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South China Center of Craniofacial Stem Cell Research, Guanghua School and Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, ChinaDepartment of Anatomy and Cell Biology, University of Pennsylvania, School of Dental Medicine, Philadelphia, PennsylvaniaResearch and Development Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, China
South China Center of Craniofacial Stem Cell Research, Guanghua School and Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China
South China Center of Craniofacial Stem Cell Research, Guanghua School and Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China
Southern Center for Biomedical Research, Fujian Key Laboratory of Developmental and Neural Biology, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian, China
South China Center of Craniofacial Stem Cell Research, Guanghua School and Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, ChinaDepartment of Anatomy and Cell Biology, University of Pennsylvania, School of Dental Medicine, Philadelphia, Pennsylvania
Address requests for reprints to Prof Songtao Shi, University of Pennsylvania, School of Dental Medicine, 240 South 40th Street, Levy 421, Philadelphia, PA 19104.
South China Center of Craniofacial Stem Cell Research, Guanghua School and Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, ChinaDepartment of Anatomy and Cell Biology, University of Pennsylvania, School of Dental Medicine, Philadelphia, Pennsylvania
Postnatal stem cells critically maintain tissue homeostasis and possess immense potential for tissue regeneration. These stem cells in the orofacial system were not identified until early 2000s when they were first found in the dental pulp and termed dental pulp stem cells (DPSCs). Isolated from either permanent or deciduous teeth, DPSCs were characterized to be highly clonogenic with multidifferentiation and neurovascular properties. Subsequent studies suggested that the origin of DPSCs may be associated with neural crest-derived cells and localized adjacent to neurovascular bundles as indicated by specific surface markers. DPSCs serve as key contributors to pulp homeostasis and injury repair. Mechanistic studies have revealed a fine-tuning regulatory network composed of both extrinsic and intrinsic factors that orchestrate fates of DPSCs. These findings have shaped our understanding of their biological nature as niche responsive progenitors. As we explore the potential of DPSCs in pulp regeneration, preclinical studies have developed diverse DPSC transplantation-based strategies, among which preconditioned DPSCs and DPSC aggregates have shown particular promise. Confirmed by recent clinical advances, DPSC transplantation after pulpectomy has successfully rebuilt the physiological pulp structure in situ functionalized with neurovascularization, indicating a novel regenerative approach for treating pulp diseases. Here, we summarized the 20-year golden journey on DPSCs from the unprecedented discovery to current clinical breakthroughs, while also suggesting future directions and challenges regarding expansion of regenerative applications and evaluation of in vivo DPSCs in diseases and therapies. The historical perspective of this field will provide a blueprint for the stem cell research and enlighten principles for de novo organ regeneration.
This article reviews DPSCs from discovery to clinical application, indicating the promise of pulp regeneration. Particularly, complete three-dimensional tissue pattern reconstruction and functional recovery based on neurovascular restoration have been achieved by the strategy of DPSC constructs in empty root canals, with virtually no risks of tumorigenesis or other side effects. DPSC transplantation is well on the way to becoming accepted clinical therapeutic approach in future endodontics.
Oral health is recognized as a key overall health indicator by the World Health Organization, but the preservation of complete and vitalized dental tissues remains as significant clinical challenges in modern dentistry
. Particularly, dental pulp, the only soft connective tissue in the tooth amid highly mineralized structures, plays an indispensable role in maintaining tooth homeostasis, while also being vulnerable to trauma and infection that may lead to irreversible pulpitis or necrosis
. At present, the most common clinical treatment of pulp diseases, known as root canal therapy, is based on pulpectomy, resulting in a permanently devitalized tooth more susceptible to structural and esthetic failure and secondary infection
. Thus, to overcome these shortcomings we would continue to advance the regenerative endodontic technology, aiming at preserving and regenerating the vitality and health of the dental pulp
, increasing efforts in stem cell research have shaped our perception of these cells as critical contributors to organogenesis and adult tissue homeostasis, as well as demonstrating their particular regenerative promise that led to the development of tissue engineering technologies near the end of the 20th century
. It has long been known that the tooth is capable of forming reparative dentin after injury; therefore it was considered that resident stem cell populations exist in the dental pulp
Temporal and spatial expression of c-jun and jun-B proto-oncogenes in pulp cells involved with reparative dentinogenesis after cavity preparation of rat molars.
