REVIEW ARTICLE

https://doi.org/10.5005/jp-journals-10015-2244
World Journal of Dentistry
Volume 14 | Issue 6 | Year 2023

Calprotectin, S100A8, and S100A9: Potential Biomarkers of Periodontal Inflammation: A Scoping Review

Annie K George¹ https://orcid.org/0000-0002-3795-2633, Sankari Malaiappan² https://orcid.org/0000-0002-4146-2477, Betsy Joseph³ https://orcid.org/0000-0002-2925-1706, Sukumaran Anil⁴ https://orcid.org/0000-0002-6440-8780

¹Department of Periodontics, Pushpagiri College of Dental Sciences, Thiruvalla, Kerala, India

^2-3Department of Periodontics, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, Tamil Nadu, India

⁴Oral Health Institute, Department of Dentistry, Hamad Medical Corporation, Doha, Qatar

Corresponding Author: Annie K George, Department of Periodontics, Pushpagiri College of Dental Sciences, Thiruvalla, Kerala, India; Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, Tamil Nadu, India, Phone: +91 9847440665, e-mail: dranniekitty121@gmail.com

Received on: 09 May 2023; Accepted on: 10 June 2023; Published on: 22 August 2023

ABSTRACT

Aim: This scoping review aims to explore evidence regarding the expression of calprotectin and its constituent homodimers in periodontal health and disease and to evaluate their potential role as biomarkers of periodontal diseases.

Methodology: Prominent databases such as PubMed/Medical Literature Analysis and Retrieval System Online (MEDLINE), PubMed Central, Scopus, and Google Scholar were searched from January 1998 to May 2022 to collect evidence regarding the expression of calprotectin in periodontal health and disease and the potential use of this heterodimer and its constituent homodimers as biomarkers of periodontal diseases.

Results: Cross-sectional studies have reported that calprotectin levels in gingival crevicular fluid, saliva, and serum are elevated in periodontal diseases than in health. It was found that calprotectin levels in oral fluids increased during the early stages of experimental gingivitis and may predict degrees of future periodontal breakdown. Experimental evidence also points out that calprotectin and its constituent homodimers in oral fluids may mirror the degree of periodontal inflammation and predict therapeutic response and disease susceptibility.

Conclusion: Within the limitations of this scoping review, it can be concluded that calprotectin and its constituent homodimers have an altered expression pattern in periodontal diseases, and calprotectin in gingival crevicular fluid or saliva may be a promising biomarker of periodontal inflammation and granulocyte activity. Further, longitudinal studies are required to understand the precise role of constituent proteins of calprotectin—S100A8 and S100A9 during periodontal inflammation.

Clinical significance: The diagnosis and management of periodontal diseases can be enhanced by identifying and validating a biomarker or panel that can detect disease susceptibility, early disease activity, and therapeutic response. This review elaborates on the potential of calprotectin and its subfractions as biomarkers of periodontal inflammation.

How to cite this article: George AK, Malaiappan S, Joseph B, et al. Calprotectin, S100A8, and S100A9: Potential Biomarkers of Periodontal Inflammation: A Scoping Review. World J Dent 2023;14(6):559-567.

Source of support: Nil

Conflict of interest: Dr Anil Sukumaran is associated as the Associate Scientific Editor member of this journal and this manuscript was subjected to this journal’s standard review procedures, with this peer review handled independently.

Keywords: Biomarker, Calprotectin, Inflammation, Periodontal diseases.

INTRODUCTION

Periodontal diseases affect >60% of the global population, with 10% affected by severe forms of this disease.¹^,² Most current measures of periodontal disease severity, such as probing pocket depth or clinical attachment loss, are indicators of past periodontal destruction. There is a well-recognized need for a biomarker that can be used as a screening agent to detect the onset of disease, indicate current disease activity, and predict future destruction/or response to therapy.³

Calprotectin or S100A8/A9 or myeloid related protein (MRP) 8/14 is a heterodimer constituted by protein subunits S100A8 (MRP8) and S100A9 (MRP14). The S100s belong to a group of over twenty small calcium-binding proteins encoded by genes on chromosome 1q21.⁴ This protein complex was initially isolated in 1965 from the bovine brain by Moore et al. These acidic proteins are named S100s as they are soluble in a 100% saturated ammonium sulfate solution.⁵ Calprotectin and its subunits are principally expressed by myeloid cells, hence known as myeloid-related proteins (MRP8) (S100A8) and MRP14 (S100A9). The constituent homodimers of this protein complex are also known as calgranulins A (S100A8) and B (S100A9) due to their calcium-binding nature.⁶

