Abstract
The paradigm shift in the endodontic field from replacement toward regenerative therapies has witnessed the ever-growing research in tissue engineering and regenerative medicine targeting pulp-dentin complex in the past few years. Abundant literature on the subject that has been produced, however, is scattered over diverse areas of knowledge. Moreover, the terminology and concepts are not always consensual, reflecting the range of research fields addressing this subject, from endodontics to biology, genetics, and engineering, among others. This fact triggered some misinterpretations, mainly when the denominations of different approaches were used as synonyms. The evaluation of results is not precise, leading to biased conjectures. Therefore, this literature review aims to conceptualize the commonly used terminology, summarize the main research areas on pulp regeneration, identify future trends, and ultimately clarify whether we are really on the edge of a paradigm shift in contemporary endodontics toward pulp regeneration.
Impact statement
Endodontic tissue regeneration is a hot research topic among tissue engineers and dentists. It aims to regenerate the lost dental pulp tissue to maintain tooth vitality. Nevertheless, the terminology and concepts are not consensual among the scientific community. This literature review aims to summarize the terminology commonly used in dentistry, summarize the main research areas on pulp tissue engineering and regeneration, and identify future trends toward the clinical application of regenerative endodontic therapies.
Introduction
Around 3.5
The cRCT includes removal of inflamed or infected pulp tissue, followed by the root canal disinfection and replacement of pulpal tissues by an inert material.7–9 A systematic review that quantified the rates of success of primary cRCT using clinical data from 2003 to 2020 indicates success rates of 92.6% after application of loose success criteria (decrease of the size of existing periapical lesion) and 82% following strict success criteria (complete resolution of periapical lesion). 10 It has been described that cRCT enables recovery of important tooth functions such as aesthetics, chewing, phonation, and swallowing. 9 Nevertheless, the general homeostasis mechanisms, such as sensitivity, defense to recurrent infections, and deposition of tertiary dentin, are compromised, which might impair the long-term prognosis of teeth that have undergone endodontic treatment. 11
The restoration of these mechanisms could return the lost properties, as long as the regenerated tissue has the closest possible characteristics to the lost organ. Numerous and diverse techniques have been applied in recent years; some have sought the reparation of specific functions, while others have attempted the complete restitution of the pulp-dentin complex (Table 1). In this way, “regenerative endodontic procedures” (REPs) were implemented in cases of pulp necrosis with immature teeth. 12 This therapy is called “revascularization” or “revitalization” (Table 1) and mainly consists of inducing bleeding into the root canal.13,14
Definitions of Common Terms Used in the Field of Endondontic Regeneration
MTA, mineral trioxide aggregate.
Nowadays, it is possible to recognize two main therapy types within the REPs pursuing full restitution of dental pulp: the induction of bleeding into the root canal, which has been introduced into clinics many years ago, and tissue engineering (TE) applied to endodontics (uses cells, scaffolds, and/or signaling molecules), which is still being studied and is not yet applied clinically. Since then, some areas of knowledge have joined in the search for a treatment, which achieves the desired restitutio ad integrum of the dental pulp, bringing with it techniques, materials, and terms that generated, as a consequence, confusion and the dispersion of the achieved results in the broad field of the investigation.
Currently, it is not clear whether both (or any) of the REPs are prone to induce the regeneration (Table 1) of tissues morphologically and physiologically identical to native endodontic tissue. In fact, an overview of the histological data supporting the outcomes of REPs reported in the literature suggests that the conclusions are often too optimistic about the effective regenerative potential of the therapies. Therefore, herein, we will seek to propose a suitable terminology, critically present the most recent advances in regenerative therapies, and identify the future challenges for REP.
Biological, Histological, and Physiological Basis of Pulp Repair/Regeneration
Dental pulp particularities
The dental pulp is a mesenchymal-origin soft tissue, rounded by specialized cells, the odontoblasts (Table 1), arranged in intimate contact with the dentin matrix. The connection established between odontoblasts and dentin explains that the pulp and dentin should be regarded a functional unit composed of histologically different elements, which is also called the pulp-dentin complex. 15 Consequently, when the pulp is under a carious attack, the dental pulp could produce reactionary or reparative dentin to hamper the bacterial penetration in the dentin (Table 1).
If bacteria continue penetrating, the damaged pulp triggers localized acute inflammation or abscess formation. 16 Meanwhile, the bacterial colonies and necrotic tissues are surrounded by immune-inflammatory cells, thus preventing the spread of pulp infection. 16 Nevertheless, histological evidence has shown that inflammation is confined to a limited pulp area within the exposure site. 17 Therefore, considering that dental pulp is entirely surrounded by the mineralized dentin matrix, except in the apical end, allowing blood supply only through the narrow apical foramen and lateral foramina, 18 in theory, the healthy pulp can be saved and remain functional if the infected pulp is removed. 16
The dental pulp, as a connective tissue, retains many characteristics of the other connective tissues of the body, including an amorphous extracellular matrix (ECM), connective tissue fibers, axons, vascular tissue, and cellular components. 9 Some particularities are noticeable in the dental pulp's ECM since it is constituted of collagenous proteins like collagen type I (56%), type III (41%), type V (2%), and type VI (0.5%). 19 Other noncollagenous proteins are also present, including osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OC), fibronectin, and osteonectin.
Moreover, dentin sialophosphoprotein (DSPP), dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix acidic phosphoprotein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) are detected only in case of compensatory mechanisms or during the formation of reparative dentin. 19 Also, growth factors as bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-β type IA and II receptors, and activin are present. 19 Moreover, four types of glycosaminoglycans are present in dental pulps: hyaluronic acid, heparan, dermatan, and chondroitin. 20
Immersed in the ECM, the other pulp elements are distributed particularly in four topographic zones: (1) the odontoblastic zone; (2) the subodontoblastic layer; (3) the cell-rich zone; and (4) the pulp core. The odontoblastic zone is the outermost layer of the dental pulp and is composed of aligned cellular bodies of odontoblasts localized immediately below the predentin. This layer also contains some capillaries and nerve fibers (Fig. 1A, B). 15 Directly below the odontoblastic zone, the subodontoblastic layer (around 40 μm) is relatively acellular, but is crossed by blood capillaries, amyelinic nerve fibers, and pulp fibroblast extensions. It may be absent in young pulps or in old pulps where reparative dentin is produced.15,19
Histological sections and schematics of normal, reparative dentin/pulp complex and regenerated dentin-pulp complex.
