1. IntroductionThe true concept of “biomimicry or biomimetics” is to develop manmade design while taking inspiration from nature [1]. Biomimicry is a Latin word (bios, meaning life, and mimesis, meaning to imitate), envisioned as a completely or partly induced biological phenomenon [2]. In the medical, dental, biotechnological, and pharmaceutical fields, the failure of conventional materials is due to the lack of the ability of these materials to follow a cellular pathway to fit in with biological systems [3].In the 1950s, Otto Schmitt a biomedical engineer introduced the term “biomimetic” [4,5]. It is the Latin word “bio” meaning life, and “mimetic” is related to simulating or mirroring nature. The objective besides biomimetics was to produce biological materials and procedures that mimic nature [4,6]. Accumulation of inorganic ions with organic protein molecules is the basic concept of novel biomimetic approaches [7,8]. Therefore, the biomimetics approaches have involved the multi-translational areas of bioengineering, biology, chemistry, and materials sciences. Moreover, in the fabrication of various biomimetic materials, nanotechnology plays a major role [5,7] Clinically, biomimetics refers to mimicking the physiognomies of a natural tooth repair of affected dentition through biomimetic procedures and materials [7,9]. For example, to improve the osseointegration of dental implants, biomimetic-dental-implant coatings of calcium phosphate (CaP) and hydroxyapatite (HA) have been investigated and implemented [10,11]. Similarly, the biomimetic process is applied in adhesive-restorative materials that demonstrated esthetics mimicking natural teeth and tooth morphology. During the last decades, the restorative approach has steadily evolved, progressing from mechanical retention to advanced adhesion. Composite-resin materials and adhesive dentistry have become valuable tools on this context. The principles of biomimetic dentistry impose introduction of advanced composite-restorative materials to clinical practice, which should align with the nature and integrity of the tooth tissues [12,13]. Regeneration of oral tissues demonstrated promising results in tissue-engineering approaches [14,15,16]. Moreover, various endodontics procedures, including formation of dentin barrier by pulp-capping, root formation during apexogenesis or apexification, apical healing by root-end fillings, and pulp regeneration by cell-homing strategies [17,18], involve biomimetic approaches in endodontology.Biomimetic dentistry is the art and science of restoring or repairing damaged teeth with various approaches that mimic natural dentition in terms of aesthetics and function. These approaches involve minimal invasive-dental management by the use of bioinspired materials to achieve remineralization [5]. Regenerative endodontics and tissue engineering are emerging and have the potential to repair damaged or partially developed teeth with normal pulp-dentin tissue [19]. This concept works by offering a natural extracellular matrix (ECM) simulating environment, signaling molecules, stem cells, and scaffolds. Consequently, the absence of pathology, pain, and the formation of root dentine is well-evident which indicates clinical success [20]. Contemporary endodontic regeneration involves a revascularization process in which the root-canal system is disinfected using the intracanal medicaments and a blood clot is formed by stimulating the tissues of the root apex. The presence of blood clots mimics a natural scaffold inside the root canal that facilitates the proliferation and differentiation of the pulp-dentin stem cells [20,21]. Moreover, the current concept of cell-homing supports the recruitment of pulp-apex tissue by endogenous-mesenchymal-stem cells [22,23]. In addition, several macromolecules are investigated to recruit endogenous-pulp cells by different approaches, including chemo-attractants, platelet-rich plasma, and ECM molecules [24]. Ideally, dental biomimetic materials should mimic the properties and functions of different parts of the tooth [25], sharing a common goal to recreate biological tissues and emphasizing using materials that simulate the biological effects of oral structures [26]. Commonly used biomaterials for dental-pulp-tissue engineering are collagen or poly(lactic) acid and hydrogel scaffolds. It is difficult to administer collagen into narrow pulp space. Moreover, to engineer dental pulp, various bioactive biomaterials, including synthetic and natural hydrogels, have been investigated for suitability [27], which are discussed in the following sections.The ultimate outcome of regenerative endodontics is enhanced patient management which could be done by various strategies that translate the biological aspects of the regeneration of pulp into the clinical aspects. These clinical protocols varied from relating the natural ability of the pulp to heal to regenerating the affected pulp-dentin complex or achieving revascularization of the empty-root canal [20]. Therefore, the aim of this paper is to thoroughly review and discuss various biomimetic approaches for pulp regeneration and endodontic applications. In addition, current trends and future research prospects for translating biomimetic approaches to clinical endodontic applications are elaborated. 2. Development of Regenerative Endodontic Procedures (REP)In 1961 Nygaard-Østby explored, for the first time, the concept of treatment of necrotic pulp by regenerative endodontics [28]. The definition of regenerative endodontic procedures has been given by Murray and Gracia as “events based on biological design to substitute missing, diseased, underdeveloped or damaged components of the tooth structures including root and dentine structures to restore physiological functions of pulp dentine complex” [29,30]. The complete assembly involved in the regenerative endodontics procedures are stem cells, signalling molecules, and scaffolds harvested on the extracellular matrix (ECM) [20,21,31,32]. The essential goal in REP is to promote pulp-tissue regeneration, development of roots, and proliferation of the progenitor-stem cells from the bone/tooth region [33]. In the apical papilla, these osteo/odonto-progenitor-stem cells prevent the infection and necrosis of the root that is caused due to the proximity of the periodontal-blood supply [34]. In addition, REP may influence angiogenesis, cell survival, differentiation migration, and proliferation. Using mesenchymal-stem-cells-markers, regenerative endodontics procedures have been shown to have diverse potentials [35,36]. During the differentiation of endothelial-progenitor cells and the revascularization process, the immunostaining technique was used to identify the abundance of CD31/collagen-IV and vascular endothelial growth factor (VEGF), R2/Collagen-IV (10). Despite of lack of clinical trials of regenerative endodontics procedures in the literature, this modality of treatment is appreciated by clinicians globally. In dental-pulp regeneration, the necessary cells can be