Expression of gingival crevicular fluid markers during early and late healing of intrabony defects after surgical treatment: a systematic review

ORIGINAL ARTICLE Expression of gingival crevicular fluid markers during early and late healing of intrabony defects after surgical treatment: a systematic review V. P. Koidou 1 &G. S. Chatzopoulos 1 &I. Tomas 2 &L. Nibali 1 &N. Donos 1 Received: 16 April 2019 /Accepted: 22 September 2019 /Published online: 7 November 2019 Abstract Background 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. Objective 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. Methods 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. Results 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. Conclusion 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. Clinical relevance 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. Keywords Gingival crevicular fluid .Markers .Guided tissue regeneration .Open flap debridement Introduction 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 [1]. 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 [2]. While nonElectronic supplementary material The online version of this article (https://doi.org/10.1007/s00784-019-03088-4) contains supplementary material, which is available to authorized users. *V. P. Koidou [email protected] 1 Centre for Oral Immunobiology and Regenerative Medicine and Centre for Oral Clinical Research, Institute of Dentistry, Queen Mary University London (QMUL), London, United Kingdom 2 Oral Sciences Research Group, Health Research Institute Foundation of Santiago (FIDIS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain Clinical Oral Investigations (2020) 24:487–502 https://doi.org/10.1007/s00784-019-03088-4 #The Author(s) 2019 surgical periodontal therapy is effective in improving the clinical parameters, such as probing pocket depth (PPD) and clinical attachment levels (CAL) [3], 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) [8–11]. 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 [12]. The expression of such markers postoperatively may define whether the healing process moves towards a regenerative or a reparative direction [12].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 MetaAnalyses) guidelines [13](Supplemental Material 1) and was registered with PROSPERO under the ID number CRD42018115794. PICO question 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?” Eligibility criteria 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. Population 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 [16] and contributing a minimum of 1 intrabony defect. Intervention and comparison Intrabony defects treatedwithGTRemployingamembrane 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). Outcome measures 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 “intrabony”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” 488 Clin Oral Invest (2020) 24:487–502 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. Study selection 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, followup 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 [17]. 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 [18].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 0–2points 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. Results Study selection The flowchart of the study selection and inclusion process is shown in Fig. 1. The initial search identified 68 MEDLINE, 110 EMBASE, 59 Cochrane database and 1 LILACS titles, with a total of 163 after duplicates’removal. One additional title was identified through hand search for a total of 164 titles. Following the screening of titles and abstracts by the two reviewers, 10 articles qualified for full text screening and all 10 met the inclusion criteria. The kappa value for interreviewer agreement was 0.99 at first round and 1.00 at second round. Study characteristics The characteristics of the included studies are presented in Table 1. All 10 included articles were in English. The study samples ranged from 12 [19]to29[20] patients. Seven of the included studies were randomised controlled clinical trials [11,19–24] and the remaining 3 were prospective cohort studies [12,25,26]. The characteristics of the included intrabony defects ranged from PD ≥5mm[12,20,24–26]toPD≥6mm[11,19, 21–23] and accompanying radiographic defect depth ≥ 3mm[11,12,19,21,24], ≥4mm[20,23]orunspecified [22,25,26]. The defects included were variations of 1-, 2-, 3wall defects [19,24], only 2or 3-wall defects [11,21]ornonspecified in the majority of the investigations [12,20,22,23, 25,26]. The types of procedures included in the test group were GTR [11,12,21,22,24–26]GTRwithEMD[19,23],and minimally invasive surgical technique (MIST) with EMD [20]. Non-resorbable membranes that were removed 6 weeks postoperatively were utilised in 3 studies [12,25,26]. Clin Oral Invest (2020) 24:487–502 489 Five of the included studies utilised Periopaper for the sampling of the GCF [12,19,20,22,23],3studiesutilisedpre-cut chromatography strips (Whatman 3MM) [24–26] and 2 studies utilised micropipettes [11,21].TheGCFsamplingtimepresented significant variation across studies, ranging from 30 s [12,20, 