The subodontoblastic layer zone covers a cell-rich pulp zone wrapping the bulk of the pulp. It is rich in pulp fibroblasts and contains macrophages, dendritic cells, and a population of mesenchymal stem cells (MSCs), termed dental pulp stem cells (DPSCs). Finally, the innermost, and the largest zone, is the pulp core, which contains fibroblasts predominantly, less abundant than in the cell-rich zone, immersed in a loose connective tissue with larger capillaries and nerves. 15 Among the different cells found in the pulp, odontoblasts are the most characteristic ones. Lin et al. indicated that “a loose connective tissue without odontoblasts cannot be defined as dental pulp tissue.” 21
The odontoblasts
Odontoblasts are tall cylindrical neural crest-derived cells (NCCs) with a long lifespan, considered “the most characteristic and specialized cell of the pulp-dentin complex.”19,22 The odontoblasts synthesize the type I collagen-rich matrix that constitutes predentin and later mineralize it. 23 Odontoblasts produce biomolecules that remain sequestered in the dentin matrix during the formation of the tooth germ and can be solubilized or exposed when cariogenic bacteria or dentin conditioning during dental treatment demineralize the dentin. The bioactive molecules released and capable of diffusing through dentin following dentin demineralization are the TGF-β family, including TGF-β1 and 3, and BMP-2, 4, and 7, among others. The importance of these bioactive molecules lies in the fact that they could promote the deposition of dentin matrix when in contact with odontoblasts or odontoblast-like cells and when implanted in conditioned dentin in in vivo models.24–26
During dentinogenesis, the odontoblasts leave a cell process embedded in the mineralized dentin matrix that contributes to the dentinal tubule formation, while the cell body remains outside the mineralized matrix.22,27 Histologically, odontoblasts are distributed in palisade (Fig. 1A, B), forming a monolayer at the healthy pulp's periphery immediately subjacent to the predentin, typically in the vicinity of dentinal tubes, which contain the odontoblast cell processes (Fig. 1A, B). Their nuclei are distributed at different heights, forming a three- to five-cell high layer.22,28 They display a large nucleus at the basal end of the cell, an abundant Golgi complex, a well-organized rough endoplasmic reticulum (RER), and abundant mitochondria. 22
Moreover, in response to the invasion of cariogenic bacteria, odontoblasts induce the innate immune response and act as mechanosensory cells in nociception processes.15,19,29,30 Odontoblasts cannot be cultivated alone after isolation, requiring the presence of other pulpal elements to survive and function. 27 Organ cultures involving the entire pulp-dentin complex have been required to maintain their growth in vitro. 31
Odontoblasts can be found immunohistologically by the expression of alkaline phosphatase (ALP), 32 aquapontin 4 (AQP4)33–35 type 1 collagen alpha 1 chain (COL1), 36 DMP1,32,37 DPP, 38 DSP, 39 DSPP,32,37 enamelysin (MMP20),32,40 nestin, 41 osteoadherin (OSAD), 42 OPN, 43 OC, 44 and phosphate-regulating gene with homologies to endopeptidases on X-chromosome (Phex). However, it is necessary to consider that these markers may also be present in other neighboring cells, such as osteoblasts and cementoblasts (Tables 1 and 2).
Distribution of Odontoblast Markers in Other Cells
Symbols denote (+) expression, (+/−) low expression, (−) no expression.
ALP, alkaline phosphatase; AQP4, aquapontin 4; COL1, type 1 collagen alpha 1 chain; DMP1, dentin matrix acidic phosphoprotein 1; DPP, dentin phosphoprotein; DSP, dentin sialoprotein; DSPP, dentin sialophosphoprotein; ECM, extracellular matrix; MMP20, enamelysin; OC, osteocalcin; OPN, osteopontin; OSAD, osteoadherin; Phex, phosphate-regulating gene with homologies to endopeptidases on X-chromosome.
Odontoblasts have some similarities with osteoblasts and cementoblasts. They produce a matrix containing fibrillar collagen, noncollagenous proteins, and proteoglycans that are subsequently mineralized. 22 The main differences are related to their shape and the relationship with their produced matrices. While the odontoblasts are columnar cells with a single cytoplasmic process, osteoblasts and cementoblasts possess a polygonal to cuboidal shape (Fig. 1C, D).22,31 The cell bodies of odontoblasts differ from those of osteoblasts and cementoblasts in that they are not confined within the ECM they have secreted. 30
Currently, the restitution of the odontoblastic zone is one of the main challenges in pulp-dentin regenerative therapies. This odontoblast restitution naturally occurs in response to the processes of infection and trauma. The following section will summarize and critically review the main research findings and opinion trends related to this process of odontoblast regeneration.
Pulp response to injury and disease: the tertiary dentin
The pulp can respond to the environmental injury triggering the production of tertiary dentin that varies according to the noxa type and intensity. 30 Tertiary dentin formation results from the dentin response to abnormal stimuli, such as caries, cavity preparation, restorative materials, and exposure. 30 Two types of tertiary dentin have been described. After mild stimuli, the secretion of matrix is increased by original odontoblasts, resulting in the production of “reactionary dentin.” When the stimulus is moderate or severe and destroys the odontoblastic layer, recently differentiated odontoblast-like cells secrete a less tubular and more irregular matrix called “reparative dentin.”22,30
It has been suggested that these newly differentiated odontoblasts could be derived from any cell from the dental papilla that reaches the basal membrane (BM) of the internal dental epithelium during the pre-eruptive period.63,64 However, the accepted paradigm that MSCs are recruited and replace damaged odontoblasts after injury is still under discussion. 30
In fact, histological evidence suggests that after a moderate injury in aged pulps, the lost odontoblasts are not replaced by new ones. Instead, new cuboidal cells derived from the pulp fibroblasts replace the lost odontoblasts and deposit a “swiss-cheese” type of reparative dentin.22,30 This fact validates Hand's position, which indicates that odontoblast-like cells (Hand's fibroblast-like pulp cells or pulpoblasts), derived from the mesenchyme of the first branchial arch and the frontonasal process, are implicated in reparative dentin formation, pulp stones, or diffuse mineralization. 19 Nevertheless, after odontoblast destruction by an injury in young pulps, formation of new odontoblasts after differentiation is still possible and is proportional to the presence of NCCs.65–67
However, NCC population progressively diminishes in the pulp throughout odontogenesis, maybe due to apoptosis or higher proliferation of neighboring non-NCCs, remaining in cell niches of perivascular cells from the cell-free and cell-rich zone (subodontoblastic layer) (Fig. 1A, B) within the postnatal dental pulp and periodontal tissues, leaving open the possibility for regeneration of odontoblasts. 68
Revascularization: the commencement of confusion
To clarify, revascularization is not a treatment. It is a term born in dental traumatology to refer to healing processes that happen after replantation or an avulsed tooth with incomplete root formation, involving the recovery of the original pulp because of the reconnection of a new pulp vascularization that allows the replacement of necrosed pulp tissue. 71 Iwaya et al., in 2001, continued to use the term revascularization to refer to the process of reconnection of blood vessels during pulp healing after root canal disinfection and the use of antimicrobials in cases of pulp necrosis. 14
After Iwaya, some authors still use revascularization to refer to the reconnection of the pulp and periodontal blood vessels after an avulsion. Di Fiore and Hartwell, in 2017, reported an accidental traumatic avulsion of an immature permanent tooth (ca. 60% of regular root length formed) over 1 h. Briefly, the treatment consisted of cleaning the tooth root and the alveolar socket, and removing any surface clot. Next, the tooth was reimplanted and stabilized by attaching it to the adjacent incisors with composite resin.
