Efficacy of biologics for alveolar ridge preservation/reconstruction and implant site development: An American Academy of Periodontology best evidence systematic review

Abstract Background The use of biologics may be indicated for alveolar ridge preservation (ARP) and reconstruction (ARR), and implant site development (ISD). The present systematic review aimed to analyze the effect of autologous blood‐derived products (ABPs), enamel matrix derivative (EMD), recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB), and recombinant human bone morphogenetic protein‐2 (rhBMP‐2), on the outcomes of ARP/ARR and ISD therapy (i.e., alveolar ridge augmentation [ARA] and maxillary sinus floor augmentation [MSFA]). Methods An electronic search for eligible articles published from January 2000 to October 2021 was conducted. Randomized clinical trials evaluating the efficacy of ABPs, EMD, rhBMP‐2, and rhPDGF‐BB for ARP/ARR and ISD were included according to pre‐established eligibility criteria. Data on linear and volumetric dimensional changes, histomorphometric findings, and a variety of secondary outcomes (i.e., clinical, implant‐related, digital imaging, safety, and patient‐reported outcome measures [PROMs]) were extracted and critically analyzed. Risk of bias assessment of the selected investigations was also conducted. Results A total of 39 articles were included and analyzed qualitatively. Due to the high level of heterogeneity across studies, quantitative analyses were not feasible. Most studies in the topic of ARP/ARR revealed that the use of biologics rendered similar results compared with conventional protocols. However, when juxtaposed to unassisted healing or socket filling using collagen sponges, the application of biologics did contribute to attenuate post‐extraction alveolar ridge atrophy in most investigations. Additionally, histomorphometric outcomes were positively influenced by the application of biologics. The use of biologics in ARA interventions did not yield superior clinical or radiographic outcomes compared with control therapies. Nevertheless, ABPs enhanced new bone formation and reduced the likelihood of early wound dehiscence. The use of biologics in MSFA interventions did not translate into superior clinical or radiographic outcomes. It was observed, though, that the use of some biologics may promote bone formation during earlier stages of healing. Only four clinical investigations evaluated PROMs and reported a modest beneficial impact of the use of biologics on pain and swelling. No severe adverse events in association with the use of the biologics evaluated in this systematic review were noted. Conclusions Outcomes of therapy after post‐extraction ARP/ARR and ARA in edentulous ridges were comparable among different therapeutic modalities evaluated in this systematic review. Nevertheless, the use of biologics (i.e., PRF, EMD, rhPDGF‐BB, and rhBMP‐2) in combination with a bone graft material generally results into superior histomorphometric outcomes and faster wound healing compared with control groups.