delivered either by cell transplantation or by cell homing [37,38]. A study conducted by Torabinejad et al. [39] found that, for successful pulp regeneration after the revascularization procedure, the presence of a 1–4 mm uninflamed tissue was beneficial. The study was conducted on immature- animal teeth [39]. Complete regeneration of pulp tissue with capillaries and neuronal cells have been found in the regeneration of canine pulp within 14 days in 2009. Iohara et al. [40] transplanted scaffolds loaded with collagen fibers (type I and III) and dental-pulp- stem cells. Moreover, after transplantation of the scaffold alone, there was “no engraftment at the pulpotomy site” that has been found [40]. Souron et al. [41] used rats’ molars in their study. They transplanted rats’ pulp cells in a scaffold comprised of type-I rat collagen. After one month of implantation, the living and mitotically-active fibroblasts, new vessels, and nerve fibers were observed where the pulp was seeded with cells, whereas a lack of cell colonization was found where the pulp was seeded with lysed cells [41].In another study conducted by Jia et al. [42], simvastatin was injected, which is an inhibitor of the competitive 3-hydroxy-3-methylglutaryl coenzyme-A reductase. The scaffold used in their study was a gelatin sponge together with dental-pulpal-stem cells on extirpated pulps [42]. Simvastatin boosted the mineralization process and regeneration of the pulp and dentin after 10 weeks of implantation [42]. A combination of poly(l-lactic acid)/Matrigel scaffold with bone-marrow-mesenchymal-stem cells were placed on the extirpated pulp in a study conducted by Ito et al. [43] on immunosuppressed rats. A total of 15% EDTA and 1.5% sodium hypochlorite were used for rinsing the pulp chamber. After 14 days of implantation, complete pulp regeneration along with nestin-expressing odontoblast-like cells beneath the dentin was demonstrated. Nestin is a type VI intermediate-filament protein originally present in neural-stem cells [43]. To answer this controversy in today’s dentistry, American Association of Endodontics (AAE) considered regenerative endodontics as the most thrilling innovative expansion [35,44,45]. 3. Revascularization or RevitalizationTeeth with apical periodontitis and immature root apex having periapical infection underwent the revascularization process in 1971 [46]. However, due to limitations in materials, instrumentation, and techniques, this attempt failed. However, with the constant innovations and developments of techniques, materials, and instruments now, several case reports [47,48] have used and incorporated this technique into everyday use with success. The process of revascularization technique is different from both apexification and apexogenesis [47,48]. Apexification is defined as ‘an apical barrier to avert the route of toxins and bacteria into periapical tissues from root canal” [5,49]. In most pulp-diseases scenario and apical periodontitis, calcium hydroxide is used. Due to its improving success rate, easy availability for the clinician and affordability for patients, it is considered one of the most important medicaments that have shown promising results [50,51]. Traditional apexification procedures were the only option for clinicians to treat pulpal necrosis of immature teeth before 2004 which presents a unique challenge to the dentist. Calcium-hydroxide dressing was considered the primary material to be used in these traditional apexification-treatment procedures. Apexification has proven to be highly foreseeable [5]. However, the disadvantage of this procedure is that over a period of months, it requires multiple appointments in addition to the higher incidence of cervical fracture [19]. ProRoot Mineral Trioxide Aggregate (MTA) is used in the artificial-apical- barrier technique to facilitate root-canal-obturation procedures [49]. When the pulp is inflamed with an incompletely developed tooth, apexogenesis is carried out [52]. Apexogenesis is a technique that discourses the inadequacies involved with capping the inflamed dental pulp. The objective of apexogenesis is the conservation of vital pulp tissue so that continuous development of roots with apical closure may occur. Calcium-hydroxide paste is placed as a wound dressing after removing most or all of the coronal pulp [53]. In recent years the treatment of necrotic-immature teeth has been changed due to the various pros and cons of apexification and artificial-barrier procedures. Revascularization is the terminology that is used to describe the treatment of immature-necrotic teeth which involves the proliferation of the tissues in the pulp space of the involved tooth [33]. When canal space is induced with bleeding, undifferentiated mesenchymal-stem cells accumulate significantly [54]. Thibodeau et al. and Wang et al. conducted various animal studies treating immature teeth with triple antibiotic paste and using the blood-clot technique in which the histopathological evaluations of the canal space have shown cementum and bone formation [55,56].When the conventional apexification and apoxgenesis methods were compared with the regenerative endodontics in a retrospective study on immature-necrosed teeth, the survival rate of the revascularization-treated teeth was the highest [57]. However, other studies concluded that due to weak root structure in a significant number of cases the reliability, the success rate of these procedures was significantly poor [58]. Kahler et al. [59] concluded that the outcomes of 16 clinical cases [59] in which they compared conventional disinfection approaches with regenerative blood-clot induction. In this study, the authors found that continued root maturogenesis was reported in only two cases when observed radiographically. In the blood clots, Gomes-Filho et al. [60] incorporated bone-marrow aspirate, platelet-rich plasma, and artificial-hydrogel scaffold along with a basic fibroblast-growth factor. They took infected, fully developed, and over-instrumented teeth and found that the addition of PRP and bone-marrow aspirates into debrided root canals did not significantly improve tissue ingrowth. Moreover, they concluded that revascularization procedures in humans did not enhance the results by the addition of an artificial hydrogel scaffold combined with the basic fibroblast-growth factor [61]. A permanently immature tooth having apical periodontics and a sinus tract was treated with a revascularization technique in contrast with the apexification process; a positive enhancement of the results was demonstrated by Iwaya et al. [48,62]. In the case of necrotic pulp, an endodontic procedure was carried out to rejuvenate tooth vitality known as “revitalization”, while the replacement of lost or damaged pulp-dentin tissue complex is known as “regeneration [29]. However, the underlying mechanism for regeneration of the dentine-pulp complex is poorly understood. Instead, root-canal therapy may undergo a repair/healing process [63]. 3.1. Advantages of the Revascularization Approach

