The Applications of Enamel Matrix Derivative in Implant Dentistry: A Narrative Review

Enamel matrix derivative (EMD) has been successfully used for periodontal regeneration in intrabony defects. Recently, its use for peri-implant bone regeneration has also been hypothesized. The aim of this paper is to review preclinical and clinical studies investigating the use of EMD in correspondence with titanium implants, alone or as an adjunct to other biomaterials. Clinical trials and case series with more than five cases were included. Seven in vitro studies evaluated the effect of EMD, placed on titanium surfaces: An increase in proliferation and viability of osteoblasts was observed in all but two studies. An increase in TGF-β1 and osteocalcin production, alkaline phosphatase activity, and angiogenesis was also reported. Nine animal studies investigated the use of EMD at implant placement or for bone regeneration of peri-implant bone defects, and some of them reported a significant increase in bone formation or bone-to-implant contact. In four of eleven clinical trials on humans, EMD was successfully used at implant placement. The other seven evaluated the use of EMD in protocols for the treatment of peri-implantitis. In conclusion, the results of EMD seem promising, but further randomized clinical trials are needed to evaluate its efficacy.


Introduction
Enamel matrix derivative (EMD) is a purified acid extract of proteins extracted from porcine enamel, that were introduced after a long period of testing as a biological medium for enhancing periodontal tissue regeneration [1,2]. The major component (more than 95%) of EMD is represented by amelogenins, a family of hydrophobic proteins that constitute the unique active component of the product [3]. Amelogenins are involved in a number of biological functions that are related to the regulation of fibroblasts and osteoblasts, enhancing their activation and, ultimately, their activity [3]. Such proteins are normally present during the development of the attachment apparatus of the tooth and have been proven to promote regenerative responses in the periodontal ligament. The mechanisms have not been fully explained, but it is known that amelogenins, when applied to a conditioned root surface, precipitate to form an insoluble extracellular matrix with high affinity for hydroxyapatite and collagen, which can interact with the surrounding cells and thus initiates the periodontal regeneration [3].
The use of EMD was widely validated in periodontal surgery by a number of systematic reviews of the literature [4][5][6][7]. The effect of such biomaterial was demonstrated for the regeneration of periodontal intrabony defects, alone or as an adjunct to bone substitute material, even when using mini-invasive surgical approaches [6]. Indeed, in one recent systematic review of the literature EMD performed better than platelet derivatives and better than the bone substitute alone in regenerative/reconstructive procedures [6].
Interestingly, a growing evidence supported the use of EMD in the surgical treatment of gingival recessions, associated or not with connective tissue graft [4]. The systematic review published in 2021 on the use of EMD to treat Miller class I or II gingival recessions proved the beneficial effect of EMD application for all the techniques tested [4].
Since the effects of amelogenins on cells involved in periodontal regeneration found substantial support in the literature, the hypothesis that EMD could stimulate osteoblast and bone regeneration has been studied both in recently published animal [8,9] and human studies [10,11]. Jung et al. [9] compared the effect of EMD-liquid as an adjunct to biphasic calcium phosphate (BCP) in a rabbit calvarial model, as evaluated through micro-CT and histomorphometric analysis. When EMD was applied, new bone formation was significantly higher and the material area was significantly lower, indicating accelerated graft degradation. A higher amount of newly formed bone at 6 months was also observed in a human RCT investigating the effect of EMD in association with deproteinized bovine bone mineral (DBBM) as compared to DBBM alone for maxillary sinus floor elevation [10]. The same results were obtained at 4 months in another RCT testing the same biomaterials for ridge preservation after maxillary anterior teeth extraction [11]. A recent in vitro study [12] investigated the effect of EMD on human osteoclasts precursors after interaction with activated endothelium, finding a concentration-dependent inhibition of osteoclastogenesis. This suggests that EMD could affect bone resorption, thus promoting bone regeneration during periodontal therapy.
The aim of the present study was to present a narrative review of the scientific literature about the use of EMD in correspondence of titanium implants, also evaluating laboratory studies with methods compatible with the purpose of the research.

