Autologous Platelet Concentrates in Treatment of Furcation Defects—A Systematic Review and Meta-Analysis

Background: The aim of this review was to evaluate the adjunctive effect of autologous platelet concentrates (APCs) for the treatment of furcation defects, in terms of scientific quality of the clinical trials and regeneration parameters assessment. Methods: A systematic search was carried out in the electronic databases MEDLINE, SCOPUS, CENTRAL (Cochrane Central Register of Controlled Trials), and EMBASE, together with hand searching of relevant journals. Two independent reviewers screened the articles yielded in the initial search and retrieved the full-text version of potentially eligible studies. Relevant data and outcomes were extracted from the included studies. Risk of bias assessment was also carried out. The outcome variables, relative to baseline and post-operative defect characteristics (probing pocket depth (PPD), horizontal and vertical clinical attachment loss (HCAL, VCAL), horizontal and vertical furcation depth (HFD, VFD) were considered for meta-analysis. Results: Ten randomized trials were included in this review. Only one study was judged at high risk of bias, while seven had a low risk, testifying to the good level of the evidence of this review. The meta-analysis showed a favorable effect regarding all outcome variables, for APCs used in adjunct to open flap debridement (p < 0.001). Regarding APCs in adjunct to bone grafting, a significant advantage was found only for HCAL (p < 0.001, mean difference 0.74, 95% CI 0.54, 0.94). The sub-group analysis showed that both platelet-rich fibrin and platelet-rich plasma in adjunct with open flap debridement, yielded significantly favorable results. No meta-analysis was performed for APCs in combination with guided tissue regeneration (GTR) as only one study was found. Conclusion: For the treatment of furcation defects APCs may be beneficial as an adjunct to open flap debridement alone and bone grafting, while limited evidence of an effect of APCs when used in combination with GTR was found.


