Identification of Gingival Crevicular Fluid Sampling, Analytical Methods, and Oral Biomarkers for the Diagnosis and Monitoring of Periodontal Diseases: A Systematic Review

Table 3

Summary of absorption techniques used in GCF sampling.

Reference	Analysis	Markers	Aim	Main findings
[36]	Enzyme assay (fluorimetric MMP kit)	MMP-1, -2, -3, -8, -9, -12, -13	To measure the levels of MMP in children with and without aggressive periodontitis (AgP).	The levels of MMP were raised in AgP sites compared to nondiseased sites in the same subjects.
[37]	ELISA	Myeloid related protein (MRP) 8/14, MRP14, total protein	To determine if the total protein, MRP14, and MRP8/14 in GCF can differentiate healthy from periodontitis sites in CP patients and if they could differentiate healthy subjects from CP patients.	These markers could not differentiate healthy from periodontitis sites in CP patients, but their levels in CP patients were higher than in healthy subjects.
[38]	Bradford method	Protein carbonyl (PC)	To assess GCF and serum levels of PC in patients with CP.	There was an increase in PC levels among CP patients, more than in healthy subjects.
[39]	ELISA, automatic colorimetric method	Total oxidant status (TOS), RANK ligand (RANKL), osteoprotegerin (OPG)	To explore the levels of total oxidant status (TOS), OPG, and RANKL levels in GCF and serum in different periodontal disease stages.	TOS, OPG, and RANKL levels increased with the severity of periodontal disease.
[40]	ELISA	Calprotectin, osteocalcin, cross-linked N-terminal telopeptide (NTx)	To evaluate the levels of osteocalcin, NTx, and calprotectin in GCF among healthy, G, CP, and generalized aggressive periodontitis (GAgP) patients.	Calprotectin level in GCF was considered as a marker for periodontal disease, while osteocalcin and NTx levels could indicate abnormal bone turnover.
[41]	ELISA	IL-1β, IL-6, IL-11, OSM, leukemia inhibitory factor (LIF)	To determine the concentrations of IL-1β, IL-11, IL-6, OSM, and LIF in GCF and plasma among periodontally diseased patients.	IL-1β, IL-11, and IL-6 GCF levels increased, but not plasma levels. They were considered dependable inflammatory biomarkers in periodontal diseases.
[42]	ELISA	Soluble triggering receptor expressed on myeloid cells 1 (sTREM-1)	To evaluate sTREM-1 levels in GCF of subjects with and without GAgP or CP and their association with subgingival plaque bacteria.	Elevated sTREM-1 levels at diseased sites and their positive association with clinical and microbiologic parameters strengthen the correlation of this marker with periodontitis.
[43]	ELISA, quantitative time-resolved immunofluorometric assay (IFMA)	MMP-8, MMP-13, tissue inhibitor of matrix metalloproteinase- (TIMP-) 1	To compare GCF levels of MMP-13 and -8 and TIMP-1 in periodontitis patients with and without RA.	RA did not affect the clinical periodontal parameters.
[44]	ELISA	RANKL, OPG	To determine the level of OPG and RANKL in GCF after nonsurgical periodontal treatment.	It could be a good indicator of treatment success.
[45]	ELISA	IL-33	To determine if IL-33 levels in GCF, saliva, and plasma could be used to differentiate between healthy and CP patients.	IL-33 levels could not be used as a biomarker for periodontal disease.
[46]	Electrochemiluminescence; technique	Osteocalcin	To measure saliva, plasma, and GCF osteocalcin levels and correlate them with osteoporosis and periodontitis.	GCF osteocalcin levels were associated with periodontal disease only.
[47]	ELISA, multiplexed bead immunoassay (MPBI), SPM	IL-1β, -18, elastase, MMP-8, -9	To evaluate the effect of scaling and root planning on periodontal status and on the levels of IL-1β, elastase, MMP-9, and MMP-8 in patients with and without T2DM.	Scaling and root planning reduced the levels of IL-1β, elastase, MMP-9, and MMP-8 in both groups.