Fifteen months later, the tooth was not symptomatic on clinical percussion and palpation tests, showed grade I dental mobility and undamaged gingival attachments, and responded positively to pulp vitality tests. Radiographically, root growth and normal apical development were observed, as healthy periapical and periradicular bone, small reduction of the root canal, and incomplete pulpal calcification. 72 These signs and symptoms indicate the presence of a pulpal revascularization response consequent to dental reimplantation treatment.
Considering that revascularization is not a treatment, we recommend using the term exclusively to refer to the blood vessel reconnection during the healing process, as was used by Strobl et al. in 2003, who suggested using laser Doppler flowmetry to diagnose the revascularization accurately on avulsed maxillary incisors after replantation and splinting. 73
Biology-Based Endodontic Therapies
After observing the importance of bleeding in bone healing, Nygaard Ostby started pioneer works testing the use of blood clots as a regenerative method, focusing on the root development in cases of pulp necrosis five decades ago. 74 Ostby proposed complementing conventional endodontic therapy with mild canal irrigation using a mix of EDTA with cetyl-trimethyl-ammonium bromide accompanied by formaldehyde disinfection.
After that, he proposed the induction of bleeding and a root canal filling using a gutta-percha point coated with kloroperka (Table 1), leaving the apical third unfilled to allow space for the clot, and thus facilitating tissue ingrowth. 75 His findings showed that the blood clot is replaced by fibrous tissue complemented by cellular cementum deposited on the walls of the root canal, even in teeth diagnosed with necrosis of the pulp. The similarity of these outcomes with those found after a partial pulp extirpation on vital cases encouraged him to implement this protocol. 74 Nevertheless, those results were severely contested a few years later due to the noticeable differences between the obtained tissues and the normal radicular pulp. Moreover, dentin resorption lacunae in some studied cases complemented the rejection.76,77
These facts led to the hypothesis's oblivion for decades until Banchs and Trope resumed it in 2004 using the blood clot as regenerative therapy in pulp necrosis cases with apical periodontitis. 78 For this treatment, the root canal was abundantly irrigated, and its disinfection was supplemented with a mixture of three antibiotics to eliminate pathogens until the signs of periapical inflammation were reduced. Later, performing a mechanical irritation, root canal bleeding was induced to the amelocemental junction level. After 2 years, remission of the periapical lesion and root development completion were observed. Iwaya named this therapy “revascularization,” 14 adopting the name from the clinician-intended goal of re-establishing blood flow in replanted teeth after dental avulsion or dental auto-transplantation.79–82
Huang and Lin rejected the term revascularization, assuming that vascularity was not the only process re-established in this type of healing, 82 and adopted the term “revitalization” from earlier studies13,83 to encompass all reparation therapies previously excluded. This fact leads to the use of revascularization, revitalization, regenerative endodontics, and even TE as synonymous or interchangeable terms in the endodontic literature;84,85 according to our conception, they are not interchangeable.
The aim of cRCT is to prevent or cure periapical diseases to ensure long-term dental survival. 86 In opposition, the therapies that aim to maintain or restore pulp vitality and function, exploring biological principles, namely tissue self-healing properties, biomaterials, or cells, were called REPs.21,87 The REPs were defined as “biology-based procedures designed to repair (Table 1) or replace damaged tissues, including dentin and root structures, as well as cells of the pulp-dentin complex.” 88
We propose to term “biology-based endodontic therapies” the treatments that promote the healing, induction, reparation, or regeneration of the pulp-dentin complex and the root structures (Fig. 2). This definition encompasses therapies that use inert materials such as calcium hydroxide or hydraulic calcium silicate cement, like mineral trioxide aggregate (MTA) (Table 1) or Biodentine (Septodont, Saint-Maur-des-Fosses, France), despite not having a biological origin, but can elicit a desirable biological response on the remnant mesenchymal cells.
Classification proposal of the biology-based endodontic therapies, adapted from Galler. 89 Bioceramic materials are inked in electric blue. Skin color represents the connective tissues like dental pulp and periodontium; the beige represents restoration materials. Fuchsia represents the gutta-percha. Blood is schematized in red. Stem cells were drawn in different colors because of their versatility of differentiation, while biomaterials were represented in a mixture of cyan, green, and yellow to include most of the variate options. The dark points represent the signaling molecules. Color images are available online.
Vital Pulp Treatments
Vital pulp treatments aim to maintain the vitality of tooth and support the inherent capability of repair and regeneration of the pulp to prevent additional injury.89,90 Vital pulp therapy is mainly directed at permanent teeth diagnosed with reversible pulpitis with incompletely formed roots. 91 However, recently, some studies have demonstrated the possibility of preserving pulp vitality in cases of irreversible pulpitis,16,92,93 if there is still a remnant of healthy pulp tissue on which the procedures can be performed. 93 Consequently, a new clinical pulp diagnosis terminology was proposed. 94
Pulp capping, pulpotomy, and partial pulpotomy
Pulp capping involves applying a dental material directly on exposed dental pulp, caused mechanically or traumatically, aiming to promote the interaction of endothelial cell with inflammatory cells and stem cells with pulp fibroblasts to obtain successful clinical healing. 22 The pulp capping procedures are indicated for pulp exposures caused by caries removal, trauma, or tooth preparation. Calcium hydroxide was the first option to perform pulp capping due to its high alkalinity, producing necrosis and an inflammatory reaction in the tissues. Consequently, proinflammatory cytokines and chemokines triggered the recruitment of stem cells, which differentiate into secondary odontoblasts. These secondary odontoblasts produce an adjacent, mineralized barrier that displays a tubular structure with fewer curved tubules. According to Galler, “the application of calcium hydroxide thus has several effects that enable healing by regeneration.” 89
Over time, for a pulp capping treatment, calcium hydroxide has been replaced by hydraulic calcium silicate cements, which have shown superior thickness of newly formed mineralized tissue, inducing dentinogenic mineralization and a better capability to seal the cavities. 95 Moreover, using hydraulic calcium silicate cements produces “a less distinct necrotic zone, hyperemia, and inflammatory reaction than calcium hydroxide.” 89 In the same way, Giraud et al. evaluated and reported that calcium silicate cements such as Biodentine and MTA showed a better performance than calcium silicate materials modified with resins, such as TheraCal, which formed a disorganized pulp tissue without dentin bridge formation. 96
Partial pulpotomy (Cvek pulpotomy) consists of “removing a small portion of the vital coronal pulp to remove necrotic or potentially inflamed and irreversibly damaged tissue and preserving the remaining coronal and radicular pulp tissues.” 22 Partial pulpotomy is nowadays used as an alternative to pulpectomy to treat permanent teeth with irreversible pulpitis targeting the preservation of the sound surviving coronal and radicular pulp tissues,91,93 which has shown promising results even in mature teeth.16,93 Moreover, partial pulpotomy using Biodentin has been shown to be an effective treatment for symptomatic carious exposures, even in tooth with irreversible pulpitis, for up to a year. 97
On the other hand, complete pulpotomy involves removing the entire coronal portion of the vital pulp to preserve the surviving root portion and to provide temporary relief of symptoms or as therapeutic measures. In this study, the coronal pulp is removed, and the vital pulp tissue is capped at the entrance level of the root canal orifice. In fact, in 2018, Taha and Abdelkhader reported very high clinical and radiographic success in sixty-four patients between 19 and 69 years of age, after complete pulpotomy and capping with a thin layer of Biodentine, 98 thus demonstrating that a complete pulpotomy is a plausible alternative.
