Tooth Repair and Regeneration: Potential of Dental Stem Cells

Abstract

Tooth defects are an extremely common health condition that affect millions of individuals. Currently used dental repair treatments include fillings for caries, endodontic treatment for pulp necrosis, and dental implants to replace missing teeth, all of which rely on the use of synthetic materials. In contrast, the fields of Tissue Engineering and Regenerative Medicine and Dentistry (TERMD) use biologically based therapeutic strategies for vital tissue regeneration, providing the potential to regenerate living tissues. Methods to create bioengineered replacement teeth benefit from a detailed understanding of the molecular signaling networks regulating natural tooth development. Here, we discuss how key signaling pathways regulating natural tooth development are being exploited for applications in TERMD approaches for vital tooth regeneration.

The mouth as the gatekeeper for overall systemic health.

Teeth are complex, highly specialized organs that are crucial for human health and wellbeing. They are essential for an individual’s ability to effectively talk and communicate, to eat and provide nutrition for the body, and also are very important for proper facial aesthetics, and interpersonal and psychosocial health and well-being. Despite the importance of teeth for overall human health, an individual’s oral and dental health can often be compromised by a variety of insults including dental caries in both deciduous (baby) and adult permanent teeth, periodontal disease and associated gum inflammation and bleeding, the high incidence of oral cancers and the subsequent surgeries required to effectively remove all cancerous tissues, and damage caused by accidental injuries incurred from sports, or those incurred by military personnel on the battlefield. Both hard and soft tooth tissue defects can compromise the natural protective properties and defenses provided by the mouth and oral cavity, often resulting in systemic, chronic health diseases. In healthy individuals, the natural balance of microbiota in the mouth consists of healthy biota that promote the health and vitality of dental tissues in the mouth as well as that of all tissues and organs in the rest of the body. In contrast, severe periodontitis is often associated with chronic bacterial infections that can compromise the natural protective properties of the oral flora, resulting in severe inflammation, pain and tooth loss. Importantly, since the oral cavity is intimately connected to the rest of the body through swallowed saliva, food and breathing, an unhealthy oral environment can seriously harm quite distant parts of the body including the heart, which can accumulate deleterious biofilms on the heart valves, and stomach and intestinal disorders including in severe cases systemic sepsis. As such, oral health is of utmost importance to overall health, especially in individuals with other health issues such as diabetes, heart disease, high blood pressure, ulcers, and immunocompromised conditions. Our ability to maintain proper oral hygiene, and to promptly and effectively repair oral and dental tissues as needed, is of vital importance for the overall health of populations worldwide.

Teeth consist of highly organized diverse hard and soft tissue types, including highly calcified enamel, dentin and cementum, and soft tissues including dental pulp and periodontal ligament [1]. Currently used therapies to repair tooth damage and loss rely on the use of synthetic materials that restore the structural defects but serve no biological functions such as promoting blood and nerve supplies. As mentioned earlier, severe dental tissue damage can lead to malnutrition caused by difficulties in eating and chewing, and can compromise an individual’s self-esteem, leading to serious psychosocial and mental health issues. Tooth development, also known as odontogenesis, is initiated by interactions between two types of dental tissues – the dental epithelium (DE) and the dental mesenchyme (DM) - similar to the epithelial/mesenchymal cell interactions directing the formation of many other ectodermal organs such as hair, sweat glands, and finger and toe nails [2]. A thorough knowledge and understanding of the molecular mechanisms regulating natural tooth development and dental stem cell differentiation is essential in order to devise effective approaches for adult dental tissue regeneration, including whole teeth. Furthermore, the tooth provides a superb model system to study the molecular and cellular mechanisms governing organogenesis. The easy anatomical accessibility of the tooth makes it a highly useful model to study both organ development and regeneration.

The importance of these studies is underscored by the fact that an estimated two out of three Americans have one or more missing teeth, and that >50% of the population aged 70 years of age or older are edentulous - meaning that they have no remaining natural teeth [3]. With an aging population estimated by 2030 to consist of greater than 50% individuals 65 years of age or older, significant health issues are anticipated due to the increased incidence of tooth loss, periodontal disease, and oropharyngeal cancers in these individuals [4]. The high incidence of tooth loss and associated oral health problems in older Americans recently prompted the American Associate of Retired Persons (AARP) to declare the “Over 50 Dental Crisis” (https://blog.aarp.org/thinking-policy/something-to-chew-on-highlights-of-findings-froman-aarp-oral-health-survey-of-older-americans). In 2006, the journal Dental Economics reported that greater than 30 million Americans were missing all of their teeth in one or both jaws (https://www.aaid.com/about/Press_Room/History_and_Background.html). (See Text Box 1). In this review we first discuss current research on developmental and stem cell biology of dental tissue and whole tooth regeneration, and subsequently how these fundamental research discoveries can be used to guide the development of new, more effective therapies to regenerate teeth and dental tissues for improved oral health.

