Expression of gingival crevicular fluid markers during early and late healing of intrabony defects after surgical treatment: a systematic review
Surgical treatments such as guided tissue regeneration (GTR) and access flap surgery are widely employed for the treatment of intrabony defects. However, little is known regarding the postoperative expression of gingival crevicular fluid (GCF) markers.
The aim of this systematic review was to compare the expression of GCF markers following treatment of periodontal intrabony defects with guided tissue regeneration or access surgery. The association of the markers’ expression with the clinical outcome was also assessed.
An electronic literature search was conducted in MEDLINE, EMBASE, OpenGrey, LILACS and Cochrane Library up to December 2018 complemented by a manual search. Human, prospective clinical studies were identified. The changes from baseline up to 30 days (early healing) and 3 months (late healing) were assessed.
A total of 164 publications were identified and reviewed for eligibility. Of these, 10 publications fulfilled the inclusion criteria. The included studies evaluated 15 different GCF markers with a follow-up time between 21 and 360 days postoperatively. PDGF, VEGF and TIMP-1 changes were often investigated in the included studies; however, contrasting results were reported. Two studies agreed that both GTR and OFD lead to similar OPG level changes. TGF-β1 is increased early postoperatively, irrespective of the surgical technique employed.
There is limited evidence available on the expression of GCF markers after surgical interventions of intrabony periodontal defects. However, OPG and TGF-β1 tend to increase early post-operatively, irrespective of the surgical technique employed, irrespective of the surgical technique employed.
More well-designed, powered studies with sampling periods reflecting the regenerative process are needed, and future research should focus on employing standardised protocols for collecting, storing and analysing GCF markers.
KeywordsGingival crevicular fluid Markers Guided tissue regeneration Open flap debridement
Periodontitis is a chronic inflammatory disease caused by bacterial biofilm that leads to a progressive destruction of the supporting apparatus of a tooth and eventually to tooth loss. The prevalence of periodontitis, according to the 2009–2010 data from the National Health and Nutrition Examination Survey (NHANES), reaches 46% in US adults .
As periodontal disease progresses, it results in bone loss that can be horizontal or vertical or a combination of both. The loss of supporting bone vertically results in the formation of intrabony defects that progressively worsen and are associated with an increased probability of tooth loss . While non-surgical periodontal therapy is effective in improving the clinical parameters, such as probing pocket depth (PPD) and clinical attachment levels (CAL) , surgical approaches are more effective—in particular for PPD of more than 6 mm [4, 5]. Currently, intrabony defects are identified as sites favourable for periodontal regeneration [6, 7] with the most commonly used techniques being guided tissue regeneration (GTR) and enamel matrix derivatives (EMD) presenting with similar clinical outcomes which are superior to open flap debridement (OFD) and osseous surgery (OS) [8, 9, 10, 11].
However, irrespective of the regenerative modality employed for the treatment of intrabony defects, little is known regarding the processes and sequences involved in the periodontal regeneration and consequently, in the postoperative expression of angiogenesis, regeneration and inflammation markers in the gingival crevicular fluid (GCF) that accompany these processes . The expression of such markers postoperatively may define whether the healing process moves towards a regenerative or a reparative direction . Understanding the cellular and biological events in periodontal wound healing can possibly provide useful information in identifying predictable regenerative treatment for the periodontium.
The aim of this systematic review was to investigate the healing patterns of intrabony defects after surgical interventions (GTR, OS, OFD, EMD) by means of angiogenesis, regeneration and inflammation markers detected in the GCF before and early (≤ 30 days) or late (3 months) after the surgical intervention. Furthermore, the association of the expression of the GCF markers with the clinical outcome was investigated.
Materials and methods
Protocol and Registration
The present systematic review followed the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analyses) guidelines  (Supplemental Material 1) and was registered with PROSPERO under the ID number CRD42018115794.
The PICO question (patient, intervention, comparison and outcome) formulated was: “In patients with periodontal intrabony defects, does the expression of GCF markers for angiogenesis, regeneration and inflammation differ when treated with GTR employing a membrane and/or bone graft and/or biologics (e.g. EMD) (test group(s)) compared with intrabony defects treated with access surgery [OFD or OS or apically positioned flap (APF)] (control group) early (≤ 30 days) and late (3 months) after the surgical intervention?”