In the protein complex calprotectin/S100A8/A9, S100A8 is constituted of 93 amino acid residues and S100A9 by 113 amino acids with molecular weights of 10.8 and 13.2 kDa, respectively. In low calcium concentrations and stable physiologic conditions, S100A8 and S100A9 form noncovalently bonded heterodimers. Although S100A8 and S100A9 occur mostly as S100A8/A9 or calprotectin, tetramerization of these proteins can occur at higher Ca⁺ concentrations.⁷

Calprotectin has an antimicrobial role in its metal-binding structure. Each of the homodimers, S1008, and S100A9, have two EF hand-type Ca⁺ binding sites. Calprotectin plays a key role in innate immune responses and binds to metal ions such as zinc, iron, and manganese, depriving bacteria of their nutritional requirements.⁸

Calprotectin constitutes over 50% of the cytosolic constituents of neutrophils and monocytes.⁹ In inflammatory micro-environments, calprotectin and its constituent proteins S100A8 and S100A9 are chiefly expressed by neutrophils, monocytes, and immature macrophages.¹⁰^,¹¹

Upon activation of the phagocytes, calprotectin is expressed as danger signals, “alarmins” or damage-associated molecular patterns.¹² The three principal classes of receptors on which calprotectin binds are the toll-like receptor 4 (TLR-4), extracellular matrix metalloproteinase inducer, and receptor for advanced glycation end products.¹³ The binding of calprotectin to these cell surface receptors starts a series of cell signaling events, leading to the recruitment of granulocytes and activation of transcription factor—nuclear factor κβ (NF-κβ) and elaboration of cytokines in sites of inflammation.¹⁴

Intracellular calprotectin is essential for calcium ion-mediated cytoskeletal and plasma membrane interactions and cytoskeletal stabilization of the phagocytes during physiologic conditions.¹⁵ Calprotectin plays important roles in cellular proliferation and differentiation, apoptosis, oncogenesis, immunity, and inflammation.¹⁶ In addition to its well-recognized role in innate immune response, calprotectin also has an essential role in the modulation of adaptive immunity¹⁷ (Fig. 1).

Fig. 1: Calprotectin in inflammation

CALPROTECTIN: AN INFLAMMATORY BIOMARKER

Serum calprotectin concentrations in a healthy state range from 0.1 to 1.6 µg/mL.¹⁸ This protein has also been isolated from saliva, gingival crevicular fluid, plasma, urine, sputum, feces, dental calculus, cerebrospinal fluid, and synovial fluids.¹⁹ Calprotectin was identified in keratinocytes from healthy²⁰ to inflamed oral mucosa.²¹ The presence of calprotectin was identified in dental calculus by immunoblotting and immunohistochemistry.²² The amount of calprotectin in gingival crevicular fluid (GCF) was first measured by Miyasaki et al.²³ and ranged from values that were nondetectable to 715 µg/mL. The amounts of GCF calprotectin were much higher than that reported from parotid saliva.²⁴ It is relatively stable and easy to measure. Calprotectin has been studied extensively in the gastrointestinal tract’s inflammatory conditions and is used as a biomarker to differentiate between inflammatory and irritant bowel disorders. It has also been extensively studied in chronic infections such as tuberculosis and autoimmune conditions like rheumatoid arthritis and psoriasis.²⁵ It has been recently recognized that higher serum calprotectin values predict poor coronavirus disease 2019 outcomes regarding the need for mechanical ventilation and mortality.²⁶ Evidence points out that it may be an early biomarker of systemic inflammation, appearing earlier than other well-recognized markers of systemic inflammation, such as C-reactive protein.²⁷

Emerging evidence indicates that calprotectin and its constituent homodimers may be biomarkers in periodontal diseases.

This scoping review aimed to explore the existing literature on the expression of calprotectin (S100A8/9) and its constituent homodimers—S100A8 and S100A9 in periodontal diseases and collate evidence that explores the potential of calprotectin and its components as biomarkers of periodontal inflammation.

METHODOLOGY

The focused question for the review was, “can calprotectin (S100A8/9) and its constituent homodimers—S100A8 and S100A9 be potential biomarkers of periodontal inflammation?”

Eligibility Criteria

Articles published from January 1998 to 31 May 2022 were searched to select appropriate studies using the following population, concept, and context framework. Population: presence of periodontal diseases; concept: experimental and observational studies; context: association of calprotectin and its constituents with periodontal inflammation. Experimental and observational studies reported in the English language were included in this review. However, commentaries, review articles, case reports, in vitro studies, in vivo studies, editorials, letters to the editor, conference papers, and consensus papers were excluded.