Text Box 1: The Dental Implant Market.

According to the American Academy for Implant Dentistry, over 15 million people in the U.S. undergo bridge and crown replacement therapies for missing teeth every year, emphasizing the significant demand for dental implants as an alternative therapy [82]. The global dental implant market was valued at USD 4.6 billion in 2019, and is expected to register a Compound Annual Growth Rate (CAGR) of 9.0% over the forecast period [82]. The increased use of dental implants in many dental specialties, combined with an increased demand for prosthetics, are some of the key factors expected to boost the market growth for dental implants [82]. As such, there is great potential for the dental implant market, and perhaps also for even better alternative therapies including bioengineered teeth.

Important Characteristics of Tooth Development

The composition of tooth types (incisors, molars, premolars, etc.) and number varies dramatically among species [5]. Numerous studies using a variety of animal models have helped us to better understand how teeth initially form and then develop to perfectly occlude (meet) apposing teeth in a growing jaw [6]. Rodent molar teeth, such as those present in mice and rats, and human molar teeth are all characterized as brachydont teeth [7] meaning that they share similar properties of low crown-to-root ratio, and do not exhibit additional crown growth after tooth eruption [7]. Based on the close similarities between tooth development in mice and humans, the majority of research on the molecular mechanisms regulating tooth development have been conducted in mice [8]. Another advantage of the mouse model is the ability to easily generate targeted mutant and transgenic mouse lines to better decipher the functions of individual molecular signaling pathways in tooth formation [9]. Moreover, in contrast to molars, rodents exhibit continuously erupting incisors that provide invaluable tools with which to study the dental stem cell niche – the environment where dental progenitor cells reside until they are recruited to regenerate dental tissues - and the microenvironments that regulate the fates of stem cells resident therein [10]. The next section will highlight recent findings on tooth development that have been made using human, mouse and rat models.

Any aberrant expression of tooth related signaling molecules can lead to abnormal tooth formation. As such, fully understanding the signaling pathways that guide natural tooth crown and root formation is essential in order to devise effective tissue engineering methods to regenerate teeth (See Text Box 2).

Text Box 2: Signaling cascades regulating tooth development.

Tooth development begins as a thickening of the oral epithelium, which subsequently proliferates and invaginates into the underlying dental mesenchyme (DM). The dental mesenchyme, which is derived from neural crest cells (NCC, see Glossary), condenses around the dental epithelium to form a structure called the tooth bud (TB, see Glossary) (Figure 1) [83]. Next, a structure called the enamel knot (EK) forms, a transient signaling center that directs the morphology of the tooth crown [84]. Signaling molecules secreted in a temporo-spatial manner by primary and secondary EKs direct the proliferation and differentiation of the dental epithelium (DE) and the DM in the vicinity of the EKs, ultimately regulating the formation of tooth cusps [85, 86]. Tooth initiation, formation and morphogenesis is mediated by precisely regulated temporospatial signaling between DE and DM tissues. The major signaling pathways that regulate tooth development include: 1) wingless/integration 1 (Wnt); 2) bone morphogenetic protein (BMP); 3) fibroblast growth factor (FGF); 4) sonic hedgehog (Shh); and 5) Notch [87, 88]. Tooth root formation begins after the completion of the tooth crown. Along with the formation of periodontal tissues and tooth root elongation, the tooth eventually erupts and anchors to the surrounding alveolar bone via the periodontal ligament (PDL) [1, 89]. The Hertwig’s epithelial root sheath (HERS), located in the cervical loop formed by the inner and outer DE cell layers, is considered to be the signaling center for tooth root formation [90, 91].

Strategies to Repair Damaged Dental Tissues via Dental Stem Cell-Based Therapies

Dental Stem Cells

Highly specialized Biomedical Engineering (BME, see Glossary) based, three dimensional (3D) in vitro tissue culture systems can serve as valuable platforms to recapitulate and study the molecular and cellular interactions that regulate tooth development and regeneration, including those regulating dental stem cell (DSC)-based dental tissue homeostasis, regeneration and repair [11]. Based on the DSC source, two approaches have been widely explored to study dental tissue renewal: 1) DSC transplantation; and 2) DSC homing approaches that recruit a patient’s own endogenous stem cells.