Technically simple approach.

There is no need of using expensive biotechnology due to currently available instruments and medicament techniques.

There are almost negligible chances of immune rejection as this approach relies on the patient’s own blood.

Bacterial microleakage can be eliminated through the induction of stem cells into the root canal space, followed by the intra-canal barrier, inducing a blood clot.

The concerns of restoration retention need to be overcome.

When this approach is applied to immature teeth, it reinforces their root walls.

As the avulsed immature tooth has necrotic-pulp tissue along with an open apex, and short and intact roots; therefore, the newly formed tissue will easily reach the coronal-pulp horn because proliferation in a short distance is required. Therefore, the strategy behind the development of new tissue is to maintain the balance between the pulp-space infection and the proliferation of new tissue.

Additional growth of open-apex root takes place due to minimum instrumentation that will preserve viable pulp tissue.

The potential to regenerate more stem cells and the rapid capacity to heal the tissue in young patients needs to be recognised (Table 1). 3.2. Disadvantages of the Revascularization Approach

The origin of where the tissue has been regenerated from is yet to be known.

According to researchers, effective composition and concentration of cells are mandatory for tissue engineering. However, these cells are entombed in fibrin clots; therefore, researchers do not rely on blood-clot formation for tissue engineering function.

Treatment outcomes will be variable by the variations in the composition and concentration of the cells [64,65,66,67] (Table 1). 3.3. Prerequisites for Revascularization Approach (Figure 1)

Revascularization studies have established the following prerequisites:

There should be open apices and necrotic pulp secondary to trauma.

In addition, open apex should be less than 1.5 mm.

The following agents can be incorporated to remove microorganisms from the canal.

○

Antibiotic paste

○○

The coronal seal should be effective.

There should be a matrix or the growth of new tissues.

When trying to induce bleeding, anaesthesia should be used without a vasoconstrictor [70].

Canals should not be instrumented.

Sodium hypochlorite should be used as the irrigant.