Search Strategy

Prominent literature databases such as Medical Literature Analysis and Retrieval System Online (MEDLINE)/PubMed, Scopus, and Google Scholar were extensively searched starting from January 1998 up to 31 May 2022. The search was conducted based on “calprotectin” and “periodontal inflammation,” which are the key concepts of the research question. Literature that contained the MeSH terms, keywords, and other free terms related to terms “calprotectin,” “periodontal diseases,” “S100A8,” “S100A9,” and “biomarker” were used with Boolean operators., were included in the initial screening. In addition, a manual search into the references of these articles was done, and grey literature was also searched. The selected articles’ full text was read in detail, and evidence was corroborated in this review.

A preliminary search identified 77 eligible studies, duplicates were removed using the Rayyan tool, and 59 original research articles were obtained. A total of 19 articles were excluded during the title and abstract screening based on eligibility criteria. Two reviewers examined the remaining 27 articles in full length. In case of disagreement, a third reviewer was contacted, who resolved the differences through discussion and finally decided to include all 27 studies (Flowchart 1).²⁸^-³⁴^,³⁶^-⁵²^,⁵⁴^-⁵⁶

Flowchart 1: Preferred Reporting Items for Systematic Reviews and Meta-Analyses flow diagram

RESULTS

This scoping review included 27 original research articles, of which 15 were cross-sectional observations, and 12 were longitudinal studies. Among the 15 cross-sectional studies, 10 studies investigated calprotectin levels in GCF/saliva/serum²⁸^,²⁹^,³¹^-³³^,³⁹^,⁴³^,⁴⁵^-⁴⁷ and five studies investigated the levels of the constituent monomers—S100A8 and/or S100A9.⁵⁰^-⁵²^,⁵⁴^,⁵⁵ Calprotectin levels in GCF/saliva/serum before and after periodontal therapy were evaluated in eight³⁰^,³⁶^-³⁸^,⁴⁰^,⁴¹^,⁴⁸^,⁴⁹ of the longitudinal studies. Three studies assessed the influence of periodontal therapy on the sub-fractions of calprotectin.³⁷^,⁴⁴^,⁵⁶

A total of 22 studies employed enzyme-linked immunosorbent assay (ELISA) to quantify proteins.²⁸^-³⁴^,³⁶^,³⁷^,³⁹^-⁴³^,⁴⁵^-⁴⁹^,⁵⁴^-⁵⁶ Reverse phase microbore high-performance liquid chromatography (HPLC),⁵¹ Two-dimensional electrophoreses, (2D-PAGE) matrix-assisted laser desorption ionization-time of flight mass spectrometry⁵⁰ (MALDI-TOF-MS) and shotgun liquid chromatography-tandem mass spectrometry⁵² were the other proteomic approaches employed by investigators.

Review results are depicted in tables. Tables 1 and 2 show the included clinical studies, cross-sectional, and longitudinal studies, respectively.