Adult human teeth possess very little capacity to repair, due to the fact that human teeth contain only limited amounts of stem cells. Currently in the clinic, biological based therapies to restore damaged dental tissues mainly focus on the repair of two types of dental soft tissues – the dental pulp and the periodontal ligament (PDL). Clinical therapies to regenerate soft dental tissues have successfully been used for a number of years now, where it is widely accepted that stem cells residing within dental soft tissues play important roles in dental tissue repair and regeneration [12]. A better understanding of how DSCs are affected by inflammatory-proliferative-remodeling signals will provide useful guidelines for successful dental tissue engineering approaches and methodologies.

Dental pulp capping is the most commonly used therapy to repair dental pulp that has been partially damaged by caries or trauma [13]. Pulp capping therapy focuses on removing damaged pulp, and then regenerating vascularized dental pulp tissue and the coordinated formation of a dentin bridge over the defect site (referred to as the dentinpulp complex), which serves to seal off the pulp from the oral cavity, thereby protecting it from future infections [14]. In some cases, successful pulp capping in immature teeth can support further tooth development including tooth root formation [15]. To promote regeneration of the dentin-pulp complex, it is necessary to stimulate dental pulp stem cells (DPSCs) residing within the pulp to divide and produce “daughter stem cells” destined for pulp regeneration, while remaining in place to maintain the stem cell niche. The newly formed daughter stem cells can then migrate away from the stem cell niche into the defect area and participate in new dental pulp and dentin bridge formation [16]. DPSCs were first isolated and characterized from postnatal human dental pulp in the year 2000 [17]. Subsequently, DPSCs were successfully isolated from human deciduous (baby) teeth, where they were named “Stem Cells from Human Exfoliated Deciduous” (SHED) [18]. Both types of stem cells showed high proliferation rates and the ability to generate dentin-pulp complex like tissues when implanted subcutaneously [19]. Since that time, significant effort has been made to identify the origin of DPSCs, but this question still remains obscure. One theory is that DPSCs originate from pericytes, cells that reside in the perivascular niche of postnatal dental pulp [20, 21]. Over the past several years, reports have indicated that NCC derived MSCs contributing to tissue repair and regeneration also come from nerves [22]. Peripheral nerve associated glial cells could give rise to DPSCs via a glial-to-MSC transition during tooth development, and also could participate in dental pulp regeneration [23]. Lineage-tracing analyses using an inducible, Cre-loxP in vivo fate-mapping approach showed that αSMA-tdTomato⁺ progenitor cells migrated from their perivascular locations to the site of injury, and differentiated into both odontoblast (dentin producing) and osteoblast (bone forming) like cells [24]. These results demonstrated that DSCs contributing to reparative dentin formation were recruited from both the vasculature as well as from peripheral nerves [24]. Another unanswered question is what triggers the migration of DPSCs to the injury site in the first place? Several cytokines and growth factors contributing to tooth development also appear to trigger DPSC migration and differentiation, including TGF-β [25], the Wnt-responsive gene Axin2 [26], and TNFα [27]. Although additional studies are clearly needed in order to fully understand the mechanisms regulating these processes, we are beginning to build an understanding of the important players. As described earlier, DPSCs are derived from the neural crest, one of the four embryonic tissue types that form all of the tissues of the vertebrate body (endoderm, mesoderm, ectoderm, neural crest) [28]. In addition to odontoblasts, DPSCs can also differentiate into functional neurons and glial cells when provided with the appropriate environmental cues [29], and exhibit the distinctive ability to secrete neurotrophic factors that guide neurite outgrowth [30]. DSCs isolated from the tooth apical papilla, named stem cells of the apical papilla (SCAP), have been demonstrated to exhibit properties very similar to DPSCs, including the ability to support tooth root formation [31].

Functional dental pulp regeneration requires not only the ability to form dentin, or dentinogenesis, but also the ability to restore dental pulp vasculature (blood vessels) and nerves, in order to ensure proper cell nutrition and viability, and to restore and maintain proper defenses against any potential pathogens encountered in pathophysiologic states such as pulpitis, periodontal disease and trauma [32]. Vital pulp therapies to repair partially damaged dental pulp have been used with some success in the dental clinic, where regenerative endodontics has focused on revitalizing dental pulp and maintaining continued root development in immature permanent teeth [33]. For now, the gold standard material for vital pulp capping is mineral trioxide aggregate (MTA), first introduced to the dental market in 1998 [34]. More recently, Biodentine (Septodont, Inc.,Saint-Maur-de-Fosses, France) has shown great promise for applications in pulp capping, based on the fact that it is a calcium silicate based product with mechanical properties similar to natural dentin tissue, and its ability to stimulate dental pulp cell proliferation and the formation of tertiary dentin [35].

