Changes and clinical significance of four biomarkers in gingival crevicular fluid after nonsurgical periodontal therapy

doi:10.12016/j.issn.2096-1456.2021.12.005

Abstract

Abstract:

Objective To study the changes in levels of interleukin (IL)-6, IL-10, tumor necrosis factor-alpha (TNF-α), and alkaline phosphatase (ALP) in the gingival crevicular fluid (GCF) of patients with severe chronic periodontitis before and after nonsurgical periodontal therapy and to explore the relationship among the levels of these four biomarkers in GCF, their periodontal status and their clinical significance to evaluate the effect of nonsurgical periodontal therapy and periodontitis activity. Methods In total, 30 patients with severe chronic periodontitis were enrolled in a 1-year longitudinal pilot study (Chinese Clinical Trial Registry: ChiCTR-OCH-13004679). At baseline and 1, 3, 6, and 12 months after nonsurgical therapy, the periodontal clinical indicators plaque index (PLI), probing depth (PD), clinical attachment loss (CAL), sulcus bleeding index (SBI) were recorded. Filter paper strips were used to collect two deep-pocket (probing depth ≥ 6 mm) and two shallow-pocket (probing depth ≤ 4 mm) periodontal sites for each patient and weighed. The levels of interleukin IL-6, IL-10, TNF-α, and ALP in GCF were assessed using enzyme-linked immunosorbent assay. Meanwhile, 30 healthy sites of 15 subjects with healthy periodontium were used as the baseline controls for patients with severe chronic periodontitis. Results At the baseline, the TNF-α, ALP and IL-6 levels in GCF of the disease sites of patients with periodontitis were significantly higher than those in healthy periodontal sites of the control group (P < 0.001), and the levels of IL-10 were significantly lower than those in the control group (P < 0.001). In patients with severe chronic periodontitis, the levels of TNF-α, ALP and IL-6 in GCF at deep-pocket sites were significantly higher than those at shallow-pocket sites (P <0.001), and the IL-10 levels were significantly lower than those at shallow-pocket sites (P < 0.001). 1, 3, 6, and 12 months after nonsurgical treatment, the levels of TNF-α and ALP in GCF at the shallow- and deep-pocket sites in patients with chronic periodontitis significantly decreased, the level of IL-10 significantly increased (P < 0.005), and the level of IL-6 in GCF at the deep-pocket sites significantly decreased (P < 0.005). However, there was no significant difference in IL-6 level at shallow-pocket sites (P > 0.05). 1, 3, 6, and 12 months after nonsurgical treatment, the periodontal clinical indicators were improved compared with the baseline. In addition, there was a significant correlation between the levels of these four biomarkers and the periodontal clinical parameters (P < 0.05). During the two follow-up visits after nonsurgical periodontal therapy, the sites with more than 2-mm increase in attachment loss had significant differences in the levels of the four biomarkers in the GCF compared with the previous visit time (P < 0.005). Conclusion The detection of the levels of these four biomarkers in GCF has strong clinical significance for assessing the severity of periodontitis and the efficacy of nonsurgical periodontal therapy. Increased levels of TNF-α, ALP, and IL-6 and decreased IL-10 levels in GCF may indicate periodontitis progression at this site.

Key words: biomarkers, IL-10, TNF-α, IL-6, ALP, gingival crevicular fluid, nonsurgical periodontal therapy, severe chronic periodontitis, clinical trial