**Table 1:** Evidence from cross-sectional studies on calprotectin and its constituent subunits as potential biomarkers of periodontal diseases
Author year, and country	Participants included in the analysis	Case definition for the diagnosis of PDL disease and periodontal disease category disease: nondisease subjects disease: nondisease sites	Evaluation of calprotectin, method, and type of biological fluid	Principal findings
Kido et al. 1998, Japan²⁸	100	Periodontitis defined based on PD. 58 P: 42 H participants, 74 D: 43 H sites.	GCF by immunoblotting and ELISA, respectively.	Calprotectin level in disease vs healthy subjects: 2.15 µg/mL: 0.59 µg/mL. Calprotectin concentration at diseased sites was 3.9-fold more than in healthy sites.
Kido et al. 1999, Japan³²	110	Periodontitis defined by PD and BOP 65 P:2 LJP: 43H participants 130 sites, shallow or deep	GCF by ELISA	Calprotectin content and concentration at pockets with PD-10 mm = 5.3 µg/site and 3.36 µg/µmL, PD-2 mm = 0.19 µg/site and 0.57 µg/µmL.GCF calprotectin concentration at BOP+ sites = 2.91 µg/mL, BOP-sites = 2.02 µg/mL.
Nakamura et al. 2000, Japan³¹	54	Periodontitis is defined by PD. 90 D sites.	GCF by ELISA	A significant positive correlation was present between GCF calprotectin concentration and GI, GI-3 sites = 2.03 µg/µL, GI-1 sites = 1.12 µg/µL, calprotectin content and concentration at sites, With disease-2.6 µg/site and 2.16 µg/µL. Healthy sites: 0.24 µg/site and 0.70 µg/µL.
Kojima et al. 2000, Switzerland⁵⁰	19	Periodontitis is defined by CAL and RBL. 10 P:4 H: three edentulous subjects: two newborns.	2D-PAGE and MALDI-TOF-MS of GCF.	2-D protein pattern of GCF of P and H subjects showed four protein spots which were identified to be S100A8 and S100A9. 2D gel image quantification showed an average of percentage volume higher in GCF from P than H.
Lundy et al. 2001, United Kingdom⁵¹	20	Gingivitis and periodontitis were defined based on clinical and radiographic measures. 15 P: 5 H participants, 5 G sites and 5 P sites, and 4 H sites from P patients and five sites in H subjects.	GCF by microbore HPLC	MRP 8 levels were: 457 ng in P,413.5 ng in G, 14.6 ng in H sites of P patients, 18.6 ng in H controls. MRP 8 was 20-fold higher in sites of periodontal inflammation compared to H sites.
Becerik et al. 2011, Turkey²⁹	80	Cases defined according to AAP 1999 classification. 20 G:20 CP:20 GAP:20H participants, 40 G:40 CP:40 GAP:40 H sites	GCF assay by ELISA	The total amount of calprotectin in GCF was highest in the P > G >H groups.
Sun et al. 2011⁴⁶	227	GAP is defined by AAP 1999 classification. 139 GAP: 88 H participants	Serum by ELISA	GAP = 2.17 mg/L, H = 1.72 mg/L Serum calprotectin concentration is significantly higher in GAP than in H controls
Kajiura et al. 2014, Japan³⁹	78	Periodontitis is defined by PD, CAL, and GI, 20 DM and P: 23 P:25 H:10 DM patients.	GCF and serum by ELISA	Calprotectin amount/sample in P = 257.4 ng/site H = 108 ng/site, Is > 2-fold higher in P and DM-P groups than in those without P.
Haririan et al. 2016⁴⁷	100	Periodontitis is defined based on AAP 1999 classification. 21GAP:35CP: 44 H participants	Saliva and serum assay by ELISA	Salivary calprotectin GAP = 62.37 ± 43.26 µg/mL, CP = 47.80 ± 43.29, H = 16.17 ± 8.76 Serum levels, GAP = 2.02 ± 1.22, CP = 1.71 ± 0.73, H = 1.33 ± 0.47 Salivary and serum calprotectin levels in P were significantly higher than in the H group.
Kajiura et al. 2016 Japan³³	34	Healthy and inflamed sites of participants defined based on PD and GI. 34 Inflamed :34 healthy sites	GCF assay by ELISA	GCF calprotectin was higher in inflamed sites than in healthy sites. In the ROC curves for GCF calprotectin determining presence or absence of periodontitis, AUC was 0.83.
Lira-Junior R et al. 2017 Sweden⁴⁵	40	Periodontitis is defined as per AAP 1999 classification. 15GAP:15G:10H participants	Saliva and serum assay by ELISA	Serum levels of calprotectin were 1.43-fold and 20.06-fold in G and GAP, respectively. Salivary calprotectin levels were 1.91 and 1.98-fold higher in G and GAP, respectively.
Shin et al. 2019 Korea⁵²	72	Periodontitis is defined as per the 5^th European Workshop in Periodontology. 36P:36H participants	Saliva assay by shot-gun proteomics and ELISA	S100A8 had the highest relative total value of abundance of 16.89 ± 11.05, and S100A9 of 4.536 ± 1.99, S100A8 in P = 11,163.58 ± 9415.63 pg/mL, H = 430.48 ± 224.74 pg/mL, S100A9 in P = 2045.12 ± 1170.26 pg/mL, H = 811.52 ± 604.0 pg/ mL
Holmström et al. 2019, Sweden⁴³	436	Participants were grouped based on BOP, PPD, and loss of alveolar bone. PD -, PD, and PD + groups BOP ≤ 20%: > 20%: 185:251 PPD < 4 mm: 1–9% sites PPD ≥ 4 mm: ≥ 10% sites PPD ≥ 4 mm: 97:228:111 PD-: PD: PD+: 302:89:45	Saliva by ELISA	Salivary calprotectin levels were 1.37-fold higher in participants with a higher percentage of sites with BOP and 1.35-fold higher in subjects with deeper pockets
Karna et al. 2019, Korea⁵⁴	326	Periodontitis staging according to the definition as per the modification of the new international periodontitis classification. 218 P:108 H participants	Saliva by ELISA	P when compared to H participants, had a higher level of S100A8 (3694 vs. 6757 ng/mL, but a lower level of S100A9 (1341 vs. 1030 ng/mL), Periodontitis screening capacity of both S100A8 and A9: - c-statistics of 0.69, 0.67 for S100A8, 0.63 for S100A9.
Kim et al. 2021, Korea⁵⁵	149	Staging of periodontitis is done as per the definitions provided by the new international classification of periodontitis. 99 P:50 H participants	Saliva, GCF, and blood assayed for S100A8 and S100A9 by ELISA	S100A8 in saliva in P was higher than in H(NPERIO) S100A8 and S100A9 in GCF were higher in NIPERIO than in P Salivary S100A8 had the highest screening ability for periodontitis with c-statistics of 0.73

