The effects of local and systemic metronidazole on bone healing in mandibular defects: a rat model

Abstract

Objective

This study evaluated the effects of local and systemic Metronidazole (MTZ) on bone healing in infected mandibular defects using a rat model.

Study design

Thirty Wistar rats were divided into Control, Local MTZ (L-MTZ), and Systemic MTZ (S-MTZ) groups. Mandibular defects were treated with xenograft mixed saliva from periodontitis patients. Bone regeneration was evaluated using micro-CT and histological analyses. Bone morphogenic protein-2 (BMP-2), alkaline phosphatase (ALP), receptor activator of nuclear factor kappaB ligand (RANKL), osteocalcin (OCN), and beta-catenin (β-catenin), high sensitivity C-reactive protein (hs-CRP) levels evaluated.

Results

Increased bone formation and reduced osteoclast counts were detected in the L-MTZ group compared to the Control. β-catenin expression was significantly higher in the L-MTZ group, BMP-2 and Runx2 levels were elevated in both L-MTZ and S-MTZ groups. ALP and OCN levels were the highest in the L-MTZ group, with no significant difference between the L-MTZ and S-MTZ groups. hs-CRP levels were significantly lower in MTZ-treated groups. Micro-CT analysis revealed the highest bone volume/total volume (BV/TV) ratio in the S-MTZ group among all groups.

Conclusion

Local MTZ application enhanced bone regeneration by promoting osteoblast activity, activating β-catenin and BMP-2/Runx2 signaling, and reducing inflammation. Systemic MTZ also improved bone healing, particularly in volumetric aspects.

Background

Alveolar bone defects can arise from various causes, and their treatment has been widely discussed in the literature [1–3]. Such defects present aesthetically and functionally problematic outcomes [4]. Bone defect regeneration is one of the most significant challenges in dentistry. The management of patients with maxillofacial defects should be guided by the principles of improving the quality of life and functional preservation of patients. Currently, a variety of graft materials are utilized for this purpose.

Autogenous bone grafts are the gold standard for bone regeneration. However, they have limited practical applications due to potential morbidity and volume restrictions. As a result, synthetic grafts and xenografts have been developed as alternatives [5]. For instance, xenografts provide bone osteoconductive strength and exhibit biomechanical properties analogous to those of bone while also serving to prevent complications [6].

Infection represents a significant factor influencing the success of the surgical procedure. In the presence of infection, the acidity and alkalinity of the environment drop below pH 2, which results in the rapid resorption of all graft materials. Bacteria gain access to the graft and induce local inflammation and a reduction in bone formation. Consequently, infection in the region intended for grafting results in graft infection during or following the procedure, accompanied by inadequate bone formation and loss [7]. Utilizing treatment protocols that aim to facilitate healing by inhibiting inflammation is a common practice in oral and maxillofacial surgery and other medical specialties [8]. Reducing inflammatory processes results in a regression of edema in the affected tissue and an acceleration of the healing process in the bone [9].

Antibiotics are agents used to inactivate or eliminate microorganisms for therapeutic or prophylactic reasons. However, their known bactericidal and bacteriostatic effects also have essential immunological properties on other biological, physiological, and host defense mechanisms [10]. Various treatment methods and agents are used for this purpose. In addition to their antibacterial properties, antibiotics are also believed to facilitate bone healing through their anti-inflammatory activity [11–13]. The impact of antibiotics, commonly employed in maxillofacial surgery, on the bone healing process in the context of inflammation remains unclear [14]. However, the superiority of local or systemic use of antibiotics over each other has not been fully proven. Combining bone grafts and antibiotics is an approach to the clinical management of infected bone defects [15, 16]. Local antibiotics are applied directly to the desired area, creating a high concentration dose there [17]. In this regard, Buchholz and Engelbrecht added antibiotics into bone grafts, stating that they would release high concentrations for a long time [18].

Metronidazole (MTZ) is a nitroimidazole group antibiotic for treating protozoa infection and is known to be particularly effective against black-pigmented Bacteroides [10]. Since anaerobic bacteria usually cause periodontal diseases, the mechanism of action of MTZ in the oral environment has been investigated with single and combined treatments. A sudden increase in plasma concentration of MTZ was observed within 1–2 h after systemic administration. Its density measured in the gingival crevicular fluid is slightly lower than that of plasma [19, 20]. The bactericidal effect of MTZ is achieved through the inhibition of DNA synthesis. Once internalized by the anaerobic organism, the drug is converted into various short-lived intermediates. These products induce bacterial death by interacting with DNA and other macromolecules deleteriously. MTZ exerts its bactericidal effect exclusively on the obligate anaerobic portion of the oral microbiota, which encompasses Porphyromonas gingivalis (P.gingivalis) and other black-pigmented Gram-negative organisms, except Aggregatibacter actinomycetemcomitans, a facultative anaerobe [19, 21]. There is a question about the use of antibiotics and whether the disadvantages are greater than the benefits because using antibiotics has been shown to increase antibiotic-resistant bacteria and select for bacterial strains that are inherently resistant/tolerant. While MTZ resistance is found in certain types of bacteria (anaerobic Gram-positive cocci; Peptostreptococci and Parvimonas micra and rods; Clostridium spp., Propionibacterium spp., Bifidobacterium spp.), Gram-negative anaerobic isolates resistant to MTZ are found to have low or no prevalence [22, 23]. MTZ (400 mg bid for 10 days) given after periodontal treatment was found to result in a low level of antibiotic resistance in the subgingival microbiota, with anaerobes dominating after 5 years [24].

