Dental stem cells in tooth repair: A systematic review [version 1; peer review: 2 approved with reservations]

Abstract Background: Dental stem cells (DSCs) are self-renewable teeth cells, which help maintain or develop oral tissues. These cells can differentiate into odontoblasts, adipocytes, cementoblast-like cells, osteoblasts, or chondroblasts and form dentin/pulp. This systematic review aimed to summarize the current evidence regarding the role of these cells in dental pulp regeneration. Methods: We searched the following databases: PubMed, Cochrane Library, MEDLINE, SCOPUS, ScienceDirect, and Web of Science using relevant keywords. Case reports and non-English studies were excluded. We included all studies using dental stem cells in tooth repair whether in vivo or in vitro studies. Results: Dental pulp stem cell (DPSCs) is the most common type of cell. Most stem cells are incorporated and implanted into the root canals in different scaffold forms. Some experiments combine growth factors such as TDM, BMP, and G-CSF with stem cells to improve the results. The transplant of DPSCs and stem cells from apical papilla (SCAPs) was found to be associated with pulp-like recovery, efficient revascularization, enhanced chondrogenesis, and direct vascular supply of regenerated tissue. Conclusion: The current evidence suggests that DPSCs, stem cells from human exfoliated deciduous teeth, and SCAPs are capable of providing sufficient pulp regeneration and vascularization. For the development of the dental repair field, it is important to screen for more effective stem cells, dentine releasing therapies, good biomimicry scaffolds, and good histological markers.


Introduction
Regenerative dentistry is designed to recover dental anatomy and function. Regenerative endodontics procedures (REPs) of a damaged tooth are a series of biological processes aimed to restore the dental pulp's physiological functions, cure periapical lesions, and substitute pulp-dentin complex cells and dentin 1,2 . Three components are involved in these techniques: stem cells, growth and bio-materials, which are often known as scaffolds or templates 3 The dental pulp consists of nerves, blood vessels and connective tissue to maintain teeth's integrity. The nerves of the pulp can mediate pain, blood flow control, recruit immunocompetent cells, and act as a mesenchymal stem cells (MSCs) niche 4 . Loss of tooth pulp stops the development of permanent root teeth that can weaken the periodontal connection and lead to teeth loss. Recent animal studies indicate that vascular dental pulp can be regenerated by cell-based therapy 5 .
Dental stem cells (DSCs) are self-renewable teeth cells, which help maintain or develop oral tissues 6 . In the literature, there are various types of dental adult stem cells, such as dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells or stem cells of the developing root apical papilla (SCAPs), dental follicle stem cells (DFSCs), and dental MSCs (DMSCs) 7,8 . Such cells can differentiate into dentine/pulp, odontoblast, adipocyte, cementblast-like, osteoblast, and chondroblast cells. DSC regenerative potential is explained through both natural and experimental conditions 9,10 . Differentiated dentinoblasts, also called secondary odontoblasts, produce new dentine in response to dental cell injury. This regenerative process is called reparative tertiary dentinogenesis 11 . This process of dentinogenesis was suggested to be used in the recruitment of endogenous DSCs. Most recent animal studies have investigated the role of DSCs in the regeneration of dental pulp tissues.
Bakhtiar et al. 12 conducted a systematic review on 47 studies that investigate the role of stem cell therapy in regeneration of dentine-pulp complex; the current systematic review aimed to update this previous systematic review, presenting 57 articles, and summarizes the current evidence regarding the efficacy of dental stem cells in dental pulp regeneration in animal models.

Methods
We report this manuscript following the preferred reporting items systematic reviews and meta-analysis (PRISMA statement) guideline 13 . All methods used in this review were conducted in strict accordance with the Cochrane Handbook for Systematic Reviews of Interventions 14 .

Literature search strategy
We searched the following databases from January 2000 to June 2019: PubMed, Cochrane Library, MEDLINE, SCOPUS, Sci-enceDirect, and Web of Science using the following keywords (((Dental pulp stem cells OR DPSCs OR stem cells OR human exfoliated deciduous teeth OR SHEDs OR Periodontal ligament stem cells OR developing root apical papilla OR SCAPs OR dental follicle stem cells OR DFSCs OR dental mesenchymal stem cells OR DMSCs) AND (pulp OR pulpal tissue OR pulp treatment OR pulpal therapy) AND (endodontic treatment OR deciduous teeth OR permanent teeth OR primary teeth OR dentition))) to identify the relevant studies.
Study selection process and eligibility criteria Two authors screened the titles and abstracts of retrieved literature records. For titles and abstracts that deemed relevant to the research question, the full-text articles of these records were obtained and screened for eligibility according to the following criteria: We included studies that meet the following PICOS criteria: 1) Population: Both in vitro and in vivo studies that investigate the endodontic regeneration following treatment with dental pulp stem cells.
2) Intervention/Comparator: studies that use all of the following types of stem cell in the regeneration of dental pulp tissue: DPSCs, SHEDs, SCAPs or DMSCs.
3) Outcomes: pulpal regeneration or repair. 4) Study design: All in vivo, in vitro, animal, or human studies.
We excluded all of the following studies: 1) Case reports and case series; and 2) non-English studies. In the case of multiple reports for the same study population, we analyzed data from the most updated dataset. Any discrepancies were resolved by discussion and consensus between reviewers.