Introduction
Furcation involvement is defined as bone resorption and attachment loss in the inter-radicular space that results from plaque associated periodontal disease [1]. The treatment of periodontal disease associated with furcation represents a challenge for the clinician, due to the complexity in anatomy and morphology of such area. The unfavorable anatomic feature of the furcation restricts adequate instrumentation for proper debridement, thereby limiting the prognosis of the involved teeth [2].
Various treatment modalities, including surgical and non-surgical therapy, have been proposed to improve the prognosis based on the degree of furcation involvement. Several classifications have been proposed over the years (Table 1), based either on the severity of horizontal probing depth into the furcation defect or on the vertical amount of alveolar bone loss within the defect [3]. The most popular one was developed by Glickman, which divides furcation defects into four grades [4]. Non-surgical strategies such as scaling and root planing, furcation-plasty, etc. are employed to treat the furcations with Grade I initial involvement which restores the gingival health. Conversely, surgical procedures including regenerative and resective approaches, are performed for the treatment of more advanced lesions, to allow access to the internal complex areas of furcations. The traditional resective approach may negatively affect the long term prognosis of the treated teeth, however, it is considered as the treatment of choice for grade III and IV furcation lesions, aiming at facilitating maintenance of the furcation area.
Regenerative approaches are aimed at furcation closure by the formation of new bone, cementum and periodontal ligament in the involved inter-radicular space. Thorough debridement with adequate instrumentation following surgical exposure of furcation involved area, is one of the earliest and most well-documented treatment protocols to achieve regeneration in grade II furcation lesions [5]. In addition, various studies were carried out in the recent past, using bone substitutes, barrier membrane, autologous, and recombinant growth factors in order to provide evidence of improved bone fill and attachment gain in treating grade II furcation lesions [6,7].
The use of biologic agents consisting of growth and differentiation factors like rhBMP2 (recombinant human bone morphogenetic protein-2), rhPDGF (recombinant human platelet-derived growth factor), and TGF-β (transforming growth factor beta), had proven to promote osteogenic induction in cases of furcation treatment, in animal studies [8][9][10]. Additionally, the use of autologous platelet concentrates (APCs) is gaining popularity as a source of multiple growth factors in high concentrations, for regenerative treatments in many clinical applications. The contribution of blood-derived platelets to the bone healing process is mainly based on the growth factors stored in their granules and released upon activation. Autologous platelet concentrates are advantageously used as a cost-effective adjunct to surgical regenerative therapy, even in combination with bone grafts and barrier membranes. Several randomized controlled trials have reported on the efficacy of the use of these APCs when used alone or in combination with various regenerative strategies and other biologic agents, suggesting improvement of post-operative soft and hard tissue healing, and improved bone fill and attachment gain [6,[11][12][13][14][15][16][17]. Different types of APCs are available, each with peculiar features, the most popular being platelet-rich plasma (PRP), along with platelet-rich fibrin (PRF), plasma rich in growth factors (PRGF) and concentrated growth factors (CGF).
A recent systematic review and meta-analysis [18], evaluating the effect of use of platelet-rich fibrin (PRF) in adjunct to open flap debridement, included two clinical trials in treatment of grade II furcation with nine month follow-up and concluded favorable results with the use of PRF in terms of clinical attachment level gain (mean difference 1.25 CI 95% [0.82, 1.65], p = 0.07) and bone fill (mean difference 1.52 CI 95% [1.18, 1.87], p = 0.05). Another systematic review [19] included two split-mouth clinical trials evaluating the effect of platelet-rich plasma and reported no consistent evidence regarding the effect of PRP in treatment of furcation defects.
The aim of this systematic review is to evaluate the adjunctive effect of APCs in treatment of furcation defects both qualitatively and quantitatively, in terms of scientific quality of the clinical trials and regeneration parameters assessment. 12 Carnevale, G. et al. [30] 2012 Degree I: Horizontal attachment loss < 1/3; Degree II: Horizontal attachment loss > 1/3; Degree III: Horizontal through-and-through destruction.
Vertical Component 1 Tal, H. et al. [31] 1982 Furcal rating 1: Depth of the furcation is 0 mm; Furcal rating 2: Depth of the furcation is 1-2 mm; Furcal rating 3: Depth of the furcation is 3 mm; Furcal rating 4: Depth of the furcation is 4 mm or more. Class III: Horizontal "through-and-through" loss.

Study Characteristics
A total of ten studies were included in this systematic review after independent screening of titles and abstracts from a pool of 153 articles retrieved from the search platforms. The systematic flow chart of the study selection process is provided in Figure 1. Out of 21 eligible studies, 11 studies were excluded with reasons provided in Table 2. The general information and the study characteristics of the included studies are detailed in Table 3.
The general comparison was between a group that received APC as an adjunct to surgical treatment (experimental group), and a group that received surgical treatment alone (control group). Three different types of comparisons were assessed, based on the treatment type. Five studies reported the comparison of open flap debridement (OFD) + APC versus OFD alone (Bajaj 2009 study evaluated the adjunctive effects of two different types of APCs in the same study, compared to OFD alone as the control group). Four studies reported the comparison of bone graft (BG) + APC versus BG alone, and only one study reported the comparison of guided tissue regeneration (GTR) + APC versus GTR alone. The results of these studies were separated for types of platelet concentrate data to facilitate subgroup meta-analysis for OFD + APC versus OFD comparison. However, it was impossible to undertake the subgroup analysis for the other two comparisons due to the lack of enough studies for sub-grouping. The risk of bias of all included studies is synthesized in Figure 2.

Study Characteristics
A total of ten studies were included in this systematic review after independent screening of titles and abstracts from a pool of 153 articles retrieved from the search platforms. The systematic flow chart of the study selection process is provided in Figure 1. Out of 21 eligible studies, 11 studies were excluded with reasons provided in Table 2. The general information and the study characteristics of the included studies are detailed in Table 3.