22]to1min[23]and2min[24–26], while some studies allowed the insertion of the strip or the pipette until 5 μhe pipette until [11, 21] with one study not reporting on the sampling time [19]. Regarding the storage of the samples, great variation was also observed: 2 studies stored their GCF samples at −80 °C [19,23], 1studyat−76 °C [21], 4 studies at −70 °C [22,24–26], 1 study at −26 °C [11]and2studiesat−20 °C [12,20]. Eight of the included studies analysed the GCF samples using enzyme-linked immunosorbent assay (ELISA) [11,12,19–21,23,25,26], 1 study used reverse-phase high-performance liquid chromatography with fluorimetric detection [22] and 1 study used multiplex beads assay [24]. Several GCF markers were investigated in the included studies. For facilitating the reader, the GCF markers were categorised under factors related with the healing of the epithelium, the connective tissue, the bone and others, even if some overlap might exist (Table 2). The follow-up of the expression of GCF markers ranged from 21days [12] to 360 days [24], while the follow-up of the clinical parameters after treatment ranged from 90 [23] to 360 days [24]. 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 [21], 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) [24]. 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. GTR Regarding GTR, 7 studies reported on the expression of GCF markers postoperatively [11,12,21,22,24–26]. Both Gamal, 2011 [11] and Gamal 2016 [21] 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 analysedwithELISA.Theconcentrations 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 [24]. Records idenfied through database searching (n = 238 ) Screening Included Eligibility Idenficaon Addional records idenfied through other sources (n = 1 ) Records aer duplicates removed (n = 164 ) Records screened (n = 164 ) Records excluded (n = 154 ) Full-text arcles assessed for eligibility (n = 10 ) Full-text arcles excluded, with reasons (n = 0 ) Studies included in qualitave synthesis (n = 10 ) Fig. 1 PRISMA flow diagram 490 Clin Oral Invest (2020) 24:487–502 Table 1 Characteristics of the included studies. (RCT: randomized controlled clinical trial, M male, Ffemale, 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, dday, wweek, 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) Study design Primary unit of analysis Number of patients completed (m/f), age (mean) Number of defects Non-surgical protocol Characteristics of the defects Type of procedure Details of technique Agrali et al 2016 (Turkey) RCT, parallel arm, not blinded Defect 12 (6/6), 44.17 30 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 vs EMD + autograft vs OFD Sulcular incisions, full-thickness flap reflection buccal and lingual Ribeiro et al 2011 (Brazil) RCT, parallel arm, blinded examiner Patient 29 (NR), 47.10 29 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 ≥5mm,BOP(+), radiographic depth ≥4 mm and width ≥2mm, number of walls NR MIST + EMD vs MIST Minimally invasive technique (MIST) (Cortellini 2007), 4 mg dexamethasone 1 h prior Kuru et al 2004 (UK) Prospective cohort, examiner blinding NR Defect 27 (10/16), 38.1 27 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 Pellegrini et al 2017 (Italy, USA) Prospective cohort, multi-centre, examiner blinding NR Patient 25 (9/16), NR 25 Non surgical periodontal treatment 4–6months 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 ≥6mm 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 Gamal et al 2011 (Egypt) RCT, split mouth, blinded examiner Defect 12 (NR), 38.2 24 OH + SRP and reevaluation 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 >4mm,radiographicdepth>3 mm, premolars/molars GTR vs OFD Sulcular incisions, full-thickness flap reflection. For GTR, periosteum pedicle serving as membrane Gamal et al 2016 (Egypt) RCT, parallel arm, blinded examiner Defect 29 (NR), 31.5 29 OH + SRP and re-e valuation at 4 weeks PI 0.2 for test and control groups, single, 2-walled or 3-walled defect, PD ≥6 mm, CAL ≥5mm, radiographic defect ≥3mm, 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 Clin Oral Invest (2020) 24:487–502 491 Table 1 (continued) subject to 1 mm perforations with a pin Keles et al 2006 (Turkey) RCT, split mouth, blinded examiner Defect 15 (6/9), 42.27 30 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 Kuru et al 2005 (UK) Prospective cohort, examiner blinding NR Analysis for defect and patient as unit of analysis 26, (11/24), 39.6 NR 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 Okuda et al 2001 (Japan) RCT, split mouth, double-blind Patient 16 (NR), NR 36 (18 + 18), 2 patients contributed with2pairs 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, oneor two-paired defects, PPD ≥6mm,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 Rakmanee et al 2018 (UK) RCT, split mouth, blinded examiner Patient 16 (NR), NR 32 OH + SRP and reassessment 6 weeks later FMPS 21.4%, FMBS 24.2%, bilateral defects, PPD ≥5mm, radiographic evidence of bone loss ≥3mm, variation of 1-, 2-, 3-walls included GTR vs access flap Minimally invasive technique–simplified papilla preservation flap (Cortellini 1999) Investigator, year (country) Postoperative medication GCF sampling, storage, analysis