Exploring the Potential of Ornidazole as an Alternative Treatment for Pulpitis: Effects on Dental Pulp Cells and Macrophages

doi:10.21203/rs.3.rs-4692321/v1

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Exploring the Potential of Ornidazole as an Alternative Treatment for Pulpitis: Effects on Dental Pulp Cells and Macrophages

https://doi.org/10.21203/rs.3.rs-4692321/v1

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Objectives: This study aimed to explore the potential of ornidazole as an alternative treatment for pulpitis, focusing on its effects on dental pulp cells (DPCs) and macrophages. We assessed the cytotoxicity of various concentrations of ornidazole, its safety and effectiveness in treating inflammatory DPCs, and its regulatory impact on inflammatory markers during inflammation.

Materials and Methods: Inflammation in dental pulp cells was induced using lipopolysaccharides (LPS), and varying doses of ornidazole were introduced. Cell proliferation, migration, regulation of inflammation, and promotion of dentinogenesis under inflammatory conditions were evaluated. Additionally, macrophages were cultured with different doses of ornidazole to analyze inflammatory gene expression.

Results: Ornidazole concentrations below 10 µg/mL showed no significant cytotoxic effects on DPCs over 7 days. The toxicity of ornidazole was concentration- and time-dependent. Ornidazole decreased the expression of IL-6 and TNF-α while enhancing IL-Ra and IL-8 expression. It also suppressed alkaline phosphatase (ALP) activity but increased the levels of odontogenic differentiation markers at both genetic and protein levels in the presence of inflammatory stimuli. Furthermore, ornidazole demonstrated immunomodulatory effects.

Conclusions: Low concentrations of ornidazole were safe for use in pulp cells. Ornidazole modulated the expression of inflammatory markers (IL-6, TNF-α, IL-8, IL-Ra) in inflamed DPCs and regulated odontogenesis-related markers. Low concentrations of ornidazole enhanced macrophage immune regulation, showing potential as a medication for treating pulpitis.

CLINICAL RELEVANCE: This study investigates ornidazole’s affinity for dental pulp cells, its effectiveness in reducing inflammation, and its regulatory effects on odontogenic processes. By examining the impact of different concentrations of ornidazole on pulp inflammation-related cells, we aim to assess its therapeutic potential for pulpitis and provide a reference for its clinical application.

ornidazole

dental pulp cells

antibiotics

cytocompatibility

The dental pulp, a loose connective tissue encased by dentin, plays a crucial role in the vitality and health of teeth. It is essential for sensory perception, immune defense, tissue repair, and regeneration following tooth damage, maintaining the balance of the pulp chamber environment[1, 2]. Pulp necrosis disrupts the supply channels of dental tissue, increasing the fragility of dentin and enamel. Pulpitis, the leading cause of pulp necrosis, involves chemotactic leukocytes increase lysosomal enzymes that lead to tissue damage[3]. High levels of endotoxins and a more complex microbiota are commonly found in teeth with pulp symptoms. All teeth with irreversible pulpitis exhibit severe acute inflammation, necrosis, microabscesses, and bacterial infection in the pulp chamber. Bacteria were also observed in the vascular lumen around the necrotic lesion[4, 5].

Microorganisms, especially bacteria, are the primary cause of periapical and pulpal diseases[6]. Streptococcus mutans is the primary bacterium initiating pulpal necrosis, while anaerobic or facultative anaerobic bacteria, such as lactobacilli, exacerbate the progression of pulpal necrosis[7]. Most anaerobic bacteria reaching the pulp lead to periapical disease[6]. These bacteria and their toxins can trigger pulpitis, ultimately resulting in pulp death[8]. Root canal therapy is the traditional treatment, using disinfectants and antibacterial drugs like calcium hydroxide and triple antibiotic paste (TAP). Calcium hydroxide inhibits bacterial proliferation due to its weak alkaline effect and is bioinert. However, calcium hydroxide is easily affected by apical tissue fluid, is soluble outside the root canal, and its long-term use adversely affects tooth fracture resistance[9]. Its antibacterial efficacy against bacteria in severely infected pulp diseases is limited and difficult to remove[10]. In endodontic therapy, antibiotics are used to treat severe apical infections due to their strong anti-infection capabilities. TAP, a combination of quinolones, tetracyclines, and nitroimidazoles, is frequently used in endodontic therapy and has a wide range of clinical applications. However, research has confirmed that tetracycline drugs in TAP can cause tooth discoloration, and combining multiple antibiotics enhances the drug's cytotoxicity. The combination and concentration of antibiotics result in different effective bactericidal concentrations on infected root canals, making drug concentration particularly important in exploring antibiotic treatment of pulp tissue[11].