There should be blood-clot formation (Table 1). Figure 1. Requisite preconditions for pulp regeneration (root canal disinfection and enlargement of the apical foramen) [71]. Figure 1. Requisite preconditions for pulp regeneration (root canal disinfection and enlargement of the apical foramen) [71]. 4. Postnatal Stem Cell TherapyBone, buccal mucosa, fat, and skin are the common sources of postnatal-stem cells. After the apex is opened, the disinfected root-canal system is injected with postnatal-stem cells. This treatment is considered the simplest technique [72]. There are numerous benefits of this type of tissue-engineering technique. Postnatal-stem cells are rationally easy to harvest, and these cells can persuade the regeneration of the pulp. Moreover, these cells are easy to deliver by syringe. In addition, application of these stem-cell therapy is used in regenerative medicine since past many years, for example, bone-marrow replacement and endodontic applications [73]. However, low survival rates are one of the major disadvantages of this technique. Moreover, these cells can migrate into different locations of the body, which presents peculiar forms of mineralization [74]. For the development of dental tissues by the differentiation of stem cells, bioactive-signalling molecules, growth factors, and scaffolds are required [75]. Consequently, with only stem cells that exclude the growth factors or scaffolds, the chance of pulpal regeneration of new tissues is very low. In this approach, the chief identification of a postnatal-stem-cell source that must be able to differentiate into the diverse cell population can be obtained [74]. However, this technique is not approved yet. 4.1. Pulp ImplantationIn this procedure, after cleaning and shaping the root canal, the substituted pulp tissue is transplanted. Purified pulp-stem-cells line is among one the sources of the pulp tissue. This pulp tissue can also grow in the laboratory by cell biopsy. For this invitro technique, pulp tissues can be cultured by biodegradable-polymer nanofibers. Moreover, these tissues can be obtained from collagen I or fibronectin-extracellular-matrix proteins [76]. It has been found that further investigations are required for the proteins, such as vitronectin and laminin. However, it has been proved that for growing pulpal cells, collagens I and III are not fruitful [77]. In the root-canal system, the localization of postnatal-stem cells is a major advantage of pulp implantation. However, there are several disadvantages to this technique. It is a restriction of this technique that the apical portion of the root canal should be harvested with pulp cells. The reason behind this concept is that the sheets of the extracellular matrix are very thin, fragile, and they lack vascularity. Therefore, a scaffold that must have cellular proliferation is required for coronal delivery. If the cells are located 200 μm from a capillary- blood supply which is the maximum oxygen-diffusion distance, these cells are in danger of anoxia and necrosis. Further, in vivo investigation and controlled clinical trials are needed to explore the success rates and outcomes of functioning pulp tissue and concerns over immune responses, although this technique presents a low possibility of health risks to patients [78]. 4.2. Scaffold ImplantationFor vascularization and cell organization, pulp-stem cells must be systematized into a three-dimensional assembly. This objective can be achieved by seeding pulp-stem cells with a porous-polymer scaffold [79]. Distribution of therapeutic medicines to precise tissues can successfully be accomplished by these nano scaffolds [80]. Moreover, the biological and mechanical properties needed for proper functioning are also provided by these scaffolds [81]. In teeth that have pulp exposure, dentin chips have been introduced which accelerate dentin-bridge formation [82]. These dentin chips aid in the reservoir of growth factors and they offer a matrix for the attachment of pulp-stem cells [83,84]. In reaction to the dentine chip and the use of scaffolds, the regeneration of the pulp-dentin complex occurs. To provide structural support to the tooth it is not necessary to have a tissue-engineered pulp in the root-canal systems [85]. Polymer hydrogel, a soft three-dimensional injectable scaffold matrix, will be administrated by syringe in tissue-engineered pulp tissues [86]. They are easy to deliver into the root-canal systems and are non-invasive. Theoretically, this hydrogel provides a substrate for organized tissue structure as they are involved in cell proliferation and differentiation [87]. Recent advancements in these techniques overcome the problems associated with hydrogels. These issues comprised limited control over tissue development and formation [88]. However, further clinical trials and research are needed to explore these techniques as hydrogels are at an initial phase of exploration. Nowadays, researchers are focussing on photo-polymerizable-hydrogel development. The main advantage of these photo-polymerizable hydrogel is that the rigidity is enhanced by placing them into the tissue site [89]. 