Vital pulp treatments take advantage of the remaining tissues. The potential of pulp tissues to repair is determined by the regenerative ability of the resident stem cells, depending on aging and epigenetic changes, and microenvironmental factors.16,89 However, it is unknown what circumstances are necessary to initiate the regenerative mechanisms. 89 The available information indicates that “after vital pulp therapy, reversible inflammatory processes within the pulp tissue are expected to heal by regeneration, while, if increasing the stimulus intensity and delayed intervention, healing will more likely occur as repair.” 89
Nonvital Pulp Treatments
Nonvital pulp treatments are biology-based procedures that procure to restitute the dental pulp after its complete ablation. This term includes different reparative endodontic treatments, such as apexification or revitalization, and regenerative treatments, including the use of stem cells, biomaterials, or combinations.
Repair by substitution
The current category includes the endodontic therapies focused on inducing healing of periapical disease and stimulating immature roots' development without achieving the restitutio ad integrum. In this study, the newly formed tissue lacks the classic arrangement described in previous sections (topographic zones); above all, the absence of a correctly formed odontoblastic layer is noticeable. However, the deposition of hard tissues such as dentin, bone, or cement on the walls of the root canal can increase the resistance to fracture and reduce mechanical stress when biting or in trauma, due to the observed increase in mineralized root tissue. 70
Apexification
Apexification is a treatment carried out to close the open apex of an immature permanent tooth that has lost its pulp vitality. 13 This therapy aims to induce apical closure by formation of a hard tissue barrier due to application of calcium hydroxide or a hydraulic calcium silicate cement before filling the canal conventionally. 99
Calcium hydroxide has been used for apexification due to its antimicrobial properties since the release of hydroxyl ions can damage bacteria. 13 Moreover, the high pH may stimulate the formation of hard tissue. 100 Calcium hydroxide apexification involves repeated intracanal dressing to form a mineralized barrier in contact with the apical tissue over time. Nonetheless, obtaining an apical barrier is necessary to refresh the calcium hydroxide paste every 3 months, often for up to 20 months. 101 Thus, calcium hydroxide apexification is no longer the method of choice, due to problems associated with multiple visits and clinical costs. Moreover, due to its high alkalinity, when it is in contact with dentin over weeks or months, it degrades the organic matrix of dentine, compromises its mechanical strength, and increases the risk of root fractures, which has favored the search for new therapeutic alternatives. 13
On the other hand, hydraulic calcium silicate cements have shown satisfactory results on apexification treatments.102,103 The formation of a calcified barrier that can be maintained over time has been reported as a sign of clinical success (Fig. 2).104,105 However, no histological comparison has been reported between these treatments. 103 Silujjai and Linsuwanont, in 2017, reported success rates of MTA apexification of 81% after 1 year, following 29 patients. However, fracture was found to be the origin of failure in teeth treated with MTA. 106 Fractures can occur in teeth treated with the barrier technique, depending on the stage of root development at the time of pulpal necrosis due to a lack of growth and strengthening of the root walls. 107
Apexification goals never focused on regenerating the pulp tissues, but on promoting an apical barrier in an open apex tooth to receive a filling material within the root canal space (Huang 13 ).
Revitalization
Revitalization consists of eliciting bleeding into the root canal after treating a tooth that suffered from pulp necrosis and/or apical periodontitis. This procedure is known to enhance vascular tissue ingrowth and the migration of periapical progenitor cells, mainly from periodontal and alveolar bone tissues.108,109
Silujjai and Linsuwanont, in 2017, reported a success rate of 76% on the application of a revitalization treatment to 17 patients after 1 year. The main advantage of revitalization treatment is the increasing root length compared to the MTA. In contrast, unsuccessful cases exhibited apical periodontitis, which was caused by a persistent infection, attributable to the lack of mechanical cleaning recommended in this treatment modality.106,110
Some years ago, most authors referred to revitalization as regenerative therapy since the evidence showed clinical success according to the signs and symptoms.15,21,64,111 However, at the moment, histological evidence has not been reported, confirming the presence of dentin-pulp-like tissues. Instead, periodontal tissues, such as cementum, periodontal ligament, and bone, have been found (Fig. 1C, D).76,77,109 Moreover, teeth with abnormal root development after revitalization have been reported. 112 For this reason, this review has included revitalization in the repair by substitution group.
Moreover, some years back, some authors considered revitalization as TE, arguing that when bleeding is induced into the root canal, it is being filled by stem cells, scaffolds (blood clot), and growth factors,15,113 the characteristic tools of TE.114,115 It is well known that within the dental pulp, between 1% and 9% of total cells are MSCs. 30 Research has shown the stemness characteristics of cells that flow into the canal after evoking bleeding. 111 Nevertheless, its specific multipotentiality has not yet been elucidated. The reengineering of complex human anatomical structures such as dental pulp cannot be achieved with individual cells or masses of unorganized cells in a scaffold. 116
A pile of bricks does not make a functional building. Likewise, in our opinion, the formation of a blood clot is not a TE scaffold. To produce a functional TE construct, a smart design must engineer the adequate porosity, architecture, and physicochemical properties that mimic the target tissue. The blood clot can modulate the wound healing cascade, promoting the formation of reparative tissue. However, the outcomes are fairly variable, depending on defect size and individuals, ranging from nearly identical to original tissues to hypertrophic/keloid scars or nonhealing in individuals suffering from some metabolic diseases (e.g., diabetes). At the moment, the influence of erythrocyte degradation, the release of chemical mediators by platelets, or the effect of specific metabolic conditions on the formation of pulp-like tissues after evoking bleeding as therapy in endodontics is not known.
When referring to the root apex's growth after evoking bleeding into the root canal, in our perspective, the radiographic evaluation is not enough to determine whether both the mineralized and loose connective tissue resulting from revitalization therapies have the same original histologic structure and function as a dental pulp. 87 Nevertheless, revitalization potentially leads to greater tooth survival than treatment options that do not stimulate root development, such as apical plugs with hydraulic cement or calcium hydroxide apexification. 117
Repair by TE
Endodontic therapies that seek repair by TE aim to completely restore the form and function of the dental pulp based on root canal filling using scaffolds or biomaterials (with or without cells), pursuing pulp tissue regeneration (Fig. 3). That means restoring the respective vascular, nerve, fibrillar, amorphous, and cellular pulp elements, among others, in the same location and function as they had in the native pulp tissue.