Types of studies
Human, prospective clinical studies assessing the expression of angiogenesis, regeneration and inflammation markers in the GCF were considered. Only studies with at least ten patients per group were included. No language restriction was set.
Systemically healthy individuals with chronic periodontitis (CP) with at least one tooth with PPD ≥ 5 mm, CAL and evidence of radiographic bone loss or aggressive periodontitis [14, 15] or periodontitis stages III or IV  and contributing a minimum of 1 intrabony defect.
Intervention and comparison
Intrabony defects treated with GTR employing a membrane and/or bone graft and/or with biologics (e.g. Emdogain) (test group(s)) and intrabony defects treated with access flap surgery (OFD or OS or APF) (control group). No restriction related to the flap technique (minimally invasive or not) was applied to avoid omitting potentially relevant data. Intrabony defects treated with adjunct growth factors e.g. EMD were included in the test group(s).
The primary outcome of this review was the change in the expression of angiogenesis, regeneration and inflammation markers in the GCF during early healing (from baseline up to 30 days) and during late healing (from baseline to at least 3 months postoperatively). Secondary outcomes considered were the association of the expression of GCF markers (early and/or late healing) with the clinical outcome, assessed with the use of surrogate measures such as PPD and/or CAL.
Information sources and electronic search
An electronic search was conducted by two independent reviewers (VK and GC) in MEDLINE, EMBASE, Cochrane Library, LILACS and OpenGrey for publications up to 10 December 2018. Combinations of controlled terms (MeSH and EMTREE) and keywords were utilised:
(“infrabony” or “intrabony” or “infra-bony” or “intra-bony” or “angular defect” or “periodontal defect”) and (“guided tissue regeneration” or “GTR” or “periodontal regeneration” or “periodontal surgery” or “open flap debridement” or “OFD” or “access surgery”) and (“gingival crevicular fluid” or “crevicular fluid” or “GCF” or “inflammatory marker” or “marker” or “growth factor” or “inflammatory mediator” or “biomarker”)
Additionally, a manual search of periodontology-related journals including Journal of Dental Research, Journal of Clinical Periodontology, Journal of Periodontal Research and the Journal of Periodontology was performed from 2015 to 2018. The list of references in the publications included in this review as well as the list of references in relevant reviews were screened for potential additional publications fulfilling the inclusion criteria.
The search results were initially screened for relevancy by means of title, keywords and abstract, independently and in duplicate by two reviewers (VK, GC). Irrelevant records were excluded at this stage. Any conflict was resolved with discussion. At the second round of screening, the full text of the publications remaining after the first round was assessed for inclusion in this review against the eligibility criteria described previously. The level of agreement between the two reviewers was calculated using Kappa statistics.
Data collection process/data items
The characteristics of the included publications were extracted by two reviewers (VK, GC). Among the details extracted were study characteristics (authors, journal of publication, year, country), number of patients, their demographics and risk factors (age, gender, smoking), diagnosis, number of intrabony defects, history of non-surgical treatment of the sites and time elapsed, characteristics of the included defects, surgical procedure employed (GTR, OFD), biomaterials used in the test group(s), postoperative care protocol, exposure rate, follow-up period, expression levels of the GCF markers, clinical outcomes (PPD, CAL), details of the methodology employed for the GCF sampling, storage, processing and detection of the markers, information regarding the main study outcome and power calculation of the study. When data from the included studies were missing, the authors of the publication were contacted through email.
Risk of bias assessment
The risk of bias of the included publications was assessed by the two reviewers independently and in duplicate. For the RCTs included, the quality of the selected publications was assessed according to the Cochrane Collaboration’s tool for assessing risk of bias . The selected publications were assessed for seven domains: sequence generation, allocation concealment, blinding of the participants and personnel, blinding of the outcome assessment, incomplete outcome data, free of selective outcome reporting and other sources of bias. For each of the individual domains, studies were classified as low, unclear or high risk of bias. Observational studies were assessed using the MINORS tool . Studies were assessed in 12 items including clarity of the aim, inclusion of consecutive patients, prospective data collection, appropriateness of end points, unbiased assessment of study end points, appropriateness of follow-up time, inclusion of loss to follow-up rate, prospective calculation of the study size, comparable control group, contemporary control groups, baseline equivalence of groups on several factors and adequate statistical analysis. Each study may receive 0–2 points for each item and the total score ranges from 0 to 24 points. Studies with fewer than 16 points are considered of low quality, while high-quality studies need to have a score of greater than or equal to 16.