4.3. Three-Dimensional Cell PrintingThe three-dimensional cell printing technique is considered the final approach for the replacement of pulp tissues [90]. This approach can be used to position cells precisely [91]. This technique mimics the natural pulp-tissue structure. In tissue-engineering technique, to maintain and repair dentine, odontoblastoid cells should be positioned around the periphery of the pulp. Moreover, the fibroblasts support the vascular and nerve cells and should be positioned inside the pulp core. This technique required great expertise and careful orientation as during this procedure apical and coronal asymmetry is the prerequisite during the placement of the pulp tissue into the shaped and cleaned root-canal system. However, currently, this technique is not available clinically and there is a dearth of literature regarding the functionality of the three-dimensional cell-printing technique [92]. 4.4. Gene TherapyIn regenerative endodontics, gene delivery has been discussed in a recent review [73]. To promote tissue mineralization, mineralizing genes would be delivered into the pulp tissues. However, Rutherford worked on this specific field of gene delivery into the pulp tissues, although there is a dearth of literature in this context [93]. He suggested further research to improve the possible gene therapy inside the pulp after he failed in his work when he transduced pulps of ferret animals with cDNA-transfected mouse BMP-7. Researchers used the electroporation method to insert mineralizing genes into the pulp space by culturing of pulpal-stem cells. Initially, the FDA approved the gene therapy research on terminally ill humans; however, after the development of numerous tumours in a nine-year-old boy, the FDA withdrew this decision in 2003. Gene therapy arising from the use of vector systems is posing serious health hazards in contrast to gene expressions [94,95]. According to the literature, tooth development can be enhanced with bone morphogenetic proteins (BMPs). Bone morphogenetic protein-2 (BMP2) is increased during the terminal differentiation of odontoblasts expression [96]. Dentin sialophosphoprotein (DSPP) has been produced by the implantation of human recombinant BMP2 on the dental papilla. The ultimate role of this DSPP is to produce the differentiation markers of odontoblasts as well as dentin-matrix proteins. An in vivo study on the amputated pulp, a large amount of reparative dentin is also induced by the BMP2 [96]. Clinically, cell-specific and safe-gene therapy is required to accurately control this gene therapy. 4.5. Nitric OxideAmong many wound healing and pathological processes, angiogenesis is considered an important process. The most potent and critical inducer of angiogenesis is the vascular endothelial growth factor (VEGF). A variety of stimuli take part in the regulation of gene expression of VEGF. The transcription factor is a key factor for hypoxia-mediated VEGF- gene upregulation, which is achieved by hypoxia-inducible factor 1 (HIF-1). Nitric oxide (NO) is a potent vasodilator. Nitric oxide (NO) can simply pervade natural membrane obstacles because it is lipophilic in nature. This VEGF regulates the amount of nitric oxide [97]. Hypoxia as well as nitric oxide upregulate the VEGF genes by enhancing HIF-1 activity. Moreover, dendrimers are released by nitric oxide which acts as antibacterial agents [98,99]. In one case, authors conducted a study in which they evaluated dendrimers with and without nitric oxide against Gram-positive and Gram-negative pathogenic bacteria. They used polypropylene imine (PPI) dendrimers that contained nitric oxide, which was compared with controlled PPI dendrimers that did not release nitric oxide. They found that >99.99% of bacterial strain was killed by dendrimers that contained nitric oxide. They further stated that there was minimal toxicity to mammalian fibroblasts with these nitric oxide-containing X dendrimers [98]. The most necessary clinical results of regenerative endodontics can be obtained by successful disinfection along with complete endodontic-tissue revascularization and regeneration. This was studied by Moon et al. [100]. However, there are many limitations of the contemporary regenerative endodontic procedure (REP). To improve the efficiency of regenerative endodontic procedure (REP), antibiotics and nitric oxide (NO) releasing biomimetic-nanomatrix gels have been developed very recently. The gel contains many functional groups as it is made up of peptide amphiphiles. This biomimetic- nanomatrix gel was mixed with antibiotics, ciprofloxacin (CF), and metronidazole (MN), and released nitric oxide. Multispecies-endodontic bacteria were used to evaluate the antibacterial effects