Materials and Methods
Although the present is a narrative review of the literature, we aimed at performing a thorough review of the existing literature by combining appropriate keywords (MeSH terms/emtree terms and free text strings) (Appendix A) in order to identify all the studies published about the behavior, in preclinical and clinical studies, of enamel matrix derivative alone or as an adjunct to other biomaterials when used in association with dental implants. Moreover, we searched for studies investigating the application of enamel matrix derivative for the treatment of peri-implant inflammatory diseases (peri-implant mucositis and periimplantitis).
We decided to include laboratory in vitro studies, animal studies, and clinical studies on humans.
The primary outcome we would like to assess was the efficacy in the experimental settings of their use, as compared with other treatment options, in similar clinical conditions. As for clinical studies, randomized controlled clinical trials, prospective and retrospective studies with an adequate study design were included. Case reports and case series with less than five cases were excluded.
Due to the relatively low number and the heterogeneity of the design of studies dealing with the predetermined focus of the present review we decided to present the results of the studies included in a narrative and critical manner, avoiding performing a meta-analysis or quantitative synthesis.
The authors reported the results of the studies separately depending on the type of the study included (in vitro, animal studies, and clinical studies on humans). For each study, the following parameters were extracted and considered: Name of the authors and year of publication, tested cells (in vitro study), characteristics of the implant, animal model (in animal studies) surface, characteristics of the population, methods and synthesis of the results obtained.

Results
The search we performed resulted in 27 papers to be considered, that are described analytically in the following paragraphs.

In Vitro Studies
Seven in vitro studies observed the behavior of enamel matrix derivatives on titanium surface, evaluating the ability of inducing and promoting osteoprogenitor cells [13][14][15][16][17], primary gingival fibroblasts [18] or endothelial cells [19] (Table 1). The study by Schwarz et al. published in 2004 investigated the effects of EMD on human osteoblasts-like cells attachment, viability, and proliferation on titanium implants with sand-blasted acid-etched surfaces (SLA), evaluating the samples for 6 days [13]. The authors found that cell proliferation and viability were improved by EMD exposure following a concentration-dependent pattern, having better outcomes after 6 days.
On the other hand, one study published in 2007, where rat osteoblasts were cultivated on either phosphate and non-phosphate titanium discs with or without the adjunct of EMD [14], did not reveal any significant advantage of one group over another one, even though it was observed that EMD could initiate an early TGF-β1 production, without any difference in the medium and long period.
In one similar study performed by Miron et al. in 2010, rat calvarial osteoblasts were cultured on titanium surfaces with or without EMD up to 4 weeks [15]. In the particular conditions of the research EMD proved to be effective in significantly augmenting cell spreading at 2 and 4 h and the proliferation of osteoblasts after 3 to 7 days, independently from the titanium surface substrate (smooth or SLA). The authors also registered an increase in alkaline phosphatase activity and in osteocalcin gene expression.
The same increase in alkaline phosphatase activity and osteocalcin production was found in one study by Qu et al. in 2011 [16], where MG-63 osteoblast-like cells were seeded on disks of SLA titanium and cultured for 7 days, and then they were 'stimulated' with EMD for 48 h. However, the authors reported a decrease in osteoblasts proliferation with high EMD concentrations within this period of observation.
The study by Shi et al. published in 2017 reported a similar decrease in cell proliferation on acid-etched and SLA surfaces after 48 h. Moreover, the authors presented data on the ability of EMD to stimulate angiogenic behavior on titanium disc surfaces (smooth, acid-etched or SLA) of cultivated endothelial cells [19]. Interestingly, EMD appeared to stimulate angiogenesis on SLA titanium.
Gingival fibroblasts proliferation on SLA and smooth surfaces coated with EMD was studied in the paper published in 2016 by Wang et al. [18]. The preparation of EMD was similar to what was presented in previous studies [15,16]. Cell adhesion, proliferation, and morphology was examined for up to 5 days after exposure. On all the surfaces examined, EMD was able to increase cell proliferation of gingival proliferation on SLA and smooth surfaces.
More recently, the study by Ramenzoni et al. published in 2020, aimed at evaluating the response of MC3T3 osteoblast-like cells cultivated on SLA titanium disks with EMD or dentin matrix derivative (DMD) or left uncoated [17]. Interestingly, EMD and DMD acted similarly in the experimental conditions, both increasing the osteoblasts proliferation and activity.