Characteristics of the included studies. (RCT: randomized controlled clinical trial, M male, F female, NR not reported, PI plaque index, FMPS full mouth plaque score, FMBS full mouth bleeding score, PD probing depth, PPD probing pocket depth, CAL clinical attachment level, BOP bleeding on probing, OFD open flap debridement, GTR guided tissue regeneration, EMD Emdogain, MIST minimally invasive surgical treatment, OH oral hygiene instruction, SRP scaling and root planing, BID two times a day, TID three times a day, d day, w week, CHX chlorhexidine, EGF epithelial growth factor, KGF keratinocyte growth factor, TGF-β1 transforming growth factor β1, PDGF platelet-derived growth factor, VEGF vascular endothelial growth factor, FGF fibroblast growth factor, MMP-1 matrix metalloproteinase-1, MMP-8 matrix metalloproteinase-8, TIMP-1 metallopeptidase inhibitor-1, Ang-1 angiopoietin-1, OPG osteoprotegerin, OCN osteocalcin, BMP-2 bone morphogenetic protein-2, BMP-7 bone morphogenetic protein-7, PAF platelet activating factor, sICAM-1 soluble intercellular adhesion molecule-1, sLFA-3 lymphocyte function-associated antigen-3)
Investigator, year (country)
Primary unit of analysis
Number of patients completed (m/f), age (mean)
Number of defects
Characteristics of the defects
Type of procedure
Details of technique
GCF sampling, storage, analysis
GCF follow-up (days)
Clinical follow-up (days)
PD change from baseline to final follow-up
Agrali et al 2016 (Turkey)
RCT, parallel arm, not blinded
12 (6/6), 44.17
OH and SRP 8 weeks before surgery
PI < 1, full mouth BOP < 20%, PD ≥ 6 mm, radiographic depth ≥ 3 mm, variation of 1-, 2-, 3-walls included
EMD + autograft vs OFD
Sulcular incisions, full-thickness flap reflection buccal and lingual
Amoxicillin + potassium clavulanate 1000 mg BID/7d, naproxen sodium 550 mg BID/7 days, 0.12% CHX BID/4 weeks
Periopaper for unspecified time, storage–80 °C, ELISA
Baseline, 7, 14, 30, 90, 180
For OFD: from 7.6 to 3.2 mm.
For EMD: from 8.3 to 3.3 mm.
For EMD + autograft: from 7.93 to 3.22 mm.
Ribeiro et al 2011 (Brazil)
RCT, parallel arm, blinded examiner
29 (NR), 47.10
OH and SRP 6 months before surgery
FMPS 16.91% for test and 15.79 for control, FMBS 11.99% for test and 9.33 for control, PD and CAL ≥ 5 mm, BOP(+), radiographic depth ≥ 4 mm and width ≥ 2 mm, number of walls NR
MIST + EMD vs MIST
Minimally invasive technique (MIST) (Cortellini 2007), 4 mg dexamethasone 1 h prior
Paracetamol every 6 h for 2 days, 0.12% CHX BID for 15 days
Periopaper for 30 sec, storage–20 °C, ELISA
Baseline, 15, 90
For MIST: from 7.12 to 3.57 mm.
For MIST + EMD: from 7.09 to 3.53 mm.
Kuru et al 2004 (UK)
Prospective cohort, examiner blinding NR
27 (10/16), 38.1
Initial periodontal therapy and reassessment
PI, FMPS, BOP, FMBS NR, PPD and lifetime cumulative attachment loss ≥ 5 mm, radiographic bone loss after initial periodontal therapy, number of walls NR
GTR vs OFD
For GTR: sulcular incisions, full-thickness flap reflection, PTFE membrane removed at 6w
For OFD: reverse bevel incisions, full-thickness flap reflection
0.2% CHX BID for 2 weeks
Pre-cut chromatography strips (Whatman 3MM) at crevice entrance for 2 min, storage–− 70 °C, ELISA, pooled samples
Baseline, 14, 28, 42, 49, 84, 182
For GTR: from 7.73 to 3.33 mm.
For OFD: from 7.20 to 3.90 mm.