INTRODUCTION
Decades of investigation have demonstrated that dental implants are a predictable and effective therapy for the rehabilitation of partially and completely edentulous patients. 1,2 However, insufficient or inadequate bone volume derived from pathological processes (e.g., chronic disease progression), congenital conditions, undesirable events (e.g., trauma), or therapeutic interventions (e.g., tooth extraction or resective surgical procedures) often represents a common challenge in clinical practice. The presence of limited bone volume may interfere with ideal positioning of the implant and, subsequently, compromise the ability to achieve and maintain optimal long-term peri-implant health, function, and esthetics. Alveolar ridge preservation (ARP) or reconstruction (ARR) and implant site development (ISD) techniques are used to correct and overcome these limitations. Under the umbrella of ARP/ARR and ISD there are a variety of procedures and techniques that share a common objective, the provision of a recipient site that is adequate for implant placement in the ideal position. More specifically, ARP aims at attenuating post-extraction dimensional changes in intact or mostly intact sites, while ARR is indicated in extraction sites presenting extensive alveolar bone damage. On the other hand, ISD aims at the correction of hard and soft tissue deficiencies in healed, edentulous alveolar ridges.
Regarding hard tissue, horizontal and vertical alveolar ridge augmentation (ARA), as well as maxillary sinus floor augmentation (MSFA) arguably represent the core ISD interventions in contemporary clinical practice. These interventions, along with ARP/ARR, can be performed with a variety of techniques and materials, each presenting specific distinctions and limitations. Absorbable and non-absorbable barrier membranes, particulate bone replacement graft materials with different origins, and autologous bone blocks are among the most frequently employed materials for bone augmentation in ISD and ARP. While proven successful in multitude of investigations, [3][4][5][6] all bone preservation and augmentation protocols present with drawbacks and limitations, potentially including, but not limited to, complications during the healing phase (e.g., infection), reduced amount of new bone formation, and delayed healing. The use of biologics has been proposed with the purpose of overcoming these limitations and increase the predictability of therapy.
Biologics are a group of agents or mediators that exert a biological effect through various mechanisms to promote tissue regeneration. Biologics promote a variety of essential cellular events in wound healing including deoxyribonucleic acid (DNA) synthesis, chemotaxis, cell differentiation, mitogenesis, and matrix biosynthesis. 7,8 Consequently, these biologics have been used to enhance the outcomes of bone regeneration procedures. 9,10 Also, biologics have attributed to a variety of additional beneficial properties such as reduced local inflammation and reduced postoperative pain, among others. 11,12 The use of biologics in periodontics and implant dentistry has been extensively studied. Nevertheless, there is still controversy regarding their true potential and clinical indications. Consequently, in alignment with the purpose of the American Academy of Periodontology (AAP) Best Evidence Consensus (BEC) on the use of biologics in contemporary clinical practice, the aim of this systematic review was to investigate the effect of commonly used biologics (i.e., autologous blood-derived products [ABPs], enamel matrix derivative [EMD], recombinant human platelet-derived growth factor-BB [rhPDGF-BB], and recombinant human bone morphogenetic protein-2 [rhBMP-2]) on the outcomes of different ARP/ARR and ISD modalities (i.e., ARA, and MSFA) by addressing the following focused question: Does the use of ABPs, EMD, rhPDGF-BB, or rhBMP-2, either as a monotherapy or in combination with scaffolds or graft materials, render superior outcomes after the performance of ARP/ARR and ISD procedures compared with a control group with standard treatment protocols not involving the use of biologics?

MATERIALS AND METHODS
The protocol of this study was designed in accordance with the Cochrane Handbook for Systematic Reviews of Interventions 13 and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) 2020 guidelines. 14

Population, Intervention, Comparison, Outcome (PICO) question
• Population: Adult individuals • Intervention: Use of ABPs, EMD, rhBMP-2, or rhPDGF-BB in ARP/ARR, ARA, or MSFA. • Comparison: Conventional ARP/ARR and ISD modalities not involving the use of biologic mediators. All three treatments (ARP/ARR, ARA, and MSFA) were evaluated individually. • Outcomes: 1. Primary: Bone changes (dimensional changes compared with baseline records [linear and/or volumetric measurements obtained before the grafting procedure] and histomorphometric data). 2. Secondary: Clinical, implant-related, digital imaging, safety, and patient-reported outcome measures (PROMs). Clinical outcomes involved structural and biological assessments performed during direct or indirect clinical examination. Digital imaging refers to the assessment of bone and soft tissue via radio-graphs, digital imaging, and communications in medicine (DICOM) and/or stereolithography (STL) files. Histologic evaluation involved the use of qualitative (descriptive histology) and/or quantitative measurements (e.g., histomorphometric). PROMs are assessments performed by the patients.

Eligibility criteria
Human randomized clinical trials (RCTs) with parallelarm or split-mouth design published in the English language after January 1, 2000 were screened. Eligibility criteria were: (1) surgical treatment of adult patients (≥18 years of age) presenting single or multiple extraction sites or edentulous areas in need of implant-supported/retained rehabilitation; (2) minimum of 10 sites per study arm; (3) minimum follow-up of 2 months for ARP/ARR; (4) minimum follow-up of 4 months for MSFA and ARA; (5) one study arm involved the use of a biologic (i.e., ABPs, EMD, rhBMP-2, or rhPDGF-BB), either as a monotherapy or combined with other modalities of treatment while another arm consisted of conventional therapy without the use of biologics; and (6) report at least one of the following outcomes of interest: dimensional bone changes or histomorphometric data. Moreover, a less specific screening using non-MeSH index terms was conducted to expand the search scope. This included the "type of intervention" AND "a biologic" (e.g., ridge augmentation AND platelet-derived growth factor). A similar strategy was used in EMBASE and Cochrane library using the filter for randomized clinical trials.

