Association between serum cytokine levels with pulp necrosis and apical periodontitis in sickle cell disease

Abstract

This study’s objective was to assess the relationship between serum cytokine levels and the occurrence of pulp necrosis (PN) and apical periodontitis (AP) in adolescents with Sickle Cell Disease (SCD). A total of 52 adolescents, including 29 individuals diagnosed with SCD, participated in the study. Serum cytokine levels including IL-1, IL-2, IL-4, IL-6, IL-1β, IL-8, IL-10, IL-17, and TNF-α were analyzed. Radiological and intraoral dental examinations were conducted on the same day. Electric pulp testing and cold tests were conducted on all intact crowned teeth to assess the presence of PN. Periapical index scores were derived from panoramic and periapical radiographs to identify teeth exhibiting AP. Statistical analyses were carried out using Student’s t-test, Chi-square test, Spearman correlation test, and the Mann–Whitney U test. In the SCD group, the levels of IL-2, IL-4, IL-6, IL-8, IL-17, and TNF-α were significantly higher compared to the control group (p < 0.001, < 0.001, < 0.001, < 0.001, < 0.001, and = 0.003, respectively). No statistically significant association was observed between the groups and PN status (p = 0.06). Among individuals with SCD, IL-8 and TNF-α levels were significantly higher in those with apical periodontitis (AP+) compared to those without apical periodontitis (AP−) (p < 0.05). Statistically significant differences in IL-8 and TNF-α levels were observed between the AP (+) SCD group and the AP (+) control group (p = 0.004 for both). Additionally, significant differences in IL-2, IL-4, IL-6, IL-8, and IL-17 levels were found between the AP (-) SCD group and the AP (-) control group (p = 0.001 for all comparisons). IL-8 and TNF-α may be associated with the presence of apical periodontitis in SCD. In adolescents with SCD, disease-related serum cytokine levels may be associated with apical lesions. Additionally, these apical lesions may not be the primary cause of PN. As with other oral and dental conditions, early detection of apical lesions is crucial, and appropriate treatment plans should be developed. Collaborative efforts between dentists and hematologists are essential in managing these cases effectively.

Background

Sickle cell disease (SCD) is a common and life-threatening monogenic hematological disease characterized by hemoglobin synthesis disorder, affecting millions worldwide. SCD is more common in some parts of the world, especially in sub-Saharan Africa, eastern Saudi Arabia, and Mediterranean countries such as Turkey and some parts of central India, than in other regions¹.

SCD is caused by abnormal beta-globin alleles carrying sickle mutations in the beta-globin gene (Glu6Val, βS). The most common and severe form of this disease is the homozygous form, which is inherited from both parents and allows the formation of the pathological sickle hemoglobin tetramer (α2βS2, HbS)². Its pathogenesis begins with the polymerization of HbS inside the cells of erythrocytes. HbS polymerization increases cellular rigidity and distorts the erythrocyte membrane, leading to erythrocyte sickling, dehydration, impaired rheology, and premature hemolysis. This process is reversible if it has just started. If the triggering factors persist, this will be permanent due to the destruction of the cell membrane. Sickled erythrocytes lead to increased blood viscosity, slowed circulation, and a hypoxic environment, especially in the small vessels. This process also disrupts pulpal microcirculation³.

SCD leads to pathological consequences in mineralized and connective tissues in the body, such as kidneys, spleen, heart, lung, and oral tissues, with various acute complications such as vaso-occlusion, infection, anemia, and infarction^2,4. Among these complications, vaso-occlusive crises (VOC) are the most prominent feature of SCD. This local vaso-occlusion, which occurs due to the increased adhesion of sickle-shaped cells to the vascular endothelium, causes severe pain episodes and damage to vital organs⁵. VOC-induced pain is hyperalgesia induced by the activation of peripheral nociceptors^6,7. It can manifest as pulpitis and jawbone pain in the oral and maxillofacial regions. The most important endodontic effect of these crises on the pulp is the impairment of its oxygenation. Asymptomatic pulp necrosis (PN) occurs due to VOC in clinically sound, untraumatized, caries-free, and periodontally healthy teeth (with intact crowns). The incidence of these complications depends on the amount of vaso-occlusion created by the sickled cells that have accumulated in the pulpal circulation^4,8.