Pellegrini et al 2017 (Italy, USA)
Prospective cohort, multi-centre, examiner blinding NR
25 (9/16), NR
Non surgical periodontal treatment 4–6 months before surgery
FMPS 5.5% for test and 6.2% for control, FMBS 3.4% for test and 3.8% for control, PPD > 5 mm, CAL ≥ 6 mm
OFD: ≤ 3 mm intrabony
GTR: > 3 mm intrabony, number of walls NR
GTR vs OFD
For GTR: simplified or modified papilla preservation, Ti-dPTFE membrane, removed at 5–6weeks
For OFD: modified Widman flap
Ibuprofen 600 mg pre-op and 6 h later and then if needed, 0.12% CHX TID for 3–4 weeks
Periopaper, 1 mm in crevice, until resistance for 30 sec, storage–20 °C, ELISA
FGF-2, MMP-1, TIMP-1, BMP-7, OPG
Baseline, 3 to 5, 7, 14, 21
For GTR: from 8.1 to 4.1 mm.
For OFD: from 5.6 to 2.9 mm.
Gamal et al 2011 (Egypt)
RCT, split mouth, blinded examiner
12 (NR), 38.2
OH + SRP and re-evaluation at 4 weeks
PI 0.3 for test and 0.5 for control groups, matched 2-walled or 3-walled defects, PD > 6 mm, CAL > 4 mm, radiographic depth > 3 mm, premolars/molars
GTR vs OFD
Sulcular incisions, full-thickness flap reflection. For GTR, periosteum pedicle serving as membrane
Amoxicillin 500 mg TID/7d, 0.12% CHX TID/2 weeks
Micropipette, mesio-facial line angle to maximum depth 2 mm until 5 μm until 5te, mesio-fa− 26 °C, ELISA
2, 3, 7, 14, 30
90, 180, 270
For GTR: from 6.1 to 2.6 mm
For OFD: from 5.6 to 4.1 mm.
Gamal et al 2016 (Egypt)
RCT, parallel arm, blinded examiner
29 (NR), 31.5
OH + SRP and re-evaluation at 4 weeks
PI 0.2 for test and control groups, single, 2-walled or 3-walled defect, PD ≥ 6 mm, CAL ≥ 5 mm, radiographic defect ≥ 3 mm, premolars/molars
GTR (occlusive) vs
GTR (perforated) vs OFD
Internal bevel incisions, full thickness flap reflections. Occlusive is the standard collagen membrane. The perforated membrane is subject to 1 mm perforations with a pin
Amoxicillin 500 mg
Micropipette, mesio-facialline angle to maximum depth 2 mm until 5 μl until 5 μl collected, storage -76 °C, ELISA
1, 3, 7, 14, 21, 30
90, 180, 270
For GTR (occlusive): from 6.1 to 3.5 mm.
For GTR (perforated) from 6.8 to 2.3 mm.
For OFD: from 7.1 to 4.5 mm.
7 patients excluded due to postoperative exposures of the membranes
Keles et al 2006 (Turkey)
RCT, split mouth, blinded examiner
15 (6/9), 42.27
OH + SRP and re-evaluation at 4–6 weeks
PI 0.65 for test and 0.63 for control, paired, vertical interproximal osseous defects PD ≥ 6 mm, number of walls NR
GTR vs flap surgery
Sulcular incisions, full-thickness flap reflection, for GTR: absorbable polylactide membrane
Periopaper, in crevice until mild resistance for 30s, storage -70 °C, reverse-phase high performance liquid chromatography with fluorimetric detection
Baseline, 42, 84, 168
45, 90, 180
For GTR: 4.5 mm change from baseline to 6 m.
For flap surgery: 4.7 mm change from baseline to 6 m.
Kuru et al 2005 (UK)
Prospective cohort, examiner blinding NR
Analysis for defect and patient as unit of analysis
26, (11/24), 39.6
Non surgical periodontal treatment prior
PI/FMPS and BOP/FMBS NR, PD > 5 mm, lifetime cumulative CAL > 5 mm, radiographic evidence bone loss after initial periodontal therapy, number of walls NR
GTR vs flap surgery
Details of surgical procedures NR, for GTR: ePTFE membrane removed at 6 weeks
Pre-cut chromatography strips (Whatman 3MM) at entrance of crevice for 2 min, storage –70 °C, ELISA, pooled samples
Baseline, 14, 28, 42, 49, 84
Okuda et al 2001 (Japan)
RCT, split mouth, double-blind
16 (NR), NR
36 (18 + 18), 2 patients contributed with 2 pairs of defects each
OH + SRP, occlusal adjustment if needed and re-evaluation 6 weeks later
PI 0.28 for test and 0.39 for control, BOP 89% for test and 83% for control, bilateral, one- or two-paired defects, PPD ≥ 6 mm, CAL ≥ 6 mm, osseous defect depth ≥ 4 mm with sounding or radiographically, minimum 2 mm keratinised gingiva, number of walls NR
EMD vs OFD
Sulcular incisions, vertical release incision 1 tooth away, full thickness flap reflection
Cefaclor 750 mg/5 days, 0.12% CHX TID/6 weeks
Periopaper, inserted until resistance for 60 sec, storage -80 °C, ELISA and one-step sandwich enzyme immunoassay
Baseline, 14, 28, 84
For EMD: from 6.33 to 3.61 mm.