摘要：

目的研究牙周基础治疗前后重度慢性牙周炎患者不同牙位龈沟液(gingival crevicular fluid,GCF)中白细胞介素6(interleukin 6,IL-6)、白细胞介素10(interleukin 10,IL-10)、肿瘤坏死因子α(tumor necrosis α,TNF-α)、碱性磷酸酶(alkaline phosphatase,ALP)水平的变化,探讨以上4种生物标志物GCF水平与牙周状态的关系以及对牙周基础治疗效果、牙周炎活动性评估的临床意义。方法共30例重度慢性牙周炎患者参与了为期1年的纵向试验研究(中国临床试验注册中心：ChiCTR-OCH-13004679)。在术前和基础治疗后1、3、6、12个月,记录牙周临床指标(菌斑指数、龈沟出血指数、探诊深度、临床附着丧失),并用滤纸条收集每例患者2个深袋牙位(探诊深度 ≥ 6 mm)和2个浅袋牙位(探诊深度 ≤ 4 mm)的GCF并称重,ELISA法测定GCF中IL-6、IL-10、TNF-α和ALP的水平。选取15名牙周健康者的30个健康牙位为重度慢性牙周炎患者的基线对照。结果基线时,重度慢性牙周炎患者的疾病位点GCF中TNF-α、ALP、IL-6水平显著高于对照组的牙周健康位点(P<0.001),IL-10水平显著低于对照组(P < 0.001);重度慢性牙周炎患者深袋位点的GCF中TNF-α、ALP、IL-6水平显著高于浅袋位点(P<0.001);深袋位点IL-10水平显著低于浅袋位点(P < 0.001)。相较于基线,基础治疗后1、3、6、12个月,重度慢性牙周炎患者深袋、浅袋位点GCF中的TNF-α、ALP水平显著降低,IL-10水平显著升高(P<0.005),深袋位点GCF中IL-6水平显著降低(P < 0.005),浅袋位点的IL-6无统计学意义上的改变(P > 0.05)。治疗后1、3、6、12个月,4项牙周临床指标较基线均有改善,以上4种生物标志物水平与牙周临床指标之间均存在显著相关性,与IL-6、TNF-α、ALP呈正相关,与IL-10呈负相关(P < 0.05)。在基础治疗后的2次随访期间临床附着丧失增加超过2 mm的位点,GCF中4种生物标志物水平与上一次相比,差异有统计学意义(P<0.005)。结论 GCF中TNF-α、ALP、IL-6、IL-10这4种生物标志物的水平检测,对于判断牙周炎的严重程度、牙周基础治疗效果具有较强的临床意义。GCF中TNF-α、ALP、IL-6水平升高、IL-10水平降低可能预示着该位点出现牙周炎进展。

关键词: 生物标志物, 白细胞介素-10, 肿瘤坏死因子-α, 白细胞介素-6, 碱性磷酸酶, 龈沟液, 牙周基础治疗, 重度慢性牙周炎, 临床试验

CLC Number:

ZHANG Qian,CHEN Bin,YAN Fuhua. Changes and clinical significance of four biomarkers in gingival crevicular fluid after nonsurgical periodontal therapy[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(12): 828-835.

张倩,陈斌,闫福华. 牙周基础治疗后龈沟液中4种生物标志物水平变化及临床意义[J]. 口腔疾病防治, 2021, 29(12): 828-835.

Figures/Tables 5

Table 1 Levels of biomarkers in GCF at periodontitis sites in patients with severe chronic periodontitis at baseline and healthy sites in the control group Median (IQR)

Group	IL-6 (pg/site)	IL-10 (pg/site)	TNF-α (pg/site)	ALP (pg/site)
Control	0.30(0.21,0.61)	1.34(0.62,1.74)	1.65(0.62,2.65)	129.7(22.31,192.8)
SPP	2.05(1.16,2.82)¹⁾	0.62(0.29,0.89)¹⁾	2.90(2.45,4.16)¹⁾	272.1(210.3,519.8)¹⁾
DPP	6.32(3.50,9.42)¹⁾²⁾	0.31(0.10,0.61)¹⁾²⁾	6.51(1.45,9.82)¹⁾²⁾	905.2(457.9,1744)¹⁾²⁾

Figure 1 Levels of IL-6, TNF-α, IL-10, and ALP in the GCF of deep-pocket and shallow-pocket sites at the baseline and 1, 3, 6 and 12 months after nonsurgical periodontal therapy a: IL-6; b: IL-10; c: TNF-α; d: ALP; *: compared with the baseline, P < 0.005. 1, 3, 6, and 12 months after nonsurgical therapy, the levels of TNF-α and ALP in GCF at the shallow- pocket (PD ≤ 4 mm) and deep-pocket (PD ≥ 6 mm) sites significantly decreased, the level of IL-10 significantly increased (P < 0.005), and the IL-6 level in GCF at the deep-pocket sites significantly decreased. IL-6: interleukin-6; IL-10: interleukin-10; TNF-α: tumor necrosis factor α; ALP: alkaline phosphatase; PD: probing depth

Figure 2 Clinical indicators of deep pocket and shallow pocket sites at the baseline and 1, 3, 6 and 12 months after nonsurgical periodontal therapy a: PD; b: CAL; c: PLI; d: SBI; *: compared with the baseline, P < 0.005. 1, 3, 6, and 12 months after nonsurgical therapy, PD and CAL in the GCF at the shallow-pocket (PD ≤ 4 mm) and deep-pocket (PD ≥ 6 mm) sites significantly decreased, and PLI and SBI in the GCF at the deep pocket sites significantly decreased; PD: probing depth; CAL: clinical attachment loss; PLI: plaque index; SBI: sulcus bleeding index; GCF: gingival crevicular fluid