The sickled erythrocytes bind to the adhesive molecule on the surface of vascular endothelial cells and activate the endothelial cells. By activating both in and out of crisis periods, inflammatory cytokines such as tumor necrotizing factor alpha (TNF-α), Interleukin (IL)−1, and IL-8 are released from leukocytes⁵. Serum cytokines, which are known to be secreted at every stage of SCD, are also observed during the development of apical periodontitis (AP) characterized by the destruction of bone tissue surrounding the root tips of teeth caused by dental infections. The role of serum cytokines in the development of AP has been demonstrated in detail, with particular emphasis on IL-6, IL-8, and TNF-α, which are known to play a potential role in both AP progression and the pathophysiology of SCD^3,9.

The primary cause of AP, irrespective of systemic conditions, is generally the presence of bacteria within necrotic pulp tissue. The inflammatory response elicited by bacteria and their by-products results in the release of cytokines, chemokines, leukotrienes, and prostaglandins at the lesion site. During this process, PN and serum cytokines are also associated with the development of AP in otherwise healthy individuals^10,11.

To date, there is a lack of studies in the literature that have examined the relationship between PN and serum cytokines in patients with SCD. Additionally, only one study⁹ has investigated periapical cytokine release in individuals with AP in the context of SCD, and no studies have explored the relationship between serum cytokine levels in both healthy individuals and SCD patients, with and without AP. This study aims to investigate the association between serum cytokines, AP and PN in adolescents with SCD.

Materials and methods

This cross-sectional study was conducted in compliance with the applicable guidelines and institutional regulations. The ethic committee of the Mersin University at Turkey approved the study protocol in 2019(Date:22/05/2019; Approval Number:221). The study was planned to include all the SCD patients in control of the Mersin University Pediatric Hematology Department. A total of 52 adolescents, including 29 with SCD who were in a stable (steady-state) condition and voluntarily agreed to participate. SCD patients who had received a blood transfusion, experienced a VOC, used penicillin for prophylaxis, or took non-steroidal anti-inflammatory drugs (NSAIDs) in the prior month, as well as those with other chronic diseases, were excluded from the study. Clinical data, including the number of VOC episodes in the previous year, were obtained from hospital records. The control group, consisted of systemically healthy children who presented to the Department of Child Health and Diseases for routine health check-ups. These children had no abnormalities in their physical or medical examinations and voluntarily agreed to participate in the study. In both groups adolescents whose root development in all teeth were not completed in all teeth excluded from the study. Adolescents and their parents were thoroughly informed about the study’s objectives, scope, and potential risks. Separate informed consent forms were appropriately obtained from both parties.

Biochemical evaluation

On the day of the intraoral examination, 10 mL of peripheral blood was collected from all participants into pre-planned tubes. The blood samples were then centrifuged at 1300 g for 10 min, and the serum was separated for the evaluation of serum inflammatory markers (IL-1, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17 and TNF-α). The serum samples were stored at −80 °C until the day of analysis. Serum inflammatory markers were measured using Enzyme-Linked Immunosorbent Assay (ELISA) kits, following the protocol provided by the manufacturer (Elabscience, China). The concentrations of the cytokines in each sample were determined by plotting a standard curve and using the corresponding optical density values from standards with known concentrations for each cytokine analysis.

Dental examination

Participants who provided blood samples for biochemical analysis underwent dental examinations at the Faculty of Dentistry, University of XXX, on the same day. Initially, a single examiner performed electric pulp sensitivity testing (EPT) (Digitest II, Parkell, Inc., Edgewood, NY) and a cold test (Endo-Ice, Coltene/Whaledent, Altstätten, Switzerland) on all intact crowned teeth to identify teeth with no sensitivity to pulp tests, indicative of PN. Teeth that did not respond to both pulp sensitivity tests conducted on intact crowns were documented and classified as having PN.