For OFD: from 6.22 to 4.28 mm.
Rakmanee et al 2018 (UK)
RCT, split mouth, blinded examiner
16 (NR), NR
OH + SRP and reassessment 6 weeks later
FMPS 21.4%, FMBS 24.2%, bilateral defects, PPD ≥ 5 mm, radiographic evidence of bone loss ≥ 3 mm, variation of 1-, 2-, 3-walls included
GTR vs access flap
Minimally invasive technique–simplified papilla preservation flap (Cortellini 1999)
Ibuprofen 600 mg or paracetamol 500 mg, 0.2% CHX BID/6 weeks
Pre-cut chromatography strips (Whatman 3MM) at crevice entrance for 2 min, storage -70 °C, Multiplex Beads Assay
Baseline, 3-5, 7, 14, 28, 42, 84, 180, 360
For GTR: 2.4 mm change from baseline to 12 m.
For access flap: 2.5 mm change from baseline to 12 m.
13/18 exposures: 2 major (> 4 mm), 2 removals, 2 minor required antibiotics (metronidazole 400 mg TID/2 weeks)
The characteristics of the included intrabony defects ranged from PD ≥ 5 mm [12, 20, 24, 25, 26] to PD ≥ 6 mm [11, 19, 21, 22, 23] and accompanying radiographic defect depth ≥ 3 mm [11, 12, 19, 21, 24], ≥ 4 mm [20, 23] or unspecified [22, 25, 26]. The defects included were variations of 1-, 2-, 3-wall defects [19, 24], only 2- or 3-wall defects [11, 21] or non-specified in the majority of the investigations [12, 20, 22, 23, 25, 26].
The types of procedures included in the test group were GTR [11, 12, 21, 22, 24, 25, 26] GTR with EMD [19, 23], and minimally invasive surgical technique (MIST) with EMD . Non-resorbable membranes that were removed 6 weeks postoperatively were utilised in 3 studies [12, 25, 26].
Summary of the conclusions of the included studies. (CAL clinical attachment level, PD probing depth, PPD probing pocket depth, OFD open flap debridement, MIST minimally invasive surgical treatment, GTR guided tissue regeneration, vs versus, EGF epithelial growth factor, KGF keratinocyte growth factor, TGF- βG transforming growth factor β1, PDGF platelet-derived growth factor, VEGF vascular endothelial growth factor, FGF fibroblast growth factor, MMP-1 matrix metalloproteinase-1, MMP-8 matrix metalloproteinase-8, TIMP-1 metallopeptidase inhibitor-1, Ang-1 angiopoietin-1, OPG osteoprotegerin, OCN osteocalcin, BMP-2 bone morphogenetic protein-2, BMP-7 bone morphogenetic protein-7, PAF platelet activating factor, sICAM-1 soluble intercellular adhesion molecule-1, sLFA-3 lymphocyte function-associated antigen-3)
The follow-up of the expression of GCF markers ranged from 21days  to 360 days , while the follow-up of the clinical parameters after treatment ranged from 90  to 360 days . However, there was rarely coincidence of the sampling times for the GCF markers with the clinical assessments postoperatively.
Finally, only 2 studies [21, 24] reported the postoperative occurrence of exposures. Gamal et al excluded the exposed sites from the study , while Rakmanee et al reported that 13 out of the 18 sites presented exposure of the membrane that was treated either with removal of the membrane (2 sites, classified as major exposure with size > 4 mm) or with administration of antibiotics (2 sites, classified as minor) .