[48]	ELISA	TNFα, RANKL	To measure the TNFα and RANKL concentrations in GCF of patients with AgP, after photodynamic or nonsurgical periodontal treatment.	Both types of treatment had the same influence on TNF-α and RANKL concentrations.
[49]	MPBI	IL-6, -4, -10, -13, -17, TNFα, macrophage inflammatory protein- (MIP-) 1α, MIP-1β, MCP1, regulated on activation normal T-cell expressed and secreted (RANTES)	To determine the effect of adjunctive sub-antimicrobial-dose doxycycline (SDD) on the local inflammatory response through chemokine and cytokine levels in GCF samples from CP patients.	SDD aided nonsurgical periodontal therapy.
[50]	ELISA	MMP-8, TIMP-1	To determine the effect of azithromycin in addition to scaling and root planning in the treatment of periodontal disease.	Azithromycin did not present any advantage over scaling and root planning.
[51]	ELISA	Mucosa-associated epithelial chemokine (CCL28), IL-8, IL-1β, TNF-α	To determine the concentrations of CCL28, IL-1β, IL-8, and TNF-α in GCF among study groups.	CCL28, IL-1β, IL-8, and TNF-α concentrations were elevated in accordance with the severity of periodontal disease.
[52]	Flow cytometry	TNF-α, IL-1β, -6, -8, -10, -12p70	To estimate the outcome of periodontal treatment on GCF and serum concentrations of many cytokines related with periodontal disease and premature birth.	GCF cytokine level reduced significantly after periodontal treatment.
[53]	MPBI, ELISA	Pentraxin 3, IL-10, -1β, -6, -8, TNFα	To estimate the correlation between clinical periodontal measurements and the concentrations of six cytokines.	There was a strong correlation between periodontal status and PTX3 or IL-1β levels in GCF.
[54]	ELISA	MDA, SOD, melatonin	To determine GCF concentrations of superoxide dismutase (SOD), malondialdehyde (MDA), and melatonin in GAgP and CP patients as oxidative stress biomarkers.	SOD, melatonin, and MDA could be used to differentiate between GAgP and CP patients.
[55]	Fluorometric kits	MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12, MMP-13	To measure GCF MMPs levels after localized aggressive periodontitis (LAgP) treatment.	LAgP treatment with SRP and systemic antibiotics was active in reducing local levels of specific MMPs.
[56]	ELISA	MMP-8, MMP-9, MMP-13	To evaluate whether the presence of periodontitis and metabolic syndrome was related to MMP in GCF in the Korean community.	MMP (-13, -8, -9) individually correlated to the presence of periodontitis and metabolic syndrome.
[57]	Western immunoblot	MMP-13	To determine the role of GCF MMP-13 in adult CP patients.	There was significant increase in MMP-13 action in advanced periodontal disease.
[58]	ELISA	IL-23	To determine GCF IL-23 levels in healthy subjects and patients with periodontal disease.	IL-23 levels increased correspondingly to periodontal disease progression.
[59]	ELISA	TNFα, soluble TNF receptors 1 and 2	To evaluate TNF-α level, soluble TNF receptors 1 and 2 in serum and GCF of healthy and CP patients.	The levels between the two TNF receptors were disproportionate.
[60]	ELISA, immunoturbidimetric analysis	Stem cell factor (SCF), high-sensitivity C-reactive protein (hs-CRP)	To determine the relation between GCF and serum concentration of hs-CRP and SCF of two CP groups of which one is with T2DM and the other is without.	SCF and hs-CRP concentrations increased in patients with T2DM.
[61]	ELISA	Calprotectin	To evaluate the levels of calprotectin in GCF in GAgP patients prior to and after periodontal treatment.	Levels of calprotectin were indicators of disease activity in both subject and site levels.
[62]	ELISA	Myeloperoxidase (MPO), calprotectin	To observe calprotectin levels in GCF during therapy for GAgP.; To determine a correlation between the MPO and calprotectin which were also determined.	Levels of calprotectin in GCF correlated to periodontal disease severity and decreased in concentration after treatment.