Therapeutic Relevance of Dental Tissue Engineering Strategies

Each year, approximately 22.3M endodontic procedures are performed in the United States alone [36]. In the past, Regenerative Endodontics Therapies (RETs) have focused on the partial regeneration of the dentin-pulp complex, with the goal of protecting the continuous growth of immature tooth roots, and to reduce the risk of fracture associated with traditional apexification procedures in thin and weak roots [37]. More recently, RETs have focused on whole pulp regeneration, in an attempt to replace currently used synthetic materials based endodontic treatments that devitalize the tooth by filling the pulp chamber with gutta-percha (GP) or another synthetic material that blocks vital tissue formation [38]. In contrast to partial vital pulp therapy, whole pulp regeneration is focused on total pulp regeneration and revascularization of fully developed teeth, by facilitating DPSC derived odontoblast differentiation and pulp regeneration [39]. An experimental tooth slice model, widely used to analyze pulp regeneration [40], has been used to demonstrate pulp like tissue and mineralized dentin formation when filled with stem/progenitor cells from the apical papilla, the dental pulp [41], or SHED [42], and implanted subcutaneously in mice. DSCs have also successfully been isolated from inflamed dental pulp tissues, supporting the feasibility of whole pulp regeneration using autologous cell sources from infected dental pulp [43]. Kim and colleagues demonstrated regeneration of the pulp-dentin complex via cell homing [44], while another study compared the ability of Biodentine, MTA, calcium hydroxide and Xeno III adhesive resin to induce reparative dentin synthesis and transforming growth factor beta 1 (TGF-β1) expression [45]. Of these, Biodentine and MTA were found to be superior in terms of improved early odontoblastic differentiation, mineralized tissue formation, improved dentin synthesis and significantly improved TGFβ1 expression [45].

Clinical Trials using Dental Stem Cells

While highly debated in the past, due to safety concerns [46], cell transplantation therapies have finally been implemented in the dental clinic. A recently published clinical trial demonstrated successful pulp regeneration and continued root development in immature single-root permanent teeth treated with autologous SHED [47] (See Clinician’s Corner). Twenty six teeth from 26 patients received autologous SHED implantation, and 10 teeth from 10 patients received standard apexification treatment. Although regeneration of dentin-like structures was limited, all patients who received cultured SHED aggregates regenerated highly vascularized dental pulp [47]. In addition, tooth root development continued in all SHED seeded samples, while no tooth root maturation was observed in any of the control treated teeth.

Clinician’s Corner.

• Recent developments in the fields of Stem Cell biology and Tissue Engineering and Regenerative Medicine and Dentistry have provided new and exciting biomedical engineering approaches and promising therapies to repair a variety of tissue defects, including dental and craniomaxillofacial tissues.

• Compared to challenges facing clinical therapies to repair essential and vital tissues and organs such as the heart and brain, tooth loss is not an acute lethal condition. Rather, oral health is inextricably linked to long term overall health, and is a significant health concern, particularly for underserved populations who may not have sufficient access to regular dental care.

• In vitro manipulation of progenitor stem cells - whether they are autologous (from an individual themselves) or allogeneic (from another individual) - may introduce potential risks including immuno-rejection by the recipient patient.

• Currently, the majority of therapies used in the Dental Clinic for dental defect repair focus on the structural repair of tooth defects and are limited to the use of synthetic materials.

• The use of novel dental therapies that employ dental progenitor stem cells combined with biocompatible scaffolds could be highly efficacious, particularly when repairing large craniomaxillofacial defects by creating living replacement tissues.

• Wisdom teeth provide an excellent source of dental stem cells, since many, if not most individuals, need to have them extracted before they become impacted. Stem cells isolated from the dental pulp of extracted wisdom or other permanent teeth are called dental pulp stem cells (DPSCs). Wisdom teeth can also be used to harvest periodontal ligament stem cells (PDLSCs) for applications in guided periodontal tissue regeneration. Stem cells isolated from the dental pulp of deciduous (baby) teeth are called “stem cells from human exfoliated deciduous teeth” (SHED), indicating that they are a younger dental cell source as compared to DPSCs.