Regenerative endodontic therapies.
Stem cell therapy
Currently, one of the most critical challenges to achieve the regeneration of dental pulp includes the creation of a microenvironment that recreates and guarantees a highly vascularized ECM for transplant survival. Dissanayaka et al. consider that “most current scaffolds fail to mimic the essential functions of natural ECM.” 122 Moreover, some authors argue that scaffold degradation induces the release of degradation by-products (remnants) that hamper tissue regeneration occasionally.118,123,124 Therefore, scaffold-free stem cell therapies have been investigated with the objective of inducing cells to create their own microenvironment and cell-cell interactions in the absence of any influence from an artificial biomaterial. 18
Stem cell therapy in the dental pulp has shown satisfactory results in in vivo studies.69,118 In an investigation carried out by Itoh in 2018, a scaffold-free three-dimensional cell construct was prepared by forming sheet-like aggregates of DPSCs using a mold (Fig. 3). 118 After that, the DPSC constructs were placed into treated human tooth root canals and then transplanted into immunodeficient mice subcutaneously. Histologic evaluation revealed that teeth were invaded by host tissue and were filled with fibrous tissue containing multiple blood vessels. Immunofluorescence showed DSPP+ cells close to the dentin walls, and a regenerated tissue containing STRO-1+ cells, located exclusively in the central part of the root canal, and CD31+ cells.
In another stem cell therapy study, DSPCs were arranged into scaffold-free microtissue spheroids and then prevascularized with human umbilical vein endothelial cells. The spheroids were then placed into the root canal of tooth root slices and implanted subcutaneously into immunodeficient mice. 122 This strategy yielded tooth slices containing pulp-like tissues that were vascularized, whereas the control group (consisting of empty tooth root slices) was filled with subcutaneous fat tissue. 122
Moreover, immunohistochemistry results showed a layer of odontoblast-like cells adjacent to dentin, positive for nestin and DSP. These cells showed cellular processes penetrating the dentinal tubules with DSP expression, indicating that they are functioning odontoblast-like cells producing dentin. 122 In a similar study, Kuang et al. used microspheres to deliver hypoxia-primed human DPSCs in 3 ways: ectopic (subcutaneous injection into 6 nude mice), semiorthotopic (root canal injection into 16 rabbit molars implanted to 4 pockets of 4 nude mice’ backs), and orthotopic (injected in treated maxillary first molars of 6 nude rats).69,122
In the ectopic assay, much greater angiogenesis was observed in hypoxic groups than in control groups. In the semiorthotopic assay, Hematoxilin and Eosin (H&E) staining showed a thick layer of cells at the pulp–dentin interface, while CD31 staining confirmed the presence of abundant microvessels. After 4 weeks of the orthotopic assay, histological analysis confirmed that hypoxia-primed DPSCs on nanofibrous microspheres could fill root canals with pulp-like tissue integrated with native dentin. According to the semiorthotopic assay, the dentin–pulp interface stained positively for DSPP in immunohistochemical staining, and CD31 showed greater vascularity than the control group. It was also noticeable that most microspheres disappeared after 4 weeks. 69
In a different approach, tissue strands were obtained using tubular alginate conduits to cast cell pellets. Subsequently, an ionic decrosslinking enabled to get rid of the alginate and obtain cylindrical strands of cell aggregates. 125 Although this technique has not yet been used in endodontics, it could be a suitable alternative once an adaptation to the shape of root canals is achieved.
As described in previous lines, stem cell-based therapy has been proven successful in in vitro studies. However, its translation to the clinic has shown some drawbacks. Besides the bioethical constraints to cell manipulation and cell-based therapies translation to clinics, 126 there are some technical challenges associated with the cell survival rate, the high operative costs, and the need to apply good manufacturing practices (GMPs).18,127,128
Cell homing
Cell homing is “the recruitment of endogenous stem/progenitor cells by biological signaling factors to the injury site to induce repair.”129,130 Compared to cell-based therapies, the appeal of cell-homing strategies in clinical settings is that MSCs are not required to be isolated and delivered. Moreover, endodontic cell-homing treatments would be more straightforward and economical than cell-based therapies and could be easily applied by clinicians without special training. 131
In the past years, this concept has been combined with the classic TE paradigm, and different natural and synthetic polymers have been proposed to obtain scaffolds that mimic the native microenvironment of dental pulp (reviewed in later sections) and facilitate the migration, attachment, and proliferation of resident dental stem cells. Likewise, different growth factors, from endogenous or exogenous sources, have been proposed to recruit cells. Among these strategies, fibrin- or collagen-based scaffolds in combination with dentin- or blood-derived proteins have shown promising results to promote pulp-like tissue formation in the root canal.70,132
Biomaterials
The use of cell-free approaches aims to regenerate endodontic tissues based on the intrinsic properties of biomaterials. 18 Here, the study is focused on the biocompatible materials that provide an adequate surface and microarchitecture where the remaining resident cells could adhere, proliferate, and organize.122,133 Biomaterials are usually processed into constructs or scaffolds aimed at mimicking features of native tissue ECM to recreate cell-to-ECM specific interactions. In the particular case of endodontic applications, biomaterials must be biocompatible and biomimetic; moreover, they must adapt to the intricate anatomy of the root canal and provide a structure capable of supporting vasculogenesis, neurogenesis, and differentiation in odontoblasts. Biomaterials can be grouped according to their origin into three main groups: synthetic, natural, and host-derived scaffolds. 122
Synthetic scaffolds are mainly represented by polyglycolic acid (PGA) and polylactic acid (PLA). For example, the proliferation of human dental pulp cells (hDPCs) cultured on three different scaffolds was compared. 134 PLA scaffolds induced higher hDPC proliferation than bovine collagen or calcium phosphate bioceramic scaffolds in vitro. Nevertheless, after 30 days, PLA scaffold degradation was only 6% of the total area. 134
In the same way, in vitro hDPC proliferation onto scaffolds fabricated from PGA fibers was compared to collagen and alginate-based hydrogels. 135 PGA increased hDPC density and induced the formation of dental pulp-like tissue (DPLT), showing substantial collagen deposition. While hDPC proliferation on collagen hydrogels was only moderate, on alginate was very low. According to these authors, the remaining PGA was minimal, while collagen was present in the newly formed matrix. 135 The mechanical toughness of these biomaterials makes them easier to implant into the root canal than the scaffold-free strategies. 136 However, these scaffolds showed poor tissue regeneration abilities and incomplete formation of hard tissues, 137 did not promote odontoblastic differentiation, 134 and showed a slow degradation.134,135
A wide range of natural biopolymers from animal, vegetal, and bacterial origin138–143 has also been explored for endodontic regeneration, being the most commonly used collagen or gelatin,28,144–149 hyaluronic acid,150–152 and chitosan.143,153,154 Scaffolds produced with these biopolymers typically show better biocompatibility and biofunctionality than synthetic biomaterials due to their native ECM-mimetic structure and degradability. 18 However, the biopolymers, particularly the animal origin ones, have a higher possibility of prompting an immune response or spreading pathogens if not correctly prepared.18,133