The apical regions of the maxillar and mandibular teeth, as imaged by digital panoramic radiography (Planmeca Promax 2DS3, Helsinki, Finland) (54 KV, 1 mA, 16 S) were examined separately. Digital intraoral periapical radiographs were then taken for the teeth suspected of having AP) for further detailed evaluation. Images were obtained using an X-ray unit (ProX^®, Planmeca, Helsinki, Finland operated at 60 kVp and 7 mA, equipped with a number 2 (3 × 4 cm) photostimulable phosphor plate detector (Planmeca, Helsinki, Finland), with a pixel size of 35 μm and 16-bit color depth. The focal receptor distance was set to 20 cm, and the exposure time was 0.08 s. The images were then scanned with the Proscanner^® (Planmeca, Inc., Helsinki, Finland), stored, and analyzed using the integrated Romexis software (Planmeca, Inc., Helsinki, Finland)¹². The periapical index (PAI) scoring system encompassed five categories: (1) normal periapical structures, (2) minor changes in bone structure, (3) changes in bone structure with minimal mineral loss, (4) periodontitis with well-defined radiolucent areas, and (5) severe periodontitis with worsening features. Teeth with PAI scores of 1–2 were classified as having no AP, whereas those with scores of 3–5 were considered to have AP. For multi-rooted teeth, the highest PAI score among the roots was used for classification¹³.

Statistical analysis

Statistical analysis was performed using the IBM SPSS Statistics 22 (SPSS Inc., Chicago, IL, USA). The normality of the parameters was assessed using the Kolmogorov-Smirnov test, and it was determined that the parameters did not show a normal distribution. While evaluating the study data, in addition to descriptive statistical methods (minimum, maximum, mean, standard deviation, median, 25th percentile, 75th percentile, and frequency), the Student’s t test or Mann-Whitney U test was used in comparing the parameters between two groups in comparing the quantitative data. Chi-Square and Chi-Square exact tests were used to compare the qualitative data. Significance was evaluated at p < 0.05 level.

Power analysis

When effect sizes (Cohen’s d) and statistical power were calculated for cytokines that showed significant differences between the SCD and control groups (Table 2), the results were: IL-2 d = 0.99, power 93.5%; IL-4 d = 0.99, power 93.5%; IL-6 d = 0.99, power 93.5%; IL-8 d ≈ 0.99, power 93.5%; IL-17 d ≈ 0.99, power 93.5%; and TNF-α d = 0.83, power 84.6%. In addition, in subgroup analyses by AP status (Table 4), power estimates for significant comparisons were: IL-2 (SCD AP(−) vs. Control AP(−)) d = 1.21, power 94.8%; IL-4 (SCD AP(−) vs. Control AP(−)) d = 1.21, power 94.8%; IL-6 (SCD AP(−) vs. Control AP(−)) d = 1.21, power 94.8%; IL-8 (SCD AP(+) vs. SCD AP(−)) d = 0.88, power 80.3%; IL-8 (SCD AP(+) vs. Control AP(+)) d = 1.94, power 86.5%; IL-8 (SCD AP(−) vs. Control AP(−)) d = 1.21, power 94.8%; IL-17 (SCD AP(−) vs. Control AP(−)) d = 1.21, power 94.8%; TNF-α (SCD AP(+) vs. SCD AP(−)) d = 2.20, power 95.0%; and TNF-α (SCD AP(+) vs. Control AP(+)) d = 1.94, power 86.5%. These findings indicate high statistical power with large effect sizes, particularly for IL-2, IL-4, IL-6, IL-8, IL-17, and TNF-α.

Table 2.

Serum cytokine levels in SCD and control Groups.

	SCD (n=29)	Control (n=23)	p
	Median (Q1-Q3)	Median (Q1-Q3)
IL-1	2.050 (1.430–3.400.430.400)	0.890 (0.610–3.050.610.050)	0.12
IL-1 Beta	0.047 (0.045–0.048.045.048)	0.046 (0.045–0.048.045.048)	0.47
IL-2	0.064 (0.056–0.080.056.080)	0.049 (0.047–0.058.047.058)	< 0.001
IL-4	0.075 (0.070–0.100.070.100)	0.054 (0.051–0.064.051.064)	< 0.001
IL-6	0.080 (0.070–0.110.070.110)	0.060 (0.050–0.070.050.070)	< 0.001
IL-8	0.120 (0.090–0.170.090.170)	0.070 (0.060–0.080.060.080)	< 0.001
IL-10	0.049 (0.046–0.054.046.054)	0.047 (0.046–0.053.046.053)	0.69
IL-17	0.070 (0.060–0.100.060.100)	0.050 (0.040–0.060.040.060)	< 0.001
TNF-α	0.230 (0.060–0.540.060.540)	0.060 (0.050–0.080.050.080)	0.003

SCD: Sickle Cell Disease; IL: Interleukin; TNF-α: Tumor Necrosis Factor-Alpha.