• DPSCs, SHED and PDLSCs provide a reliable and easily accessible source of progenitor cells for use in dental tissue regenerative therapies.

• DPSCs and SHED not only have the potential to differentiate into dental tissue related cells including dental pulp cells, odontoblasts that create dentin, and osteoblasts that create jaw bone, but can also be used to create cells that can produce non-dental tissues.

• Consequently, therapeutic applications for DPSC and SHED are not limited to use in the dental field, but also can be used as a wider cell resource for a variety of applications in the fields of Tissue Engineering and Regenerative Medicine.

Clinical studies for PDL generation, also known as periodontium generation, involve the regeneration of three types of tissues – periodontal ligament (PDL), cementum and alveolar bone [48]. Guided tissue regeneration (GTR) therapy is commonly used in the clinic for PDL regeneration to treat periodontitis and other periodontal diseases [49]. The limited regenerative properties of the periodontium is thought to be due to the very small numbers of PDL stem cells (PDLSCs) present [50], which like the other MSCs, have the ability to differentiate into tissue specific dental cell types for dental tissue repair. STRO-1+/CD146+ stem cells identified in the PDL have been suggested as a source of stem cells for regenerative therapies [51]. Lineage-tracing studies in the continuously erupting mouse incisor have shown that PDL tissue was derived in part from mesenchymal Igfbp5+ and Lrig1+ progenitor cells, while others have shown that fragmented tooth root epithelial cells also contribute to PDL tissue formation [52]. Further studies are necessary to elucidate the inductive signaling pathways regulating these processes, which could then potentially be used for a variety of clinical applications for dental tissue regeneration. Several clinical trials using PDLSCs for periodontal tissue regeneration are currently being conducted worldwide. One completed clinical trial conducted on a total of 30 periodontitis patients treated with autologous PDLSCs showed no statistically significant differences between the PDLSC-treated and control groups, although an increase in alveolar bone height was observed in both groups [53].

Recently, another type of stem cell with great differentiation potency, induced pluripotent stem (iPS) cells, were created by viral delivered over expression of key stem cell factors in somatic cells [54]. Viral transfection of Lin28/Nanog/Oct4/Sox2 or c-Myc/Klf4/Oct4/Sox2 into hDPCs harvested from SHED, and from SCAP, efficiently reprogrammed these cells into iPS cells [55]. Six hDPSCs lines transfected with retroviruses expressing Oct3/4, Sox2, Kl4, and c-Myc, were used to create iPS cells from 5/6 of them [56]. To date, no clinic trials have been reported for tooth/dental tissue regeneration using iPS cells.

De Novo Tooth Regeneration

Whole tooth regeneration, similar to natural tooth development, is a complex process that requires precise temporospatial interactions and reiterative molecular signaling between the dental epithelium (DE) and the dental mesenchyme (DM) (Figure 1). Previous research has shown that early stage embryonic DE can initiate tooth development [57], while later in tooth development, the odontogenic capacity shifts to the DM, providing it with the capacity to induce tooth formation when recombined with epithelium [57, 58]. In humans, mature teeth have lost their ability to regenerate in part due to the fact that the DE is no longer present in erupted teeth [59]. Therefore, to overcome this issue, two possible theoretical approaches have been proposed for human whole tooth regeneration. One is to generate a tooth, tooth bud or tooth root in vitro, and then transplant the regenerated tooth/tooth bud back into the defect site. A bioengineered tooth root could support an artificial crown after first being securely stabilized in the jaw bone. A second approach is to create a bioengineered replacement tooth bud in vivo, at the defect site.

Figure 1: Schematic of immature tooth bud (left) and adult tooth (right).

The Enamel Organ (EO) gives rise to tooth enamel, while the Pulp Organ (PO) gives rise to all of the remaining dental tissues including dentin, dental pulp, cementum and periodontal ligament tissues.