Ornidazole, a nitroimidazole derivative, is a key component to novel TAP and widely used to treat anaerobic bacterial infections[12]. Although ornidazole is commonly used in oral formulations clinically, targeted delivery of local antibiotics can enhance the anti-infective effects while reducing side effects[13]. Some studies have shown that ornidazole incorporated into pulp capping systems and scaffolds for promoting pulp regeneration exhibits excellent anti-inflammatory, antibacterial, and anti-infective properties, with better control than ornidazole alone[14]. This highlights ornidazole as a promising drug for controlling infections in pulpitis. However, the impact of ornidazole on pulpitis cells, particularly dental pulp cells and macrophages, remained underexplored. Given its reported efficacy, this study aimed to investigate ornidazole's potential application in dental pulp therapy.

The pulp tissue comprises various cell types, including odontoblasts, fibroblasts, stem cells, immune cells, and endothelial cells[1]. Dental pulp cells are essential for pulp regeneration, capable of differentiating into odontoblasts and nerve cells for tissue repair[15]. Macrophages are crucial for vascular reconstruction and inflammation regulation, intervening by phagocytizing bacteria and releasing cytokines[16]. These cells are pivotal in tooth development, infection control, and self-repair processes[17]. Understanding the effects of ornidazole on these cells is essential for assessing its potential value in treating pulpitis.

Hence, this study investigated the impact of various concentrations of ornidazole on the proliferation of dental pulp cells, regulation of inflammation, and odontogenic differentiation under inflammatory conditions, as well as its combined effect with macrophages on inflammation regulation.

Cell isolation and culture

This experiment was completed in compliance with the Basic Code of Medical Ethics and was approved by the Ethics Committee of Jinan University (approval number: JNUKY-2022-070). Dental pulp cells (DPCs) were isolated from young permanent teeth obtained during orthodontic procedures. The teeth, isolated within 4 hours of extraction, were stored in 4°C PBS. Pulp tissue was extracted and digested using 0.3 vol.% COL-1 collagenase. The digested pulp was cut into pieces in a sterile petri dish, dispersed, and spread flatly on the culture flasks. The culture medium was changed every three days after observing cell growth. When the cell growth reached 80%-90% confluence, the cells were digested, counted, and then cultured for 4–7 passages. RAW264.7 (Procell, China) was used to serve as a macrophage model. Cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (H-DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel).

Cell viability and migration

Cells were treated with various concentrations of ornidazole. Live/dead cell staining was performed with Calcein-AM (Beyotime, China) and propidium iodide (Beyotime, China) observed by an inverted fluorescence microscope (TCS SP8, Leica8, Germany), and cell viability was assessed using a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay. Cell migration was evaluated by scratch assays with images processed using Image J 1.54h.

Alkaline phosphatase (ALP) assay and alizarin red staining

DPCs pre-induced with 1 µg/mL LPS were cultured with ornidazole. An odontogenic induction medium containing vitamin C, β-sodium glycerophosphate, and dexamethasone was used for culturing DPCs. ALP staining, Alizarin red staining, and a colorimetric ALP activity assay were conducted according to the manufacturer's instructions. For macrophage-conditioned culture, RAW264.7 was induced with LPS and ornidazole, and the conditioned medium was used for DPCs culture.

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

Cells treated with LPS and ornidazole underwent RNA isolation, reverse transcription, and RT-qPCR, following the manufacturer's instructions (Vazyme, China). The mRNA expression levels of interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-1Rα (IL-1Ra), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), stromal cell-derived factor-1 (SDF1), collagen type I (COL-1), bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2), dentin matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP) of cells were measured using a PCR instrument (CFX Connect, Bio-Rad, USA), with GAPDH as the housekeeping gene. The primer sequences are listed in Table S1.