AP, aggressive periodontitis; BOP, bleeding on probing; CAL, clinical attachment loss; CP, chronic periodontitis; D, disease; DM, diabetes mellitus; ELISA, enzyme linked immunosorbent assay; G, gingivitis; GAP, generalized aggressive periodontitis; GCF, gingival crevicular fluid, GI, gingival index; H, healthy; HPLC, high pressure liquid chromatography; LJP, localized juvenile periodontitis; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry, NPERIO, no periodontitis; P, periodontitis; PD, pocket depth; RBL, radiographic bone loss, 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis

**Table 2:** Evidence from longitudinal studies on calprotectin and its constituent subunits as potential biomarkers of periodontal diseases
Author, year, and country	Study design	Participants in analysis	Case-definitions for periodontal disease category, disease/nondisease patients and/or sites gingivitis/periodontitis.	Study design, type of biological fluid, and methodology	Principal findings
Que et al. 2004, Switzerland³⁴	Clinical trial	15	Gingivitis is defined based on PI, GI, BOP and PD, and gingival health at baseline to experimental gingivitis.	GCF assay for MRP8, MRP14, and MRP8/14 by ELISA.	Calprotectin levels at the stage of induced gingivitis were dependent on baseline levels and displayed two response patterns.
Kaner et al. 2006, Germany³⁰	Clinical trial	23	Cases defined by AAP 1999 classification. GAP patients.	GCF assay for calprotectin by ELISA.	At baseline, GCF calprotectin concentration was 2.9-fold and content 4.9-fold higher in deep than in shallow sites. After therapy, a significant decrease in calprotectin levels was seen in initially deep sites.
Haigh et al. 2010, Newzeland⁴⁴	Clinical trial	9	Defined based on PD and BOP and extent in terms of percentage of sites involved. Severe periodontitis.	Salivary proteome assay by 2D-SDS-PAGE, Protein identification was done by MS and LC.	2.31-fold change of S1008 and 1.4 to 1.9- fold change in S100A9 was seen in pre-treatment versus post-treatment samples
Anderson et al. 2010, Switzerland³⁷	Longitudinal observational	30	Cases and controls were defined based on PD and CAL. 19 P: 11 H participants.	GCF assay for MRP8/14, MRP 14, and total protein by ELISA.	The ability of MRP8/14 and MRP 14 to distinguish periodontitis from healthy subjects is shown as AUC = 0.93 and AUC = 0.7, respectively. Posttreatment, MRP8/14 was reduced from 4000 ng/15 seconds sample to 1500 ng/15 seconds.
Kaner et al. 2011, Germany³⁶	RCT	36	Cases were defined according to AAP 1999 classification. GAP patients. A PD increase of at least 0.5 mm between 3 and 6 months was defined as disease-active sites.	GCF assayed for calprotectin by ELISA.	The calprotectin amount/sample was reduced after SRP. The calprotectin concentration amount/sample was increased in future active sites. 51.8 µg/ µL vs 13.9 µg/ µL and 22.3 µg vs 3.8 µg. ROC curves showed that future increase of PD was shown by GCF calprotectin concentration (AUC = 0.793) and content/sample (AUC = 0.776).
Zhou et al. 2012⁴²	Observational	11	Periodontal disease was defined based on the plaque index, GI, PD, and bleeding index.	Saliva assay for calprotectin by ELISA.	Calprotectin concentrations increased and were maximum on the 21^st day of the trial. The salivary concentration of calprotectin was correlated positively with PI, GI, and BI.
Kido et al. 2018, Japan³⁸	Experimental	36	Cases were defined as per the classification system of the Japanese Society for Periodontology and were based on GI and BOP. 118 P: 120 H sites.	IC chip used.	The AUC for calprotectin in GCF was 0.826. Results demonstrate the utility of calprotectin on IC for distinguishing periodontal disease from health.
Afacan et al. 2020, Turkey⁴¹	RCT	60	Cases defined as per AAP 1999 classification. Severe periodontitis.	GCF calprotectin was estimated by ELISA.	An increase in GCF calprotectin was seen in treatment groups. Calprotectin may indirectly support periodontal healing.
Kim et al. 2020, Korea⁵⁶	Prospective study	149	Periodontal health and disease defined by New international classification of Periodontal diseases and conditions. 99 P: 50 H subjects.	Saliva was assayed by ELISA.	S100A8 levels in saliva were 1.9-fold higher than in the H group. It had a high screening ability for P (c-statistic of 0.73, sensitivity: 0.67, and specificity: 0.68). After NSPT level of salivary S100A8 decreased from 8554.3 pg/mL to 2461 pg/mL and was less than that in H participants.
Gao et al. 2021, China⁴⁰	Longitudinal observational	110	The definition for periodontitis cases was as per the 1999 classification and staged and graded as per the new international classification for periodontitis, 35 DM-P:32 CP:43 H participants.	GCF and serum calprotectin levels were assayed by ELISA.	Levels of GCF and serum calprotectin are highest in DM-P > CP > H and are significantly lower 3 months after NSPT. Based on the reduction of calprotectin after NSPT, a group with higher and another with a lower reduction in average calprotectin amount in GCF and serum were identified.
Lira Junior et al. 2021, Sweden⁴⁸	Longitudinal observational	195	Periodontitis cases were defined as moderate-to-severe periodontitis according to AAP 1999 classification and graded according to the new AAP-EFP classification. 72 P:63 G: 60 H participants.	Salivary levels of S100A8/9 were assessed by ELISA.	Significant positive correlation between S100A8/9 and clinical parameters of periodontal destruction. The AUC to distinguish periodontitis from healthy for S100A8/9 was 0. 738. At baseline, two distinct patient groups were identified based on the level of S100A8/9, a low-value and high-value group. S100A8/9 increased significantly at 3 and 6-months post-NSPT in the former group and significantly decreased at 1-month posttreatment in the latter.
Kamanatham et al. 2022, India⁴⁹	Clinical trial	64	Periodontitis was defined based on PD, CAL, and percentage of sites involved. 30 P: 30 DM-P.	Salivary calprotectin was done by ELISA.	Salivary levels of calprotectin are higher in the DM-P group, and the reduction of salivary calprotectin was more in the LLLT treatment groups.

