Columns: REVIEW Adult stem cell-based apexogenesis Li Y et al. ASC-based apexogenesis
Yao Li, Li-Hong Shu, Ming Yan, Wen-Yong Dai, Jun-Jun Li, Guang-Dong Zhang, Jin-Hua Yu
Yao Li, Li-Hong Shu, Ming Yan, Wen-Yong Dai, Jun-Jun Li, Guang-Dong Zhang, Jin-Hua Yu,Institute of Stomatology, Nanjing Medical University, Nanjing 213003, Jiangsu Province, China
Li-Hong Shu, Department of Stomatology, Children’s Hospital of Changzhou, Changzhou 213003, Jiangsu Province, China
Ming Yan, Guang-Dong Zhang, Jin-Hua Yu, Endodontic Department, Dental School of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Author contributions: Li Y, Shu LH, Yan M, Dai WY, Li JJ, Zhang GD and Yu JH solely contributed to this paper.
Supported by National Natural Science Foundation of China, No. 81371144; Natural Science Foundation of Jiangsu Province, No. BK20131392; and the Priority Academic Program Development of Jiangsu Higher Education Institutions PAPD, No. 2011-137
Correspondence to: Jin-Hua Yu, PhD, DDS,Associate Professor, Vice Director, Institute of Stomatology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, Jiangsu Province, China. firstname.lastname@example.org
Telphone: +86-25-86862843 Fax: +86-25-86862823
Received: November 23, 2014 Revised: January 1, 2014
Accepted: March 13, 2014
Generally, dental pulp needs to be removed when it is infected, and root canal therapy (RCT) is usually required in which infected dental pulp is replaced with inorganic materials (paste and gutta percha). This treatment approach ultimately brings about a dead tooth. However, pulp vitality is extremely important to the tooth itself, since it provides nutrition and acts as a biosensor to detect the potential pathogenic stimuli. Despite the reported clinical success rate, RCT-treated teeth are destined to be devitalized, brittle and susceptible to postoperative fracture. Recently, the advances and achievements in the field of stem cell biology and regenerative medicine have inspired novel biological approaches to apexogenesis in young patients suffering from pulpitis or periapical periodontitis. This review mainly focuses on the benchtop and clinical regeneration of root apex mediated by adult stem cells. Moreover, current strategies for infected pulp therapy are also discussed here.
When the dental pulp is infected, traditionally, the dental pulp must be replaced with inorganic materials (paste and gutta percha) via root canal therapy (RCT). However, for RCT-treated teeth, the loss of pulp vitality, which primarily provides nutrition and acts as a biosensor to detect the potential pathogenic stimuli, will bring about various problems including the decreased strength and increased fragility; teeth are destined to be dead, devitalized, brittle and susceptible to postoperative fracture. Therefore, crowns are suggested to protect the non-vital tooth, but subsequently bring about other complications, including food impaction, recurrent caries, gingivitis, coronal leakage or microleakage, etc.