Table 2 Correlation between periodontal parameters and the levels of biomarkers in GCF

Parameters	IL-6	IL-10	TNF-α	ALP
PD(mm)	P < 0.000 1	P < 0.000 1	P < 0.000 1	P < 0.000 1
	r=0.758	r=-0.633	r=0.697	r=0.676
CAL (mm)	P < 0.000 1	P<0.000 1	P < 0.000 1	P < 0.000 1
	r=0.703	r=-0.594	r=0.729	r=0.687
PLI	P=0.003	P=0.007	P=0.004	P=0.003
	r=0.374	r=-0.256	r=0.398	r=0.372
SBI	P=0.001	P=0.004	P=0.003	P=0.002
	r=0.477	r=-0.389	r=0.380	r=0.459

Table 3 Levels of biomarkers in GCF at the sites of periodontitis progression 1, 3, 6, and 12 months after nonsurgical periodontal therapy $\bar{x}±s$

Biomarkers and clinical parameters	1 month/baseline	3 months/1 month	6 months/3 months	12 months/6 months
sites/total, ratio	8/120, 6.67%	12/120, 10%	10/120, 8.33%	12/120, 10%
IL-6(pg)	6.72±1.07¹⁾/5.71±0.87	5.16±0.48¹⁾/4.18±0.45	6.31±0.89¹⁾/4.71±0.50	6.10±1.00¹⁾/4.46±0.74
TNF-α(pg)	6.99±0.93¹⁾/5.67±0.62	4.72±0.61¹⁾/3.85±0.70	5.70±0.84¹⁾/4.08±0.49	5.54±0.78¹⁾/4.33±0.86
IL-10(pg)	0.37±0.04¹⁾/0.50±0.03	0.42±0.06¹⁾/0.60±0.04	0.47±0.07¹⁾/0.77±0.10	0.80±0.17¹⁾/1.30±0.17
ALP(pg)	1 372.45±221.14¹⁾/1 071.29±208.25	1 091.54±243.48¹⁾/514.36±35.28	672.39±103.69¹⁾/441.23±70.15	642.34±130.29¹⁾/343.27±52.36
CAL(mm)	7.44±0.82¹⁾/5.31±0.75	7.12±0.61¹⁾/4.96±0.54	7.05±0.72¹⁾/4.75±0.75	7.33±0.72¹⁾/5.08±0.70