In recent studies performed by our group, we have combined natural biopolymers with platelet-rich products, a milieu of growth factors and scaffolding proteins, 155 for different TE applications.120,156–158 In the case of endodontic regeneration, we investigated an injectable hydrogel based on self-setting hyaluronic acid, supplemented with platelet lysate (Fig. 3). 120 After 14 days, injected into tooth slice organs with hDPCs, immersed in osteogenic medium, almost all hydrogel was degraded and replaced by DPLTs attached to the walls of the root canal disks. Moreover, platelet lysate incorporated in gelatin-based microspheres promoted microvascular formation in vivo, which is necessary for successful endodontic regeneration. 159
It is essential to differentiate the provisional matrix (clot) that is formed after the provoked bleeding used on the revitalization procedures of other blood-derived products. Despite starting from patient's (or compatible donor) whole blood, the blood-derived products imply external manipulation to concentrate specific blood fractions, usually platelets, into a therapeutic concentration, before activation or blending with other biomaterials to stimulate the release of specific growth factors or cytokines, or the formation of a fibrin mesh.21,30,87,155,160
Endodontic tissue engineering
TE is “an interdisciplinary field that applies engineering and life sciences principles toward creating tissues or cellular products outside the body (ex vivo), which can restore the function of missing or diseased tissues and organs, typically using combinations of stem cells, scaffolds, and signaling molecules.” Moreover, it also uses the gathered knowledge to restore, maintain, or enhance tissue functionality within the body (in vivo).116,161
In particular, research in the endodontic tissue engineering (ETE) field aims to regenerate the lost inner tooth structures based on these principles.162,163 Extensive research has been conducted on ETE both in vitro and in vivo.121,146,147,149,164–168 Moreover, it has recently been applied clinically 148 (Fig. 3).
In an in vivo study, a construct of autologous DPSCs supplemented with platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF-2), nerve growth factor (NGF), and BMP-7 seeded in a chitosan hydrogel was compared with acellular growth factor-loaded scaffolds in periodontitis-induced teeth. Radiographically, the DPSC group showed significantly better root thickening and lengthening, and apical closure than the acellular control. Histologically, the cellular group formed a loose connective tissue containing numerous blood vessels with some dystrophic calcifications, while the control group did not display any formation of tissue into the root canals. 143
In a pilot clinical trial using a TE strategy, autologous DPSCs with granulocyte colony-stimulating factor (G-CSF) in a collagen scaffold were transplanted into five patients (three male and two female; between 20 and 44 years of age) diagnosed with irreversible pulpitis and treated using conventional endodontic instrumentation and irrigation. 148 After 4 weeks, treated teeth demonstrated a robust positive electric response, functional dentin formation, and dental pulp was similar to the control tooth.
These studies have demonstrated security and efficacy for using ETE on pulp regeneration; nevertheless, its results are still unspecific. Although radiographs or tomographies demonstrate the deposit of mineralized matrix, the nerve reconnection by vitality tests, and the restitution of blood flow by laser Doppler, odontoblasts' presence is decisive to indicate that there was regeneration as such. For that reason, it is necessary to discuss if all the repairs by TE therapies are actually obtaining regeneration or reparation and if it is possible to measure and evaluate these outcomes.
Regeneration or Repair?
At the moment, there is no detailed parameter to determine when a dental pulp was repaired or regenerated for in vitro and in vivo assays, or clinical trials. Preclinical models have been instrumental in disclosing the regenerative potential of biology-based endodontic therapies. The less restrictive ethical concerns allow further evidence to anticipate the quality of the newly formed tissue, namely the histological analysis and assessment of specific marker expression.
Remarkable efforts have been made to regain the functions of the regenerated endodontic tissues, including the revascularization or the deposition of mineralized ECM (Table 2).169–171 Nevertheless, as described above, in addition to finding a vascularized and innervated loose connective tissue that produces a mineralized matrix, it is essential to have odontoblasts producing mineralized matrix deposition in a concentric and peripheric way to consider a tissue as a DPLT.
Analysis of a histological section alone cannot be used to definitively ascertain that the observed tissue is a DPLT. Typically, only H&E staining is conducted, and the criterion used to evaluate regeneration is the presence or absence of healthy and vital tissue and newly mineralized tissue.70,143,172
In the same way, Iohara tested in vivo a magnetic resonance imaging (MRI) method to assess the pulp regeneration induced by DPSCs with growth factors seeded in a chitosan hydrogel. However, as an evaluation criterion, Iohara used a resultant endodontic vascularized loose connective tissue stained with H&E as the gold standard where it was possible to observe deposits of mineralized matrix inside the connective tissue after 90 and 180 days. In our criteria, the presence of a well-vascularized loose tissue is a remarkable outcome, but is insufficient to define it as an endodontic pulp-like tissue if the distribution of regenerated cells and mineralized tissues is not in an adequate place (Fig. 1).
Moreover, in another assay, Iohara et al. supplemented their histological stainings with an immunohistochemical analysis using BS-1 lectin and PGP 9.5 as markers of blood vessel neurites, respectively. 173 Although it is essential to identify neurites in an endodontic vascularized connective tissue, it is not enough evidence to identify it as DPLT.
Some studies have tried to identify odontoblasts using markers expressed by cells that secrete mineralized matrices, such as ALP, 174 OPN, or BMP2.149,154,175 However, these markers are also expressed in bone (Table 2), raising doubts about whether the tissues obtained are actually pulp-like tissues. Other works have also included the analysis of COL1 marker to identify connective tissues, CD31 or VEGF markers for endothelial cells and blood vessels, complemented with the DMP1, DSP, or DSPP markers for the dentin ECM.69,118,176–181 Nevertheless, these markers are also expressed by cementoblasts and osteoblasts (Table 2).
Other studies have focused on evaluating blood vessels, human mitochondria, or M2 macrophages (also known as proreparative macrophages) as markers of regenerative microenvironments.146,182 However, even though M2 macrophages may indicate regeneration, it is impossible to determine which tissue is regenerating. For this reason, Ito et al. used CD68 as a pan-macrophage marker indicating a regenerative process and nestin to identify odontoblasts within endodontic samples. 183 Although a greatly innervated and vascularized endodontic connective tissue indispensably needs to have a layer of peripheral odontoblasts that deposit mineralized matrix to be considered DPLT, the mere presence of odontoblasts would be insufficient, but more approximate than the evidence mentioned above.
Some studies comply with those that we consider as minimum parameters required for a preclinical study to confirm the presence of a DPLT. Histologically, the presence of loose connective tissue is observed. Moreover, using immunohistochemistry, blood vessels were identified using the CD31 marker, the deposit of mineralized matrix with DSPP, and the presence of odontoblasts through nestin expression.109,137,184,185 Yet incomplete, these markers are the most proximal to identifying DPLT formation in preclinical assays.