This study aimed to evaluate the effects of MTZ, administered locally and systemically, on bone regeneration in infected mandibular defects. By utilizing a rat model, the study aimed to assess MTZ’s potential to enhance osteogenesis, modulate inflammatory responses, and influence key signaling pathways, such as BMP/Runx2 and Wnt/β-catenin, thereby offering insights into its clinical applicability in dentistry.

Materials and methods

Animals

Thirty male Wistar rats (4 months old, weighing 350–400 g) were used. Animals were maintained under conventional conditions (temperature of 22 ± 2 °C; 12-hour light-dark cycle; unrestricted access to tap water and regular rat diet in pellet form). Each experimental procedure was performed in accordance with the University Animal Experiments Ethics Committee (PAUHDEK-2024/04). The experimental protocol was designed according to the “NC3Rs ARRIVE Guidelines, Animal Research: Reporting of in-vivo Experiments”. In the present study, power analysis was performed with the help of the G Power 3.1.9.7 program to calculate the sample size. When effect size = 0.98, α = 0.05, and 1-β = 0.99, it was determined as 10 for each group [25].

Experimental procedure

Animals were anesthetized by intraperitoneal injection of 50 mg/kg bw ketamine (Eczacibasi Ilac Sanayi, Istanbul, Türkiye) and 5 mg/kg bw xylazine chloride (Virbaxil^®, São Paulo, Brazil). Postoperatively, animals were observed on food consumption and weight gain. Rats were divided into 3 groups: Control; local metranidazole (L-MTZ); systemic metranidazole (S-MTZ). After general anesthesia, the skin around the recipient sites was shaved and then disinfected with a 10% povidone-iodine solution, and local infiltration anesthesia was applied to the incision site with an articaine containing 1/100,000 epinephrine. A linear submandibular incision of 20 mm was made in the skin parallel to the inferior border of the mandible. Blunt dissection of the soft tissues and masseter muscle was performed. Subsequently, the periosteum was carefully elevated to expose the mandibular bone. A standardized defect was created around the first molar after exposing the buccal surface (5 × 4 × 1 mm³) around the first molar using a round dental bur at a low speed with continuous irrigation with physiological saline solution [26]. Anterior defect border 1 mm distal to mandibular anterior and coronal border 1 mm below alveolar crest [26, 27]. In the Control group, xenograft (Geistlich Bio-Oss, Switzerland) was mixed with saliva from a stage 3 periodontitis patient and then applied to the defect area. In the L-MTZ group, saliva-infected xenograft and local 0.75% metronidazole gel (ROZA, ORVA İlaç, İzmir, Türkiye) were mixed and applied to the defect area (Fig. 1) [28]. 2 mg 0.75% metronidazole gel was placed in an autoclave and heated at 121 °C for approximately 20 min. MTZ, which lost some weight and increased viscosity after autoclaving, was mixed with xenograft and applied to the defect area. In the S-MTZ group, a saliva-infected xenograft graft was applied to the defect area, and postoperatively, 235 mg/kg MTZ was mixed with 0.5 ml distilled water and given to rats three times a day for 7 days by oral gavage [29, 30]. After xenograft application in all groups, the dissected soft tissues were sutured with 4/0 resorbable suture (Pegelak, Doğsan, Türkiye). All animals were sacrificed at 8 weeks post-operating by cardiac puncture. Blood samples were collected in EDTA-coated tubes and centrifuged at 3,000 rpm for 10 min at 4 °C. Obtained serum samples were stored at -20 °C until used for enzyme-linked immunosorbent assay (ELISA) studies.

Fig. 1.

Surgical procedure images. (A, B) defect size measurement, (C) xenograft and MTZ gel, (D) filling the defect with graft

Histopathological analysis

During the necropsy, jaw samples were dissected and fixed in a 10% neutral-buffered formalin solution. Following fixation, samples were decalcified in a decalcifying solution (10% EDTA Decalcification Solution (E-IR-R112), Elabscience USA) for two weeks. They were then dehydrated in a graded ethanol series and embedded in paraffin. Five-micron serial sections were cut from the paraffin blocks. The sections were stained with hematoxylin and eosin (HE).