[63]	HPLCG	Platelet activating factor (PAF)	To determine the correlation between PAF and periodontal healing.	Alterations in PAF levels in GCF might be valuable for observing the regeneration and repair of periodontal tissues.
[64]	ELISA	chondroitin sulfate (CS), ALP	To determine the role of CS, ALP levels in estimating different periodontal disease stages.	The level of CS was better than the ALP level for determining periodontal disease stages.
[65]	ELISA	CS WF6 epitope	To evaluate GCF levels of CS WF6 epitope in healthy and periodontally diseased patients.	CS WF6 epitope levels positively correlated to the advancement of periodontal disease.
[66]	ELISA	MMP-8, -9, OPG, CRP, IL-1β	To evaluate the performance of MMP-8, -9, OPG, CRP, and IL-1β levels in GCF as a biomarker for periodontal disease.	MMP-8, -9, OPG, CRP, and IL-1β levels could support clinical parameters in the diagnosis of periodontal disease.
[67]	ELISA	IL-1β, IL-8, MMP-8	To evaluate the influence of SRP on levels of cytokines in GCF from CP patients, in relation to clinical parameters.	SRP reduced the IL-8, IL-1β, and MMP-8 GCF levels.
[68]	ELISA	MMP-8	To determine the association between MMP-8 in GCF and the severity of periodontal disease.	The level of active MMP-8 was higher in sites with deeper pocket depth.
[69]	ELISA, immunoturbidimetry	MCP-4, hs-CRP	To investigate GCF and serum levels of hs-CRP and MCP-4 among healthy and periodontally diseased patients.	hs-CRP and MCP-4 levels increased from periodontal healthy to periodontitis. hs-CRP and MCP-4 could be biomarkers of inflammation in periodontal health and disease.
[70]	ELISA	MCP-1, TNF-α	To determine and correlate GCF levels of MCP-1 and TNF-α in CP and AgP patients.	GCF levels of MCP-1 and TNF-α showed positive correlation.
[71]	ELISA, fluorometric method	PGE2, thiobarbituric acid reactive substance (TBARS)	To evaluate the effects of SRP and flurbiprofen in smokers and nonsmokers in CP patients on two GCF biomarkers.	PGE2 and TBARS levels in smokers decreased more than in nonsmokers after the flurbiprofen intake.
[72]	IFMA	MMP-8	To measure the levels of MMP-8 in GCF among two CP groups (smokers and nonsmokers).	The levels of MMP-8 could be used in the monitoring of periodontal diseases.
[73]	ELISA, IFMA	Azurocidin, chemokine ligand 5, MPO, TIMP-1 MMP-13, -14	To determine the diagnostic accuracy of GCF biomarkers.; To compare two analytical techniques used to measure MMP-8 levels.	Collagenolytic MMPs and myeloperoxidase (MPO) could be considered as good biomarkers for periodontal diseases. IFMA analytical method was more precise than ELISA.
[74]	ELISA, radioimmunoassay	25-Hydroxy vitamin D3, osteocalcin, IL-1β, IL-6	To investigate the effect of SRP on the levels of 25-hydroxy vitamin D3 and three other biomarkers in GAgP patients.	Periodontal treatment led to reduction in the levels of 25-hydroxy vitamin D3 and IL-1β.
[75]	ELISA	PGE2, IL-1, TNF-α	To estimate the effect of combining two antibacterial drugs in initial periodontal treatment on periodontal parameters and certain biomarkers in patients with aggressive periodontitis.	Both types of treatment had substantial effect on periodontal disease status.
[76]	ELISA, immunoblotting	hCAP18/LL37, CS	To quantify GCF levels of hCAP18/LL-37 and CS in healthy, CP, and AgP study groups.	A positive correlation between the CS and hCAP18/LL-37 levels was noted in CP patients only.
[77]	MMP-8 specific chair-side dip-stick test	MMP-8	To determine the accuracy of MMP-8 specific analytical techniques.	This testing method could be useful to support clinical periodontal diagnosis.