In clinical trials, confirming the success of pulp regeneration therapies has become a real challenge because of ethical issues and the impossibility of recovering the sample from further analysis. Most regenerative endodontic studies have focused on demonstrating pulp regeneration by documenting the root apex's development through dental imaging.70,128 However, it is possible to find this same result in teeth that have not received any regenerative treatment. 66 In the same way, unprecise tests have been used to demonstrate pulp regeneration clinically,70,148,166 such as cold pulp testing (positive predictive value [PPV] 0.84, negative predictive value [NPV] 0.87) and electric pulp testing (PPV 0.89, NPV 0.80). 186 Alternatively, the use of more precise techniques128,167 such as pulse oximetry (PPV 0.94, NPV 0.99) or laser Doppler (PPV 0.94, NPV 1) is more recommended. 186
In this way, it seemed convincing to demonstrate pulpal regeneration showing apical closure and pulpal vitality. However, additional tests are still necessary to confirm that the newly formed tissue is DPLT formed by odontoblast-like cells. These odontoblasts should be cubic or cylindrical with oval nuclei, aligned in a palisade at the periphery of the loose tissue and below the host tooth's mineralized dentin when viewed under the microscope using H&E staining. Above and beyond, the newly formed mineralized matrix should only be deposited in the periphery in a controlled manner, showing a tubular dentin-like structure where only cytoplasmic processes of the odontoblasts should be included.121,187
Future Perspectives
Development of reliable ex vivo and preclinical models
The biology-based endodontic therapies are typically tested in several preclinical models before clinical trials (Table 3). The in vivo and ex vivo models aim to emulate the human dental pulp's complex anatomophysiology to replicate or anticipate the complex tissue response before the preclinical experiments. One of the dental pulp's characteristics is its unique disposition within a rigid encasement of mineralized dentin, lacking a collateral source of blood supply.9,13 As a consequence of both, the tissue pressure of the pulp shows uniquely high values (10.3 mmHg),31,188 which is different from the typical cell culture setups. Although extensive efforts have been performed to create test conditions that resemble the human dental pulp real microenvironment, pulp pressure and its microcirculation have not yet been considered critical factors that could influence the final result.108,163,181
Preclinical Research of Biologically Based Endodontic Therapies Focused on Dental Pulp Regeneration During the Last 5 Years
+++: PLT A complete evidence [(1) Odontoblast-like cells formed confirmed by immunohistochemical stain or PCR using specific odontoblasts antibodies like nestin or DSPP. (2) Dentin-like tissue deposit confirmed histologically, and (3) Blood vessels formed].
++: PLT A good evidence (meet two parameters).
+: PLT A insufficient evidence (meet one parameter).
−−: Pulp-like tissue not achieved good evidence [(1) No confirmation of odontoblast-like cells histologically. (2) Cementum-like or bone-like tissues deposited into the root canal].
Set of factors: G-CSF, VEGF, PDGF, NGF, BMP7.
bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BMSCs, bone marrow stem cells; BSP, bone sialoprotein; CGF, concentrated growth factor; DPSC, dental pulp stem cell; ECFCs, endothelial colony-forming cells; FGF, fibroblast growth factor; GPF, glomerular permeability factor; G-CSF, granulocyte colony-stimulating factor; HA, hydroxyapatite; HUVECs, human umbilical vein endothelial cells; mCT, micro-computed tomography; MEPE, matrix extracellular phosphoglycoprotein; MRI, magnetic resonance imaging; NGF, nerve growth factor; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PDGFR, Platelet-derived growth factor receptor; PLG, poly-DL-lactide/glycolide; PLLA, porous poly (L-lactic acid); PLT A, pulp-like tissue achieved; PLYS, polylactic acid-polylysine; SCAPs, stem cells from the apical papilla; SDF-1, stromal cell-derived factor 1; TCP, tricalcium phosphate; TE, tissue engineering; TGF, transforming growth factor; TRHDE, Thyrotropin releasing hormone degrading enzyme; VEGF, vascular endothelial growth factor; vWF, von Willebrand Factor; WB, Western blot.
Regarding the architecture, the tooth root slice assay, one of the most common assays to evaluate endodontic regeneration, presents marked differences compared with the endodontic cavity. This assay presents two communication sides when implanted within subcutaneous tissue in semiorthotopic models. 195 Similarly, in orthotopic studies, there are substantial differences. Mice's incisors show continuous growth, while the dog and pig teeth have wide open apexes, which could influence the pressure and blood flow values differentiating it from the human pulp environment and, consequently, disparage the results of assays.109,195
How would human MSCs from the neural crest respond 196 to the paracrine factors released by pre-odontoblasts 121 in an engineered ECM similar to that of the dental papilla? 197 Only an ex vivo assay could answer that question. However, the available models barely recreate the dental pulp physiological microenvironment, compromising the anticipation of the clinical relevance. Future research should develop culturing systems that can emulate these characteristics, complementing the existing models. 198
Bioreactors have become a platform that meets previous parameters to create, evaluate, and validate new biomaterials and their potential application to regenerate dental tissues. Recently, bioreactors have been used to simulate various human body features in a highly controlled environment. Flow perfusion, strain, rotational, spinner-flask, and customize-combined bioreactors have been used to regenerate maxillofacial tissues.186,199–201 In summary, bioreactors allow for an effective exchange of essential gases and nutrients at the cellular level and for homogeneous cell colonization and matrix deposition within large TE scaffolds. 186 However, so far, bioreactors used to simulate pulpal environmental conditions have not tried to mimic it. Instead, research has focused on hypoxia 69 and microgravity, 202 which relevance to recreate the endondontic cavity is questionable.
Considering that, the dental pulp constitutes a connective tissue with particular properties due to its encasement within a mineralized matrix. The control of blood pressure and flow will allow us to manage and optimize strategies and scaffolds to overcome the initial periods of vessel sprouting and blood perfusion to continue with the phase of formation of a vascular network in ectopic and orthotopic models, avoiding adverse reactions that could be decisive when it comes to regenerating tissues in the tiny pulp spaces. 203 So, we consider that it is necessary to create an environment that emulates the complicated environment within the pulp chamber and canals.