Synthesis of results
The results and conclusions of the individual studies included are presented in Table 2. Due to the significant heterogeneity of the included studies, in relation to the methodology employed, a meta-analysis was not performed.
Regarding GTR, 7 studies reported on the expression of GCF markers postoperatively [11, 12, 21, 22, 24, 25, 26]. Both Gamal, 2011  and Gamal 2016  employed the same GCF sampling method using a micropipette inserted at 2mm depth in the sulcus and filled with 5μL of GCF. The samples were subsequently stored at -76C and analysed with ELISA. The concentrations of platelet-derived growth factor-BB (PDGF-BB) peaked during the early stages of healing (< 14 days) and decreased to baseline values by 30 days. Similarly, Rakmanee et al employing a different methodology, using pre-cut chromatography strips at the entrance of the gingival crevice for 2 min and stored at − 70C, found again increased PDGF-AB amounts 7 days postoperatively that decreased to baseline levels after 42 days . Furthermore, Rakmanee et al found similar PDGF-AB levels both after GTR and after OFD that were accompanied by a similar clinical response. However, the sites subjected to GTR were associated with high rates of exposure (13/18) that may have significantly affected the regenerative process and thus the clinical response observed.
Furthermore, Rakmanee et al reported that GCF osteoprotegerin (OPG) amounts significantly increased 2–3 days postoperatively and subsequently declined . No significant differences were noted between sites treated with GTR and sites treated with OFD. Pellegrini et al. using Periopaper inserted in the gingival crevice for 30 s found OPG levels to decrease following GTR and OFD; however, no comparison by treatment was reported for the change of the marker .
The expression levels of vascular endothelial growth factor (VEGF) were investigated by Rakmanee et al.  and Gamal et al. . The former did not detect any significant difference in the change of VEGF GCF levels between sites treated with GTR and sites treated with access surgery using pre-cut chromatography strips . However, the study by Gamal and co-workers, which used micropipettes, found that VEGF concentrations measured statistically significant higher concentrations in defects treated with OFD and GTR using a perforated membrane during the early postoperative period (days 1, 3 and 7) compared to defects treated using the occlusive membrane .
Kuru et al. 2004, using pre-cut chromatography strips at the entrance of the gingival crevice for 2 min, found increased transforming growth factor βi (TGF-β1 levels 2 weeks postoperatively, that however were not statistically significant and declined to below baseline levels by 4 weeks . The change in the TGF-β1 levels was similar both after GTR and after OFD and accompanied a similar clinical response 6 months postoperatively.
Regarding EMD, 3 studies reported on the expression of GCF markers [19, 20, 23]. Ribeiro et al., using Periopaper in the gingival crevice until resistance was felt and for 30 s, reported that TGF-β1 levels in sites treated with MIST and EMD significantly increased by 15 days postoperatively and the levels decreased after 3 months . Furthermore, the changes for TGF-β1 levels were similar for sites treated with MIST and MIST with EMD and accompanied a similar clinical and radiographic response for both treatments. In contrast, Agrali et al. using Periopaper, inserted in the gingival crevice for unspecified amount of time, reported significantly higher TGF-β1 levels for EMD-treated defects compared with OFD-treated defects 7 and 14 days postoperatively . In the same line, the authors concluded that defects treated with EMD presented a superior clinical and radiographic improvement compared with defects treated with OFD. It is however worth noting that the majority of the defects treated with EMD were localised at anterior teeth, compared with the majority of the sites treated with OFD that were localised at molar teeth.
Okuda et al. described an increase for matrix metalloproteinase-8 (MMP-8) and metallopeptidase inhibitor 1 (TIMP-1) GCF levels 2 weeks postoperatively for defects treated with EMD and OFD that thereafter declined, more dramatically for EMD-treated defects . MMP-1 levels significantly decreased from 2 to 4 weeks postoperatively for defects treated with EMD .
Ribeiro et al. using Periopaper in the gingival crevice for 30 s concluded that OPG levels increase 15 days postoperatively and similar changes are noted after both MIST and MIST with EMD . Interestingly, Rakmanee et al. concluded that the OPG amount significantly increased 2–3 days postoperatively and subsequently declined with no significant differences between sites treated with GTR and OFD . Consequently, similar changes were noted for the OPG levels after MIST and MIST with EMD or after GTR and access flap. Furthermore, both studies reported similar clinical and radiographic improvements for both treatment groups [20, 24]; thus, the similar expression patterns in the levels of expression of OPG accompanied a similar clinical response, irrespective of the surgical technique employed.