Study & Year Reason for Exclusion
Mehta et al. 2018 [38] Use of Collagen Membrane along with DFDBA in control group Wanikar et al. 2018 [39] Both Control and Experimental group use PRF Kaur et al. 2018 [40] Both Control and Experimental group use PRF Sharma et al. 2017 [41] Both Control and Experimental group use PRF Asimuddin et al. 2017 [42] Comparison between use of PRF and Allograft + GTR Salaria et al. 2016 [43] Case Report Biswas et al. 2016 [44] Comparison between PRF and Bioactive Glass. Pradeep et al. 2016 [45] Both Control and Experimental group uses PRF Sandhu et al. 2015 [46] Case Report Mellonig et al. 2009 [47] Histological assessment Lekovic et al. 2003 [48] Comparison of PRP/BPBM/GTR versus OFD alone PRF-platelet-rich fibrin, PRP-platelet-rich plasma, DFDBA-de-mineralized freeze-dried allograft, BPBM-bovine porous bone mineral, GTR-guided tissue regeneration.     The forest plot of the included studies evaluating the change in PPD shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.59, 95% CI 1.38, 1.80). The subgroup analysis is also favorable for both PRF (p < 0.001, mean difference 1. 46   The forest plot of the included studies evaluating the change in VCAL shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.24, 95% CI 1.08, 1.40). The subgroup analysis is also favorable for both PRF (p < 0.001, mean difference 1. 18   The forest plot of the included studies evaluating the change in VCAL shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.24, 95% CI 1.08, 1.40). The subgroup analysis is also favorable for both PRF (p < 0.001, mean difference 1.18, 95% CI 1.01, 1.36) and PRP (p < 0.001, mean difference 1.58, 95% CI 1.17, 2.00).
APC + OFD vs. OFD Alone ( Figure 5) The forest plot of the included studies evaluating the change in VCAL shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.24, 95% CI 1.08, 1.40). The subgroup analysis is also favorable for both PRF (p < 0.001, mean difference 1.18, 95% CI 1.01, 1.36) and PRP (p < 0.001, mean difference 1.58, 95% CI 1.17, 2.00).  APC + BG vs. BG Alone ( Figure 6) The forest plot of the included studies evaluating the change in VCAL in using APC in adjunct with BG is favorable; however, the result is not statistically significant (p = 0.62, mean difference 0.06, 95% CI −0.18, 0.30). The forest plot of the included studies evaluating the change in HCAL shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.01, 95% CI 0.89, 1.12). The subgroup analysis is also favorable for both PRF (p < 0.001, mean difference 0.93, 95% CI 0.80, 1.06) and PRP (p < 0.001, mean difference 1.50, 95% CI 1.18, 1.83). The forest plot of the included studies evaluating the change in HCAL shows evidence of an advantage of using APC in adjunct with BG (p < 0.001, mean difference 0.74, 95% CI 0.54, 0.94).  The forest plot of the included studies evaluating the change in HCAL shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.01, 95% CI 0.89, 1.12). The subgroup analysis is also favorable for both PRF (p < 0.001, mean difference 0.93, 95% CI 0.80, 1.06) and PRP (p < 0.001, mean difference 1.50, 95% CI 1.18, 1.83). APC + BG vs. BG Alone ( Figure 6)

Study or Subgroup
The forest plot of the included studies evaluating the change in VCAL in using APC in adjunct with BG is favorable; however, the result is not statistically significant (p = 0.62, mean difference 0.06, 95% CI −0.18, 0.30). The forest plot of the included studies evaluating the change in HCAL shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.01, 95% CI 0.89, 1.12). The subgroup analysis is also favorable for both PRF (p < 0.001, mean difference 0.93, 95% CI 0.80, 1.06) and PRP (p < 0.001, mean difference 1.50, 95% CI 1.18, 1.83). APC + BG vs. BG Alone (Figure 8) The forest plot of the included studies evaluating the change in HCAL shows evidence of an advantage of using APC in adjunct with BG (p < 0.001, mean difference 0.74, 95% CI 0.54, 0.94).