Mann-Whitney U test; Q1–Q3: 25th–75th percentiles; p < 0.05 considered statistically significant.

Table 4.

Comparison if serum cytokine levels based on AP status in SCD and control Groups.

	SCD		Control		p
	AP (+) (n=11) a	AP (-) (n= 18) b	AP (+) (n=4) c	AP (-) (n= 19) d	(a-b)	(a-c)	(c-d)	(b-d)
	Median (Q1-Q3)	Median (Q1-Q3)	Median (Q1-Q3)	Median (Q1-Q3)
IL-1	1.700 (0.960–03.170.960.170)	2.250 (1.510–3.440.510.440)	1.510 (0.820–1.600.820.600)	1.520 (0.890–3.350.890.350)	0.16	0.36	0.47	0.15
IL-1 Beta	0.046 (0.045–0.047.045.047)	0.047 (0.046–0.500.046.500)	0.048 (0.046–0.060.046.060)	0.047 (0.046–0.048.046.048)	0.23	0.17	0.42	0.77
IL-2	0.070 (0.060–0.110.060.110)	0.060 (0.050–0.080.050.080)	0.060 (0.050–0.240.050.240)	0.049 (0.048–0.050.048.050)	0.32	0.43	0.57	0.001
IL-4	0.080 (0.060–0.100.060.100)	0.070 (0.060–0.120.060.120)	0.050 (0.040–0.210.040.210)	0.054 (0.052–0.060.052.060)	0.79	0.15	0.63	0.001
IL-6	0.080 (0.070–0.120.070.120)	0.080 (0.070–0.100.070.100)	0.054 (0.050–0.190.050.190)	0.055 (0.053–0.070.053.070)	0.53	0.12	0.52	0.001
IL-8	0.140 (0.110–0.330.110.330)	0.100 (0.090–0.140.090.140)	0.080 (0.060–0.800.060.800)	0.070 (0.060–0.090.060.090)	0.03	0.004	0.63	0.001
IL-10	0.048 (0.046–0.050.046.050)	0.049 (0.046–0.060.046.060)	0.047 (0.046–0.600.046.600)	0.047 (0.046–0.530.046.530)	0.51	0.79	0.87	0.48
IL-17	0.070 (0.060–0.100.060.100)	0.070 (0.060–0.090.060.090)	0.060 (0.050–0.230.050.230)	0.050 (0.048–0.060.048.060)	0.42	0.30	0.49	0.001
TNF-α	0.570 (0.530–0.590.530.590)	0.070 (0.060–0.190.060.190)	0.070 (0.060–0.120.060.120)	0.060 (0.055–0.070.055.070)	<0.001	0.004	0.37	0.28

SCD: Sickle Cell Disease; IL: Interleukin; TNF-α: Tumor Necrosis Factor-Alpha; AP (+): A periapical index (PAI) score of ≥3 is indicative of Apical Periodontitis; AP (-): Individuals with the PAI score below 3 are not diagnosed with AP.

a: SCD group with AP; b: SCD group without AP; c: Control group with AP; d: Control group without AP.

The Mann–Whitney U test was applied, and results are shown as Q1–Q3 (25th–75th percentiles). Statistical significance was set at p < 0.05.

Ethics approval

The study protocol was approved by the Mersin University Clinical Research Ethics Committee (date:22/05/2019; number:221).

Consent to participate

A written informed assent was obtained by adolescents and a impartial witness and a informed consent was obtained from all adolescents’s parents prior to data collection.

Results

The mean age was 16.8 ± 1.9 years in the SCD group and 15.7 ± 2.1 years in the control group, with no statistically significant difference between the groups (p = 0.07). While 72.4% of participants in the SCD group and 52.2% in the control group were male, and 27.6% and 47.8% were female, respectively, no statistically significant association was found between gender distribution and group status (p = 0.13) (Table 1).

Table 1.

Demographic characteristics by Group.