References 35

[1]	Ramseier CA, Anerud A, Dulac M, et al. Natural history of periodontitis: disease progression and tooth loss over 40 years[J]. J Clin Periodontol, 2017, 44(12):1182-1191. doi: 10.1111/jcpe.12782. DOI URL
[2]	Tonetti MS, Jepsen S, Jin L, et al. Impact of the global burden of periodontal diseases on health, nutrition and wellbeing of mankind: a call for global action[J]. J Clin Periodontol, 2017, 44(5):456-462. doi: 10.1111/jcpe.12732. DOI URL
[3]	Wang QT, Wu ZF, Wu YF, et al. Epidemiology and preventive direction of periodontology in China[J]. J Clin Periodontol, 2007, 34(11):946-951. doi: 10.1111/j.1600-051X.2007.01139.x. DOI URL
[4]	Ma X, Wang Y, Wu H, et al. Periodontal health related-inflammatory and metabolic profiles of patients with end-stage renal disease: potential strategy for predictive, preventive, and personalized medicine[J]. EPMA J, 2021, 22:1-12. doi: 10.1007/s13167-021-00239-0.
[5]	Kalsi AS, Moreno F, Petridis H. Biomarkers associated with periodontitis and peri-implantitis: a systematic review[J]. J Periodontal Implant Sci, 2021, 51(1):3-17. doi: 10.5051/jpis.1902840142. DOI URL
[6]	Fatima T, Khurshid Z, Rehman A, et al. Gingival crevicular fluid (GCF): a diagnostic tool for the detection of periodontal health and diseases[J]. Molecules, 2021, 26(5):1208. doi: 10.3390/molecules26051208. DOI URL
[7]	Armitage GC. Development of a classification system for periodontal diseases and conditions[J]. Ann Periodontol, 1999, 4(1):1-6. doi: 10.1902/annals.1999.4.1.1. URL PMID
[8]	孟焕新. 牙周病学[M]. 4版. 北京: 人民卫生出版社, 2014: 131.
	Meng HX. Periodontology[M]. 4th ed. Beijing: People’s Medical Publishing House, 2014: 131.
[9]	Nalmpantis D, Gatou A, Fragkioudakis I, et al. Azurocidin in gingival crevicular fluid as a potential biomarker of chronic periodontitis[J]. J Periodontal Res, 2020, 55(2):209-214. doi: 10.1111/jre.12703. DOI URL PMID
[10]	Barros SP, Williams R, Offenbacher S, et al. Gingival crevicular fluid as a source of biomarkers for periodontitis[J]. Periodontol 2000, 2016, 70(1):53-64. doi: 10.1111/prd.12107. DOI URL
[11]	Tsuchida S, Satoh M, Takiwaki M, et al. Current status of proteomic technologies for discovering and identifying gingival crevicular fluid biomarkers for periodontal disease[J]. Int J Mol Sci, 2018, 20(1):86. doi: 10.3390/ijms20010086. DOI URL
[12]	Nazar MZ, Philip K, Alabsi AM, et al. Identification of gingival crevicular fluid sampling, analytical methods, and oral biomarkers for the diagnosis and monitoring of periodontal diseases: a systematic review[J]. Dis Markers, 2016: 1804727. doi: 10.1155/2016/1804727.
[13]	Bostanci N, Belibasakis GN. Gingival crevicular fluid and its immune mediators in the proteomic era[J]. Periodontol 2000, 2018, 76(1):68-84. doi: 10.1111/prd.12154. DOI URL PMID
[14]	Keles YZ, Keles GC, Avci B, et al. Nonsurgical periodontal therapy reduces salivary and gingival crevicular fluid YKL-40 and IL-6 levels in chronic periodontitis[J]. Oral Health Prev Dent, 2020, 18(1):815-822. doi: 10.3290/j.ohpd.a45086.
[15]	Stadler AF, Angst PD, Arce RM, et al. Gingival crevicular fluid levels of cytokines/chemokines in chronic periodontitis: a meta-analysis[J]. J Clin Periodontol, 2016, 43(9):727-745. doi: 10.1111/jcpe.12557. DOI URL PMID
[16]	Akram Z, Abduljabbar T, Abu Hassan MI, et al. Cytokine profile in chronic periodontitis patients with and without obesity: a systematic review and meta-analysis[J]. Dis Markers, 2016: 4801418. doi: 10.1155/2016/4801418.
[17]	Mansell A, Jenkins BJ. Dangerous liaisons between interleukin-6 cytokine and toll-like receptor families: a potent combination in inflammation and cancer[J]. Cytokine Growth Factor Rev, 2013, 24(3):249-256. doi: 10.1016/j.cytogfr.2013.03.007. DOI URL
[18]	Martínez-Aguilar VM, Carrillo-Ávila BA, Sauri-Esquivel EA, et al. Quantification of TNF-α in patients with periodontitis and type 2 diabetes[J]. Biomed Res Int, 2019: 7984891. doi: 10.1155/2019/7984891.
[19]	Cicek AV, Ilarslan YD, Erman B, et al. Statins and IL-1β, IL-10, and MPO levels in gingival crevicular fluid: preliminary results[J]. Inflammation, 2016, 39(4):1547-1557. doi: 10.1007/s10753-016-0390-7. DOI URL
[20]	Shi T, Jin Y, Miao Y, et al. IL-10 secreting B cells regulate periodontal immune response during periodontitis[J]. Odontology, 2020, 108(3):350-357. doi: 10.1007/s10266-019-00470-2. DOI URL