Horizontal Furcation Depth (HFD)
APC + OFD vs. OFD Alone (Figure 11) The forest plot of the included studies evaluating the change in HFD shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.13, 95% CI 0.85,1.41). No subgroup analysis was carried out for this outcome due to the lack of enough studies. The forest plot of the included studies evaluating the change in HFD shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.13, 95% CI 0.85,1.41). No subgroup analysis was carried out for this outcome due to the lack of enough studies.

Discussion
The use of platelet concentrates to promote periodontal regeneration has gained popularity in the last 10 years, as demonstrated by the increasing number of evidence-based randomized studies and systematic reviews [19,51,52]. A recent Cochrane systematic review [53] investigated the effect of APC for the surgical treatment of infrabony defects, reporting positive effects when APC is used in combination with OFD, OFD + BG, but not with GTR and enamel matrix derivative. The latter two treatments have a predictable and well-documented efficacy, and they are since long considered the gold standard for periodontal defects, so it can be difficult for any adjunctive therapy to further enhance the clinical outcomes. Evidence-based studies on the efficacy of APC for the regeneration therapy of furcation defects are relatively scarce as compared to infrabony defects. Our systematic review published in 2011 investigated the effects of APC on infrabony defects, gingival recessions and furcation defects but found only two studies on the latter topic, both using platelet-rich plasma [19]. The present study is the first comprehensive systematic review that was aimed at exploring and comparing the effect of various APCs for enhancing furcation treatment. It was designed according to a standard protocol, aimed at selecting only the best evidence studies, so as to provide the most reliable results. Only one of the ten included studies was judged at high risk of bias [50], while seven had a low risk, testifying to the good level of the evidence of this review. The results, derived from the analysis of different clinical outcome variables, suggested that the use of APC may be beneficial for improving the regeneration of furcation defects, when associated with OFD, in line with the above   The forest plot of the included studies evaluating the change in HFD shows evidence of an advantage of using APC in adjunct to OFD (p < 0.001, mean difference 1.13, 95% CI 0.85, 1.41). No subgroup analysis was carried out for this outcome due to the lack of enough studies.

Discussion
The use of platelet concentrates to promote periodontal regeneration has gained popularity in the last 10 years, as demonstrated by the increasing number of evidence-based randomized studies and systematic reviews [19,51,52]. A recent Cochrane systematic review [53] investigated the effect of APC for the surgical treatment of infrabony defects, reporting positive effects when APC is used in combination with OFD, OFD + BG, but not with GTR and enamel matrix derivative. The latter two treatments have a predictable and well-documented efficacy, and they are since long considered the gold standard for periodontal defects, so it can be difficult for any adjunctive therapy to further enhance the clinical outcomes. Evidence-based studies on the efficacy of APC for the regeneration therapy of furcation defects are relatively scarce as compared to infrabony defects. Our systematic review published in 2011 investigated the effects of APC on infrabony defects, gingival recessions and furcation defects but found only two studies on the latter topic, both using platelet-rich plasma [19]. The present study is the first comprehensive systematic review that was aimed at exploring and comparing the effect of various APCs for enhancing furcation treatment. It was designed according to a standard protocol, aimed at selecting only the best evidence studies, so as to provide the most reliable results. Only one of the ten included studies was judged at high risk of bias [50], while seven had a low risk, testifying to the good level of the evidence of this review. The results, derived from the analysis of different clinical outcome variables, suggested that the use of APC may be beneficial for improving the regeneration of furcation defects, when associated with OFD, in line with the above