Total healing area (mm²), defect closure rate (%), new bone formation (NFB; mm²), and the residual material area (RMA; mm²) were calculated histopathologically. The images of the histological section were merged into a single composite image covering the surgical defect. The total area was then delineated on the image. This was the cortical region of the mandibula where the defect had been made.

1) Total healing area (mm²) = Total new bone formation (mm²) + total graft material (mm²).

2) Defect closure (%) = (total new bone ingrowth, mm)/(original defect width, mm)×100 (%). The original defect width was measured and as 100% of the width to be analyzed.

4) New bone formation (mm²) = The new bone area was measured in mm².

5) Residual material area (mm²) = Graft materials in entire defect area in mm² [31, 32].

At 400 magnification, osteoblasts and osteoclasts were detected in a 1.23 mm² area [33]. An experienced pathologist blinded to the study design, analysed the histopathological changes. Each lesioned area in the rats was examined under a light microscope (Olympus CX41, Olympus Corporation, Tokyo, Japan). ImageJ (version 1.48, National Institutes of Health) used for image morphometric analysis.

Immunohistochemical method

Sections were obtained in six serial cuts for histopathological examinations and mounted on poly-L-lysine-coated slides for immunohistochemical analyses. The samples were immunostained for Alkaline Phosphatase (ALP Antibody - #DF6225), Beta-catenin (β-catenin Antibody - #AF6266), Bone morphogenic protein-2 (BMP-2 Antibody - #AF5163), Osteocalcin (OCN Antibody - #DF12303), Receptor activator of nuclear factor kappaB ligand (RANKL Antibody - #AF0313), and Runt-related transcription factor 2 (Runx2 Antibody - #AF5186), antibodies. All primary antibodies were purchased from Affinity Bioscience (Columbus Avenue, San Francisco) and diluted 1/100. Sections were incubated with the primary antibodies overnight, followed by applying a biotinylated secondary antibody and streptavidin–alkaline phosphatase conjugate. UltraVision Detection system Large Volume Anti Polyvalent, HRP (RTU) (TP-060-HL) (Thermo Scientific, UK) served as the secondary antibody, with diaminobenzidine (DAB) used as the chromogen for antigen visualization. Negative controls were prepared by omitting the primary antibody step. All evaluations were conducted on blinded samples. Slides were analyzed for immunopositivity, and a semiquantitative analysis was performed, scoring staining intensity from 0 to 3 (0: no staining; 1: slight; 2: medium; 3: marked) five different areas for each section.

After standard microscopic examination, computer-assisted histomorphometric measurements and immunohistochemical scoring were performed using an automated image analysis system (Olympus CX41, Olympus Corporation, Tokyo, Japan). Evaluation was performed using CellSens Life Science Imaging Software, with ImageJ (version 1.48, National Institutes of Health) used for image analysis.

ELISA analysis

Plasma levels of high-sensitivity C-Reactive Protein (hs-CRP) were measured using a commercially available rat hs-CRP ELISA Kit (ELK Biotechnology, USA) with detection range: 15.63–1000 pg/mL; sensitivity or lower detection limit according to the manufacturer’s instructions. A set of serially diluted standards was prepared to create a standard curve. The kit was equilibrated at room temperature. Then, 100 µL of Standard Working Buffer or sample was added to each well, and the plate was incubated at 37 °C for 80 min. The liquid was discarded, the wells were washed three times with 200 µL of 1× Wash Buffer, and the plate was patted dry. Next, 100 µL of Biotinylated Antibody Working Solution was added to each well, and the plate was incubated at 37 °C for 50 min. The liquid was discarded again, the wells were washed three times, and 100 µL of Streptavidin-HRP Working Solution was added. The plate was incubated at 37 °C for 50 min. After discarding the liquid, the wells were washed five times, and 90 µL of TMB Substrate Solution was added. The plate was incubated at 37 °C for 20 min in the dark. Finally, 50 µL of Stop Solution was added, the plate was shaken for 1 min, and the OD was recorded at 450 nm for result calculation.

Micro CT

The evaluation of new bone formation in the mandibular defect area was performed using the Skyscan 1272 micro-CT system (Bruker, Kontich, Belgium). Specimens were scanned at a voltage of 60 kV, a current of 200 µA, and a pixel resolution of 5 μm. A 0.5 mm aluminum filter was used to reduce beam hardening artifacts. The rotation step was set at 0.4°, and 360° scans were completed for each sample. Image reconstruction was carried out using NRecon software (Bruker) with settings optimized for beam hardening correction (20%) and ring artifact reduction. Quantitative analysis was performed to assess bone parameters within the region of interest (ROI), which was defined to include the entire defect area and surrounding bone, including bone volume (BV), total volume (TV), bone volume fraction (BV/TV), bone surface area (BS), bone surface density (BS/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). A global threshold was applied to accurately segment bone from surrounding soft tissue, ensuring consistent and precise analysis.