Western blot analysis

Cells were lysed with RIPA solution, and total protein concentrations were determined. Proteins were separated by gel electrophoresis, transferred to PVDF membranes, and blocked with skimmed milk. Primary antibodies (Beyotime, China) were incubated overnight, followed by secondary antibody (Beyotime, China) incubation at room temperature. Blots were visualized using a gel imager (Wealtec KETA ML, USA), and densitometry was quantitated using Image J 1.54h. The expression levels of COL-1, BSP, DMP-1, RUNX2, and DSPP proteins was detected with GAPDH as the reference standard.

Statistical analysis

Each assay was repeated at least four times, and statistical analysis was performed using SPSS 26.0. Data were expressed as the mean ± standard deviation, and one-way analysis of variance with Tukey’s test was used for comparisons, with significance set at P < 0.05.

Effects of ornidazole on the affinity of dental pulp-related cells

DPCs were cultured with varying concentrations of ornidazole adding to the culture medium to explore the bioactivity of this drug. Comprehensive analysis of the CCK-8, cell migration, and live/dead cell staining showed that concentrations below 10 µg/mL exhibited a favorable affinity for DPCs before day 7 of incubation. In contrast, concentrations higher than 50 µg/mL demonstrated concentration-dependent inhibition of DPCs with increasing incubation time. At concentrations as high as 500 µg/mL, the drugs exhibited significant cytotoxicity and minimal effect on migration, suggesting that high doses of ornidazole are significantly toxic to cells in the pulp chamber (Fig. 1).

Effects of ornidazole on inflammation regulation of dental pulp-related cells

To evaluate the Inflammation regulation capability of ornidazole, DPCs induced by LPS for 24 hours were exposed to ornidazole (1–10 µg/mL). Results detected on day 3 by RT-qPCR revealed that ornidazole reduced the expression of IL-6 and TNF-α while promoted the expression of IL-8 and IL-1Ra in LPS-induced DPCs in comparison to the control group (Fig. 2A). Ornidazole demonstrated inflammatory modulation effects on LPS-stimulated DPCs, primarily by reducing the pro-inflammatory effects of IL-6 and TNF-α. Additionally, ornidazole favored IL-8 and IL-1Ra, which exhibit potent pro-angiogenic effects, regulating potential inflammation at concentrations below 10 µg/mL[18–20].

Effects of ornidazole on odontogenic markers of dental pulp-related cells under inflammation conditions

Ornidazole's impact on odontogenesis-associated markers under inflammation conditions induced by LPS stimulation was investigated. The results revealed inhibited ALP activity and ARS staining but increased mRNA expression of markers such as DSPP, DMP-1, BSP, COL1, and RUNX2 at 1–10 µg/mL (Figs. 2B-I). Western blot analysis of three selected odontogenic representative proteins showed that, at concentrations of 1–10 µg/mL, ornidazole up-regulated the expression of DSPP, RUNX2, and COL-1 proteins to varying degrees at 14 days, with continued up-regulation at 21 days except for COL-1 (Figs. 2J-M).

Effects of ornidazole on regulating the immune microenvironment of dental pulp-related cells

RAW264.7 was used as a model to culture with varying concentrations of ornidazole under LPS-induced inflammatory conditions. Ornidazole was observed to slightly inhibit macrophage viability both during direct exposure and pre-induced with LPS (Fig. 3A). In response to prolonged inflammation stimuli, the expression of specific markers in RAW264.7 exposed to ornidazole (2 µg/mL) was assessed. RAW264.7 treated with ornidazole showed elevated expression levels of IL-10, IL-1Ra, TGF-β1, and SDF-1, while the expression of TNF-α and IL-1β was down-regulated compared to the control. Ornidazole appeared to be involved in regulating the immune microenvironment by modulating macrophage polarization, thereby enhancing the later-stage odontogenic differentiation of DPCs (Fig. 3B-N).