Mouse embryonic tooth bud derived bioengineered teeth

The embryonic mouse tooth bud model has widely been studied to explore possible methods for tooth regeneration. In one example, mouse DE and DM cell layers were created and then combined together to form an artificial tooth bud [60]. The key component of this model was the use of early embryonic stage DE tissue, either intact or dissociated, to ensure successful tooth formation. Tooth formation was observed when the DE layer was combined with dissociated DM cells, a mixture of embryonic neural stem cells harvested from embryonic spinal cords, or embryonic bone marrow cells [61, 62]. The successful regeneration of fully functional teeth was first achieved in 2007 using artificial tooth buds created, once again, from embryonic tooth bud cells [63]. In the ensuing years, the same method was again shown to successfully form fully functional teeth [64] [65], making it by far the most advanced model for functional tooth regeneration from embryonic tissues. Using this model, single cell suspensions of DE and DM harvested from embryonic mouse tooth germs were recombined within a collagen drop, in vitro cultured to develop to early bell stage, and then transplanted into an 8-week-old host mouse jaw. Quite surprisingly, this study reported the formation of full sized teeth that erupted and occluded in a manner similar to a natural tooth [64].

Adult post-natal tooth bud derived bioengineered teeth

In ongoing parallel studies, investigators took a different approach to regenerate teeth using clinically relevant cell sources, those obtained from post-natal teeth. Using this model, DE and DM cells were harvested from unerupted 6 month old pig teeth, and used to create bioengineered tooth constructs [66]. Mixed DE and DM cells, or DE and DM cells alone, were seeded onto tooth shaped, three-dimensional (3D) biodegradable polyester scaffolds and transplanted into the omentum of athymic rats. Analyses of harvested implants showed the formation of numerous small but anatomically correct tooth crowns, consisting of well-organized dentin, odontoblasts, pulp, and enamel, although no tooth root structures were observed in these bioengineered teeth [66, 67]. Subsequently, this group went on to create hybrid tooth bud and jaw bone constructs consisting of a bioengineered tooth bud created using a human DM cell seeded core surrounded by a post-natal porcine DE cell seeded scaffold, which was then combined with a bone cell seeded construct and implanted in adult rat or pig jaws [68–70]. In this model, not only were tooth crown-like structures observed, but also tooth root-like structures containing PDL tissues that were closely integrated with the surrounding newly formed bone [68–70]. Although the fact that clinically available post-natal dental tissues could be used to regenerate teeth was an extremely promising result, the bioengineered tooth crown and root structures were of variable size and shape, indicating the need for better methods to precisely regulate the formation of regenerated dental tissues.

One possible solution was to use natural tooth bud extracellular matrix (ECM) scaffolds created from decellularized post-natal tooth buds (dTB), to guide bioengineered tooth formation of specified size and shape [71]. This approach was based on previous studies showing that gentle decellularization processes could safely be used to remove immunogenic components from whole organs and tissues, while maintaining the natural ECM and its signaling components [72]. Based on the successful use of decellularized ECM scaffolds for applications in regenerative medicine, dTB-ECM (dECM) scaffolds were created from postnatal porcine tooth buds, and then re-seeded with porcine DE cells and human DPSCs, as well as human umbilical vein endothelial cells (HUVECs) to facilitate revascularization. These dTB-ECM constructs were then transplanted into fresh tooth extraction sockets of adult mini pigs, and grown for 1 or 3 months [71]. Upon harvest and analyses, it was found that these dTB-ECM constructs consistently directed the formation of organized, bioengineered teeth of comparable size to natural human teeth [71]. Excitingly, decellularized ECM scaffolds were recently approved by the US Food and Drug Administration (FDA) for therapeutic applications in humans [73], making the dTB-ECM biomimetic tooth bud model a potentially promising therapy for tooth regeneration in humans [74].

Current Challenges for Tooth Regeneration

Although the described approaches show great promise for future applications in regenerative translational dentistry, certain challenges remain. One is how to create bioengineered teeth of precisely specified size and shape. While the ability to achieve proper tooth morphology and occlusion with opposing teeth is known to be critical for proper tooth function and longevity [75], a detailed mechanistic understanding of signaling networks regulating natural tooth formation, or of how these networks could be manipulated to regulate the precise size and shape of bioengineered teeth, remains uncertain [76]. The importance of mechanical forces in regulating tooth morphogenesis has also increasingly been recognized [77]. For example, a recent study showed that tooth buds cultured in vitro without the presence of the surrounding jaw bone exhibited tooth cusp offsets, indicating the importance of jaw-tooth interactions for normal tooth morphology [78]. A major challenge for creating clinically relevant bioengineered tooth buds is the lack of appropriate human DE cell sources. As discussed above, the most promising cell source at the present time may be autologous iPS cells, which could then be differentiated to create DE cells competent to contribute to tooth formation.