by using bacterial-viability assays. Animal-model experiments were used to evaluate pulp-dentin regeneration. The concentration-dependent antibacterial effect was found in the antibiotics and NO-releasing biomimetic-nanomatrix gel. Moreover, nitric oxide without antibiotics also showed an antibacterial effect on endodontic species. Tooth revascularization has been promoted by antibiotics and NO-releasing biomimetic-nanomatrix gel by an in vivo analysis. To improve the current REP, an antibiotics and nitric oxide (NO) releasing biomimetic-nanomatrix gel was developed. For the regeneration-endodontics procedure, an optimum concentration of nitric oxide-releasing nanomatrix gel is recommended [100]. The positive or negative effects of nitric oxide may be attributed by changing the amount and concentration of nitric oxide. HIF-1 and VEGF activity was negatively affected by the release of nitric oxide. The activated endothelial nitric oxide synthase (EnoS) produces VEGF-mediated angiogenesis. Akt/PKB, Ca2+/calmodulin, and protein kinase C are the pathways by which eNOS get activated through VEGF. The NO-mediated VEGF expression as well as VEGF-mediated NO production by eNOS can be regulated by HIF-1 and heme oxygenase 1 (HO-1) activity. Angiogenesis in normal tissue can be regulated by the relations between NO and VEGF [97]. 4.6. Platelet-Rich Plasma (PRP)Special challenges are faced by clinicians for the treatment of an immature tooth with necrotic pulp and open apex. One of the strategies for its treatment is the traditional apexification procedure. This treatment process requires the formation of the apical barrier by multiple applications of calcium hydroxide. This apical barrier can also be formed by placing mineral trioxide aggregate (MTA) into the canal, which is followed by the conventional root-canal procedure [33]. Due to incomplete root formation with these procedures, the chances of root fracture are very common [19,33]. Platelet-rich plasma (PRP) has been suggested as probably the greatest platform for RET that will overcome all these problems [33,101]. Platelet-derived growth factor, transforming growth factor b, and insulin-like growth factor form an integral part of the PRP [5]. PRP can be utilized as a scaffold as it can form a three-dimensional fibrin matrix. It is easily prepared from the patient’s autologous whole blood [33,102,103,104]. Growth factors and cytokines are 4-fold higher in platelets than found in whole blood [105]. Mandibular-continuity defects, for the first time, were healed by the PRP and the placement of cancellous-bone grafts by the dental community [106]. Human dental pulp stem cells (DPSCs), when treated with PRP, resulted in an increase in the differentiation and proliferation of these cells [107].In 2008, Hargreaves and colleagues [33] in regenerative endodontics encouraged the use of PRP, and for the first time in 2011, the PRP procedure was used for regenerative endodontics in a permanent, necrotic, immature, and nonvital tooth with an open apex. Infusion of PRP into the root canal up to the cementoenamel junction, followed by triple-antibiotics paste medication, was performed by Nakashima et al. [75]. They observed the closure of the apex and healing of periapical lesions after five and a half months. In addition, they also found encouraging results in electrical-pulp-testing-cold tests [25]. In a study conducted by Torabinejad M et al. [108], the authors injected PRP and observed that, after 14 months of the treatment, extirpated soft tissue was present that was evaluated through microscopy. Pulp-like connective tissue was also found in the microscopic section [108]. A study conducted on beagle dogs by Zhu et al. [109], in which authors infused PRP in endodontically prepared root canals, found the formation of cementum-like tissue and soft tissue [109]. In contrast, in a study conducted by Torabinejad et al., researchers found no significant difference when a root canal was treated with PRP in relation to soft-tissue formation [110]. In a 39-year-old female patient with necrotic pulp who has extensive periapical radiolucency and open apex after delivering PRP in a root canal, healing of the periapical lesion after 30 months of treatment was notified by the researchers [111]. There are numerous advantages of PRP treatment. During the preparation of the PRP, erythrocytes that would be responsible for necrosis after clot formation was removed [33]. For cell migration, fibrin, fibronectin, and vitronectin are required which is obtained from the formation of PRP clots [104]. Moreover, in regenerative-endodontic procedures, the optimal level of MTA placement is mandatory which can be done by the collagen matrix present in the PRP [70,108]. Before clot formation, PRP does not release growth factor until it is activated. As soon as it is activated either endogenously or through the exogenous, such as by incorporation of calcium chloride or thrombin that acts as a clotting factor, PRP will start secreting growth factors that contribute to the repair and regeneration of the tissues [104,112]. Zhang et al. [113] concluded that histologically, no significant difference was observed between blood clots and PRP. PRP can be employed in clinical cases when little or no bleeding is observed from apical tissues. The source of stem cells, their interaction, and the role of inflammatory cells in the root canal need further exploration for the advancement of regenerative-endodontic treatment (Figure 2 and Figure 3). 