. This notion was proved when dental pulp stem cells (DPSCs) were discovered in the permanent and deciduous human teeth in 2000 and 2003, respectively (pDPSCs and dDPSCs, respectively; the latter was named as SHED)
. Such discoveries opened a new avenue of research in oral sciences. In the past 2 decades we have witnessed tremendous advancement in DPSC studies from functional characterization
Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: evidence for HDAC2 involvement.
(Fig. 1). In this review, we summarized the 20-year golden journey on DPSCs from the original discoveries to current clinical breakthroughs and discussed future directions and challenges.
Figure 1Major discoveries that shaped our understanding of DPSCs: timeline and current trends. (A) In the past 2 decades, scientific studies on DPSCs can be categorized into 3 major phases. The works from 2000 to 2003 focused on the discovery and characterization of DPSCs. From 2004 to 2012, the regulatory mechanisms of DPSCs were subsequently investigated. Meanwhile, preclinical studies using DPSCs for pulp regeneration were performed on the basis of findings regarding their neurovascular properties. From 2013 to 2019, with the advance of techniques, in vivo characterization of DPSCs was conducted, particularly elucidating their localization and surface markers. Furthermore, clinical application of DPSCs in pulp regeneration has been successfully evaluated. (B) The total number of original articles published on DPSCs continuously increases after DPSC discoveries, plotted from 2004 to 2019. Results were collected from PubMed search for “dental pulp”, “tooth pulp”, “pulp stem”, “SHED”, “DPSC”, “DPSCs”, “pulp progenitor”, and “pulp progenitors”. (C) Total number of citations about DPSC studies continuously increases after DPSC discoveries, plotted from 2004 to 2019. Results were collected from Web of Science search for “dental pulp”, “tooth pulp”, “pulp stem”, “SHED”, “DPSC”, “DPSCs”, “pulp progenitor”, and “pulp progenitors”.
The pDPSCs were the first orofacial stem cells to be isolated and identified, which possess typical characteristics as mesenchymal stem cells (MSCs), demonstrating plastic adherence and clonogenic properties with multilineage differentiation capabilities in vitro
. Compared with MSCs derived from bone marrow, pDPSCs are highly proliferative and able to differentiate into odontoblasts, generating an ectopic dentin-pulp complex after transplantation in vivo
. Subsequently, dPSCs, which exhibit even higher clonogenic, proliferative, and osteogenic capacities than pDPSCs, were discovered in the human deciduous teeth
. DPSCs were subsequently isolated from human natal teeth and tooth germs, which also possess higher proliferation rate compared with pDPSCs, confirming that earlier developmental stages provide DPSCs with functional advantages
. Enlightened by DPSC discoveries, a series of orofacial mesenchymal stem cells (OFMSCs) were successively isolated and identified in the last decades, such as periodontal ligament stem cells
Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis.
. The OFMSC populations are all easily accessible and expandable with regenerative potential, which make them ideal candidates for tissue engineering applications
. It is further noted that DPSCs are characterized by potent immunomodulatory properties, considering that they inhibit T-helper 17 cells and promote regulatory T cells for treating systemic lupus erythematosus and experimental colitis
In vivo functional characterization of DPSCs is closely related to their origin and localization. It has long been believed that OFMSCs are derived from neural crest cells in the early head development that form the ectomesenchymal tissue
. Their neurovascular properties are further supported by the neurovascular bundle (NVB), a specialized niche centered on arteries and accompanying nerves in the continuously growing mouse incisor
. DPSCs localized adjacent to NVB are regulated by the NVB-secreted Sonic hedgehog (Shh) protein contributing to development, homeostatic maintenance, and injury repair of the dental mesenchyme in vivo
In light of the above findings, general and specific surface antigens of DPSCs have been revealed. Classical MSC makers have been identified in DPSCs, such as CD90 and STRO-1
. There are also functional markers of DPSCs that have been revealed. For example, programmed cell death 1 (PD-1) has been recently reported as a spatial-restricted specific antigen for DPSCs but not bone marrow MSCs, the significance of which to DPSCs was confirmed by loss-of-function and sorting experiments showing reduced capacity of proliferation, yet with accelerated differentiation capabilities on PD-1 deficiency
. Other markers, such as CD31, CD105, and CD146, or even the side population by flow cytometry, are also used as negative or positive sorting criteria to obtain subsets of DPSCs with superior regenerative potential
. Collectively, different subpopulations of DPSCs might play heterogeneous or complementary roles in the maintenance of pulp tissue homeostasis and repair in situ, while also presenting functional implication in regenerative applications.