In this sense, combining microfluidic technologies and bioengineering has enabled the design of various tissues and organs-on-a-chip (OoC).204–206 These dynamic and miniaturized devices aim to recreate fundamental organ architecture and basic physiological mechanisms of functional units under controlled conditions. 207 In fact, microfluid-based in vitro models have rapidly evolved since their inception in the late 1990s. 208 Of these OoC, one of the most representative is the lung-on-a-chip that mimics the epithelial/endothelial interface in polydimethylsiloxane membranes. 206 Since then, the development of OoC devices has been on the rise. 209 Among these, the dentin–pulp interface has been recreated with a design comprising two microfluidic channels separated by a dentin fragment. 210
In this OoC system, one channel contains cells directly cultured on a dentin wall, while the other channel allows to flow different compounds representing the tooth surface. Although this device imitates some of the structural and functional characteristics of the pulp–dentin interface and allows to test the cytotoxicity of different dental biomaterials in a microenvironment more consistent with the in vivo conditions, adding controllable pressure, functional capillaries, immune cells, and innervation on the “pulp side” is still a challenge. Nevertheless, the evolution of this technology offers a promising route to revolutionize the way researchers study tissue and organ physiology and pathophysiology in the coming years.
Criteria for proper endodontic tissue regeneration evaluation
According to our perspective, pulpal regeneration can only be considered when there is a well-vascularized and innervated connective tissue with peripherally aligned odontoblasts capable of producing mineralized matrix centripetally outside of them.
In vitro and in vivo, we consider that when samples are retrieved after determined time points, the presence of three minimal parameters that characterize the dental pulp must be demonstrated histologically to determine success in a regenerative endodontic assay:
Confirm the presence of a well-vascularized and innervated loose connective tissue using specific markers like COL1, CD31, and PGP 9.5. Dentin-like tissue deposition must be confirmed using specific dentin markers like DSPP, DSP, or DPP. These dentin-like tissues should only be present in the pulp-like tissue peripheric zone. The odontoblast-like cells should be recognized as a peripheral alignment of cuboidal or columnar cells that mark positive for nestin or AQP4 and deposit dentin matrix outside them.
In the case of clinical trials, the primary test that could validate biology-based endodontic treatments should be the finding of histologically pulp-like tissues using teeth with orthodontic extraction. If not possible, molecular imaging could be applied, marking the neo-formed tissues with magnetic agents labeled with monoclonal antibodies as nestin or AQP4. 198 However, for now, before having more conclusive proof, clinical trials need to reach three minimal parameters to succeed in healing after a regenerative endodontic treatment (no failure):
Radiographic imaging of inner pulp spaces free of mineralized matrices.
Healing of the periapical tissues (if apply), an apical remodeling (if apply), and presence of the periodontal ligament space along the root.
Pulp vitality, confirmed by blood circulation using laser Doppler, pulse oximetry, or cold pulp test.
Translation of regenerative therapies to clinics
Even though the mechanisms, procedures, and results in preclinical assays have not been fully elucidated and only a few works showed convincing results of obtaining DPLTs (Table 4), numerous REPs have already reached the clinical trial phase in recent years. Nevertheless, only a few preclinical experiments show convincing results of obtaining DPLTs. Coincidently, these preclinical trials have not progressed toward translation to clinical practice. This fact does not mean that existing clinical trials are invalid. Some of these clinical trials have achieved a follow-up of between 28 weeks and 3 years without reporting incidents. However, there is not enough evidence to admit that a regenerative process exists in these cases.
Clinical Research of Repair by Tissue Engineering Therapies Focused on Dental Pulp Regeneration During the Last 5 Years
+++: Minimal parameters to consider success in a regenerative endodontic treatment [(1) Radiographic evidence of absence of mineralized tissues occupying the dental pulp space. (2) Presence of the periodontal ligament space along the root. (3) Blood circulation confirmed and/or pulp vitality test positive].
One of the three minimal parameters accomplished.
Two of the minimal parameters accomplished.
−−−: Pulp-like tissue not achieved complete evidence [(1) Radiographic evidence of mineralized tissues filling the pulp chamber and/or root canal. (2) Absence of the periodontal ligament space or reabsorption. (3) No signal of blood circulation and pulp vitality test negative].
Ld, laser doppler; L-PRF, leukocyte platelet-rich fibrin; Pt, electric pulp testing; Rx, radiography; SHEDs, stem cells from human exfoliated deciduous teeth.
We believe it is necessary to return to the laboratory to overcome the drawbacks of the lack of true identification of DPLTs, and after a consensus, give the translative step with the established objectives of what is sought and based on solid evidence from previous actions, or eliminate subjectivity through more precise tests demonstrating de novo regeneration of completely lost pulp through regenerative endodontic therapies.
For this reason, we call on the scientific community to first create a common language among regenerative endodontic researchers on the constituent elements and objectives of pulpal regeneration. Second, get back to the preclinical assays to understand better the developmental and regenerative mechanisms in dental pulp tissues. Third, unify criteria to standardize preclinical and clinical tests to identify favorable and unfavorable outcomes in pulp regeneration. Finally, protocolize procedures ethically and safely, requiring the use of GMPs and compliance with clinical biosafety protocols.
Enhance the communication between the players
Research on the field of dental pulp regeneration is cross-disciplinary, which will need the cooperation of all the partners to gain momentum and achieve the aimed change in the paradigm. As we have reviewed in this work, researchers with different backgrounds work in the regeneration of the dental pulp, all with their own language and evaluation parameters.
It is worth remembering the value of the standarization carried out by Ingle and Levine in 1958 and reported by Goldberg in 1990211 or the consensus of the AAE in 2009212 for the advancement in endodontics by providing simplicity in the identification processes of instruments and providing a language in diagnostic terminology. For this reason, through this review, we call on those involved in dental pulp regeneration to form a college or expanded discussion committee of clinicians and researchers, to reach a consensus in the field to consolidate the advances on regenerative endodontics to reach the clinical translation efficiently and safely.
Conclusions
The data summarized in this review suggest that stem cells, biomaterials, and growth factors have the potential to overcome the obstacles encountered in revitalization of dental pulp. However, to date, insufficient evidence has been reported to demonstrate dental pulp regeneration in any of the published clinical cases that used biology-based endodontic therapies in nonvital dental pulps. This fact is related to the absence of a confirmatory method established by consensus between clinicians and researchers.
Regarding the preclinical data, dental pulp regeneration was confirmed in three experimental assays using our criteria for proper endodontic tissue regeneration evaluation. Considering that, we believe currently we are approaching the edge of a paradigm shift in contemporary endodontics toward pulp regeneration. To date, the cell homing approach has shown promising results among all strategies applied. Nevertheless, to potentiate the awareness and the translation to the clinic of this research field, there is a need to define the concepts and mechanisms involved in pulp regeneration correctly. Moreover, unification of criteria is necessary when evaluating the clinical and preclinical outcomes determining pulp regeneration's success or failure.
Footnotes
Authors' Contributions
E.A.-O.: writing and figure preparation; P.S.B.: writing and reviewing; P.T.S.: reviewing and editing; K.M.G.: reviewing and editing; M.G.-F.: writing, reviewing, and editing; M.E.G.: reviewing and editing. All authors approved the final version of this review article.
Disclosure Statement
No competing financial interests exist.
Funding Information
We acknowledge the financial support from Research Council of Norway for project number 287953. Secretary of Higher Education, Science, Technology, and Innovation, SENESCYT (Spanish acronym) from Ecuador, reference number CZ06-000132-2017-01.