With respect to bioengineered replacement tooth formation in situ, in the jaw at the site of prior tooth loss, cell homing has been proposed as a possible means. This novel technique relies on biomaterials that can release bioactive molecules to attract stem cells in the surrounding tissues to migrate towards and populate the scaffold, and eventually contribute to the generation of new tissues [79]. In one example, investigators filled dental pulp chambers and tooth root canals of endodontic treated human teeth with growth factors, including Vascular endothelial growth factor (VEGF-2), Fibroblast growth factor (bFGF), Platelet-derived growth factor (PDGF), Nerve growth factor (NGF), and Bone morphogenetic protein-7 (BMP7) [44]. After being transplanted subcutaneously and grown in mice, the teeth were found to contain dental pulp-like tissue with erythrocyte-filled blood vessels after 3 weeks. The same group also manufactured polycaprolactone (PCL)/hydroxyapatite (HA) tooth scaffolds containing stromal-derived factor-1 (SDF1) and bone morphogenetic protein-7 (BMP7) and implanted them either subcutaneously or into the jaw bone of rats. When harvested, these implants were found to contain distinct PDL tissues and new bone, but no dentin or enamel tissues [80]. Multipotent dental apical papilla cells combined with tricalcium phosphate (TCP) scaffolds and implanted into minipig incisor tooth extraction sockets, formed both soft and mineralized tooth root tissues [61]. The advantage of this “bio-root” approach is that it could be administered in situ, without the need for stem cell isolation or ex vivo manipulation [80]. Unlike embryonic dental progenitor cells that are not available for use in translational dentistry, postnatal dental stem cells are a readily available cell source for human dental therapies.

It is important to anticipate that novel scaffold biofabrication methods and 3D bioprinting will play critical roles in advancing the fields of tissue engineering and regenerative medicine and dentistry. Sophisticated scaffold fabrication technologies are continuously evolving to more effectively meet the rigorous demands required to regenerate fully functional replacement tissues, including dental tissues. Many reports, including several mentioned above, already have provided sufficient evidence for the feasibility of such techniques for dental tissue engineering and regeneration. New advances in biofabrication techniques, including more precise control of the physical, biomimetic and degradation properties of novel scaffolds, are anticipated to eventually facilitate the creation of new tissue engineering inspired therapies to effectively repair load bearing tissues, including teeth and surrounding alveolar bone, to support a variety of clinical applications.

Concluding Remarks

The existing and extensive current body of literature has contributed to a significantly improved understanding of how natural tooth development can guide dental tissue and whole tooth tissue engineering approaches, making stem cell therapies for dental tissue regeneration an exciting prospect. Still, the use of dental stem cells for clinical applications in dental tissue regeneration remains challenging for a variety of reasons, including the need to standardize and regulate methods for dental stem cell isolation, validation, expansion, handling, storage and shipping (see Outstanding Questions). The excessive costs associated with ex vivo cell manipulation are a major concern, not to mention inherent potential liabilities associated with cell transplantation, including contamination, pathogen transmission and tumorigenesis [81]. Nonetheless, we envision a future of widespread applications ranging from repair of common tooth defects such as dental caries and dental pulp regeneration, to the replacement of whole teeth lost by injury and/or disease, and eventual applications to repair large craniomaxillofacial defects caused by trauma or genetic birth defects. It is clear that continued efforts to improve our knowledge and understanding of dental tissue development and disease, dental stem cell biology, and dental tissue regeneration will provide a solid foundation for future regenerative approaches and clinical therapies for Translational Dental Medicine.

Outstanding Questions.

• In what ways is adult bioengineered tooth development similar to natural embryonic tooth development?

• In what ways is bioengineered tooth development distinct from natural embryonic tooth development?

• How best can we use our detailed knowledge of natural tooth development to guide bioengineering approaches to successfully regenerate living, functional teeth?

• What key signaling pathways are essential in order for bioengineered teeth to develop into living, functional replacement teeth?

• How can we precisely control the size and shape of a bioengineered tooth, including proper occlusion with opposing teeth?

• How can we control bioengineered tooth eruption?

• What types of progenitor stem cells are the best sources to use to regenerate each type of dental tissue – dental pulp, dentin, enamel, cementum, PDL and alveolar bone?

• Are autologous cells the only cell type we can use for effective TERMD therapies or can we also develop methods to employ allogenic and/or xenogeneic cell sources for these purposes?

• If progenitor dental stem cells need to be expanded in vitro, how best to do this in a controlled, reproducible, safe and efficient manner?

• How best to optimize and standardize dental stem cell isolation, purification, expansion, long-term storage, and transportation?

• What is the best method for long term storage of dental stem cells? Is cryopreservation the only method to use?