4.7. Cell Homing In tissue regeneration, the first concept of cell homing was presented in Lancet in 2010. The concept was based on the delivery of transforming growth factor-b3 (TGFb3) without cell transplantation. This approach was first used for the regeneration of the articular cartilage [114]. However, for dental-tissue regeneration, the idea of cell homing was introduced in 2010 [115]. During cell homing, root canal of the extracted human teeth was shaped and cleaned followed by the delivery of the growth factors, scaffold, and stem cells. Residual proteins in the root canal or dentinal tubules were deactivated in the first phase. This can be done by sterilization of extracted teeth in an autoclave. This was followed by the infusion of collagen gel into a shaped and cleaned root canal that might be with or without basic fibroblast growth factors (bFGFs), vascular endothelial growth factors (VEGFs), platelet-derived growth factors (PDGFs), nerve growth factors (NGFs), or bone morphogenetic proteins (BMPs). The animal model that was chosen by the authors was Sprague-Dawley rats in which both the experimental and control teeth were subcutaneously implanted for three to six weeks. The authors in this model observed the endodontically treated root for the dental pulp-like tissue having blood vessels. Enzyme-linked immunosorbent assay (ELISA) was performed on the soft tissues isolated from the root canal of both treated and control teeth. Various biomolecules, including dentin sialoprotein, NGF2, and von Willebrand factor, were quantified using the ELISA. Moreover, blood vessels, dentin-like tissue, and neural-like tissue are all found in human teeth after growth-factor delivery. These procedures do not involve ex-vivo cultivation or in-vivo transplantation [115]. The prime difference between the cell homing and cell transplantation approaches is that, in the latter case, for dentine/pulp regeneration, the isolated cells (stem/progenitor) from the host are transplanted into the root canal of the host. Dental pulp-like cells have been differentiated in the cell-homing approach when growth factors are recruited into the root-canal system. Cell-homing-technique-dental-organ regeneration presents a harmonizing and/or balancing approach to cell-transplantation technique and, at the same time, this strategy has shown auspicious results in animal models [23,115,116]. Hematopoietic-stem cells were militarized and transferred to different tissues or organs using active navigation in the cell-homing approach. The ultimate outcome of this process is pulp-dentin re-cellularization and revascularization. Numerous growth factors along with cell homing will result in pulp-dentin regeneration. Tissue revascularization and regeneration-cell homing consist of two distinctive cellular processes. They are differentiation and recruitment [117,118].Migration of the cells to the defective site is referred to as recruitment. However, the presence of a mesenchymal stem/progenitor having the ability to differentiate into cells that form pulp and dentine is mandatory [117,118]. When stem/progenitor cells are transformed into mature cells, this process is known as differentiation. During pulp and dentine regeneration, odontoblasts and pulp fibroblasts are formed by the differentiation of the stem/progenitor cells. These processes need to persuade the budding of endothelial cells and neural-fibril cells for angiogenesis. However, more literature is needed to establish the fact that endothelial cells are formed directly by the differentiation of the dental pulp stem/progenitor [117,118]. In a study conducted by Kim et al. [115], the researchers implanted endontically treated human teeth into mouse dorsum. Fibroblast growth factor and/or vascular endothelial growth factor (bFGF and/or VEGF) were delivered [115]. After evaluation, they found that dentinal walls of the root canal were integrated with recellularized and revascularized connective tissue. Moreover, it was found that when bFGF, bone morphogenetic protein-7 (BMP7), platelet-derived growth factor (PDGF), VEGF, and nerve growth factor (NGF) were delivered in combination, it resulted in vascularized and cellularized tissues. These results were obtained with some endodontically treated teeth in which dentine formation and VEGF antibody were found in the dentinal wall of the root canal.