		SCD (n=29)	Control (n=23)	p
Age (Mean ± SD)		16.8±1.9	15.7±2.1	0.07 †
Gender, n (%)	Male	21 (72.4)	12 (52.2)	0.13 †††
	Female	8 (27.6)	11 (47.8)
PN, n (%)	(-)	24 (82.8)	23 (100)	0.06 †††
	(+)	5 (17.2)	0 (0.0)
AP, n (%)	(-)	18 (62.1)	19 (82.6)	0.10 †††
	(+)	11(37.9)	4 (17.4)

SCD: Sickle Cell Disease; SD: Standard Deviation; PN: Pulp Necrosis; AP: Apical Periodontitis.

†Student’s t-test; ††Chi-square test; p < 0.05 was considered statistically significant.

Although IL-1, IL-1β, and IL-10 levels were higher in the SCD group compared to the control group, these differences were not statistically significant (p > 0.05). In contrast, IL-2, IL-4, IL-6, IL-8, IL-17, and TNF-α levels were significantly elevated in the SCD group (p < 0.001, < 0.001, < 0.001, < 0.001, < 0.001, and 0.003, respectively) (Table 2).

PN was observed in 17.2% of individuals in the SCD group (five individuals, affecting a total of 20 teeth), while no cases were detected in the control group. Among the teeth diagnosed with PN, ten were located in the maxilla and ten in the mandible. Of these, eight were anterior teeth, five were premolars, and seven were molars. However, the difference in PN prevalence between the SCD and control groups was not statistically significant (p = 0.06) (Table 1). Within the SCD group, no statistically significant differences were found between PN (+) and PN (-) individuals concerning any of the cytokine levels (p > 0.05) (Table 3). Among individuals with SCD, 31% reported no VOC episodes in the past year, 44.8% reported one episode, 10.3% reported two episodes, 6.9% reported three episodes, and one participant (3.4%) experienced four or five episodes. No significant correlation was observed between VOC frequency and PN status (p > 0.05).

Table 3.

Comparison of serum cytokine levels based on PN status in SCD and control Groups.

	PN (+) (n=5)	PN (-) (n=24)	p
	Median (Q1-Q3)	Median (Q1-Q3)
IL-1	3.18 (2.18–3.54.18.54)	1.98 (1.29–3.33.29.33)	0.11
IL-1 Beta	0.05 (0.04–0.05.04.05)	0.05 (0.05-0.05.05.05)	0.18
IL-2	0.06 (0.06–0.07.06.07)	0.07 (0.05–0.10.05.10)	0.98
IL-4	0.07 (0.06–0.11.06.11)	0.08 (0.07–0.10.07.10)	0.63
IL-6	0.08 (0.07–0.11.07.11)	0.08 (0.07–0.11.07.11)	0.93
IL-8	0.11 (0.09–0.27.09.27)	0.12 (0.10–0.17.10.17)	0.93
IL-10	0.05 (0.05-0.05.05.05)	0.05 (0.04–0.06.04.06)	0.56
IL-17	0.06 (0.05–0.08.05.08)	0.07 (0.06–0.10.06.10)	0.56
TNF-α	0.59 (0.06–0.62.06.62)	0.20 (0.06–0.53.06.53)	0.35

SCD: Sickle Cell Disease; IL: Interleukin; TNF-α: Tumor Necrosis Factor-Alpha; PN: Pulp Necrosis.

Mann-Whitney U test, Q1-Q3: 25%−75% quartile, p < 0.05 significant.

AP was detected in 37.9% of individuals in the SCD group and 17.4% of those in the control group. However, the difference in AP prevalence between the two groups was not statistically significant (p = 0.10) (Table 1). AP was detected in 37.9% of individuals in the SCD group and 17.4% of those in the control group. In the SCD group, AP were observed in 17 teeth, with ten located in the mandible and seven in the maxilla. Of these, three were incisors, two were premolars, and 12 were molars. Regarding dental status, ten teeth exhibited carious lesions, one had a restoration, five had root canal treatment, and one was clinically sound. In the control group, all teeth with AP were located in the mandible, comprising one incisor and four molars. Among them, three teeth were decayed and two had received root canal treatment. Notably, no periapical lesions were detected in any of the teeth with PNs in the SCD group.

Within the SCD group, no statistically significant differences were observed between AP (+) and AP (-) individuals in terms of IL-1, IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-17 levels (p > 0.05). In contrast, IL-8 (p = 0.03) and TNF-α levels were significantly higher in AP-positive patients compared to AP-negative patients (p < 0.001) (Table 4).