Statistical analysis

The data were presented as the mean ± standard deviation (SD). Statistical analysis was carried out using SPSS software version 23.0 (SPSS Inc., Chicago, IL). The Shapiro-Wilk method was employed to assess the normality of the data distribution. A one-way analysis of variance (ANOVA) post hoc Tukey test was applied for total healing area, defect closure rate, new bone formation, residual material area, osteoblast number, osteoclast number, bone volume, bone volume fraction, trabecular thickness, trabecular number, trabecular separation and ELISA results to assess differences among the three groups. Kruskall Wallis, post hoc Mann-Whitney U with Bonferroni correction was used as a non-parametric test in the data obtained by ALP, β-catenin, BMP-2, OCN, RANKL and Runx2 results. A p-value of less than 0.05 was regarded as statistically significant.

Results

Histopathologic results

Residual graft materials, connective tissue formation, and newly formed bone tissues were observed in the Control group. In L-MTZ (9.03 ± 0.11 mm²), total healing was notably more pronounced than in the Control (3.30 ± 0.05 mm²) and S-MTZ (7.71 ± 0.09 mm²). Similarly, residual graft materials, connective tissue, and newly formed bone tissues were detected in S-MTZ. The most intense bone development was observed in L-MTZ (4.11 ± 0.22 mm²) compared to the Control (3.50 ± 0.07 mm²) and S-MTZ (3.57 ± 0.04 mm²). The highest osteoclast count was recorded in Control (12.20 ± 0.32), while it was lower in L-MTZ (8.50 ± 0.22). Conversely, the lowest osteoblast count was observed in Control (9.00 ± 0.29), whereas a significant increase in osteoblast numbers was noted in L-MTZ (11.50 ± 0.34) (p < 0.05, Table 1). Local application was found to be more effective than systemic application (Figs. 2 and 3; Table 1).

Table 1.

Histopathological data for all groups

Histopathology	Control n = 10	L-MTZ n = 10	S-MTZ n = 10	p value
	Mean ± SD	Mean ± SD	Mean ± SD
Total healing area (mm2)	3.30 ± 0.05a	9.03 ± 0.11b	7.71 ± 0.09c	0.001
Defect closure rate (%)	37.91 ± 0.36a	52.17 ± 0.21b	50.62 ± 0.18b	0.001
New bone formation area (mm2)	3.50 ± 0.07a	4.11 ± 0.02b	3.57 ± 0.04a	0.001
Residual material area (mm2)	22.05 ± 0.15a	20.31 ± 0.41b	28.45 ± 0.14b	0.034
Osteoclast number	12.20 ± 0.32a	8.50 ± 0.22b	9.60 ± 0.26b	0.001
Osteoblast number	9.00 ± 0.29a	11.50 ± 0.34b	9.70 ± 0.26a	0.001

Values are presented as mean ± Standard deviation. The mean values shown with different small superscript letters within the same line are statistically significant, ANOVA = p < 0.05. x400 magnification, osteoblasts and osteoclasts were detected in a 1.23 mm² area

Fig. 2.

In situ immunohistochemical expressions and histopathological appearance of all groups. β-catenin: moderate expression (black arrow) in the Control group, marked increase in expression (black arrows) in L-MTZ, increased expression (black arrows) in S-MTZ. BMP-2: expression findings between the groups, moderate expression (black arrow) in the Control group, increase in expression (black arrows) in L-MTZ, marked expression (black arrow) in S-MTZ. Runx2: moderate expression (black arrows) in the Control group, increase in expression (black arrows) in L-MTZ, marked expression (black arrows) in S-MTZ. ALP: moderate expression (black arrow) in the Control group, marked increase in expression (black arrows) in L-MTZ, moderate expression (black arrow) in S-MTZ. OCN: moderate expression (black arrow) in the control group, markedly increased expression (black arrow) in L-MTZ, and increased expression (black arrow) in S-MTZ. RANKL: Similar RANKL expressions (black arrows) in (A) Control group, (B) L-MTZ, and (C) S-MTZ, Streptavidin biotin peroxidase method, Scale bars = 50 μm Representative histopathological images among the groups. (A) Marked fibrous tissue, moderate new bone formation (white arrow with a black border), and residual graft materials (RG) in the Control group. (B) Decreased fibrous tissue, moderate residual graft material (RG), and marked new bone formation (white arrow with a black border) in L-MTZ. (C) Decreased fibrous tissue, moderately increased new bone formation (white arrow with a black border), and residual graft materials (RG) in S-MTZ. HE, Scale bars = 200 μm

Fig. 3.