AAP-American Academy of Periodontology; AUC, area under curve; BOP, bleeding on probing; CAL, clinical attachment loss; CP, chronic periodontitis; D, disease; DM, diabetes mellitus; EFP, European Federation of Periodontology; ELISA, enzyme linked immunosorbent assay; G, gingivitis; GAP, generalized aggressive periodontitis; GCF, gingival crevicular fluid; GI, gingival index; H, healthy; IC, immunochromatographic; LC, liquid chromatography; MRP, myeloid related protein; MS, mass spectrometry; P, periodontitis; PD, pocket depth; PI, plaque index; RBL, radiographic bone loss; RCT, randomized controlled clinical trial; ROC, receiver operating characteristic; 2D-SDS-PAGE, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

DISCUSSION

A detailed review of available evidence indicates that levels of calprotectin and its constituent homodimers have an altered expression pattern in periodontal disease. Cross-sectional studies have reported higher GCF calprotectin levels in gingivitis, chronic and aggressive periodontitis patients.²⁸^,²⁹ GCF calprotectin concentrations and amount per site reported in patients with generalized aggressive periodontitis³⁰ were higher than that reported from study populations of chronic periodontitis.³¹^,³² Calprotectin levels in GCF were higher in sites with higher gingival index,³¹ in sites that bled on probing²⁹ and calprotectin concentrations correlated with probing pocket depth.³² GCF calprotectin content was proportional to the GCF volume, and there was no correlation between GCF calprotectin concentration and GCF volume.³¹ Evidence points out that calprotectin levels in GCF correlate with probable proposed biochemical markers of localized tissue destruction and disease activity, such as interleukin (IL) 1β, PGE2, collagenase, aspartate aminotransferase, and myeloperoxidase levels.³⁰^-³² GCF levels of this protein reflect periodontal inflammation and neutrophilic activity, and it may be used as a prognostic marker.³⁰ In receiver operating characteristic (ROC) curve curves plotted for GCF calprotectin determining the presence or absence of periodontitis, the area under the curve (AUC) was 0.83.³³ An AUC between 0.7 and 0.9 is indicative of moderate predictive discrimination. The authors of this study discussed that their cross-sectional design was a limitation in drawing causal interpretations and that adjustments for co-morbidities were not made in their study.³³

A longitudinal study³⁴ which quantitatively assessed the variations in GCF calprotectin and its subunits in an experimental gingivitis model³⁵ reported that calprotectin levels vary among individuals in early stages of gingivitis and the authors reported that calprotectin levels may predict response patterns to plaque accumulation and or susceptibility to plaque microbiome among individuals. Kaner et al., from their clinical trial of adjunctive local and systemic antimicrobials to nonsurgical periodontal therapy (NSPT) in patients with generalized aggressive periodontitis, concluded that GCF calprotectin levels predicted site and subject-level disease activity (in terms of an increase of >0.5 mm of probing pocket depth at sample sites between 3 and 6 months after NSPT).³⁶ The authors stated that their study results may not be generalized and that the results should be confirmed in other periodontal diseases. A longitudinal study investigating calprotectin levels in chronic periodontitis at both site and subject levels at baseline and at 3 and 6 months after NSPT found that GCF calprotectin differentiates between periodontally healthy and diseased subjects but does not differentiate between healthy or diseased sites.³⁷ A new immunochromatographic chip system was used to assess GCF calprotectin levels in an NSPT trial, the AUC for calprotectin in GCF was 0.826, and the authors concluded that evaluation of calprotectin in GCF by this chip system could be used for diagnosis and monitoring therapeutic responses in periodontitis.³⁸