As a result, a better way to treat dental pulp disease is needed. Nowadays stem cell-based therapies represent a promising potential to improve the life of patients with conditions ranging from neurodegenerative and traumatic diseases to regenerative medicine requiring replacement of complex structures such as bones and teeth[1-3]. Stem cells have the capacity of self-renewal and multiple-differentiation shown in Figure 1. They can be divided into four types, including totipotent stem cells, pluripotent stem cells, multipotent stem cells and unipotent or progenitor stem cells. Due to the ethical and legal issues, the clinical application of embryonic stem cells (ESCs) is still controversial and restricted, although they can differentiate into almost every cell type in the human body.  Adult stem cells, however, become valuable because they can be isolated from many different adult tissues and demonstrate the potential to give rise to cells of various lineages. The typical adult stem cells, like neural stem cells (NSCs), hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), are considered as multipotent because they can give birth to different cell types in the tissue of origin. NSCs present in the subventricular zone and the subgranular zone of the hippocampal dentate gyrus and are able to generate three cell types of the brain including neurons, astrocytes, and oligodendrocytes. HSCs were first named by Cohneim about 130 years ago. They can be isolated from bone marrow and differentiate into all blood cells of the myeloid and lymphoid cell lineages[3,7].HSCs are the best-studied stem cells for transplantation. They have been used in stem cell-based therapies for decades, especially in an allogeneic setting. Hematopoietic disorders or patients with other malignancies undergoing intensive chemotherapy or radiation therapy are usually treated with this kind of stem cells. Another kind of adult stem cells were first described by Friedenstein in the 1970s and were later defined as “mesenchymal stem cells” (MSCs) by Caplan and others. [10-13] MSCs have the potential to differentiate towards lineages of mesenchymal origin, including bone, cartilage, fat, connective tissue, muscle, and marrow stroma. They can be isolated from diverse organs and tissues, such as bone marrow, adipose tissue, umbilical cord blood and stroma, placenta, amniotic membrane, synovium, lung, dental pulp tissue and so on[3,5].Recently, more attention was paid to the clinical use of MSCs transplantation to treat diseases that affect the host organ, including kidney injury, liver failure, myocardial infarction, articular cartilage defect and spinal cord injury, etc.[14-18]. However, the application in the treatment of a young infected tooth remains unclear.
During tooth development, enamel is formed by ameloblasts derived from the oral epithelium, while dentin and dental pulp originate from the dental papilla. The development of tooth depends on many kinds of stem cells, some of which still exist after the formation of the root. It offers a chance for the tooth to regenerate itself when the tooth stops developing because of infection or trauma. Generally, apexification by calcium hydroxide (CH) is the most traditional method to treat the immature tooth suffering from infection, usually bringing about a calcified barrier in the root with an open apex or an incompletely formed root with necrotic pulp[19,20]. However, the root often stops developing, leading to an imbalanced crown and root containing thin root dentin and wide apex, which may cause root fracture during functional movements, and it also takes a long period to complete the treatment. Recently, another method called apexogenesis has been developed with the emergence of mineral trioxide aggregate (MTA), which refers to a vital pulp therapy procedure that encourages physiological development and formation of the root end[21,22]. Compared to apexification, teeth after apexogenesis with vital pulp therapy develop a normal thickness of dentin, root length and apical morphology with fewer follow-up appointments[23,24]. In Jung’s cases, he observed a separately growing root after the apexiﬁcation treatment, which indicated the coexist of apexification and apexogenesis. It is believed that some stem cells play important roles in the continuing root development of infected immature tooth.
ADULT STEM CELL CANDIDATES
MSCs with the capacity of self-renewal and multi-lineage differentiation are regarded as attractive progenitor cell sources for tissue engineering and regeneration.To date, several kinds of MSCs have been identified as promising candidates for dental tissue engineering in the dentistry field such as bone marrow mesenchymal stem cells (BMMSCs), dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells (PDLSCs), dental follicle precursor cells (DFPCs) and stem cells from apical papilla (SCAPs)[26-30]. But which MSCs are mostly suitable for apexogenesis? On one hand, a tooth derives from a tooth germ consisting of various MSCs which can develop certain parts of the tooth. On the other hand, MSCs of different origins may present various differentiation abilities. Previous studies have revealed that MSCs can differentiate into certain tissues after specific induction. Accumulating evidence demonstrates that MSCs have the potential to cross lineage boundaries, even able to differentiate into speciﬁc cells of tissues beyond their origin. ] (Tables 1 and 2 describe the adult stem cell candidates and their in vivo transplantation outcomes from different research groups).