Within the control group, no statistically significant differences were observed between individuals with and without AP in terms of any serum cytokine levels (p > 0.05) (Table 4).

A statistically significant difference in IL-8 and TNF-α levels was observed between the AP (+) SCD group and the AP (+) control group (p = 0.004 for both). However, no significant differences were found between the groups in terms of the other serum cytokine levels (p > 0.05) (Table 4).

Statistically significant differences in IL-2, IL-4, IL-6, IL-8, and IL-17 levels were found between the AP (−) SCD group and the AP (−) control group (p = 0.001 for all). However, no significant differences were observed in the remaining serum cytokine levels between the two groups (p > 0.05) (Table 4).

Discussion

Sickle cell disease is an inflammatory disease known to play a significant role in the pathogenesis of both acute and chronic inflammation. Numerous studies have explored the association between elevated serum cytokine levels and various organ damage and clinical manifestations of the disease^14–17. In our study, the finding that inflammatory cytokine levels were significantly higher in adolescents with SCD compared to healthy adolescents supports this notion.

It has been reported that oral manifestations may be more prevalent in individuals with SCD, attributed to the interplay of inflammation and infection, similar to other systemic conditions where overall health is closely linked to dental health from an early age^18–22. Studies evaluating oral health in patients with SCD have reported a higher prevalence of toothache without any pathology, jawbone expansion, osteoporotic changes in the mandible, gingival hypertrophy, dental caries, and pulp necrosis compared to healthy children^{3,4,8,9,23,24}. However, no studies have been identified that examine the relationship between serum cytokine levels and AP or PN in patients with SCD.

Pulp necrosis, which occurs in systemically healthy individuals, typically develops due to bacterial infection of the dental pulp originating from a carious lesion¹¹. However, in SCD, VOCs are recognized as a risk factor for PN in permanent teeth with intact crowns, even in the absence of bacterial infection^4,8. In our research, PN was detected in 5 individuals (17%) with SCD. Demirbaş et al.³ reported that 6% of the teeth in patients with SCD exhibited PN, while Costa et al.⁴ observed that PN was 8.33 times more prevalent in SCD patients than in healthy individuals. The disparity in the reported frequency of PN between studies may be attributed to differences in the patient populations. In our study, no statistically significant association was found between the frequency of VOC and the presence of PN (p > 0.05). However, it is essential to note that the study population consisted of adolescents; in older individuals, the cumulative frequency of VOC episodes may be higher, potentially contributing to an increased incidence of PN.

Although inflammation plays a critical role in the pathogenesis of PN, no significant differences were observed in serum cytokine levels between SCD patients with and without PN in our study. This result may be attributed to the limited number of patients with PN. Moreover, we were unable to identify comparable data in the existing literature. We would like to emphasize that the primary aim of this study was to investigate the relationship between cytokines and AP. Nevertheless, considering that AP may also occur in healthy individuals, that PN is a major cause of AP in otherwise healthy populations, and that PN is recognized as an intraoral complication of SCD, we considered it necessary to include PN in our analysis. Although the frequency of PN cases could not be anticipated at the outset, the relatively low prevalence observed in our study may be interpreted as a favorable finding in terms of disease prognosis.

In the control group, PN was not expected in intact crowned teeth due to the absence of bacterial load, which aligned with the study findings. Conversely, in the SCD group, PN was observed independently of bacterial presence, and no associated pain was reported in the affected teeth. These results support the conclusion that clinical and radiographic evaluations at six-month intervals constitute an appropriate approach for the early detection of tooth-related endodontic infections in this population.

Another parameter evaluated during the dental examination in this study, AP, is a condition that develops in the surrounding bone tissue as the immune system attempts to manage bacteria and their by-products in the infected necrotic pulp in systemically healthy individuals^10,11. Therefore, AP is an anticipated outcome in the progression of PN over time in the healthy population. However, in the present study, AP was not detected in any of the teeth with PN. The bacterial load, which is known to contribute to the development of AP, may not be sufficient in our SCD patients with PN. This aligns with the findings of Costa et al.²³, who demonstrated that bacteria do not play a significant role in the formation of PN in SCD patients.

