoclast precursors into mature osteoclasts.
In the BP group, which developed MRONJ—like lesions after BP administration, M1—dominant polarization of Mφ was observed, accompanied by prolonged inflammation and delayed tissue repair. In contrast, the BP + FS group exhibited M2—dominant polarization and improved tissue repair following a single local FS injection. BP is reportedly selectively internalized by osteoclasts at bone—resorptive sites and induces osteoclast apoptosis. This cytotoxic effect not only suppresses bone turnover but also affects the immune microenvironment of surrounding tissues.29 BP—induced cell injury leads to the release of damage—associated molecular patterns, which activate NF—κB signaling, resulting in sustained production of pro—inflammatory cytokines and polarization toward M1Mφ, while suppressing M2 polarization.21,30,31 Conversely, statins reportedly inhibit HMG—CoA reductase and suppress the synthesis of FPP and geranylgeranyl pyrophosphate, leading to reduced prenylation of small GTP—binding proteins such as Rho/Rac/Ras.32 This down-regulates the Rho—ROCK pathway and inhibits degradation of IκB, thereby suppressing NF—κB activation and downstream production of inflammatory cytokines.32 Statins have also been shown to activate PPARγ in Mφ, a key regulator associated with anti—inflammatory responses and M2 polarization.33 These findings suggest that FS shifts the Mφ balance toward the M2 phenotype in MRONJ—like lesions, thereby promoting tissue repair.
In conclusion, this study demonstrated that a local single administration of FS near MRONJ—like lesions, where M1Mφ are already predominant, increased the M2/M1 ratio and promoted healing. FS may also enhance soft tissue repair through epithelial and fibroblast activation, and indirectly facilitate necrotic bone resolution and new bone formation by stimulating osteoblast differentiation and RANKL production, leading to osteoclast maturation. Collectively, these findings suggest that FS promotes healing of MRONJ—like lesions by inducing a shift toward M2—dominant polarization.
The main limitation of this study is the use of a rat model, which does not fully reflect clinical conditions. Since no established diagnostic criteria for MRONJ exists in rats, bone exposure observed two weeks after tooth extraction was defined as an MRONJ—like lesion in this study.
Acknowledgments
This work was supported by JSPS KAKENHI Grant Number JP22K21016.
Funding Statement
This work was supported by JSPS KAKENHI Grant Number JP22K21016.
Footnotes
Declaration of competing interest: The authors declare no conflicts of interest relevant to this article.
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