Discussion
The use of platelet concentrates to promote periodontal regeneration has gained popularity in the last 10 years, as demonstrated by the increasing number of evidence-based randomized studies and systematic reviews [19,51,52]. A recent Cochrane systematic review [53] investigated the effect of APC for the surgical treatment of infrabony defects, reporting positive effects when APC is used in combination with OFD, OFD + BG, but not with GTR and enamel matrix derivative. The latter two treatments have a predictable and well-documented efficacy, and they are since long considered the gold standard for periodontal defects, so it can be difficult for any adjunctive therapy to further enhance the clinical outcomes. Evidence-based studies on the efficacy of APC for the regeneration therapy of furcation defects are relatively scarce as compared to infrabony defects. Our systematic review published in 2011 investigated the effects of APC on infrabony defects, gingival recessions and furcation defects but found only two studies on the latter topic, both using platelet-rich plasma [19]. The present study is the first comprehensive systematic review that was aimed at exploring and comparing the effect of various APCs for enhancing furcation treatment. It was designed according to a standard protocol, aimed at selecting only the best evidence studies, so as to provide the most reliable results. Only one of the ten included studies was judged at high risk of bias [50], while seven had a low risk, testifying to the good level of the evidence of this review. The results, derived from the analysis of different clinical outcome variables, suggested that the use of APC may be beneficial for improving the regeneration of furcation defects, when associated with OFD, in line with the above findings regarding infrabony defects. Further, it may be noted that APC in adjunct to OFD + BG also showed significant improvement in HCAL and HFL. Since only one RCT evaluated the adjunctive effect of APC when using GTR for grade II furcation defects, no meta-analysis was feasible. The results of this study, suggested that the adjunct of APC produced no significant advantage as compared to GTR alone, in line with previous findings for infrabony defects.
This review has some strengths and limitations. In recent years, there has been fierce competition among companies producing different types of platelet concentrates, all claiming that their product was superior to the others. This also introduced a number of different protocols for the preparation of APC. Indeed, very few studies comparing different types of APC have been performed in the periodontal field (as well as in other fields), so that it seems difficult to indicate if there is really a superiority of some APCs over the others for specific conditions. In the present review, we were able to perform a meta-analysis with subgroups, keeping separate different APC (PRP and PRF), only in the group considering OFD alone. The outcomes using different APC was very similar as can be seen in Figures 3-6 This can be considered a strong point of the present review. However, the precise difference in effects between different APC cannot be estimated, due to a lack of direct comparisons. The same subgroup analysis could not be performed in the OFD + BG group, due to heterogeneity among studies in the type of APC used, and the insufficient number of studies using the same type of APC. Indeed, also when different studies use the same type of APC, this does not necessarily represent a warranty of homogeneity in the protocols. For example, over 20 different types of devices producing PRP are currently available on the market, and at least five different companies produce centrifuges for PRF [54]. A recent in vitro study compared the characteristics of PRF obtained using four different centrifuge systems [55]. This study found that, even though in all cases a leukocyte-and platelet-rich fibrin is obtained after centrifugation, the centrifuge characteristics and centrifugation protocols significantly impact the cell composition and distribution, the growth factors release pattern and the fibrin architecture of the final products. So, when PRF is used in different studies, one cannot be sure to refer to a product with the same features, unless the same centrifuge system is used. In spite of the above limitations, caused by lack of homogeneity in study protocols, it can be noted that all studies investigating the effect of APC as an adjunct to OFD alone, consistently reported a beneficial effect. The latter can be considered a strength point evidenced by this review.
In addition to the regenerative properties, platelet concentrates have also been demonstrated to carry further advantages in the postsurgical healing period. Evidence-based studies in different oral surgery procedures have reported that the adjunctive use of APC is associated with an improvement of patients' quality of life and pain reduction in the post-surgical period [56,57] Unfortunately, such effects were not consistently addressed in the studies included in the present review.
Finally, though specific clinical studies have not been performed so far, there is consistent preclinical evidence that APCs have an antimicrobial effect against a number of species commonly found in the oral cavity, which suggest they may potentially represent a beneficial tool for the control of postsurgical infection [58,59]. Indications for future research: There is a huge demand for conducting more evidence-based comparative studies with wide sample size (among different APC and grafting materials and versus other biological agents), to investigate patients' quality of life, to treat various grades of furcation, in order to verify the actual beneficial effects of use of APC as adjunct with wide variety of regenerative strategies.