Studies have also reported that the amount of GCF calprotectin is higher in subjects with chronic periodontitis and those with chronic periodontitis and diabetes mellitus compared to healthy subjects with and without diabetes mellitus.³⁹^,⁴⁰ GCF calprotectin levels were evaluated in severe periodontitis patients who underwent full mouth disinfection, full mouth ultrasonic debridement, and quadrant wise scaling and root planing. Study participants were re-evaluated at 1, 3, and 6 months. A statistically significant increase in GCF calprotectin was seen in all the treatment groups. The authors pointed out that calprotectin may indirectly influence periodontal healing.⁴¹

Salivary calprotectin levels increased during the early stages of experimental gingivitis and may predict degrees of periodontal inflammation.⁴² The authors discussed that a treatment arm would have displayed alterations in the protein levels posttherapy.⁴² Calprotectin levels were higher in patients with >20% sites that bled on probing and pocket depth of >4 mm.⁴³ Salivary proteomic studies in severe periodontitis have observed marked expression of calprotectin.⁴⁴ Serum levels of calprotectin were significantly higher in patients with aggressive periodontitis compared to those with periodontal health.⁴⁵^,⁴⁶ Lira-Junior et al. discussed a small sample size and cross-sectional design as limitations of their study.⁴⁵ Haririan et al. reported that salivary and serum levels of S100A8/9 were higher in patients with periodontitis when compared with those with periodontal health. They also reported a positive correlation between salivary S100A8/9 and the presence of the periodontal pathogen Treponema denticola.⁴⁷ Salivary calprotectin levels in periodontitis patients at baseline and at 3 and 6 months after NSPT point out two distinct patient “clusters” having higher and lower protein levels at baseline. Those with higher calprotectin levels at baseline have been reported to have more periodontally inflamed surface area posttherapy.⁴⁸ Salivary levels of calprotectin were assessed in periodontitis patients with and without type 2 diabetes mellitus. The adjunctive benefit of low-level laser therapy (LLLT) over SRP alone was assessed in subgroups. Salivary calprotectin levels were higher in the group with diabetes mellitus and periodontitis, and subgroups receiving LLLT showed more salivary calprotectin reduction. The study concluded that salivary calprotectin levels might be a prognostic marker in the management of periodontitis⁴⁹ (evidence from human studies substantiating the potential use of calprotectin as a biomarker of periodontal inflammation is summarized in Tables 1 and 2).

S100A8 and S100A9 as Biomarkers

The prominent presence of S1008/MRP8 and S100A9/MRP14 was detected by mono and 2D PAGE. After periodontal therapy, a decrease in these proteins, as evidenced by a decreased density of protein spots, was also reported.⁵⁰ Lundy et al. identified one protein peak when conducting a GCF assay by microbore HPLC. Microanalytic procedures such as liquid chromatography, quadrupole mass spectroscopy, amino acid sequencing, and SDS-PAGE identified this protein as S100A8. The authors reported a 20-fold increase in S100A8 in sites with periodontal disease than in healthy sites in periodontitis patients and healthy controls.⁵¹ Subunits of calprotectin were assayed by ELISA in a longitudinal experimental gingivitis trial.³⁵ Study results indicated that calprotectin subunits are expressed in much lesser amounts than calprotectin.³⁴ In salivary proteomic studies, a relative abundance of S100A8 and S100A9 was observed in patients with severe periodontitis when compared to study participants with incipient periodontitis and subjects with healthy periodontium (PDL).⁵²