In Vivo Studies on Animal Models
Nine studies examine the behavior of EMD in the presence of titanium surfaces in animal models (Table 2) [20][21][22][23][24][25][26][27][28].  Three animal studies used evaluated the effect of EMD at implant placement through similar protocols [20,22,25]. The EMD application was evaluated in rats in the study by Shimizu-Ishiura, published in 2002, by wetting titanium mini-implants at the time of placement [20]. Rats were sacrificed after 4, 7, 14, and 30 days, finding that EMD was able to enhance trabecular bone formation around titanium implants after implantation.
No differences in bone formation between the test (implants wetted with EMD at the time of implant placement) and negative control group were found in the study published in 2003 by Franke Stenport and Johansson on 36 commercially pure titanium implants placed in rabbits [22].
In an animal study performed by Birang et al. in 2012, 12 titanium implants were positioned in Iranian dogs, half of them wetted with EMD before insertion [25]. Bone formation was observed after 4 weeks from the surgery in both groups, but the osteoblasts activity was more pronounced in the EMD group.
The application of EMD in implant placement procedures was also part of the protocol called 'socket-shield technique' described by Hurzeler et al. in 2010, presenting the results of one study in one beagle dog [24]. In detail, implants were placed with or without contact with the radicular fragment and EMD was applied: Osseointegration occurred in all implants, together with the formation of new cementum for implants placed in direct contact with the radicular shield.
The paper by Casati et al. studied the histomorphometric results of EMD (with or without GBR) application for the treatment of peri-implant bone dehiscences in mongrel dogs, created ad hoc at the time of implant placement [21]. Bone-to-implant contact (BIC) was higher in test groups than in the negative control group, but without any statistically significant difference. Interestingly, no differences could be found between GBR alone and EMD alone, thus demonstrating a substantial bone healing stimulation with EMD.
Similarly, the study by Craig et al., published in 2006 in minipig studied EMD in conjunction with dental implants, in the presence of cultivated periodontal ligament and gingival connective tissues cells, for filling ad hoc created circumferential bone dehiscences [23]. The histomorphometric results found that the amount of bone-to-implant contact could be positively influenced by the presence of EMD.
The use of EMD in conjunction with bone substitutes such as deproteinized bovine bone mineral (DBBM) and biphasic calcium phosphate (BCP), in the presence of periimplant bone dehiscences was studied in the research published in 2015 by Wen et al. [26]. Even though an effect of EMD was observed on bone regeneration, no significant differences could be observed as compared to the control groups.
BCP embedded with EMD was examined also in the study published by Lim et al. in 2016 [27]. In five mongrel dogs, peri-implant defects were created and filled with BCP alone, EMD alone or EMD and BCP, and bone regeneration was evaluated over time. No additional effect was observed for EMD and BCP-EMD groups, with regards to bone-toimplant contact and bone fill.
The ability of EMD to enhance bone regeneration when associated with DBBM for the treatment of vestibular peri-implant bone dehiscence in six beagles was studied recently by Ikawa et al., which found that EMD could improve significantly the augmented area and the bone-to-implant contact [28].