Extracellular matrix with disconnected cells is found on neo-pulp tissues. This new pulp tissue appears to be dense and contains erythrocyte-filled blood vessels having endothelial-like cell lining. When bFGF, VEGF, PDGF, NGF, and BMP7 are delivered and the entire root canal is filled with dental pulp-like tissues found in microscopic images. Von Willebrand factor, dentin sialoprotein, and NGF are expanded in the ELISA after the combinatory delivery of bFGF, VEGF, or PDGF, with basal NGF and BMP7.

Figure 2. Clinical procedure for dental-pulp regeneration using a cell-free approach [119]. Figure 2. Clinical procedure for dental-pulp regeneration using a cell-free approach [119].

Figure 3. Summary of the advantages and disadvantages of the regenerative approaches in endodontics.

Figure 3. Summary of the advantages and disadvantages of the regenerative approaches in endodontics.

7. Clinical Outcomes of Regenerative Endodontics ProcedureThe clinical success of regenerative endodontic procedures, as defined by The American Association of Endodontists, is evaluated by the following three outcomes [327].

Primary goal: This is an essential goal. It consists of elimination of signs, symptoms, and bony healing.

Secondary goal: This is a desirable goal. In this, there will be increased root length and root-wall thickness.

Tertiary goal: Vitality testing is positive.

It has been demonstrated that the primary goal of regenerative endodontics is generally achievable with high probabilities (91–94%) of success [328,329]. The failures might be due to minimal filing and disinfection protocols [330]. In the majority of studies, the secondary goal of regenerative endodontics is related to the narcotic pulp of immature permanent teeth that demonstrates the thickening of the canal walls and/or continued root development. However, it has been shown that these findings are not always predictable after RET treatment. Although it has been believed that, to strengthen the fragile immature permanent teeth and restore the vitality of the damaged tissue, RET was capable of regenerating the pulp–dentine complex by improving the thickness of the canal walls and the canal space [310,328]. However, the concept that the thickening of the canal walls after RET significantly reinforce the immature permanent teeth is not established by studies. It is just a radiographic assumption. Nevertheless, in a study conducted by Zhou et al., the researchers elaborated that fracture resistance of thickened canal walls was increased in an animal study [331]. The mechanism involved is that cell migration occurs from the apical papilla and remaining residual pulp to the disinfected canal space. This will lead to the deposition of dentine on canal walls and root apex which increases the thickness of the canal wall and root length. It was suggested that cells from the remaining residual pulp [47,68] or cells from the apical papilla [332] might migrate to the disinfected canal space to deposit dentine on the canal walls and the root apex, and thus increasing the thickness of the canal walls and length of the root [68]. 9. Conclusions

It appears that in-vitro and animal experiments regarding biomimetic approaches in regenerative endodontics are widely performed, but they are still at the inception stage. The findings related to stem-cell therapy, pulp implant, scaffold implant, 3D-cell printing, and gene therapy are quite promising as positive features with regard to pulp regeneration and tissue mineralization have been observed. Despite the aforementioned advantages, future developments in pulp-dentin tissue regeneration are needed to demonstrate the functional tissue regeneration and the ultimate favorable clinical benefits. In addition, some bioactive materials seem to be favorable as they promote osteoconduction and osseointegration, and are capable to proliferate, differentiate, and mineralize the human-dental-pulp cells. However, physico-mechanical characteristics of some materials are not satisfactory and warrant further investigations. Enamel matrix and dentine matrix derivatives have also been researched and their role in the dentine regeneration is encouraging, but there is a lack of scientifically validated data.