• For dental cell homing approaches for dental tissue and whole tooth regeneration, what are the best cues to guide proper progenitor stem cell migration and differentiation?

• What are the best scaffolding materials for dental tissue and whole tooth tissue engineering?

• What are the best fabrication methods to create cell seeded tooth scaffolds?

• Is it possible to fabricate off-the-shelf scaffolds that can reliably be used to bioengineer replacement teeth of precisely specified size and shape?

Figure 2: Methods for whole tooth regeneration.

A) Cells harvested from embryonic Enamel Organ (EO) and Pulp Organ (PO) tissues are recombined in a collagen gel drop and cultured in vitro. The reconstituted embryonic tooth germ is then transplanted and grown in the jawbone of an adult host animal. B) Cells harvested from postnatal EO and PO tooth buds seeded onto a decellularized tooth bud ECM (dTB-ECM) scaffold and transplanted into the jawbone of an adult host animal.

Highlights.

• The importance of oral health, and its essential link to systemic health, is a serious health concern worldwide.

• Current therapies used to repair craniomaxillofacial (CMF) defects largely rely on the use of synthetic materials, that lack many essential features of natural CMF tissues.

• The ability to regenerate living, hard and soft dental tissues to repair dental tissues damaged by disease, trauma, genetic and other disorders, would be a significant improvement over the currently used methods.

• Recent progress in the fields of Tissue Engineering and Regenerative Medicine and Dentistry has helped to devise more effective methods to regenerate vital dental tissues.

• Novel TERMD therapies are anticipated to significantly improve our ability to effectively repair CMF tissues.

• These new therapies are anticipated to have a significant impact on the dental therapeutics market.

Glossary

Biomedical engineering (BME)

is the field that combines engineering and biomedical science approaches to use scaffolds, cells and growth factors to guide the formation of tissues and organs

Decellularized Extracellular matrix (dECM) Scaffolds

ECM scaffolds created by gently decellularizing natural tissues in a manner that retains instructive ECM molecules and gradients that can guide tissue and organ regeneration of reseeded cells. Dental pulp, PDL tissue and immature tooth buds are the most commonly used dECM scaffold sources used in dental field at this time

Dentin-Pulp Complex (DPC)

Mineralized dentin and non-mineralized predentin, and the dental pulp, a type of loose connective tissue containing blood vessels, nerves and dental stem cells, and dentin secreting odontoblasts that line the inner dentin surface and extend cellular processes into the dentin tubules. The integrated and dynamic nature of the dentin-pulp complex is such that injury to the dentin can affect the dental pulp, and reciprocally that injury to the dental pulp can in turn affect the quantity and quality of the dentin

Dental Pulp Stem Cells (DPSCs)

Dental stem cells isolated from dental pulp

Enamel Knot (EK)

The EK signaling center is a compact group of cells in the tooth enamel organ, specifically in the inner enamel epithelium. The number of cusps and the shape of tooth are controlled by the numbers and locations of EK signaling centers

Gutta-percha (GP)

GP is a plastic material obtained from the Malaysian percha tree, commonly used in the dental clinic for root canal treatment (RCT) of teeth with severe pulp damage. RCT involves removal of the pulp, thorough cleaning and disinfection of the root canal, followed by filling the root canal with GP

Guided tissue regeneration (GTR)

A technique commonly used in the dental clinic for PDL regeneration and repair

Neural crest cells (NCC)

NCCs are specialized cells present in the embryonic ectodermal germ layer, which give rise to most of the craniofacial tissues including craniofacial bones and teeth, as well as other tissues such as peripheral and enteric neurons and glia

Periodontal Ligament Stem Cells (PDLSCs)

PDLSCs are resident stem cells present in periodontal ligament tissues

Regenerative Endodontics Therapies (RETs)

RETs are biological material based endodontic therapies aiming on dental pulp revascularization and revitalization

Tissue Engineering and Regenerative Medicine and Dentistry (TERMD)

is the use of biomedical engineering techniques to engineer functional, living, replacement tissues and organs

Tooth Bud (TB)

TBs are highly specialized rudimentary tooth organs consisting of dental epithelial and dental mesenchymal cells, which develop into mature teeth

Stem Cells of the Apical Papilla (SCAP)

SCAP are dental stem cells residing in the apical papilla of immature tooth roots

Stem cells from Human Exfoliated Deciduous Teeth (SHED)

SHED are stem cells present in the dental pulp of human exfoliated deciduous (baby) teeth