Regulatory Mechanisms of DPSC Behaviors
Understanding the regulatory landscape of DPSC behaviors is necessary to decipher their roles in maintaining pulp tissue homeostasis and to improve the efficacy in tissue regeneration. To date, accumulating studies have indicated a concerted action of both extrinsic cues and intrinsic factors that coordinately determine fates of DPSCs (Fig. 1).
Because DPSCs localize at specific anatomic sites of perivascular and perineural pulp regions, they are connected in the root canal to the external environments, which encompass humoral, neural, gaseous, and mechanical factors together constituting a milieu for a tight modulation of DPSC function
. A great amount of humoral regulators have been unraveled that affect DPSCs, including those from bacteria (eg, lipopolysaccharides), the immune system (eg, tumor necrosis factor alpha), and surrounding dental cells (eg, preameloblast-derived factors)
. In addition, because physiological levels of oxygen in vivo range between 3% and 6% in concentrations, DPSCs may preserve a quiescent state but demonstrate high rates of proliferation under ambient oxygen tensions at 21% in culture
. Besides, there are a number of dentin-derived signaling molecules like bone morphogenetic protein 2 (BMP2) that may contribute to pulp regeneration and tissue repair. It was reported that BMP2 plus dentin matrix carrier stimulated pulp cell differentiation into odontoblasts and induced formation of tubular dentin
Intrinsic mechanisms for DPSC regulation generally involve signaling pathways, transcriptional factors, and epigenetic modulators, which form fine-tuning cascades in response to extrinsic cues to orchestrate DPSC function as a molecular network. It has been reported that the nuclear factor-kappa B pathway and the mitogen-activated protein kinase pathway respond to lipopolysaccharide and/or tumor necrosis factor activated by the inflammatory microenvironments, resulting in functional alterations of DPSCs
. Studies have also documented the WNT signaling, which plays an important role in maintaining stemness properties, interacts with Notch and c-Jun N-terminal kinase signaling pathways, and critically regulates differentiation of DPSCs
. Furthermore, epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNAs, are indispensable for regulating self-renewal and stemness of DPSCs, the targets of which involve crucial signaling pathways controlling lineage commitments of DPSCs, such as the phosphatase and tensin homolog/phosphoinositide 3-kinase/AKT pathway
Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: evidence for HDAC2 involvement.
. Taken together, the bona fide regulatory mechanisms of DPSCs have increasingly been uncovered in the past decades, which further provide great benefits for functional modulation of DPSC behaviors in regenerative applications.
Applications of DPSCs in Dental Pulp Regeneration
On the basis of functional characterization and mechanistic investigations of DPSCs, attempts to achieve pulp regeneration by using DPSCs have been extensively tested in preclinical studies. The regeneration strategies can be categorized into 4 types of applications: DPSCs with scaffolds, DPSCs with scaffolds and cytokines, DPSCs with cytokines, and DPSC constructs (Table 1).
Table 1Preclinical Studies of Pulp Regeneration Using DPSCs
Application strategy
Stem cells
Scaffolds
Cytokines
Species
Implantation site
Regenerative outcomes
Reference
Stem cells + scaffolds
pDPSCs
Synthetic PLG scaffolds
None
Mouse
Subcutaneous in root canal
Pulp-like tissues with established vascularity after whole pulpectomy
Trophic effects and regenerative potential of mobilized mesenchymal stem cells from bone marrow and adipose tissue as alternative cell sources for pulp/dentin regeneration.