[21]	de Aguiar MC, Perinetti G, Capelli JJ. The gingival crevicular fluid as a source of biomarkers to enhance efficiency of orthodontic and functional treatment of growing patients[J]. Biomed Res Int, 2017: 3257235. doi: 10.1155/2017/3257235.
[22]	Abduljabbar T, Vohra F, Kellesarian SV, et al. Efficacy of scaling and root planning with and without adjunct Nd: YAG laser therapy on clinical periodontal parameters and gingival crevicular fluid interleukin 1-beta and tumor necrosis factor-alpha levels among patients with periodontal disease: a prospective randomized split-mouth clinical study[J]. J Photochem Photobiol B, 2017, 169:70-74. doi: 10.1016/j.jphotobiol.2017.03.001. DOI URL
[23]	Ito H, Numabe Y, Hashimoto S, et al. Correlation between gingival crevicular fluid hemoglobin content and periodontal clinical parameters[J]. J Periodontol, 2016, 87(11):1314-1319. doi: 10.1902/jop.2016.160092. DOI URL
[24]	Kellesarian SV, Malignaggi VR, Abduljabbar T, et al. Efficacy of scaling and root planing with and without adjunct antimicrobial photodynamic therapy on the expression of cytokines in the gingival crevicular fluid of patients with periodontitis: a systematic review[J]. Photodiagnosis Photodyn Ther, 2016, 16:76-84. doi: 10.1016/j.pdpdt.2016.08.009. DOI URL PMID
[25]	Armitage GC. Analysis of gingival crevice fluid and risk of progression of periodontitis[J]. Periodontol 2000, 34:109-119. doi: 10.1046/j.0906-6713.2002.003427.x. DOI URL
[26]	Segarra-Vidal M, Guerra-Ojeda S, Vallés LS, et al. Effects of photodynamic therapy in periodontal treatment: a randomized, controlled clinical trial[J]. J Clin Periodontol, 2017, 44(9):915-925. doi: 10.1111/jcpe.12768. DOI URL PMID
[27]	Singh N, Chandel S, Singh H, et al. Effect of scaling & root planing on the activity of ALP in GCF & serum of patients with gingivitis, chronic and aggressive periodontitis: a comparative study[J]. J Oral Biol Craniofac Res, 2017, 7(2):123-126. doi: 10.1016/j.jobcr.2017.03.006. DOI URL
[28]	Türer ÇC, Durmuş D, Balli U, et al. Effect of non-surgical periodontal treatment on gingival crevicular fluid and serum endocan, vascular endothelial growth factor-a, and tumor necrosis factor-alpha levels[J]. J Periodontol, 2017, 88(5):493-501. doi: 10.1902/jop.2016.160279. DOI URL
[29]	Kurgan Ş, Önder C, Balcı N, et al. Gingival crevicular fluid tissue/blood vessel-type plasminogen activator and plasminogen activator inhibitor-2 levels in patients with rheumatoid arthritis: effects of nonsurgical periodontal therapy[J]. J Periodontal Res, 2017, 52(3):574-581. doi: 10.1111/jre.12425. DOI URL PMID
[30]	Wu Y, Chen L, Wei B, et al. Effect of non-surgical periodontal treatment on visfatin concentrations in serum and gingival crevicular fluid of patients with chronic periodontitis and type 2 diabetes mellitus[J]. J Periodontol, 2015, 86(6):795-800. doi: 10.1902/jop.2015.140476. DOI URL
[31]	Gonçalves TE, Zimmermann GS, Figueiredo LC, et al. Local and serum levels of adipokines in patients with obesity after periodontal therapy: one-year follow-up[J]. J Clin Periodontol, 2015, 42(5):431-439. doi: 10.1111/jcpe.12396. DOI URL PMID
[32]	Balli U, Ongoz Dede F, Bozkurt Dogan S, et al. Chemerin and interleukin-6 levels in obese individuals following periodontal treatment[J]. Oral Dis, 2016, 22(7):673-680. doi: 10.1111/odi.12520. DOI URL PMID
[33]	Bostanci V, Toker H, Senel S, et al. Evaluation of IL-1β, IL-1ra, and IL-10 levels and outcome of periodontal therapy in chronic periodontitis with familial Mediterranean fever[J]. Clin Oral Investig, 2017, 21(1):469-475. doi: 10.1007/s00784-016-1816-1. DOI URL
[34]	De GD, Tiosso-Tamburi R, Furletti DV, et al. Clinical and immunoinflammatory evaluation of one-stage full-mouth ultrasonic debridement as a therapeutic approach for smokers with generalized aggressive periodontitis: a short-term follow-up study[J]. J Periodontol, 2016, 87(9):1012-1021. doi: 10.1902/jop.2016.150632. DOI URL
[35]	Bunaes DF, Mustafa M, Mohamed HG, et al. The effect of smoking on inflammatory and bone remodeling markers in gingival crevicular fluid and subgingival microbiota following periodontal therapy[J]. J Periodontal Res, 2017, 52(4):713-724. doi: 10.1111/jre.12438. DOI URL PMID