Material and Methods
This systematic review and meta-analysis were carried out based on preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines. The protocol of this systematic review was registered on the international prospective register of systematic reviews (PROSPERO) with registration number CRD42019100015.

Research Question
What is the effectiveness of autologous platelet concentrates used as an adjunct to different types of surgical techniques for the treatment of furcation defects, compared to the same surgical techniques alone?

•
Randomized clinical trials (RCT), either of a parallel group or of a split-mouth design; • Presence of at least one experimental group in which APCs were clinically applied as an adjunct to surgical procedures alone or in combination with bone grafting materials or GTR procedures for the therapy of furcation defects; • Presence of an appropriate control group, in which the same therapeutic procedures as those employed in at least one experimental group were clinically applied for the treatment of furcation defects, without the adjunctive effect of APCs; • Patients included in the RCT should present with maxillary/mandibular Grade 2 or 3 furcation defects; • Patients included in the RCT should have no systemic diseases nor taking medications that could potentially influence the outcome of periodontal therapy; • The follow-up period had to be at least 6 months.

Selection of Studies
Following the electronic search in all the respective databases, the records were imported into EndNote 13 software (EndNoteX3; Thomas Reuters, New York, NY, USA) and the duplicates were sorted to be removed from the pool of titles. A total of 153 titles and abstracts (if available) were independently screened by two reviewers (MDF, SP) to exclude all articles clearly not meeting the inclusion criteria. Of all the eligible articles, full texts were obtained and were thoroughly assessed.
Only articles fulfilling the inclusion criteria were considered. In cases of disagreement between the two reviewers, a third reviewer (LF) was consulted. Detailed reasons were stated for all excluded studies.

Data Extraction and Management
The relevant data of the included studies were extracted using an Excel spreadsheet (Microsoft, Radmond, WA, USA). Data were independently extracted by two review authors (MDF, FG) and recorded on predetermined spreadsheets. In case of missing or unclear information, the authors of the included studies were contacted by email for providing clarification or missing information.
The following data were recorded for each included report:

Risk of Bias Assessment
Risk of Bias was assessed by two independent reviewers (ACD, AS) for all the included clinical trials and the discrepancies were resolved by discussion and in consent with a third reviewer (MK). The domains of the study were graded under high, unclear or low risk, based on the following categories: Selection bias (random sequence generation and allocation concealment), performance bias (blinding), detection bias (assessor blinding), attrition bias (incomplete outcome data), reporting bias (selective reporting), sample size calculation and number of surgeons involved. Based on the domains, the studies categorized as low risk of bias if all domains were at low risk; high risk of bias if two or more domains were at high risk; or medium risk of bias if one domain were at unclear or high risk.

Data Synthesis
Data of the various outcomes were extracted from each included study. Parallel group and split-mouth studies were combined in the meta-analysis of treatment effects. For all the outcomes, mean differences and 95% confidence interval (CI) were used to summarize the results for each included study. The meta-analysis was performed using Review Manager 5.3 software (RevMan 5.3, Version 5.3.5 Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014) using the fixed or random effects models, as appropriate. Fixed effects meta-analysis was used when the heterogeneity was small (i 2 < 60%, p > 0.05). When the heterogeneity was large (i 2 > 60%, p < 0.05), a random-effects model analysis was undertaken.

Conclusions
In conclusion, the evidence available in the literature for the beneficial effects of platelet concentrates in periodontal furcation defects has been increasing in recent years. Platelet-rich plasma and platelet-rich fibrin may be advantageously used as an adjunct to open flap debridement alone and additional grafting procedures, while there is no evidence of an effect of APC when used in combination with GTR, for the treatment of furcation defects.