Bone marrow mesenchymal stem cells
Bone marrow mesenchymal stem cells (BMMSCs) are the first isolated mesenchymal stem cells with spindle-shaped morphology which have the ability to adhere to a plastic surface with high proliferative potential. BMMSCs possess the self-renewal capacity to form colonies in vitro and are capable of differentiating into multiple mesenchymal cell lineages such as osteoblasts, adipocytes, chondrocytes, muscle cells, tenocytes, or nerve cells[33-35].However, BMMSCs are limited to a growth potential of 30 to approximately 50 population-doublings (PDs) following ex vivo expansion. BMMSCs express the Oct-4, Nanog, STRO-1, CD73, CD90, CD105, CD146[36,37] and are negative for CD14, CD34, CD45 and HLA-DR[38-40]. Based on their multi-lineage differentiation potential and their high proliferative capacity, BMMSCs lead to a great potential for stem cell-based regenerative therapies. For instance, the intracoronary transplantation of autologous BMMSCs for ischemic cardiomyopathy has shown the promising results.Furthermore, after transplantation of BMMSCs into regions of central nervous injury, an improved functional recovery was observed in the injured rodent brain or spinal cord. Ohazama et al have reported that the combination of adult BMMSCs and embryonic oral epithelium can stimulate an odontogenic response in BMMSCs, and transfer of the complex into adult renal capsules can result in the development of tooth structures and associated bone. Li et al also have demonstrated that the combination of oral epithelial cells from rat embryos with BMSSCs can generate tooth-like structures expressing dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1) surrounded by bone and soft tissue.Kawaguchi et al have shown complete regeneration of periodontal defects after BMMSCs transplantation, and histologically produced cementum, periodontal ligament (PDL), and alveolar bone.
Dental pulp stem cells
Dental pulp stem cells (DPSCs) were first isolated and characterized from dental pulp tissue by Gronthos et al in 2000. Similar to MSCs, DPSCs are positive for CD29, CD44, CD59, CD90, CD106, and CD146, and negative for CD34, CD45, and CD11b. DPSCs are described as a highly proliferative cell population with the self-renewal ability and multi-lineage differentiation potential. DPSCs possess mesenchymal stem cell properties such as a fibroblast-like morphology, adherence to a plastic surface, and the ability to form colonies when cultured in vitro and they are able to differentiate into chondrocytes, adipocytes, odontoblasts and neural-like cells under appropriate inductive conditions. Previous studies have shown that DPSCs are capable of differentiating into odontoblastic lineages in vitro[44,45] and form ectopic pulp-dentine like tissue complexes lined with odontoblast-like cells expressing dentin sialophosphoprotein (DSPP) when transplanted subcutaneously into immunocompromised mice in vivo. DPSCs can also form bone-like tissues when transplanted into immunocompromised mice. Some studies have demonstrated that DPSCs are able to differentiate into endothelial-like cells and express blood vessel markers and neural markers, but the in vivo differentiation potential is still under debate[46-48]. Due to their easy obtainment and the potential of multi-lineage differentiation, DPSCs are thought to be an ideal cell source for tissue regeneration and engineering.
Stem cells from apical papilla
Apical papilla means the soft tissue at the apices of developing permanent teeth and Stem cells from apical papilla (SCAPs) are a population of mesenchymal stem cells residing in the apical papilla of incompletely developed teeth. The surface makers are similar to BMMSCs and DPSCs, but CD24 is only detected in SCAPs. The expression of CD24 is down-regulated following osteogenic induction. It has been reported that SCAPs display a higher proliferation rate than DPSCs, probably because they are derived from a developing tissue. Similar to DPSCs, SCAPs are able to differentiate into a variety of cell types, but appear to have greater dentinogenic potential than DPSCs. An in vivo study has shown that SCAPs with hydroxyapatite/tricalcium phosphate particles that was transplanted into immunocompromised mice can generate a typical dentin structure with a layer of dentin tissue formed on the surface of the hydroxyapatite/tricalcium phosphate along with connective tissue.SCAPs also demonstrate the capacity to undergo adipogenic differentiation after inductionand express neural markers with or without stimulation when cultured in vitro. Besides, SCAPs are able to form a bio-root with the use in combination with PDLSCs. All the above suggest that SCAPs can be used for tissue regeneration and engineering. Based on previous findings, SCAPs appear to be the source of primary odontoblasts that are responsible for the formation of root dentin.This may explain the clinic phenomenon that apexogenesis can occur in infected immature permanent teeth suffering from apical periodontitis or abscess[23,51].