Preclinical experiments on dental pulp regeneration started with using scaffolds, because it was anticipated that scaffolds may exert beneficial effects on the seeded stem cells
materials into partial or whole pulpectomized root canals, confirming that pulp-like tissues with morphologic characteristics resembling the normal dental pulp can be generated in subcutaneous and in situ experiments. Further studies have evaluated effects of certain cytokines on DPSCs transplanted with scaffolds. One of the representative cytokines is granulocyte-colony stimulating factor (G-CSF), which possesses anti-inflammatory and antiapoptotic effects while inducing neurogenesis and angiogenesis
Trophic effects and regenerative potential of mobilized mesenchymal stem cells from bone marrow and adipose tissue as alternative cell sources for pulp/dentin regeneration.
have been applied, aiming to provide extra neurogenesis or vascularization in pulp regeneration. Consequently, pulp-like tissue was also regenerated after subcutaneous implantation or implantation in pulpectomized root canals in situ.
It was proposed that the specific usage of functional DPSC subsets would improve their regenerative applications. Accordingly, functional sorting to obtain side population (SP) cells of the dental pulp was applied to stimulate angiogenesis in tissue regeneration
. All these functional DPSC subsets were delivered with scaffolds and achieved pulp-like tissue regeneration. Nevertheless, it is noticeable that all the regeneration with scaffolds failed to restore the physiological structure of the dental pulp because of the putative interruption of natural microenvironments.
Transplantation of DPSCs without scaffolds but pretreated with cytokines is further reported
, suggesting that beneficial microenvironments for DPSC function are crucial for pulp regeneration. In this regard, it has been initially reported that a dentinogenic microenvironment influences DPSC capacity for an improved dentin-pulp complex regeneration
. There are also several studies adopting the exogenous cell-free strategy for pulp regeneration with only cytokine treatments, such as using basic fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, nerve growth factor, and BMP7, intending to induce autologous stem cell migration
. However, although connective tissues with abundant cells indeed formed, the cytokine-only strategy is hard to interpret because unidentified cells are recruited with unpredictable outcomes
In view of the findings and limitations of previous preclinical research, recent studies have documented that application of only DPSC constructs, a regeneration strategy without scaffolds or cytokines, might even be more effective
. These approaches aim to take advantage of the self-assembling niche by DPSCs, which are able to provide plenty of the natural extracellular matrix resembling the physiological niche structure
. Expectedly, complete three-dimensional tissue pattern reconstruction and functional recovery have been achieved on the basis of neurovascular restoration in empty root canals
. Taken together, the above preclinical results are instructive, and the strategy of DPSC constructs may serve as feasible methodology for dental pulp regeneration in the clinical practice.
Clinical Findings Using DPSCs for Pulp Regeneration
Since the regenerative potential of DPSCs was initially proposed and was subsequently supported by preclinical experiments on in situ pulp regeneration, 2 clinical studies on pulp regeneration have been launched within the past several years that have achieved breakthroughs in humans
in 2017, 5 patients with irreversible pulpitis were enrolled and received in situ transplantation of pDPSCs pretreated with G-CSF. After 24 weeks, 4 of the 5 patients showed positive responses to the electric pulp test, a widely used method in clinics to examine neurologic reactions, indicating neural recovery during pulp regeneration
. However, only 3 of the 5 patients demonstrated dentin formation according to cone-beam computed tomography, suggesting unexpected pulp regenerative outcomes using G-CSF–preconditioned pDPSCs
in 2013–2018, with higher success rates and convincing evidence using dDPSC aggregates. This clinical trial enrolled 36 patients with pulp necrosis in a 12-month follow-up, among which 26 teeth were transplanted with dDPSC aggregates in situ, leaving 10 teeth as controls
. Histologic evaluations demonstrated successful regeneration of three-dimensional whole dental pulp tissues by dDPSC aggregates, which contain an odontoblast layer, connective tissues, blood vessels, and even neuronal markers
. More surprisingly, cone-beam computed tomography detected increased length of the root and reduced width of the apical foramina in recipient immature permanent teeth, proving that the regenerated dental pulp possessed normal function to maintain continued root development
represents the first human organ successfully regenerated in randomized clinical trials, which further shaped our perception of true regeneration together with its applicative principles. To obtain true organ regeneration with physiological patterns and function, both optimal stem cell “seeds” and suitable “soils” are in demand. For consideration of stem cells, it has been reported that DPSCs have partially methylated domains mimicking the inner cell mass and placental methylome, which helps to explain why DPSCs possess immense regenerative potential
. Furthermore, the reason underlying more powerful capabilities of dDPSCs in pulp regeneration can be attributed to the fact that dDPSCs are derived from underage individuals
. Modulating of recipient microenvironments might further be necessary in advanced pulp regeneration in complicated conditions, because inflammation serves as a detrimental factor for tissue regeneration
. Future possible environmental engineering strategies may also include establishing developmental niche characteristics through introducing appropriate humoral and mechanical cues. Collectively, the first successful experience of organ regeneration in human history would be an important reference for future regeneration studies. With regard to the potential risks of DPSC transplantation for treatment of necrotic root canals, tumorigenesis is currently not detected, whereas either remaining inflammation or complete disinfection may affect stem cell survival and expansion
. Despite the risks of failure, pulp regeneration still holds great benefits and promise to achieve functional vitalized pulp regeneration as an advantage over traditional root canal therapy.