A 12-week-long animal experiment (8 weeks for developing periodontitis and 4 weeks of treatment) reported that S1008, S100A9, and S100A8/9 levels were upregulated in periodontitis and had a higher diagnostic ability. Posttreatment values of S100A8/9 returned to values observed in periodontal health, but S100A8, S100A9, and matrix metallopeptidase 9 (MMP-9) in periodontal stability remained higher than values in health, suggesting the screening ability of these proteins.⁵³ A cross-sectional salivary proteomic study conducted in a Korean population reported that S100A8 and A9 had higher screening ability for periodontitis than the individual protein subunits alone. S100A8 levels were increased, and S100A9 levels decreased in periodontitis.⁵⁴ They proposed a screening model for periodontitis using both S100A8 and S100A9. Another recent study that examined salivary, GCF and serum levels of S100A8 and S100A9 among adults with and without periodontitis reported that salivary levels of S100A8 could be used to screen patients for periodontitis.⁵⁵ Both of the above-mentioned authors⁵⁴^,⁵⁵ discussed that storage of saliva samples for >1 month prior to ELISA was a limitation for their study as prolonged storage may affect the stability of salivary proteins. From their prospective NSPT clinical trial, Kim et al. reported that salivary S100A8 and MMP-9 had significant diagnostic power for periodontitis and that S1008 had an excellent prognostic ability. Salivary S100A8 levels in saliva were significantly higher in periodontitis, and this protein had a higher potential for screening periodontitis than MMP-9.⁵⁶

In vitro studies have contributed evidence for the plausible mechanistic role of calprotectin and its constituent homodimers on cells of the PDL. The effects of calprotectin, S100A8, and S100A9 on human gingival fibroblasts (HGF) were studied in vitro. The authors reported that calprotectin, S100A9, and to a lesser extent, S100A8 caused the release of monocyte chemoattractant protein 1, IL-6, tumor necrosis factor-α (TNF-α). S100A9 caused increased expression of IL-1β and TNF-α in HGF than calprotectin, and they proposed a different role for S100A9 in periodontal pathogenesis. The expression of these proinflammatory mediators was mediated by TLR-4 activation and downstream signaling via the NF-κβ and mitogen-activated protein kinase (MAPK) pathways.¹⁴

Gao et al. also reported that calprotectin acts via its subunit S100A9 and causes the expression of proinflammatory cytokines IL1-β, IL-6, and IL-8 from HGF. S100A9 upregulated IL-6 via NF-κβ, c-Jun amino-terminal kinase (JNK) 1/2, and p38 MAPK signaling and induced IL-8 through NF-κβ, JNK1/2, p38, and extracellular-regulated kinase (ERK) 1/2 MAPK signaling cascade.⁵⁷ The same authors also investigated the effects of calprotectin and its subunits on periodontal ligament (PDL) cells and reported that S100A9 acts on PDL cells by binding to TLR -4. S100A9 induced IL-6 expression in PDL cells via NF-κβ and p38 pathways and caused IL-8 release via the NF-κβ, ERK1/2, JNK1/2, and p38 signaling pathways. The proinflammatory effects of S100A9 on HGF and PDL cells were ROS-dependent.⁵⁷^,⁵⁸

Zheng et al. studied the effects of recombinant human calprotectin (rhS100A8/A9) and its homodimers rhS100A8 and rhS100A9 on PDL cells. They reported that rhS100A8/A9 had significant apoptotic activity. rhS100A9 and rhS100A8/A9 induced expression of IL-6, IL-8, TNF-α, and cyclooxygenase 2 via the NF-kB pathway.⁵⁹ The soluble form of IL-6 needed for IL-6 function in HGF has been reported to be upregulated in THP-1 monocytes treated with calprotectin.⁴⁶ This may cause the progression of periodontitis mediated by a gingival fibroblast and macrophage cross-talk.⁶⁰ These study results highlight the diverse roles of S100A8 and S100A9 in periodontal pathogenesis.

This scoping review identifies GCF and salivary levels of calprotectin and their sub-fractions as potential biomarkers of periodontal inflammation. However, there are a few limitations. Case definitions for periodontal diseases and detection methods of the proteins were diverse among these studies. GCF studies suffer from the major limitation of miniscule sample volumes for assay. Salivary samples are easily obtainable in sufficient volumes, but sample processing methods were not standardized among the studies. Methodological considerations such as salivary flow rate and total protein content have not been adequately addressed. Microchip devices for chairside diagnosis and the employment of bioinformatics and artificial intelligence for periodontal disease diagnosis are the future.

CONCLUSION

The diagnosis and management of periodontal diseases can be enhanced by identifying and validating a biomarker or panel that can detect disease susceptibility, early disease activity, and therapeutic response. Calprotectin, S100A8, and S100A9 have altered expression in periodontal diseases. Elevated calprotectin levels in GCF and/or saliva indicate periodontal inflammation. Review results point out that salivary and/or GCF calprotectin levels may mirror periodontal disease susceptibility. S100A8 and S100A9 may be used as screening agents for periodontal diseases.

ORCID

Annie K George https://orcid.org/0000-0002-3795-2633

Sankari Malaiappan https://orcid.org/0000-0002-4146-2477

Betsy Joseph https://orcid.org/0000-0002-2925-1706

Sukumaran Anil https://orcid.org/0000-0002-6440-8780

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