Data extraction
The following data were extracted and recorded in duplicate by two independent reviewers (F.S.L.A. and A.M.): (1) citation, and year of publication; (2) study location: country and type of setting (e.g., private practice, university, military, or dental hospital); (3) type of procedure and approach; (4) characteristics of participants (i.e., sample size [initial and final number of participants per arm], sex and age distribution per arm); (5) characteristics of interventions: test and control groups; (6) outcome measures of interest; and (7) source of funding.

Methodological quality and risk of bias assessment
The assessment of methodological quality and risk of bias of each included RCT was performed in duplicate using the Cochrane risk-of-bias tool for randomized trials (RoB1) 21 which provided guidelines for the following parameters: (1) random sequence generation; (2) allocation concealment method; (3) blinding of participants and personnel; (4) blinding of outcome assessment; (5) incomplete outcome data; (6) selective reporting; and (7) other bias.

Data synthesis
Data were collated into evidence tables and presented according to the objective/indication of the surgical intervention of interest. The descriptive analysis was structured by type of ISD procedure and divided into the following categories: study characteristics, population characteristics, intervention characteristics, and effect of biologic on treatment outcomes. In addition, based on the criteria established by the adapted version 22 of the American Dental Association (ADA) Clinical Practice Guidelines Handbook (see Tables S1-S3 in online Journal of Periodontology), 23 critical assessment of the literature and strength of recommendation were applied to the extracted data and results presented in this systematic review. These recommendations were presented according to the following set of criteria: • Clinical comparisons and main findings: Description of the comparisons (i.e., therapies involving the use of biologics vs. controls) and outcomes of interest, based on the main findings of individual studies and pooled estimates (if available). This description was structured as described above: by type of intervention divided into four different categories. • Level of certainty: Assessment of the extent to which there is confidence in the estimate of the effect of therapy considering the best available evidence. Briefly, this assessment is dictated by the following domains: (a) risk of methodological bias; (b) applicability of evidence; (c) inconsistency or unexplained heterogeneity of results; (d) imprecision (wide confidence intervals); and (e) high probability of publication bias (e.g., selective reporting). Level of certainty may be classified as: high, moderate, or low (see Tables S1 and S2 online Journal of Periodontology).

F I G U R E 1 PRISMA flowchart
• Net benefit rating (benefit-harm estimation): Whether the expected benefits outweigh the potential for harm. • Adverse events and complications: Relevant adverse events and complications. • Strength of clinical recommendation: This assessment reflects the extent to which one can be confident that adherence to the treatment recommendation will be more beneficial than harmful, considering the strengths and weaknesses of the best available evidence. Strength of clinical recommendation may be classified as: strong, in favor, weak, expert opinion for/supports, expert opinion questions the use, expert opinion against, or against (see Table S3 in online Journal of Periodontology).

RESULTS
The PRISMA flowchart for literature selection is depicted in Figure 1. In summary, 3044 records were identified after removal of duplicates. Among them, 90 were assessed for full-text and 39 were included in the qualitative synthesis (18 in ARP/ARR, nine in ARA, and 12 in MSFA). A summary with the characteristics of the included investigations is presented in Tables 1, 2, and 3. The most frequent reason for exclusion based on full-text evaluation was insufficient sample size (n = 20) followed by inadequate report of the primary outcome (n = 15). The complete list of excluded articles is displayed in Table S4 in the online Journal of Periodontology.
Due to the significant heterogeneity across articles (e.g., discrepancies between experimental and control groups, diversity of biologics used, and different grafting procedures), a quantitative synthesis of the data reported in the included studies and, consequently, a meta-analysis could not be completed. Instead, a descriptive but thorough analysis of the reported outcomes was performed. It is important to highlight that certain biologics were used off label in some of the selected studies.