Conclusion and Perspectives
Indeed, DPSC studies in the past 20 years represent a golden journey that has achieved dramatic progress and remolded our recognition of stem cell and oral sciences, as well as paving an avenue for opportunities and challenges (Fig. 1). From the discoveries of DPSCs as typical postnatal stem cell populations
. The fates of DPSCs are regulated by a series of endogenous and exogenous synergies, encompassing signaling ligands, receptors, pathways, and epigenetics
. Of particular importance, the DPSC research has proceeded beyond the preclinical stage of pulp regeneration with convincing results in pilot and randomized clinical practices
. The envisioned workflow is as follows: selection for patients suitable for pulp regenerative therapy; first admission for removal of dental infection; stem cell culture from healthy teeth to harvest DPSC aggregate; readmission for DPSC implantation; conduction of corresponding tests of outcomes; and risk assessments
. Together with the supporting patents, established oral stem cell banks, and the recently approved DPSC solution for injection (Fig. 1), the field will continue to grow as a blueprint for future stem cell research and therapy.
Concurrently, we are facing a dualistic reality that both frontiers and challenges remain. On the one hand, despite recent advances having characterized DPSCs in vivo, knowledge on how functional alterations of in situ DPSCs putatively contribute to developmental, traumatic, and infectious pulp diseases is limited. Furthermore, there is still lack of methodologies for targeting DPSCs in situ for pulp disease treatments. In this regard, bioinformatic approaches would be applied for analyzing potential molecular targets in disordered DPSCs and combined with aptamer-based delivery systems for specific gene editing in vivo
. On the other hand, there is an urgent need for improving clinical practice of DPSCs, including the long-term stability of regenerative effects, the risk and quality control, and the industrialization. Notably, recent studies have revealed that DPSC-derived extracellular vesicles possess usage-friendly characteristics of easy acquisition and better storage, and they show immense potential as a cell-free next-generation approach to optimize DPSC cytotherapy
. In consideration of all the available evidence and visions, DPSCs are well on their way to becoming accepted therapeutic protocols, which will provide an innovative and promising perspective for de novo organ regeneration in the next decades.
Acknowledgments
This work was supported by grants from a Guangdong Financial Fund for High-Caliber Hospital Construction, the Postdoctoral Innovative Talents Support Program of China (BX20190380 to B.S.), the Young Elite Scientist Sponsorship Program by CAST of China (2019QNRC001 to B.S.), and the General Program of China Postdoctoral Science Foundation (2019M663986 to B.S.).
The authors deny any conflicts of interest related to this study.
Temporal and spatial expression of c-jun and jun-B proto-oncogenes in pulp cells involved with reparative dentinogenesis after cavity preparation of rat molars.
Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: evidence for HDAC2 involvement.
Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis.
Trophic effects and regenerative potential of mobilized mesenchymal stem cells from bone marrow and adipose tissue as alternative cell sources for pulp/dentin regeneration.
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