GTR and EMD
Regarding the combination of GTR and EMD, Agrali et al. reported on the levels of TGF-β1. The combination-treated defects, similarly distributed to anterior and posterior teeth, presented similar changes in the TGF-β1 levels as the EMD-treated defects in the first 2 postoperative weeks . The similar changes of TGF-β1 levels accompanied a similar clinical response for EMD and EMD with GTR, that was superior to OFD. However, as discussed previously the majority of the defects treated with OFD were localised in posterior teeth.
Finally, regarding OFD alone, there is agreement between two investigations that an increase in TGF-β1 levels is initially observed, accompanied by a return to baseline levels by 14 days [19, 26]. For PDGF, conflicting results are presented; Gamal and co-workers reported an initial increase and a decrease by 7 days postoperatively to below baseline levels . In contrary, another investigation from the same group reported a decrease for the PDGF levels that continued until 30 days postoperatively . Rakmanee et al. noted an increase in the PDGF amount after OFD that continued until 3 months postoperatively .
For the remaining markers and for more detail regarding the expression of the investigated GCF markers after the surgical treatments, the reader is referred to the detailed Table 2.
Risk of bias assessment
Risk of bias assessment of observational studies using the MINORS tool. Studies were assessed in 12 items and can receive 0–2 points for each for a total score ranging from 0 to 24 points. Studies with fewer than 16 points are considered of low quality while high quality studies need to have a score of greater than or equal to 16
Kuru L, J Periodontol. 2005
Kuru L, J Clin Periodontol. 2004
Pellegrini, J Periodont Res 2017
A clearly stated aim
Inclusion of consecutive patients
Prospective collection of data
End points appropriate to the aim of the study
Unbiased assessment of the study end point
Follow-up period appropriate to the aim of the study
Loss to follow-up < 5%
Prospective calculation of the study size
An adequate control group
Baseline equivalence of groups
Adequate statistical analyses
This systematic review identified 15 GCF markers expressed after surgical treatment of intrabony defects (GTR, OS, OFD). For 7 of those, most of which are related with the healing of connective tissue, TGF-β1, PDGF, VEGF, FGF, MMP-1, TIMP-1 and OPG, data was available from more than one investigation. While for the majority of factors a definitive conclusion cannot be reached, robust suggestions can be drawn regarding the OPG levels in regenerative surgeries. In two investigations, employing different GCF sampling and storing techniques, the OPG levels after MIST or MIST with EMD and after GTR or access flap similarly increased within the two postoperative weeks and thereafter declined [20, 24]. Furthermore, both studies reported similar clinical and radiographic improvements; thus, the similar expression patterns of OPG likely accompanied a similar clinical response. OPG acts as a soluble decoy receptor, binding to the receptor activator of nuclear factor-kappa B (RANKL) and inhibiting the osteoclastogenic action . Therefore, OPG has been identified as a critical factor in bone formation and the regulation of bone resorption.
The finding of this review however comes in contrast with a human polymerase chain reaction (PCR) study assessing the gene modulation 21 days following treatment of intrabony defects with either GTR with an expanded polytetrafluorethylene (ePTFE) membrane or flap surgery . Among others, OPG mRNA levels were significantly higher in GTR sites, compared with access flap sites. Furthermore, in an investigation of the gene expression profile of cells derived from GTR subjected defects (regenerating-tissue derived cells–RTCs), a differential and highlighted expression of the gene encoding OPG (TNFRSF11B) was found compared with matched periodontal ligament mesenchymal cells (PLCs) . These contrasting results may be due to the high rate of exposures (13/18 GTR sites) in the study by Rakmanee et al. that may have significantly affected the regenerative process and thus the OPG expression . In addition, the Ribeiro et al. investigation, as most of the included studies, was not powered for GCF and could therefore lack statistical power to detect differences in the expression levels between treatments .
Interestingly, Okuda et al. found that the use of EMD in intrabony defects resulted in an early postoperative increase for MMP-8 and TIMP-1, followed by an accelerated return to baseline levels, when compared to OFD . This highlighted reduction may associate with an EMD-induced accelerated pattern of wound healing and resolution of inflammation moving towards regeneration rather than repair.