[78]	ELISA, SPM	MMP-8, -9, TIMP-1, -2, MPO	To determine GCF levels of five biomarkers in healthy and CP patients before and after treatment.	The biomarker levels were greater in CP groups. Their levels reduced after treatment.
[79]	ELISA	LL-37	To evaluate GCF LL-37 levels in control and periodontally diseased groups and the degree of LL-37 by GCF elements.	LL-37 was detected in both study groups. There was high degradation of LL-37 level, mainly in Porphyromonas gingivalis positive sites.
[80]	MPBI	Granulocyte macrophage colony stimulating factor (GM-CSF), interferon-γ (INF-γ), IL-10, IL-1β, IL-2, IL-6, TNF-α	To determine the outcome of periodontal treatment by monitoring the alterations in cytokine levels from GCF samples in GAgP patients.	The periodontal treatment led to an increase in IL-10 levels and reduced IL-1β and GM-CSF levels.
[81]	ELISA	8-Hydroxydeoxyguanosine	To evaluate the effect of nonsurgical periodontal treatment on 8-hydroxydeoxyguanosine levels in GCF and saliva.	8-Hydroxydeoxyguanosine in GCF could reveal the severity of periodontal disease.
[82]	MPBI	INF-γ, IL-4, IL-33, thymic stromal lymphopoietin (TSLP)	To measure GCF levels of TSLP, IFN-γ, IL-4, and IL-33 in healthy and periodontally diseased patients.	Levels of IFN-γ related to the site stage and not on the disease stage IL-4. TSLP levels were detected in a few patients, while IL-33 was not detected.
[83]	SPM	ALP	To explain the effect of nonsurgical periodontal treatment on ALP action in GCF among CP patients.	ALP showed high activity following periodontal treatment, but after 60 days the ALP action reduced.
[84]	ELISA	IL-1β, TNF-α, MMP-8, MMP-9	To determine the effect of nonsurgical periodontal treatment together with photodynamic therapy (PDT) on periodontal conditions in CP patients.	The use of PDT did not show any benefit in nonsurgical periodontal treatment.
[85]	ELISA	Visfatin	To identify the existence of visfatin in serum and GCF.	The level of visfatin increased in relation to the severity of periodontal disease.
[86]	ELISA	8-Isoprostane	To measure 8-isoprostane concentrations in GCF in different periodontal diseases.	8-Isoprostane concentrations elevated in accordance with periodontal disease progression.
[87]	ELISA, RANDOX analyzer	Progranulin, hs-CRP	To measure GCF and serum levels of progranulin and hs-CRP in control subjects, CP and CP with T2DM patients.	CP with T2DM patients showed more hs-CRP and PGRN levels than the other groups.
[88]	ELISA	MMP-9, MMP-8	To measure GCF MMP-9 and MMP-8 levels in healthy subjects and patients with periodontal disease.	GCF MMP-9 and MMP-8 showed elevated levels in periodontally diseased patients.
[89]	ELISA	MMP-2, MMP-8	To measure GCF levels of MMP-9 and MMP-2, and the MMP-8 levels in saliva among control subjects and patients with periodontal diseases.	All the types of MMP were found to be associated with clinical parameters.
[90]	ELISA, Western blot radioimmunoassay	IL-1β, MMP-8, bone resorption marker carboxyterminal telopeptide cross-link fragment of type I collagen (ICTP), total collagenase activity	To discover the association between specific biomarkers in GCF with bone resorption clinical parameters.	The biomarkers were associated with clinical attachment loss.
[91]	MPBI	IL-1α, -1β, -6, -10, TNF-α	To measure the total GCF levels of six cytokines in patients with periodontal disease before and after nonsurgical periodontal therapy.	Nonsurgical periodontal treatment resulted in reduced IL-1β, -1α and IL-6 levels. Nonetheless, TNF-α or IL-10 levels were not decreased.