ELISA and histopathologic results of groups. Serum hs-CRP; * p < 0.05, Control vs. other groups. Osteoblast number; *p < 0.05, L-MTZ vs. other groups. Osteoclast number; *p < 0.05, Control vs. other groups. Defect closure rate (%); *p < 0.05, Control vs. other groups. New bone formation area (mm²); * L-MTZ vs. other groups. Residual material area (mm²); *p < 0.05 Control vs. other groups. ANOVA = p < 0.05

The effect of MTZ on the Wnt/β-catenin signaling pathway in the mandible

The expression of β-catenin in the mandibular defect area was examined using immunohistochemistry. Results revealed that significantly higher expression of Wnt/β-catenin signaling pathway was detected in L-MTZ than in Control, with no significant differences compared to S-MTZ, as observed in Table 2; Fig. 2.

Table 2.

Immunohistochemical scores of groups

Immunohistochemistry scores	Control n = 10	L-MTZ n = 10	S-MTZ n = 10	p value
	Mean ± SD	Mean ± SD	Mean ± SD
β-catenin	1.50 ± 0.16a	2.70 ± 0.15b	2.20 ± 0.13ab	0.001
BMP-2	1.80 ± 0.20a	2.60 ± 0.16b	2.40 ± 0.16ab	0.019
Runx2	1.70 ± 0.15a	2.60 ± 0.16b	2.50 ± 0.11b	0.003
ALP	1.50 ± 0.16a	2.30 ± 0.21b	2.00 ± 0.21ab	0.034
OCN	1.70 ± 0.15a	2.50 ± 0.16b	2.40 ± 0.16b	0.007
RANKL	2.20 ± 0.20a	2.40 ± 0.16a	2.30 ± 0.21a	0.791

Values are presented as mean ± Standard deviation. The mean values shown with different small superscript letters within the same line are statistically significant. Kruskal Wallis = p < 0.05. Scores were recorded in five different areas under a ×40 lens, and the average was calculated

BMP-2/ Runx2signaling pathway

The lowest BMP-2 and Runx2 expression was observed in the Control group. Significantly increased BMP-2 and Runx2 expression was observed in L-MTZ and S-MTZ groups compared to the Control group (p < 0.05) (Table 2; Fig. 2).

Bone formation and resorption markers

The highest ALP activity was observed in the L-MTZ group. The S-MTZ group showed higher immune activity than the Control group and lower immune activity than the L-MTZ group, but the results did not gain statistical significance. The lowest OCN immunostaining was observed in the Control group. L-MTZ and S-MTZ groups had significantly higher OCN immunoreactivity than the Control group (p < 0.05). No significant difference was found between the L-MTZ and S-MTZ groups regarding both OCN and ALP immunostaining. RANKL is a soluble membrane protein of the tumor necrosis factor (TNF) family and is produced by osteoblastic cells and T cells, stimulating osteoclast activation and differentiation and inhibiting osteoclast apoptosis. In our study, no significant difference was found between the groups regarding RANKL immunohistochemistry (Table 2; Fig. 2).

Serum hs-CRP results

Higher serum hs-CRP values were observed in the Control group (2209.78 ± 269.76 pg/ml) than in L-MTZ (1056.71 ± 121.20 pg/ml) and S-MTZ (613.00 ± 90.57) with no significant differences between L-MTZ and S-MTZ (Fig. 3).

Micro-CT findings

The S-MTZ group was significantly higher bone volume than the Control (p < 0.05). The highest value in BV/TV, which could predict the strength and stiffness of normal and pathologic cancellous bone, was found in the S-MTZ group and the lowest in the Control group with significantly different (p < 0.05). There was no difference between the groups in terms of Tb.Th and Tb.N. A significant difference was found between S-MTZ and Control in Tb.Sp (p < 0.05). In terms of BS and BS/TV, the lowest values were found in the Control group, the highest values were found in the S-MTZ group, and there was a significant difference between all groups in both parameters (p < 0.05) (Table 3; Fig. 4).

Table 3.

Micro-CT results of all groups

Micro-CT parameters	Control n = 10	L-MTZ n = 10	S-MTZ n = 10	p value
	Mean ± SD	Mean ± SD	Mean ± SD
BV	8.24 ± 0.61a	9.64 ± 1.01a, b	17.67 ± 1.51b	0.003
TV	22.56 ± 0.89a	22.67 ± 1.06a	27.42 ± 0.67b	0.009
BV/TV	36.52 ± 0.83a	42.08 ± 0.12a, b	64.43 ± 0.69b	0.002
Tb.Th	0.24 ± 0.04	0.23 ± 0.39	0.28 ± 0.39	0.184
Tb.N	1.48 ± 0.75	1.99 ± 0.86	2.25 ± 0.40	0.230
Tb.Sp	0.36 ± 0.71a	0.27 ± 0.63a, b	0.18 ± 0.63b	0.008
BS	138.08 ± 0.63a	164.35 ± 0.82b	222.91 ± 1.44c	0.002
BS/TV	6.11 ± 0.18a	7.25 ± 0.39b	8.12 ± 0.20c	0.001

Values are presented as mean ± Standard deviation. The mean values shown with different small superscript letters within the same line are statistically significant, ANOVA = p < 0.05. BV, bone volume; TV, total volume; BV/TV, bone volume fraction; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; BS, bone surface; BS/TV, bone surface density

Fig. 4.