Previous studies have demonstrated an association between AP and a wide range of systemic conditions, including diabetes, cardiovascular diseases, osteoporosis, inflammatory bowel diseases, hereditary coagulopathies, and the use of biologic agents^25,26. In the present study, although not reaching statistical significance, the incidence of AP was found to be higher in SCD patients compared to the control group. To the best of our knowledge, there are no data in the existing literature directly comparing the prevalence of AP in SCD patients with that in the healthy population. Our study is among the first preliminary investigations to explore the association between the presence of AP and serum cytokine levels in patients with SCD. We believe that future studies with larger patient populations will build upon our initial findings and provide further insights into this subject.

In our study, when comparing subgroups of SCD patients, those with AP (+) exhibited significantly higher levels of IL-8 and TNF-α compared to AP (–) patients. These findings suggest that, in addition to the chronic inflammation inherently caused by SCD, local dental inflammation may also contribute to the elevation of these cytokines.

Bacteria in necrotic pulp tissue stimulate the host immune defense system, leading to the release of cytokines, chemokines, leukotrienes, and prostaglandins. The roles of IL-1 beta, IL-2, and IL-4 in the development of AP have been demonstrated¹¹. Furthermore, IL-6, IL-8, and TNF-α serum cytokines are recognized as key contributors to both the pathogenesis of SCD and the progression of AP^5,6,27–29. Ferreira et al.⁹ reported that the mRNA expression of TNF-α, IL-1α, IL-10, and IL-17 in the periapical interstitial fluid of APs was significantly higher in the SCD group compared to the control group. Palafox et al.³⁰, in a longitudinal study investigating acute periapical lesions in a healthy population, reported elevated levels of TNF-α and IL-6 cytokines compared to the control group. In our study, a comparison of serum cytokine levels between SCD patients with and without AP revealed no statistically significant differences in the levels of IL-1, IL-1 beta, IL-2, IL-4, IL-6, IL-10 and IL-17. However, IL-8 and TNF-α levels were found to be significantly elevated in SCD patients with AP. Although variations in findings are anticipated due to differences in body fluids analyzed and measurement methods employed across studies, our results, alongside related studies, highlight the significant role of TNF-α in host defense mechanisms in both acute and chronic periapical lesions.

Furthermore, IL-8, similar to TNF-α, plays a significant role in endothelial cell and leukocyte activation, macrophage stimulation, the affinity of leukocyte surface molecules for endothelial receptors, and leukocyte chemotaxis in SCD patients^10,13,31. Notably, aside from this study, there is no data in the existing literature linking elevated IL-8 serum levels to AP in SCD patients. Based on our findings, we suggest that IL-8 and TNF-α, which are recognized as key contributors to the pathogenesis of SCD, may also play a role in the development of AP in this patient population.

A limitation of this study is the small number of SCD patients, especially those with PN. In our country, nationwide hemoglobinopathy control programs implemented by the Ministry of Health—such as mandatory premarital SCD carrier testing and early diagnosis and treatment strategies aimed at reducing disease severity—have contributed to a gradual decline in its prevalence. Therefore, SCD is already considered a rare condition in our population. In addition, the strict exclusion criteria applied in our study and the specific age range selected further limited the number of patients included. This study included all SCD patients under follow-up in the Pediatric Hematology Department of our university who met the inclusion and exclusion criteria. However, the resulting sample size remained limited.

Cytokine levels can also fluctuate with subclinical infections, stress, or recent minor illnesses, which were not reported or controlled for. these levels may be influenced by stress and subclinical infections; however, given the difficulty of controlling such factors, their assessment was not feasible in the present study and has been acknowledged as a limitation. Nevertheless, we made every effort to include as exclusion criteria those conditions that could potentially alter inflammatory cytokine levels. Indeed, similar exclusion criteria have also been applied in many clinical studies investigating the relationship between sickle cell anemia and inflammatory cytokines.

While PN prevalence was higher in SCD patients, the lack of statistical significance makes it speculative to conclude. The small sample likely masked true associations. However, all eligible individuals were included, and the sample size exceeded that of similar studies. Future cohort studies with larger populations can be conducted to investigate the findings further.