With respect to PDGF, three isoforms exist (AA, AB, BB). Two studies included in this review [11, 24] demonstrated that GTR and access flap lead to similar changes: an initial increase of PDGF-BB  and PDGF-AB  during the early healing period (up to 7 days), accompanied by a decrease to baseline levels. Two PDGF receptors exist, the PDGF-Rα and the PDGF-Rβ who binds PDGF-AB with low and PDGF-BB with high affinity . In contrast with the included in this review studies, a significant upregulation of PDGF-Rβ in regenerating periodontal tissues has been observed  suggesting that the ligands are involved in the early cascade of events involved in regeneration.
Furthermore, in the only study powered for GCF markers , perforated PTFE membranes were shown to result in significantly higher VEGF during the early healing period (1, 3 and 7 days) when compared to occlusive membranes. In an animal model of GTR using porcine extracellular matrix (ECM), membrane cells recruited early postoperatively into the membrane compartment result in highlighted expression of, among other factors, VEGF at the RNA level. The VEGF expression was significantly highlighted 3 days postoperatively and thereby decreased by 28 days . This VEGF upregulation, along with other regenerative molecules, early postoperatively in the mRNA and the protein level, may suggest that the membrane itself acts as a bioactive compartment guiding the regenerative process and not solely as an active barrier.
TGF-β1 is a connective tissue cell signalling protein that plays a critical role in several stages of wound healing, as it promotes the mitogenic activity of gingival and periodontal ligament cells and the upregulation of extracellular matrix components [33, 34]. With regards to TGF-β1, the existing literature is conflicting. The studies included in this review studies suggest that the clinical and radiographic outcome may be related to the TGF-β1 level changes [20, 26]. Ribeiro et al and Kuru et al showed that GTR or OFD and MIST or EMD treatments exhibited similar TGF- β1 increase early post-operatively, as well as similar clinical and radiographic improvements [20, 26]. However, the significant clinical and radiographic improvement following EMD in Agrali’s study was associated with a significant TGF- β1 increase for EMD at anterior teeth, in contrast with OFD at mainly posterior teeth . When OFD was employed alone for the treatment of intrabony defects, two investigations agreed that an increase in TGF-β1 levels is initially observed, accompanied by a return to baseline levels by 14 days [19, 26]. In the same line, in an immunocytochemistry investigation in biopsies, a highlighted increase was noted for TGF-β1 receptor in regenerating periodontal tissues (6 weeks), while the receptor was almost undetectable in healthy tissues . The highlighted receptor presence in regenerating tissues may suggest that the corresponding TGF-β1 plays a pivotal role in the early healing.
As it became evident, an important limitation was that only one of the ten included studies  was powered to detect significant differences in the GCF markers, whereas the remaining nine studies were either powered for the clinical outcomes or did not report any power calculation. Furthermore, inclusion of intrabony defects with varying number of defect walls does not allow for meaningful conclusions as regeneration is more likely to occur in 3-walled defects and to be accompanied by a different array of GCF markers compared to a 1-wall defect. In addition, reportedly, a large variation across investigations was observed in the methodology employed for the GCF sampling, storage and detection. These variations would introduce confounders if a meta-analysis was attempted. For example, the sampling methods (Periopaper, micropipette), the duration of collection (30 s, 2 min, until a specific volume is collected), the depth of strip insertion (entrance of the pocket or full depth), the storage (temperature) or the preparation of the samples (processing individual or pooled samples) introduce variations that would affect the conclusions drawn and their generalisability. Furthermore, it becomes imperative that more well-designed, powered studies with sampling periods reflecting the regenerative process are needed. Future investigations should employ standardised protocols for GCF sampling, processing and storage.
There is limited evidence available on the expression of markers of angiogenesis, regeneration and inflammation in the GCF in the early and late healing after surgical interventions of intrabony periodontal defects
OPG is increased early postoperatively, irrespective of the surgical technique employed
A trend is noted for TGF-β1 increase early postoperatively, irrespective of the surgical technique employed. A highlighted increase is noted after use of EMD at anterior teeth that may relate with an improved clinical outcome.
More well-designed, powered studies with sampling periods reflecting the regenerative process are needed
Future research should focus on employing standardised protocols for collecting, storing and analysing GCF markers and establishing adequate statistical power to reach conclusions that may shed light in the biological events involved in the early periodontal wound healing and thus facilitate the development of predictable regenerative treatments
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
For this type of study, formal consent is not required.
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