GCF markers GCF follow-up (days) Clinical followup (days) PD change from baseline to final follow-up Membrane exposures Agrali et al 2016 (Turkey) 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 TGF-β1 Baseline, 7, 14, 30, 90, 180 180 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. None reported Ribeiro et al 2011 (Brazil) Paracetamol every 6 h for 2 days, 0.12% CHX BID for 15 days Periopaper for 30 sec, storage–20 °C, ELISA TGF-β1 OPG OCN Baseline, 15, 90 90, 180 For MIST: from 7.12 to 3.57 mm. For MIST + EMD: from 7.09 to 3.53 mm. None reported Kuru et al 2004 (UK) 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 T GF-ELas Baseline, 14, 28, 42, 49, 84, 182 180 For GTR: from 7.73 to 3.33 mm. For OFD: from 7.20 to 3.90 mm. None reported 492 Clin Oral Invest (2020) 24:487–502 Table 1 (continued) Pellegrini et al 2017 (Italy, USA) 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 T GF-ELas E-cadherin, EGF, VEGF, FGF-2, MMP-1, TIMP-1, BMP-7, OPG Baseline, 3 to 5, 7, 14, 21 180 For GTR: from 8.1 to 4.1 mm. For OFD: from 5.6 to 2.9 mm. No exposure Gamaletal2011 (Egypt) 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 PDGF-BB 2,3,7,14,30 90,180,270 ForGTR:from6.1to2.6mm For OFD: from 5.6 to 4.1 mm. No exposure Gamaletal2016 (Egypt) Amoxicillin 500 mg Micropipette, mesio-facialline angle to maximumdepth2mm until 5 μl until 5 μl collected, storage -76 °C, ELISA PDGF-BB VEGF 1, 3, 7, 14, 21, 30 90, 180, 270 For GTR (occlusive): from 6.1to3.5mm. For GTR (perforated) from 6.8to2.3mm. For OFD: from 7.1 to 4.5 mm. 7 patients excluded due to postoperative exposures of the membranes Keles et al 2006 (Turkey) None Periopaper, in crevice until mild resistance for 30s, storage -70 °C, reverse-phase high performance liquid chromatography with fluorimetric detection PAF 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. None reported Kuru et al 2005 (UK) None Pre-cut chromatography strips (Whatman 3MM) at entrance of crevice for 2 min, storage –70 °C, ELISA, pooled samples sICAM-1 LFA-3 Baseline, 14, 28, 42, 49, 84 NR NR None reported Okuda et al 2001 (Japan) Cefaclor 750 mg/5 days, 0.12% CHX TID/6 weeks Periopaper, inserted until resistance for 60 sec, storage -80 °C, ELISA andone-stepsandwich enzyme immunoassay MMP-1 MMP-8 TIMP-1 Baseline, 14, 28, 84 90 For EMD: from 6.33 to 3.61 mm. For OFD: from 6.22 to 4.28 mm. NR Rakmanee et al 2018 (UK) 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 Ang-1 VEGF bFGF BMP-2 OPG TIMP-1 KGF PDGF-AB Baseline, 3-5, 7, 14, 28, 42, 84, 180, 360 180, 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) Clin Oral Invest (2020) 24:487–502 493 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 2–3dayspostoperatively and subsequently declined [24]. 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 [12]. The expression levels of vascular endothelial growth factor (VEGF) were investigated by Rakmanee et al. [24]andGamal et al. [21]. 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 [24]. 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 [21]. 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 [26]. The change in the TGF-β1 levels was similar both after GTR and after OFD and accompanied a similar clinical response 6 months postoperatively. EMD 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 [20]. 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 [19]. 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 Table 2 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, TGFβGtransforming 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 protein2, BMP-7 bone morphogenetic protein-7, PAF platelet activating factor, sICAM-1 soluble intercellular adhesion molecule-1, sLFA-3 lymphocyte function-associated antigen-3) FACTORS RELATED WITH THE HEALING OF EPITHELIUM GCF marker Investigato r, Year Is periodontal surgery leading to significant changes in the expression of GCF markers? Is there any significant difference in the expression of GCF markers for intrabony defects treated with GTR vs OFD? Is the study powered for GCF markers, for clinical outcomes or both? Is there any significant difference in the clinical response for intrabony defects treated with GTR vs OFD? Is the expression of GCF markers directly associated with clinically significant changes following treatment? E-cadherin Pellegrini et al 2017 No significant change observed for Ecadherin for any group Not reported No power calculation reported Higher percentage of ‘better responders’ in GTR vs OFD. No conclusion can be drawn for the association of Ecadherin with the clinical outcome EGF Pellegrini et al 2017 EGF levels significantly increased post-op in GTR Not reported No power calculation reported Higher percentage of ‘better responders’ in GTR vs OFD. No conclusion can be drawn for the association of EGF with the clinical outcome KGF Rakmanee et al 2018 KGF amounts increased (nonsignificantly) at 7 days and decreased to baseline levels for GTR and access surgery No significant differences in KGF amount between GTR and access surgery. Powered for clinical outcomes No significant differences for CAL gain, PPD reduction, radiographic bone fill and radiographic defect resolution for GTR or access