Maxillary sinus floor augmentation
Year of publication ranged from 2003 to 2020. A total of 12 articles were selected of which six 47-52 reported split-mouth and six [53][54][55][56][57][58] parallel-arms studies ( Table 3). All the studies, but one that was performed in private practice, 48 were conducted in university settings. The most frequent method of assessment was histomorphometry of bone biopsies. [48][49][50][51][52][54][55][56][57][58] The second most prevalent method of assessment was three-dimensional radiography. 47 analyzed. It is important to highlight that two different articles by Stumbras and colleagues reported different outcomes of the same sample of patients. 28,29 Most studies reported a mean age for the subjects evaluated, generally ranging from 40 to 60 years. Only one investigation presented with great discrepancy from the above-mentioned range, reporting a mean of 22.62 ± 2.44 years. 38

Alveolar ridge augmentation
In total, 231 patients were evaluated. These contributed to 320 sites. Only four dropouts from one study were noted. 10 The age ranged from 19 to 76 years. Females contributed slightly higher to the sample when compared with males. Light smokers (≤10 cig</day) were included in one study. 12

Maxillary sinus floor augmentation
Overall, 323 patients for a total of 502 maxillary sinuses, were evaluated. Only two dropouts from one study were noted. 53 Males and females contributed equally to the total sample. While two studies 54,55 did not provide information on the inclusion of smokers, one study 49 stated that light smokers (≤10 cig</day) were included.
Unassisted healing was included as a control group in 13 investigations, [24][25][26][27][28][29][30][31]34,[36][37][38][39] while two studies compared DBBM alone versus DBMM in combination with EMD, 9,11 and three studies involving the use of rhBMP-2 reported the sole use of a collagen sponge, β-TCP + HA, or DBM gel as control groups. 32,33,35 Most studies did not attempt to obtain primary closure; nor did they use additional materials for socket sealing other than sutures. 24,25,27,31,[36][37][38][39] Nevertheless, it is important to mention that multiple investigations studying ABPs also used this biologic as a membrane to cover the socket orifice. Other studies involved the use of collagen membranes, 11,28-30,33 a rapidly absorbing collagen sponge in combination with cyanoacrylate, 26 or a free mucosal graft 9 to cover the socket for one or more of the included groups.

Alveolar ridge augmentation
Overall, seven studies 10,[40][41][42][43]45,46 explored the effects of biologics on horizontal ridge augmentation (HRA), while two studies 12,44 evaluated HRA and vertical ridge augmentation (VRA). In three studies, 10,41,45 conventional guided bone regeneration (GBR) by means of an absorbable barrier membrane was performed in the test and control groups. In one study, GBR was only applied in the test group, while the control group consisted of autogenous block grafts harvested from the mandibular ramus. 42 Further, one study 40 tested the effect of ABPs in combination with intraoral autogenous block grafts compared with the same intervention, but grafted simultaneously with anorganic bovine bone mineral and covered with a resorbable barrier membrane. The only study that explored the effectiveness of platelet-rich plasma (PRP) on HRA and VRA used a titanium-mesh and anorganic bovine bone mineral. 12 It is worth noting that one study assessed an envelope approach for regeneration using DBBM and rhBMP-2, but no barrier membrane. 44 In terms of implant placement stage, one study 41 reported the use of the biologic with simultaneous implant placement, while all the other included studies involved delayed implant placement.

Maxillary sinus floor augmentation
Only one study 53 aimed at testing the effect of biologics on transalveolar sinus floor elevation. In this study, a special drilling system incorporating hydraulic properties to lift-up the membrane was used. Hence, the intervention for the vast majority of the studies was MSFA via lateral window approach, as described elsewhere, 60 with the osteotomy performed with either a rotatory bur or a piezoelectric instrument. Concerning the bone replacement graft material, six studies combined the biologic with a bone substitute (4 studies [49][50][51]54 with anorganic bovine bone mineral and two studies 47,55 with β-tricalcium phosphate), one with autogenous bone harvested from the iliac crest, 48 and in one study the biologic was used per se. 53 Seven studies tested the effect of ABPs, 47