[92]	MPBI	IL-1β, IL-4, IL8, elastase activity	To observe differences in clinical, immunologic, and microbiologic responses to SRP in patients with different periodontal diseases.	SRP resulted in nonsignificant differences between severe forms of CP and GAgP.
[93]	ELISA	IL-1β, IL-8, MMP-8, MMP-9	To measure the concentration of specific biomarkers in GCF and the bacterial compositions in dental plaque in patients with and without type 1 diabetes (T1DM).	IL-1β and MMP-8 concentrations were found to be more elevated in patients with T1DM.
[94]	ELISA	IFN-γ, IL-23, IL-4, IL-17, TNF-α, OPG, RANKL	To determine the outcome of complete mouth SRP and noncomplete mouth SRP on cytokines levels and on clinical parameters over a twelve-month period.	Both types of treatment showed improvement in clinical parameters and the same changes in cytokines at twelve months.
[95]	ELISA	RANKL, OPG	To determine OPG and RANKL levels in GCF in patients with CP and AgP, as well as healthy subjects.	RANKL was present in periodontitis sites, especially in moderate periodontitis patients, whereas OPG was not noticeable in some sites with bleeding on probing.
[96]	IFMA, MMP-8 specific chair-side dip-stick test, DentoAnalyzer Device, ELISA	MMP-8	To compare 4 techniques used for MMP-8 analysis.	DentoAnalyzer Device, IFMA and chair-side dip-stick test had the same detection ability, while dip-stick test appeared to be better.
[97]	ELISA	OPG, sRANKL	To determine GCF levels of the soluble RANKL and OPG in smokers with periodontal disease.	Smoking suppressed OPG production and led to increased sRANKL∖OPG.
[98]	Checkerboard immunoblotting	IL-1β, IL-8, MMP-8	To investigate GCF levels of three cytokines and the microbial composition of the subgingival biofilm in control group and patients with periodontitis.	There were more cytokines and bacteria in the nondiseased sites in patients with periodontal diseases than there were in healthy individuals.
[99]	MPBI	GM-CSF, IL-2, -10, -13, -6, -1β, TNF-α, IFN-γ	To observe the relation between subgingival bacterial species and GCF cytokine concentrations in periodontal health and GAgP.	GAgP patients showed elevated ratio of IL-1β/IL-10 compared to the control group.
[100]	ELISA, Erels’ colorimetric method	IL-1β, TOS, total antioxidant status (TAS)	To investigate the smoking outcome on the relationship between oxidation and IL-1 in periodontitis patients and response to nonsurgical periodontal therapy.	SRP impacted IL-1β concentrations in GCF, while no effect was detected on the TAS and TOS.
[101]	ELISA	hs-CRP	To measure the concentrations of hs-CRP in GCF and serum in periodontally diseased patients in the presence and absence of coronary artery disease (CAD).	Both periodontally diseased groups showed higher Hs-CRPHs-CRP concentrations than did the control group.
[102]	MPBI	IL-2, 12(p70), -3, -4, -5, -10, -13, -1α, -1β, -6, -12(p40), -8, -7, -15, IP-10, MCP-1, MIP-1α, RANTES, eotaxin, IFN-γ, GM-CSF, TNF-α	To investigate the existence of GCF biomarkers among smokers and nonsmokers with and without periodontal disease.	Periodontitis patients showed increased biomarker profiles. Smoking led to a reduction in many chemokines and cytokines.
[103]	ELISA	Cystatin C, IL-1β, TNF-α	To determine cystatin C levels, IL-1β, and TNF-α in the GCF and saliva of periodontally healthy children (PHC) and children with gingivitis.	GCF and saliva cystatin C levels were higher in PHC, but there was no correlation between cystatin C levels and TNF-α or IL-1β levels in GCF or saliva.
[104]	ELISA	TNF-α, IL-4, INF-γ, IL-23, IL-17, sRANKL, OPG	To determine GCF levels of six biomarkers in CP patients with and without T2DM.	CP patients with T2DM showed more biomarker levels than did nondiabetic patients.