Micro-CT images of groups

Discussion

The repair of bone defects can be challenging for both the patient and the physician in case of infection. Antibiotic resistance or systemic effects of antibiotics can also limit the use of antibiotics. In our study, the effect of MTZ applied locally and systemically in the treatment of bone defects was investigated. According to our results, both topical and systemic application of MTZ increased osteogenic markers while decreasing hs-CRP levels. MTZ treatment enhanced osteogenesis, confirmed by histopathological and radiographic analyses compared to the Control group. Local and systemic administration of MTZ stimulated the Wnt/β-catenin and BMP-2/Runx2 signaling pathways, promoting osteogenesis.

Infections such as osteomyelitis, septic arthritis, and periodontitis significantly jeopardize the integrity of our bones, joints, and oral tissues, leading to substantial bone loss [34]. These conditions may develop spontaneously or emerge as complications following surgical interventions. Upon the body encounters these infections, a pronounced inflammatory response is initiated, which can manifest either in localized areas or, more broadly, throughout the organism. This inflammatory response activates osteoclasts—cells responsible for bone resorption—while concurrently inhibiting the activity of osteoblasts, which are essential for bone formation. Consequently, this imbalance culminates in a reduction of bone density [35]. While antibiotics serve a critical function in the management of infections, it is essential to acknowledge that certain classes, including gentamicin, cephalosporin, and tetracycline, may adversely affect the activity of osteoblasts, thereby impeding the crucial processes of bone healing [36–38]. The prospect of utilizing an antibiotic that not only effectively controls infections but also promotes bone formation presents a transformative opportunity within orthopedics and dentistry. Such an advanced antibiotic could mitigate the dependence on additional medications, facilitate faster patient recovery by enhancing bone tissue healing, and significantly reduce the risks of complications, including non-union and the necessity for revision surgeries.

The bacteria in saliva are those shed from biofilms on oral tissues and appear to represent the overall oral microbiome, and oral health conditions greatly influence the bacterial composition of the salivary microbiome [39]. Avoiding saliva when performing any procedure in the oral cavity is impossible. The oral microbiota changes in any case of dysbiosis, and there is an increase in pathogenic microorganisms in the red complex (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola) [39–41]. MTZ is effective against periodontal anaerobic pathogens, including P. gingivalis [42]. Although MTZ is an antibiotic, it has been emphasized that it may also have anti-inflammatory effects [43]. MTZ is used to treat skin conditions such as rosacea and acne. The way it works is thought to be anti-inflammatory rather than antibacterial [44].

There are conflicting statements in the literature on this subject. According to the conclusion of a review published by Suarez et al., the potential anti-inflammatory effect of MTZ in the treatment of periodontitis has been demonstrated [45]. MTZ treatment in children with giardiasis reduced high TNF-α, sIL-2R, IL-1, IL-6, IL-8, C-reactive protein (CRP), and nitric oxide levels [46]. In the in vivo step of a study, applied with 5% and 10% MTZ immobilized PHB/gel nanofibrous scaffold, it was found that MTZ caused an increase in TGF-β and a decrease in IL-6 and accelerated secondary wound healing [47]. In a study into MTZ in patients with pneumonia caused by the coronavirus, 44 people were divided into two groups. One group received the standard treatment of hydroxychloroquine (200 mg twice a day), lopinavir/ritonavir (400/100 mg twice a day), and ribavirin (1200 mg twice daily). The other group did the same treatment plus MTZ (250 mg orally every 6 h for 7 days). The authors reported that those taking MTZ had reduced erythrocyte sedimentation rate (ESR) and IL-6 levels by day 7. They also observed reductions in ferritin and CRP, concluding that MTZ has anti-inflammatory effects in patients with lower airway inflammation due to COVID-19 [46]. Rizzo et al. [47] investigated the impact of MTZ on high-performance liquid chromatography (HPLC) treated with or without lipopolysaccharide (LPS) from P. gingivalis at concentrations of 2.5, 25, 250, and 2500 µg/ml. The results showed that MTZ had no cytotoxic effect on HPLC and inhibited the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-12, and TNF-α). CRP is an acute-phase protein synthesized and secreted by the liver in response to signals from the body (i.e., infection, trauma, or tissue damage) [48]. At the molecular level, production of CRP is induced by pro-inflammatory cytokines IL-1, IL‐6, and IL‐17 in the liver [48]. In our study, hs-CRP was measured as an inflammatory marker. The highest levels were measured in the control group, and higher hs-CRP levels were observed in the group with locally mixed MTZ in the graft than in the group with systemic MTZ. The lowest levels were detected in the group using systemic MTZ. Our study concluded that MTZ use affects systemic inflammation and decreases CRP levels in accordance with the studies.