Alveolar ridge preservation
Eighteen studies reported dimensional and/or histomorphometric changes occurring after tooth extraction.
Fifteen investigations evaluated dimensional changes, six of them through clinical measurements or casts analysis 26,27,31,32,36,38 and the remaining 10 used threedimensional radiography (note that Coomes et al. used both methods). 9,11,25,28,[32][33][34][35]37,39 These investigations studied the dimensional changes at different locations, including, but not limited to, vertical collapse at mesial, distal, mid-buccal, and mid-lingual aspects, as well as horizontal (width) changes at different levels from the alveolar crest. In general, selected investigations failed to demonstrate superior outcomes in association with the use of biologics when compared with conventional approaches. 9,11,26,28 Nevertheless, biologics (alone and/or in combination with other graft materials) did contribute in most investigations to attenuate the resorption process that typically occurs after tooth extraction as compared with unassisted healing or with the sole use of an ACS. [25][26][27][28]31,32,34,36,39 It is important to note that the differences between groups, when present, were mostly associated to changes in both ridge height as well as width in the most coronal aspects of the socket. 26,28,39 Other investigations reported on alternative methods for assessment such as radiographic bone fill 27,31,37,39 and evaluation of the so-called "alveolar bone area". 30 These alternative analyses typically resulted in more favorable outcomes for the test group. 30,31,37,39 Two investigations also reported on early soft tissue wound healing 11 and dimensions of socket orifice, 38 both demonstrating no differences between groups.
Histomorphometric assessment of bone biopsies was performed in six investigations. 9,[24][25][26]29,30 Overall, the use of biologics (i.e., ABPs and EMD) seems to have a beneficial effect on mineralized tissue formation with all six studies reporting superior percentages for the groups involving the use of biologic mediators as a monotherapy or in combination with graft materials. These comparisons reached statistically significant differences (to at least one other group) in five investigations. 9,25,26,29,30 On the other hand, these differences seem to be more modest for non-mineralized tissue and the diminished presence of residual graft material. Nevertheless, biologics contributed to the remodeling of allogenic and xenogenic grafting materials reporting in general a lower percentage of residual graft that occasionally reached statistical significance. 9,26,30 Notably, the only study reporting histomorphometric assessment with the use of EMD demonstrated statistically significant differences for all three parameters (greater percentage of mineralized tissue, less residual graft, and less soft tissue marrow spaces) favoring the test group. 9 It is important to mention that the above-mentioned comparisons were made to a variety of "control" groups that sometimes involved the use of bone replacement grafts. None of the studies included in this review evaluated the histomorphometric outcomes in extraction sockets treated with rhBMP-2.
Only three investigations assessed PROMS. Kumar et al. reported more favorable outcomes regarding postoperative pain with the use of PRF, although swelling was more prevalent in one of the groups involving the use of this biologic, likely due to the additional use of calcium sulfate in this particular group. 27 Temmerman et al. also found differences in terms of postoperative pain favoring the use of L-PRF. 39 Lee and colleagues failed to observe differences in pain and swelling severity, but demonstrated statistically significant differences favoring the use of EMD for the duration of pain and swelling. 11 Overall, the use of biologics appears to be associated with more favorable outcomes regarding postoperative pain; however, these differences seem to be minimal and last only for a limited period of time.
Regarding implant-related outcome measures, two investigations evaluated the feasibility of implant placement after performing ARP/ARR with rhBMP-2 + ACS versus different control groups. Both studies reported a greater number of implants installed without the need for further augmentation in the groups involving the use of rhBMP-2. 32,34 Last, no adverse events derived from the use of ABPs or EMD were reported. On the other hand, two out of four investigations evaluating the effectiveness and safety of rhBMP-2 reported adverse events. 32,34 Coomes et al. reported that 12% of patients in the test group (vs. 0% in the control group) experiencing mild erythema and localized swelling that resolved spontaneously 7-10 days after the procedure. 32 Fiorellini and colleagues reported a total of 250 adverse events for 78 out of the 80 subjects evaluated in their investigation. These events were mostly associated with the test groups and primarily consisted of transient postoperative oral edema, pain, and erythema. 34

Alveolar ridge augmentation
In general, the use of biologics included in test groups did not show superior outcomes in terms of clinical, radiographic, or histologic parameters when compared with the control groups. Nevertheless, it must be noted that one study reported a statistically significant difference in terms of mineralized tissue formation and horizontal bone gain after 4 months of healing, favoring the PRP group. 10 Interestingly, another study demonstrated that covering the titanium-mesh with PRP in ARA procedures may lead to significantly less incidence of wound dehiscence, which in turn, may lead to reduced postoperative complications and failure of the regenerative intervention. 12 No adverse events derived from the use of biologics were reported. Importantly, the use of BMPs was proven safe and effective, but their performance was not superior to the control groups. PROMs revealed slightly enhanced outcomes in terms of postoperative pain after the use of BMPs compared with autogenous block grafts. 43