In our study, the control group was randomly selected from individuals presenting to a pediatric outpatient clinic for routine health examinations and without known systemic diseases, in accordance with established inclusion and exclusion criteria. However, similar to the references we used in this preliminary study, socioeconomic status,, dietary habits, caries experience, microbial factors^30,32 or oral hygiene³⁰ practices were not considered in the group selection process. This is another limitation of our study. The lack of complete control for these variables may have contributed to residual confounding effects, making it difficult to attribute the observed results solely to the variable under study. Consequently, some of the observed effect may have been influenced by these uncontrolled confounders. We believe that future large-scale, population-based studies that include tighter matching or statistical adjustment for such variables will allow for a more robust assessment of causal relationships.

Another limitation of this study is that in adolescents, pulp sensibility tests may have diagnostic limitations due to the ongoing root development and the possibility of less reliable responses³³. This negativity was attempted to be overcome by additionally applying the cold test and, therefore, using two pulp sensitivity tests. However, this limitation can be addressed by utilizing pulp vitality assessment methods, such as Laser Doppler Flowmetry, transmitted laser light, laser speckle imaging, pulse oximetry, transmitted light plethysmography, or dual-wavelength spectrophotometry, all of which are recommended for evaluating pulp vitality in cases of dental trauma^33–35. Unfortunately, these devices, which are not commonly used in dental clinics in our country and are costly, were unavailable in our clinic during this preliminary study. The non-use of more advanced methods could be diagnostic limitaton also.

Microbiological sampling from root canals or periapical tissues was not feasible due to ethical and procedural constraints associated with obtaining invasive specimens from this medically vulnerable population. Given that the pathogenesis of apical periodontitis is predominantly driven by microbial infection, the lack of direct microbiological assessment in our study also represents a limitation. While this preliminary study demonstrates an association between the presence of AP lesions and elevated TNF-α and IL-8 levels in SCD patients, this limitation limits our ability to determine whether the observed cytokine changes reflect a purely systemic inflammatory state or an immune response additionally triggered by a local microbial burden. Future studies integrating microbiological profiling with cytokine analysis will be important to more clearly elucidate the immunological mechanisms underlying AP.

To the best of our knowledge, this study is the first to investigate the relationship between AP and PN in individuals with SCD, representing a pioneering contribution to the field. Furthermore, future studies could be expanded to include the analysis of various oral fluids such as saliva and interstitial fluid from apical lesions, as well as systemic hematological parameters including C-reactive protein and white blood cell counts, together with microbial assessments performed through culture of root canal samples. In this way, the interaction between local and systemic inflammatory responses could be elucidated more comprehensively.

Although many cytokines were elevated in AP-positive SCD patients, these cytokines are already increased in SCD even in the absence of AP. Nevertheless, the absence of such differences between AP-positive and AP-negative individuals in the control group, and the fact that within the SCD group—despite the potential involvement of multiple cytokines—only IL-8 and TNF-α showed a clear distinction, make our preliminary findings noteworthy and worthy of further exploration in future, larger-scale studies.

Conclusion

Despite the limitations of this study, it was determined that serum cytokines, such as IL-8 and TNF-α, which are known to be released in patients with SCD outside of crises, may be associated with the presence of APs. Although no correlation was found between serum cytokine levels and PN teeth, this preliminary study is believed to serve as a foundation for future research involving SCD patients. Moreover, it is crucial for hematologists and dentists who monitor these patients to recognize the importance of oral health, which is closely linked to overall health, and the need for interdisciplinary collaboration.

Abbreviations

SCD

Sickle Cell Disease

VOC

Vaso-Occlusive Crises

ELISA

Enzyme-Linked Immunosorbent Assay

TNF-α

Tumor Necrotizing Factor Alpha

IL

Interleukin

PN

Pulp Necrosis

EPT

Electric Pulp Sensitivity Testing

AP

Apical Lesion

PAI

Periapical Index

IL

Interleukin

TNF-α

Tumor Necrosis Factor- Alfa

mRNA

Messenger Ribonucleic Acid

miRNA

Micro Ribonucleic Acid

Author contributions

M.Y conceived the idea; M.Y.and S.A. collected the dental data; and A.A.O. did the biochemical evaluation, F.K. and S.U. collected the hematological data; M.Y wrote the manuscript and E.E. was responsible for performing the statistical analyses. Both authors read and approved the final manuscript.

Funding

This project was supported by Scientific Research Projects Unit of Mersin University/Turkey with the project number: 2019-3-AP5-3803.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.