MTZ is also reported to improve wound healing by improving myofibroblast differentiation [49], promoting rapid contraction and epithelization, culminating in an early repair process [50], and promoting angiogenesis [28, 51]. Topical use of MTZ has been shown to promote wound healing via three mechanisms: reducing inflammation to minimize tissue destruction, speeding up epithelization and keratinocyte growth, and reducing bacterial load [52]. The effect of MTZ-loaded nanoparticles (25%) gel formulation on periodontal regeneration in periodontal defects in dogs was investigated. According to histological results, new cement, new alveolar bone, and new attachment formation were observed. The authors concluded that this effect was due to the antibacterial effect of MTZ [53]. Taneja et al. [54] applied platelet-rich fibrin (PRF) as a control group in patients in whom they applied PRF in combination with 1% MTZ gel, and periodontal parameters and defect filling were analyzed. In terms of periodontal parameters and defect filling, the MTZ + PRF group showed statistically significant superiority over the PRF group. The authors mentioned that MTZ is an immunomodulatory agent with osteogenic potential and antimicrobial properties. On the contrary, Needleman et al. [55] evaluated whether topical application of a slow-release antimicrobial agent could have an adjunctive effect on healing after periodontal surgery and found that applying MTZ gel to the exposed root surface provided no additional benefit. ALP is produced by osteoblasts and correlates positively with the bone formation rate measured by histomorphometry [56]. OCN is a non-collagenous protein secreted by osteoblasts and is involved in bone mineralization and remodeling by regulating osteoblast and osteoclast activity [57]. Both bone ALP and OCN are usually considered markers of bone formation.

In our study, we evaluated the effects of MTZ on bone formation and osteoclast activation. Both locally and systemically administered MTZ caused increased bone formation compared to the control group. When bone formation markers were analyzed, MTZ administration showed a significant increase in ALP and OCN in both local and systemic administration groups compared to the control group. Alain et al. [58] soaked a combination of freeze-dried bone allograft and PRF with MTZ (0.5% solution) and performed sinus and alveolar crest augmentation with this mixture. New bone formation was observed in the CBCT, which was taken 10 weeks after surgery. MTZ prevented contamination of the biomaterial and protected the early stages of bone formation from infection and inflammation, which could lead to necrosis. Duggal et al. [59], performed femoral osteotomy in ovariectomized (OVX) rats and treated rat calvarial osteoblasts (RCOs) and bone marrow stromal cells (BMSCs) with human doses of MTZ (117 mg/kg and 235 mg/kg) and MTZ in the form of a special in situ gel. MTZ increased ALP activity and stimulated mineralized nodule formation by both rBMSCs and mBMSCs. It also increased the mRNA levels of BMP-2, Runx2, and osteogenic genes.

MTZ stimulated osteogenic differentiation and mineralized nodule formation 21 days after treatment. It also stimulated the Wnt pathway in osteoblasts. In the femoral and tibial metaphyses, bone mineral density and BV decreased in OVX and significantly increased with MTZ treatment. MTZ restored Tb.Sp due to a complete repair of Tb. Th and a significant increase in Tb.N. Wnt and BMP proteins are involved in promoting bone regeneration and the differentiation of stem cells through a synergy of signaling pathways [60]. The Wnt/β-catenin signalling pathway is important in controlling the differentiation of osteoblasts to promote bone formation, inhibit bone resorption and maintain bone mass [61, 62]. The pathway influences metabolism and is expressed in the bone tissue’s primary cell types, osteoblasts, osteoclasts, and osteocytes. Early bone regeneration at the fracture site has resulted in upregulation of the Wnt pathway [63, 64]. BMP-2 is an essential marker in the BMP subfamily that regulates osteoblast differentiation by enhancing the expression of key transcription factors, including ALP, OCN, collagen type I (COL-I), and Runx2. However, Wnt and BMP ligands often act in a way that is similar or complementary in biological contexts [65]. According to the results of our study, both systemic and local MTZ use modulate the canonical WNT pathway and the BMP/Runx2 pathway.