Clinical Studies on Humans
The search retrieved eleven papers on the use of EMD in the presence of dental implants in humans (Table 3) [29][30][31][32][33][34][35][36][37][38][39]. The studies reported the results both on soft and hard tissues.  Four studies evaluated the effect of EMD at implant placement [29,32,38,39]. One clinical report by Cangini and Cornelini, evaluated EMD as compared to the bioresorbable collagen membrane for enhancing bone and soft tissue healing around implants positioned immediately after extraction [29]. In 32 patients, half of the space between the implants and socket walls was filled with EMD and half covered with the bioresorbable membrane, and periodontal parameters were evaluated after 12 months, observing that the use of membrane performed better than EMD in this particular condition.
The effect of EMD on soft tissues after implant placement was the object of one clinical study published in 2015 on a total of five patients [32]. The histologic outcomes of biopsies retrieved 14 days after implant placement showed that EMD caused an increase of microvessel density as compared to control sites.
More recently, the research by Cardaropoli et al. was focused on the EMD application at the level of the hard and soft tissues, at the time of immediately loaded screwed restoration placement [38,39]. The soft tissues esthetics were satisfactory and stable after one year, together with the height and width of the bone crest evaluated through radiographs and CBCTs.
The other papers focused on the use of EMD within clinical protocols for the treatment of peri-implantitis, with a non-surgical [33,35] or surgical approach [30,31,34,36,37]. The case series by Froum et al. in 2012 described a protocol for the treatment of peri-implantitis that involved the use of EMD and presented the results of 3-7.5 years of such treatment on 51 affected implants [30]. Even though the results of such protocol appeared encouraging, the effects of EMD on bone regeneration could not be evaluated separately. The report was updated in 2015 (on 170 implants in 100 patients with 2-10 years follow-up) with similar results [31].
In the study by Faramarzi et al. published in 2015, EMD was compared to minocycline spheres as an adjunct to nonsurgical therapy for the treatment of peri-implant mucositis [33]. A total of 64 implants (divided into three groups) were treated and then followed-up for 3 months, evaluating clinical and microbiological outcomes. Both the use of EMD and minocycline resulted in better outcomes than the control group as an adjunct to the nonsurgical treatment of peri-implant mucositis. Another study of the same research group compared, with a similar protocol, EMD plus nonsurgical treatment and nonsurgical treatment alone in 46 patients with peri-implant mucositis [35]. After 3 months, the clinical and laboratory parameters improved significantly in the EMD group as compared to controls.
Isehed et al. published the results of one randomized controlled clinical trial on the use of EMD after surface debridement and decontaminations of implants presenting a bone dehiscence due to peri-implantitis [34,36]. The study was performed on 26 subjects and they were followed-up for up to 5 years. After 12 months, the EMD group showed increased marginal bone level than the control group and after 5 years it was confirmed that EMD was appositively associated with implant survival over time.
Moreover, the treatment of peri-implantitis by means of the adjunctive use of EMD was the objective of the study by Esberg et al. [37]. The protocol was the same as used by Isehed et al., namely open flap debridement and decontamination, with or without the adjunct of EMD before flap closure. It was demonstrated that EMD is related to implant survival over time, expressing a different proteomic expression profile.