Maxillary sinus floor augmentation
The benefit of using ABPs in combination with bone substitutes was clearly demonstrated in one study (L-PRF). 51 In summary, a statistically significant difference of ≈14% that favored the test group for newly formed bone and ≈10% that favored the control group for residual bone graft was reported. This study concluded that bone healing can be accelerated by means of combining a bone graft with L-PRF and that this may lead to earlier implant placement after MSFA. Another study showed modest benefits as only a difference of 8% to 10% in terms of mineralized tissue formation could be seen in favor of the test group (PRP). 55 It is worth noting that the only study that explored the effect of ABPs versus a "true" control group (saline) resulted in superior outcomes by means of vertical bone gain ≈1mm) in favor of the test therapy. The use of rhPDGF-BB was tested in one study that revealed that mineralized tissue formation was ≈10% higher in the test group after 4 to 5 months of healing. 49 Nevertheless, at 7 to 9 months the difference was negligible. In consistency with this finding, greater mineralized tissue formation at early healing time points was observed when rhBMP-2 was used. 52,57 No adverse events derived from the use of biologics were reported.

Clinical recommendations
Based on the screened evidence and the results described in this manuscript, strength of clinical recommendation according to the American Dental Association (ADA) Clinical Practice Guidelines Handbook was established.
These recommendations were grouped by interventions as follows: •

Main findings
The demand for ARP/ARR and ISD interventions has increased in recent years due to the popularity of dental implant therapy. Nonetheless, research efforts over the last two decades have been focused on increasing predictability through minimally invasive approaches and the use of biologics to promote enhanced outcomes. The present systematic review aimed at exploring the effect of biologics on ARP/ARR and ISD interventions. Interestingly, it was observed that limited and heterogeneous high-quality evidence exist, which precluded the conduction of a meta-analysis. In this sense, it is important to emphasize that the use of certain biologics (i.e., EMD and rhPDGF-BB) for the studied interventions are considered off-label. This likely contributed to the heterogeneity of the findings, the marked differences amongst studies, the limited number of investigations, and the lack of evidence evaluating certain therapies (e.g., rhPDGF-BB for ARP or EMD for MSFA). As such, data extracted from the studies selected should be cautiously interpreted. Nevertheless, studies included in this review reported no adverse events derived from the use of biologics with the exception of rhBMP-2 in ARP/ARR. These adverse events were more frequently observed in the test groups involving the use of this biologic but were never severe and included most commonly localized edema, pain, and erythema. With regard to ARP/ARR, both ABPs and EMD provide satisfactory outcomes when combined with bone replacement graft materials. Also, ABPs alone outperformed unassisted healing in most studies with regard to dimensional changes after tooth extraction. Similarly, the usage of rhBMP-2 in combination with either a graft material or an ACS was also associated with favorable results that generally outperformed controls groups. The effectiveness of rhBMP-2 in ARP/ARR seems to be dose-dependent. Last, superior histomorphometric outcomes are associated with the use of ABPs and EMD in ARP/ARR. For ARA procedures, rhPDGF-BB, ABPs, and rhBMP-2 are effective in promoting bone formation. Similarly, ABPs may be beneficial in terms of higher rate of mineralized tissue formation and lower incidence of early postoperative complications. Regarding MSFA, rhPDGF-BB, ABPs, and rhBMP-2 are effective in promoting and accelerating bone formation during the early stages of healing compared with control therapies. The above-mentioned findings are in general terms aligned with those reported in previous systematic reviews. 17,18,[61][62][63]