As a result, increased new bone formation was observed. Although, Wnt/β-catenin signaling prevents osteoclast differentiation by reducing RANKL [63]. Our study found no difference between the groups regarding RANKL immunoexpression. However, the osteoclast number was found to be the lowest in L-MTZ, then S-MTZ, and the highest in the control group. The Osteoprotegerin (OPG)/RANKL/Reseptor activator of nuclear factor kappa-B (RANK) pathway plays a vital role in the differentiation of osteoclasts. OPG competitively inhibits RANKL/RANK interactions, blocking osteoclast differentiation, bone resorption, and apoptosis. The fact that RANKL expression did not differ between groups while the number of osteoclasts was higher in control group may due to the difference in the RANKL/OPG ratio, but the RANKL/OPG ratio was not analyzed in our study.

Systemic administration of MTZ has many adverse effects, mainly affecting the gastrointestinal tract, including diarrhea, nausea, and vomiting. It can also cause hypersensitivity and gastrointestinal intolerance. Long-term use may increase the risk of bacterial resistance [66]. Alternatively to systemic, local treatment can lead to a higher drug concentration at the target site at a lower dosage, thereby reducing side effects [66]. MTZ is a decisive surgical adjuvant for bone graft surgery, limiting biomaterial contamination by anaerobic bacteria, protecting the early bone formation phases from infection and inflammation, and counteracting possible systemic preoperative contamination [67]. In our study, we impregnated the graft with 0.75% MTZ gel (7.5 mg MTZ in 2 g gel) and achieved better bone healing than the Control group. Local application of MTZ in bone grafting procedures can be used if the systemic risks of systemic antibiotic administration are to be avoided. Microbiological analysis, antibiotic resistance research, and various inflammatory markers (such as TNF and IL-6) were not investigated in our study. In addition, the regenerative capacity of rats and their response to infection differ from that of humans. It may not always be possible to generalize the results to humans. These are the limitations of this study.

Conclusion

This study demonstrates the potential of MTZ as an effective adjunct in treating infected mandibular bone defects. Local and systemic MTZ significantly enhanced bone regeneration and modulated key signaling pathways, including Wnt/β-catenin and BMP/Runx2. Local application, in particular, showed promising results by achieving comparable bone healing outcomes to systemic administration. These findings support using locally applied MTZ as a practical and efficient approach in clinical settings. This study provides strong preclinical evidence that MTZ, particularly in local form, could be an effective adjunct in regenerative dentistry and maxillofacial surgery. Future studies should explore optimal dosing, delivery methods, and long-term effects of MTZ, as well as its anti-inflammatory properties, potential to induce M2 macrophage polarization, and ability to enhance osteoblast differentiation, to support its practical use in bone defect treatment, dental implantology, and periodontal regeneration.

Abbreviations

MTZ

Metronidazole

L-MTZ

Local Metronidazole

S-MTZ

Systemic Metronidazole

BMP-2

Bone morphogenic protein-2

ALP

Alkaline Phosphatase

OCN

Osteocalcin

RANKL

Receptor Activator of Nuclear Factor Kappa-B Ligand

RANK

Receptor Activator of Nuclear Factor Kappa-B

hs-CRP

High Sensitivity C-Reactive Protein

BS

Bone surface area

TV

Total Volume

BS/TV

Bone surface density

BV

Bone Volume

BV/TV

Bone Volume/Total Volume

Tb.Th

Trabecular Thickness

Tb.N

Trabecular Number

Tb.Sp

Trabecular Separation

IHC

Immunohistochemistry

ELISA

Enzyme-Linked Immunosorbent Assay

ROI

Region of Interest

CBCT

Cone Beam Computed Tomography

WNT

Wingless-Related Integration Site

DNA

Deoxyribonucleic Acid

TGF

Transforming Growth Factor

PRF

Platelet-Rich Fibrin

TNF

Tumor Necrosis Factor

NFB

New Bone Formation

β-catenin

Beta-catenin

RMA

Residual Material Area

Runx2

Runt-related transcription factor 2

DAB

Diaminobenzidine

COL-1

Collagen type I

ESR

Erythrocyte Sedimentation Rate

LPS

Lipopolysaccharide

HPLC

High-performance liquid chromatography

CRP

C-Reactive Protein

OVX

Ovariectomized

RCO

Rat Calvarial Osteoblasts

BMSC

Bone Marrow Stromal Cell

OPG

Osteoprotegerin

Author contributions

All authors contributed to the study conception and design. Conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, supervision, writing by A.L.A. conceptualization, data curation, formal analysis, investigation, methodology by A.K. Formal analysis, investigation, methodology, writing– original draft by Ö.Ö. Data curation, formal analysis, investigation, methodology, project administration, writing– original draft by N.Z, Z.Ö, and A.A. All authors read and approved of the final manuscript.

Funding

No funding was received to conduct this study.

Data availability

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Declarations

Ethical approval

Ethics committee approval of the study was obtained from Pamukkale University Animal Experiments Ethics Committee (PAUHDEK-2024/04).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.