Discussion
The present study aimed at understanding the efficacy and effectiveness of the use of EMD in correspondence of dental implants and peri-implant tissues in general.
The available scientific literature did not allow performing a meta-analysis, due to the large heterogeneity among the studies and to the low number of randomized clinical trials. However, we attempted to provide narratively the outcomes of the research in this particular field.
The laboratory studies included in the review were mainly about the effect of EMD on osteoblasts activity in general on titanium surfaces, in most studies SLA. The effect of EMD on cell proliferation was recognized in a substantial number of laboratory studies [40]. Indeed, it was observed that EMD in a liquid carrier could significantly improve osteoblasts and periodontal ligament cells growth and differentiation, with more expressions of genes codifying for collagen and osteocalcin [41]. Such stimulation was observed also towards the expression of connective tissue growth factors in periodontal ligament cells [42,43]. In general, the activity of EMD on the adhesion, proliferation, and activity of cells involved in bone regeneration was evaluated in addition to bone substitutes, such as DBBM, etc. [44][45][46]. With regards to the adsorption of EMD to the surfaces of the bone substitute material, a liquid carrier was demonstrated to be more efficient in the laboratory conditions, augmenting the surface of the grafting material coated by EMD [44]. In our review, the results of most laboratory studies confirmed that fibroblast and osteoblasts stimulation by the EMD application could be obtained also on titanium surfaces, with a significant effect as compared to controls, despite the fact that controversial results were obtained in two of the included studies [16,19].
Studies on animals are required to test the outcomes of bone grafting procedures performed using bone substitutes or other agents that aimed at increasing the regenerative potential [47].
Due to the heterogeneity in the materials and methods of the considered studies, it is not possible, in the present review, to find a substantial and proven effect of EMD on animal models on bone regeneration. It should be highlighted that EMD was safe, and its performances resulted at least non-inferior as compared to the control groups in two studies [20,21]. It is notable that in all animal studies, the defects were created ad hoc, and they were not of infective origin.
Several clinical studies reported the results of using EMD associated with dental implants placed in humans. As stated before, it was not possible to perform any quantitative synthesis of the results, and so the outcomes should be considered with caution, since in general the level of evidence was low.
In the studies included in the present review, EMD was tested for bone regeneration, for improving the soft tissue healing, and in protocol for the treatment of peri-implant mucositis and peri-implantitis. In general terms, EMD could improve bone-to-implant contact in immediately positioned implants even though the long-term effects on bone stability and on implant survival need to be confirmed by more studies.
The results of EMD were very promising, in the clinical studies included in the review, as an adjunct to other therapies for the treatment of peri-implant mucositis or peri-implantitis. The research performed by Froum et al., whose results were published in two papers, proposed the use of EMD in a complex surgical treatment protocol for peri-implantitis that included flap reflection, surface decontamination with chlorhexidine or a dedicated brush, platelet-derived growth factor, and guided bone regeneration with a bone substitute [30,31]. The results of the protocol proposed were similar to those presented in scientific literature after treatment of peri-implantitis and under an adequate protocol for supportive care [48]. Moreover, the promising results of the use of EMD were confirmed in one comparative clinical trial in which EMD was used associated with open flap debridement of implant surface, although the number of cases examined in the medium term (3-5 years) was relatively low [34,36,37]. Furthermore, the same positive results could be observed in the treatment of peri-implant mucositis, as an adjunct to standard mechanical therapy and biofilm removal, although there is no clear evidence of a superior effect of such protocol over the standard treatment [33,35]. As it was reported recently, it should be highlighted that nonsurgical therapy, without any additional chemical or mechanical agents, is sufficient for the resolution of peri-implant mucositis [49].
The stability and the anatomical characteristics of the so-called 'implant supracrestal complex (ISC)' are fundamental for maintaining oral hygiene and for limiting the possibility of occurring in peri-implant inflammatory diseases [50]. The EMD are adopted for increasing the stability and the health status of ISC in general, showing a substantial improvement in soft tissue healing but we have no information on the long-term clinical outcomes, since a number of factors could be more important in determining implant success over time.
Although the review found a moderate support in scientific literature on the use of EMD in the presence of dental implants, the results of the studies included should be considered with caution and the limitations of the present study have to be acknowledged. First, we had no possibility of performing any quantitative synthesis of the results, so the outcomes of each study could not be pooled together. Then, the studies included presented a substantial heterogeneity in study methods and in the protocols adopted, and this aspect does not allow proposing any standard of care. Moreover, animal studies presented a low evidence of the efficacy of EMD in laboratory conditions.

Conclusions
In conclusion, the present paper found a sparse evidence on the efficacy of the use of EMD for increasing bone regeneration and as an adjunct for the treatment of peri-implant diseases. However, in all studies, the EMD application was demonstrated as safe and not inferior to control protocols with regards to both clinical and laboratory outcomes. The promising, although limited, results might deserve to be studied further, in welldesigned controlled clinical trials with a medium to long follow-up period and adequate sample size, in order to improve bone regeneration in particularly challenging conditions as peri-implant tissue.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study is contained within the article.