Stem cells from human exfoliated deciduous teeth
Stem cells from human exfoliated deciduous teeth (SHEDs) were first isolated by Miura et al from dental pulp tissue derived from exfoliated deciduous teeth. SHEDs exhibit a higher proliferation rate than DPSCs. They express similar surface markers as compared to DPSCs and BMMSCs, but the expression of CD105 and CD146 is higher, suggesting higher capacity for differentiation. SHEDs have the potential to differentiate into neurons, adipocytes, osteoblasts, and odontoblasts as well as DPSCs. When SHEDs are subcutaneously transplanted into immunocompromised mice, they can form ectopic dentin-like tissue, but not able to regenerate a dentin-pulp-like complex. Wang et al has revealed that SHEDs exhibit an enhanced potential to form bone, while Miura et al have suggested that SHEDs can not differentiate directly into osteoblasts but only induce new bone formation. These findings imply that deciduous teeth may be involved in bone formation during the eruption of permanent teeth. SHEDs can also differentiate into neural cells after neuronal induction and express early neuronal markers. As a result, SHEDs can be a promising source of stem cells for regenerative medicine.
Periodontal ligament stem cells
The periodontal ligament (PDL) is the soft connective tissue interposed between the cementum and the inner wall of the alveolar socket,which is derived from the dental follicle. Previous studies have suggested that the PDL space may contain stem cells that exhibit osteogenic, cementoblasts, adipogenic, chondrogenic and neurogenic characteristics under certain culture conditions[30,56], which is defined as periodontal ligament stem cells (PDLSCs). When cultured in vitro, PDLSCs can form mineralized nodules, express the bone-associated markers including alkaline phosphatase (ALP) and bone sialoprotein (BSP), response to bone-inductive factors such as insulin-like growth factor 1 (IGF-1) and express high level of scleraxis, which is a speciﬁc transcription factor associated with tendon cells[58,59]. Although PDLSCs express a range of cementoblastic/osteoblastic markers, they do not form dentin and its associated haemopoietic components in vivo. Previous studies have demonstrated that PDLSCs transplanted into immunocompromised mice can generate a typical cementum/PDL-like structure, in which a thin layer of cementum-like tissue is formed on the surface of the carrier, along with condensed collagen ﬁbres with sparse cells that resemble PDL structures[56,58]. But the cemetum/PDL-like structures appeared totally different from typical bone/marrow structures generated by BMMSCs and dentin/pulp-like structures generated by DPSCs. Due to their capacity to form periodontal structures, PDLSCs can be used as a cell source for the treatment of periodontal diseases, tissue engineering and stem cell-based therapies.
Dental follicle precursor cells
Dental follicle is an ectomesenchymal tissue that surrounds the enamel organ and the dental papilla of the developing tooth germ prior to eruption. This tissue contains progenitor cells that form the periodontium including cementum, PDL, and alveolar bone. Dental follicle precursor cells (DFPCs) are isolated from human dental follicles of impacted third molars, expressing typical mesenchymal stem cell markers such as STRO-1, CD13, CD44, CD73, Notch1, and nestin[60,61]. Moreover, GoPro49, a novel Golgi protein, has been identiﬁed as a speciﬁc marker for DFPCs. DFPCs possess the ability to differentiate into osteoblasts/cementoblasts, chondrocytes, adipocytes, and neuron-like cells when grow under the appropriate culture conditions in vitro[3,60,61]. Recent studies have revealed that DFPCs cultured at 38 to 40°C demonstrate greater osteogenesis, indicating that appropriate heat-stress treatments can promote their differentiation.When DFPCs were transplanted into immunocompromised mice, a structure comprised of fibrous or rigid tissue was generated, expressing BSP, osteocalcin (OCN) and collagen type I. However, there were no dentin, cementum, or bone formation observed in the transplants in vivo. But some studies[65,66] have demonstrated that DFPCs can form cementum-like matrix or bone structures in the subcutaneous area of immunodeficient mice. More work still has to be performed to explore their potential capability during the cellular therapies of periodontal tissues.