When the dental pulp is invaded by bacteria and becomes inflamed, the functionality of dental pulp cells is compromised. Gram-negative anaerobic bacteria are considered the primary cause^[21]. These bacteria produced toxic LPS during replication or death, promoting DPCs to release various inflammatory factors such as TNF-α, IL-Ra, IL-6, and IL-8. These cytokines ultimately induce acute inflammatory reactions[22]. Controlling infection reduces the release of inflammatory factors, aiding in the recovery of dental pulp cell vitality. Infection control depends on inhibiting bacterial growth. Ornidazole, which inhibits anaerobic bacteria, has shown advantages in the enclosed environment of the oral cavity and is one of the most used drugs for treating oral diseases. However, the effects of ornidazole on dental pulp activity remain unclear. The results of this study indicated that the reasonable use of low concentrations of ornidazole did not inhibit dental pulp tissue activity. To verify the affinity between ornidazole and DPCs, different concentrations of ornidazole were added to the culture medium of inflamed DPCs, and assays such as the CCK-8, live/dead fluorescence staining, and cell migration experiments were conducted. This research concluded that the toxicity of ornidazole to DPCs depended not only on the concentration but also on the duration of exposure. Results revealed that ornidazole was biocompatible to DPCs at concentrations below 10 µg/mL and treatment durations of less than 7 days. If the drug concentration increased to 100, it was safe if the treatment duration was kept within 3 days. At concentrations greater than 500 µg/mL or treatment durations exceeded 7 days, ornidazole inhibited DPCs proliferation and promoted cells death. In the potential immune microenvironment of dental pulp, high concentrations of ornidazole inhibited cell migration and proliferation and promoted apoptosis. This might be due to the toxic effects of ornidazole’s degradation products on dental pulp cells[23]. Previous studies have shown that nitroimidazole drugs had neurotoxicity, which was concentration-dependent, supporting our hypothesis[24].

In clinical practice, it is essential to strictly control the concentration of ornidazole and the duration of contact with DPCs. As a pulp therapy drug, the concentration of ornidazole should be safe for pulp cells while retaining antibacterial effects. When using ornidazole, both its affinity for cells and its effective antibacterial concentration should be considered. Studies have shown that the minimum inhibitory concentration of ornidazole against common oral bacteria such as Porphyromonas gingivalis and Prevotella intermedia was 0.062-1 µg/mL[25]. Therefore, a concentration of ornidazole between 1–10 µg/mL is effective for inhibiting bacteria and is safe for DPCs, making ornidazole suitable for dental pulp therapy.

Qualified endodontic treatment drugs should prioritize safety and effectively regulating inflammation. Besides evaluating the safety of ornidazole on pulp cells, this study examined its ability to regulate inflammation in inflamed pulp cells. Researches showed that IL-6 and TNF-α played crucial roles in regulating pulpitis progression and influencing DPCs differentiation[26–29]. IL-6 levels in pulpitis are significantly higher than that in normal dental pulp. TNF-α is an early inflammation marker that affects pulp pain production and inhibits the survival, migration, and odontogenic differentiation of DPSCs[30–32]. IL-Ra participates in normal physiological functions using specific antibodies and acts as an anti-inflammatory agent to regulate inflammation^[33]. IL-8 has both anti-inflammatory and strong angiogenic effects, essential for DPCs survival and maintaining a robust blood supply and immune regulation[34]. Ornidazole exhibited an inflammatory gene regulatory effect on DPCs stimulated by LPS on day 3, helping controlling inflammation. Research has found that levornidazole’s immunomodulatory effects played a vital role in ameliorating intestinal inflammatory process[35]. Therefore, the modulation of pulp cell inflammation observed in this study might stem from both the inhibition of anaerobic bacteria by ornidazole and its immunomodulatory effects.

The continuous development of dental roots is driven by the ongoing differentiation of cells into dental pulp, with the primary function of dental pulp being the formation of dentin. Throughout tooth development, the proliferation and differentiation of dental pulp cells are regulated by complex signaling molecules and pathways[36]. This study found that ornidazole regulated the differentiation of dental pulp cells into dentin. The role of odontogenic genes regulated by ornidazole in DPCs was a key focus. DSPP, COL-1, and RUNX2 are important odontogenic biomarkers affecting DPCs differentiation into dentin[37, 38]. COL-1 is recognized as an early differentiation marker for cell differentiation into dentin[39]. Results showed that both COL-1 gene and protein expressions were negatively correlated with drug concentration. RUNX2, which inhibits odontoblast terminal differentiation and induces transdifferentiation of odontoblasts to osteoblasts at later stages, showed a positive correlation between RUNX2 gene/protein expression and drug concentration[40]. It can be preliminarily concluded that early dentin formation was unaffected, but ornidazole notably inhibited the expression of proteins related to later-stage dentin formation under potential inflammation. ALP, highly expressed in mineralized tissue cells, plays a crucial role in forming hard tissue by promoting mineralization and reducing extracellular pyrophosphate concentrations[41]. The difference in staining between the drug group and the control group was more significant at 21 days compared to 14 days, indicating that ornidazole significantly inhibited the mineralization of DPCs after 14 days of direct stimulation, and the inhibitory effect was time-dependent. Ornidazole inhibited ALP activity and ARS mineralization in DPCs, with the inhibitory effect being proportional to concentration and time, confirming previous results on odontogenic genes and proteins. Previous studies have been found that antibiotic treatment can compromise the mineralization of dental tissues in both clinical and animal models[42]. This is because gut microbiota aids in maintaining continuous growth of incisors in injured rodents and differentiation abilities of DPSCs, while antibiotics suppress the microbial stimulation for tooth differentiation, weakening the differentiation capacity of DPSCs[42]. Our experimental results support this viewpoint.