What is the biologic plausibility of these findings?
Biologics are molecular mediators that regulate cellular events in the wound healing process via established mechanisms of action, which include angiogenesis, osteogenesis, cementogenesis, extracellular matrix formation, and chemotaxis, among other biological processes. 8,63 Biologics are used in clinical settings to increase predictability and enhance the outcomes of therapy. Nevertheless, different biologics have diverse dominant effects and therefore, their use should be tailored according to the clinical scenario and the desired outcomes. For instance, rhPDGF-BB, a potent mitogenic agent, is naturally released by blood platelets after binding to specific cell surface receptors. 64 In vitro, rhPDGF has been shown to promote fibroblast, cementoblast, and osteoblast migration and proliferation. 65 On the other side, the rationale for the use of ABPs is primarily based on the role that platelets have in hemostasis and for being a natural source of growth factors. 66 Furthermore, it has been demonstrated in-vitro that PRF elicits an anti-inflammatory response in macrophages 67 and suppresses osteoclastogenesis. 68 EMD contains naturally occurring proteins such as enamelin, amelogenin, and ameloblastin. This biologic has demonstrated to induce the proliferation of mesenchymal stem cells, as well as enhance osteogenic differentiation by stimulating the proliferation of pre-osteoblasts and differentiation of osteoblast-like cells and osteoblasts. 69,70 Last, rhBMP-2 belongs to a group of molecules, the bone morphogenetic proteins, the largest subfamily of the transforming growth factor-β superfamily. 71 To date, 14 bone morphogenetic proteins have been identified, with rhBMP-2 and -7 being the most extensively used and investigated. These proteins are capable of inducing bone formation by guiding the differentiation of mesenchymal cells into bone and bone marrow cells. 72 Nevertheless, despite their biological properties and other evidence supporting the clinical use of these biologics, in general terms, findings from this systematic review do not strongly support the use of biologics to optimize the outcomes of ISD interventions.

Recommendations for future investigations
Properly designed RCTs aimed at evaluating the clinical, implant-related, digital imaging, histologic and patientrelated outcomes of ARP/ARR and ISD procedures involving the use of biologics in different clinical scenarios are warranted. To date, the literature is replete with articles reporting the use of biologics, more specifically, ABPs, EMD, rhPDGF-BB, and rhBMP-2 Nevertheless, the great majority of these investigations are case control, case series or case reports. 19 Although these investigations could provide valuable information, the risk of bias, mainly due to the presence of variables unaccounted for, can be very significant. Consequently, in order to establish guidelines and recommendations for the use of biologics in ARP/ARR and ISD procedures, only a high level of clinical evidence was considered in this systematic review. The strict eligibility criteria unequivocally lead to a limited selection of studies, which may have influenced the outcomes of the review. Future clinical studies should involve groups or study arms as methodologically similar as possible with the only difference being the additional use of a biologic. These studies are expected to further contribute to elucidate the true efficacy of these mediators. Also, the evaluation of PROMs should be routinely considered in future investigations.

Limitations
The main limitations of this systematic review are: (1) The marked methodological heterogeneity across selected investigations that prevented the performance of a quantitative analysis. For the same reason, comparisons between biologics were not feasible; (2) The efficacy of some biologics could not be assessed due to the lack of clinical investigations reporting their usage, for example rhPDGF-BB for ARP/ARR and EMD for MSFA; (3) Although grouped under the umbrella of biologics, these mediators greatly differ between one another and, therefore, a comparative assessment of reported outcomes should be done with caution. Moreover, although often presented as a consolidated category for the purpose of this review, it must be recognized that ABPs represent a heterogenous group of therapeutic agents. The sole variation in centrifugation protocols can affect their composition and potential for regeneration, 73 and (4) A variety of patient-and site-specific variables can affect the outcomes of therapy. Including only RCTs can contribute to reduce the likelihood of selection bias; however, some critical parameters, such as the thickness (whenever present) of the facial alveolar bone in extraction sites, 9,74,75 were not evaluated in most included investigations.

CONCLUSIONS
Current evidence does not support that the use of ABPs, EMD, rhPDGF-BB, or rhBMP-2, either as a monotherapy or in combination with alternative materials in the context of ARP/ARR and ISD, renders superior clinical and radiographic outcomes when compared with conventional interventions. On the other hand, histomorphometric results are favorably influenced by the adjunctive use of these biologics. PROMs were under-reported in the included investigations and were minimally influenced by the application of biologics. Given these findings, it is currently not possible to establish recommendations for the clinical use of ABPs, EMD, rhPDGF-BB, or rhBMP-2 in ARP/ARR and ISD interventions. Future investigations should focus on conducting well-designed clinical trials that assess clinical, implant-related, digital imaging, histologic and patient-related outcomes in relation to the use of biologics in ARP/ARR and ISD procedures versus a proper control.

C O N F L I C T S O F I N T E R E S T
The authors have no conflicts of interest related to this systematic review.