surgery The similar expression patterns of KGF accompanied a similar clinical response with GTR and access surgery 494 Clin Oral Invest (2020) 24:487–502 GCF marker Investigato r, Year Is periodontal surgery leading to significant changes in the expression of GCF markers? Is there any significant difference in the expression of GCF markers for intrabony defects treated with GTR vs OFD? Is the study powered for GCF markers, for clinical outcomes or both? Is there any significant difference in the clinical response for intrabony defects treated with GTR vs OFD? Is the expression of GCF markers directly associated with clinically significant changes following treatment? TGF-β1 Agrali et al 2016 GCF volume and TGFβ1 levels increased at 7 days post-op and then decreased to below baseline levels (by 90 days) for both EMD and EMD + autograft TGF-β1 could not be detected in 41% of OFD, 26% of EMD and 6% of EMD + autograft during the follow up (0 to 180 days). EMD and EMD+autograft showed significantly higher TGFβ1 concentrations at 7 days and TGF-β1 amounts at 14 and 180 days vs OFD. Powered for clinical outcomes Clinical and radiographic improvements noted for all groups. EMD and EMD + autograft presented statistical significantly higher CAL gain and radiographic defect fill vs OFD. No significant difference for EMD and EMD + autograft. The trend for increased TGF-β1 expression observed in EMD and EMD + autograft correlates with a superior clinical response, compared to OFD. Ribeiro et al 2011 TGF-β1 levels significantly increased after 15 days and reduced to baseline levels after 3 months for MIST and MIST + EMD. Similar changes in TGFβ1 levels were observed for both groups, MIST and MIST + EMD Powered for clinical outcomes Similar clinical and radiographic improvements were noted for both groups Similar expression patterns in TGF-β1 accompanied a similar clinical response with MIST and MIST + EMD Kuru et al 2004 TGF-β1 levels increased two-fold 2 weeks post-op (not statistically significant), declined to levels lower than baseline after 4 weeks and remained stable until 26 weeks for GTR and conventional flap TGF-β1 levels were similarly increased for GTR and conventional flap treated sites Power calculation not reported No statistically significant differences between GTR and conventional flap noted for clinical parameters 6 months post-op. Similar expression patterns in TGF-β1 accompanied a similar clinical response with GTR and conventional flap FACTORS RELATED WITH THE HEALING OF CONNECTIVE TISSUE Pellegrini et al 2017 TGF-β1 levels were decreased compared to baseline following regeneration surgery A downward trend was detected only after GTR but not after OFD (nonsignificant differences between groups) No power calculation performed A higher percentage of ‘better responders ‘in terms of PD and CAL was found in GTR vs OFD. No conclusion can be drawn for the association of TGF-β1 with the clinical outcome PDGF Gamal et al 2011 PDGF-BB concentrations peaked during the early post-op days (days 2 and 3) and decreased at 7,14, and 30 days in GTR and OFD No significant difference was found in PDGF-BB concentrations between GTR with periosteum membrane and OFD sites. Power calculation not reported GTR led to statistically significantly higher PPD reduction, CAL gain and intrabony component reduction vs OFD. No conclusion can be drawn for the association of PDGF levels with the clinical outcome Gamal et al 2016 PDGF-BB concentrations at GTR with perforated membranes and OFD sites peaked during the early stages of healing (1-14 days) and then decreased at 21 and 30 days PDGF-BB levels at GTR with perforated membranes and OFD sites showed statistically significant higher levels than GTR with occlusive membrane at 1, 3, 7, 14 days. PDGF-BB levels decreased gradually at days 21 and 30 in all groups with no significant differences. Powered for GCF markers and clinical outcomes GTR with perforated membrane showed a statistically significant improvement in PPD, CAL and intrabony defect vs GTR with occlusive membrane and OFD. GTR with occlusive membrane-treated resulted in significant PPD reduction, CAL gain and reduction of the intrabony defect vs OFD. No conclusion can be drawn for the association of PDGF levels with the clinical outcome Rakmanee et al 2018 PDGF-AB amount increased early post-op (7 days) and decreased to baseline levels after 42 days for GTR and access surgery. Similar changes for PDGF-AB were observed for GTR and access surgery. Powered for clinical outcomes No significant differences for CAL gain, PPD reduction, radiographic bone fill and radiographic defect resolution noted for GTR or access surgery. Similar expression for PDGF-AB accompanied a similar clinical response after GTR and access surgery VEGF Pellegrini et VEGF levels increased 3 Not reported No power Higher percentage of No conclusion can be Clin Oral Invest (2020) 24:487–502 495 real-time assessment of periodontal regeneration: pilot study. J Periodontal Res 52:388–396 13. Moher D, Liberati A, Tetzlaff J, Altman DG, PRISMA Group (2009) Preferred reporting items for systematic reviews and metaanalyses: the PRISMA statement. 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