PUTATIVE INDUCTIVE MATERIALS
In addition to stem cells, adequate inductive materials that can promote the differentiation of stem cells are essential to the success of stem cell-based treatment. Previous researches have investigated the effects of several materials on different stem cells, which may guide us to pair the right stem cells with the most compatible inductive material.
Calcium hydroxide (CH) (pH = 12.5) has a good antimicrobial characteristic and can inhibit tooth resorption and induce the hard tissue formation. It has been successfully utilized in various endodontic treatments, such as apexification, apexogenesis, pulp capping, pulpotomy, and routine root canal therapy in infected canals for its potential to induce hard tissue repair at the site of pulp exposure[68,69], Earlier study has suggested osteo-inductive properties of CH, however, no significant changes were observed in vitro When CH is placed at the exposed pulp site, damaged primary odontoblasts are replaced with newly differentiated odontoblast-like cells. Ji et al have exhibited that CH can increase the recruitment, migration, proliferation, and mineralization of DPSCs and PDLSCs. Besides, Ruparel’s work has suggested that CH can promote cell survival of SCAPs. Moreover, low concentrated CH induces the proliferation of pulp fibroblasts. However, there exist several disadvantages of CH used in apexification, including multiple visits, the long treatment time and the risk of root fracture as a result of long-term use of CH[67,73-75]. It is also suggested that direct contact of CH with the tissue will induces the formation of calcified tissue in the pulp space, thus preventing pulp tissue from regeneration. Another problem is that CH may damage the Hertwig’s epithelial root sheath (HERS) and thereby destroy its ability to induce the nearby undifferentiated cells to become odontoblasts.
Mineral trioxide aggregate
In 1993, Mineral trioxide aggregate (MTA) was firstly introduced into Endodontics and now has been widely used in diverse endodontic therapies, including pulp capping, pulpotomy, apical barrier formation, apexogenesis in developing teeth, repair of root perforations and root canal filling. MTA is a cement mixture that consists of different oxide compounds, including sodium and potassium oxides, calcium oxide, silicon oxide, ferric oxide, aluminum oxide, and magnesium oxide.  Compared with CH, MTA is a better choice for direct pulp capping because of its lower solubility, improved mechanical strength, better marginal adaptation, and better sealing ability, but no significant histological difference is established[77,78] Some data also suggest that MTA is more predictable with consistent hard-tissue formation as a result of the release of large number of Ca2+ ions or the secretion of bone morphogenetic protein 2 (BMP-2) and transforming growth factor-beta 1 (TGF-β1) by periodontal fibroblasts. Some studies have reported that MTA can induce the formation of hard tissue in a shorter period of time than CH[81,82]. Further study revealed that MTA can induce tissue regeneration via the promotion of mesenchymal stem cell adhesion, proliferation, and migration.Recent researches have shown that MTA stimulate the odontogenic differentiation of DPSCs, with the up-regulation of OCN and DSP[84-86]. The significantly increased level of the angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblastic growth factor-2 (FGF-2) of DPSCs are observed following the treatment with MTA in vitro[84,85], which play a critical role in tissue development, cell migration, inflammation and wound repair.
Generation of well-vascularized pulp-like tissue by using a tooth slice model has been reported. Huang suggested that DPSCs differentiate into odontoblast-like cells with a cellular process extending into dentinal tubules when seeded onto the existing dentine. Cordeiro et al have demonstrated similar findings that odontoblast-like cells arose from the stem cells and localized against the existing dentine surface in their in vivo study model. Previous studies have shown that human DPSCs, SCAPs and SHEDs, with the combination of synthetic scaffolds or human root segments, are able to generate vascularized pulp-like tissues and form dentin-like mineral structures depositing onto the existing dentinal wall in the root canal space[88-90]. The mechanism behind this phenomenon has been speculated to be related with the released growth factors by dentine, such as TGF-β, which attract and induce the differentiation of odontoblasts. Chemical disinfection of the root canal space may destroy these embedded growth factors.