Hemodynamic reconstitution and immune regulation are crucial for DPCs survival[43, 44]. Macrophages play a vital role in regulating inflammation and affecting the differentiation of DPCs[45]. Regulating macrophage polarization can regulate the inflammatory status of reversible and irreversible pulpitis[46, 47]. Macrophages are important in the early stages of the wound-healing in reversible pulpitis[48]. To understand the mechanism by which ornidazole inhibited inflammation in dental pulp-related cells, the inflammatory regulation of macrophages by ornidazole was investigated. RAW264.7 treated with ornidazole exhibited heightened expression of genes associated with anti-inflammatory responses and reduced expression of genes linked to pro-inflammatory processes. RAW264.7 interacting with ornidazole exhibited significantly higher expression of SDF-1 compared to the control, suggesting a potential enhancement of the migration of pulp-associated cells and angiogenesis[49, 50]. The staining results of conditionally cultured cells and the determination of ALP enzyme activity suggested that ornidazole could promote the differentiation of dental pulp cells through immune cell stimulation. This contrasts with previous experimental results. It was hypothesized that low concentrations of ornidazole could enhance the anti-inflammatory response of macrophages in the inflammatory microenvironment and indirectly induce differentiation into odontoblasts through macrophage secretion. However, with increased exposure time, it began to shift towards an inhibitory effect on differentiation. To verify this hypothesis, further experiments will be considered in the future.

Concentrations of ornidazole ranging from 1–10 µg/mL were not significantly cytotoxic to dental pulp cells over a 7-day period and can be safely used for therapeutic purposes. In the inflammatory immune environment, ornidazole exerted a regulatory effect on inflammatory genes by upregulating IL-8 and IL-Ra expression while downregulating IL-6 and TNF-α expression. Additionally, ornidazole affected the expression of odontogenic genes such as DSPP, DMP-1, BSP, COL1, and RUNX2, as well as proteins including DSPP, RUNX2, and COL-1 in DPCs under inflammatory conditions. However, ornidazole inhibited ALP activity and mineralization in DPCs, with the inhibitory effect being positively correlated with both concentration and duration of exposure. In a low-concentration ornidazole microenvironment, macrophage immune regulation and induction of odontogenic cell differentiation were promoted within the first three days.

ACKNOWLEDGEMENTS

Thanks are due to Medical Experimental Center, School of Medicine for test support.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Jinan University (approval number: JNUKY-2022-070).

FUNDING

This study was supported by the Science and Technology Program of Guangzhou (202102021127), the National Innovation and Entrepreneurship Training Program for Undergraduate (202110559103, 202210559062), and the Special Funds for the Cultivation of Guangdong College Students' Scientific and Technological Innovation (“Climbing Program” Special Funds, pdjh2022b0061, pdjh2023b0064).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

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No competing interests reported.

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Exploring the Potential of Ornidazole as an Alternative Treatment for Pulpitis: Effects on Dental Pulp Cells and Macrophages

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Cell isolation and culture

Cell viability and migration

Alkaline phosphatase (ALP) assay and alizarin red staining

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

Western blot analysis

Statistical analysis

RESULTS

Effects of ornidazole on the affinity of dental pulp-related cells

Effects of ornidazole on inflammation regulation of dental pulp-related cells

Effects of ornidazole on odontogenic markers of dental pulp-related cells under inflammation conditions

Effects of ornidazole on regulating the immune microenvironment of dental pulp-related cells

DISCUSSION

CONCLUSION

Declarations

References

Additional Declarations

Supplementary Files

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