Data extraction
Data extraction was performed manually and data were entered into a structured Microsoft Excel sheet (For Windows, Professional Plus version 2016). We extracted data of the following domains: 1) Characteristics of study design; 2) Baseline criteria of the included population; and 3) Study outcomes. There was not sufficient data for meta-analysis.

Search strategy results
The electronic search retrieved 4433 unique articles. After removing duplications, 2780 articles were enrolled in the title/ abstract screening. This led to the retrieval and screening of 327 full-text articles for eligibility. Studies that were not eligible with our criteria were excluded. In total 57 articles were included in the qualitative synthesis. A flow diagram of the selection process is shown in Figure 1. A summary of characteristics, models, and populations of the included studies and their key outcomes are shown in Table 1. Variation of the extracted data is reported in Table 2.

Types of stem cells
In this systematic review, we reviewed multiple types of stem cells, such as dental follicle stem cell (DFSC), bone marrow mesenchyme stem cell (BMSC), periodontal ligament stem cell (PDLSC), dental pulp extracellular matrix (DPEM), adiposederived stem cell (ADSC), DPSC, SCAP, and SHED. The majority  Dental pulp stem cells All included experiments utilizing DPSCs were isolated from human healthy pulp tissues, usually orthodontics, to be used in an animal model. Stem cells from exposed pulp have also been reported to be more likely to differentiate into osteoblastic cells than dentinogenic ones. In this review, 20 articles used DPSCs in mice models 16 However, two studies reported that DPSCs formed an inflamed pulp-like tissue 15,20 .

Stem cells from apical papilla
SCAPs were commonly isolated from immature third molars. Wang et al. 15 reported that SCAPs have greater generation of mineralized tissue than those with DPSCs and higher differentiation of osteo/odontoblast in the supplemental medium khpo4. Furthermore, SCAPs have been reported to have re-vascularizing properties, heterotopic dental pulp/dentin complex formation, faster proliferation and mineralization, and more efficient migration and telomerase than DPSCs 46,65 .

Periodontal ligament stem cells
PDLSCs demonstrated a significant role in maintenance of MSC characteristics after implantation 41 . In addition, the dentin tissue structure produced by dental follicle cell (DFC) was more complete. In the included experiments, Gao et al. 22 used PDL for regeneration of a fresh bio-root. They developed an effective bio-root of PDL tissue, using a mixture of DPSC-hydroxyapatite wrapped in a layer of PDLSCs. These freshly produced miniature pig roots, both in mineral components and biomechanical characteristics, had comparable characteristics to natural teeth, but only 20% of the samples attained success, whilst titanium implants were 100% effective.

Stem cells from human exfoliated deciduous teeth
SHEDs, which rare derived from extracted deciduous teeth, were used in mice models in four studies 43,58-60 . It was observed that the capability of mineralization of SHEDs was higher than DPSCs 43 . Casagrande et al. 60 reported that SHEDs express markers of odontoblastic differentiation (DSPP, DMP-1, MEPE).

Discussion
This is the largest and most updated systematic review aiming to investigate the role of DSCs in tooth repair. We found that multiple DSCs have a potent role in tissue regeneration and vascularization of dental pulp-like tissues 54 Several dentin therapies demonstrate further excellent outcomes, which should be followed by platelet-rich plasma/platelet-rich fibrin (PRP/PRF) or collagen gels in REPs and improved biomimicry to maintain various levels of the variables that release oral stem cell niche formation. Recently, cell survival of stem cells is much easier than in the past due to the appropriate interaction with dentine-released factors 21,23 . Thus, screening for more appropriate stem cells, dentine releasing treatments, scaffolds with good biomimicry and good histological markers is an exciting activity for future REP improvements.
Besides dental sources, non-dental cells, such as the MSCs derived from bone marrow and adipose stem cells, are able to regenerate the pulp tissue.
Generally, our study showed that adult stem cells appear to be able to regenerate dentine-pulp complexes; therefore, the criteria of selection should be considered the most cost-effective and cheapest, particularly when the main obstacle is the expense 28,32,35 . Moreover, our findings demonstrated that the human body is a wealthy source of stem cells; therefore, the third molars or any orthodontic tooth originated from a human body are excellent sources of stem cells. As regards cells circulating, these cells migrate to sites and engage in a recovery process in the presence of chemotactic gradients, as their capacity for root canal migration was shown 33 .
This study showed two limitations; 1) we could not conduct a meta-analysis due to insufficient data; and 2) we failed to find a suitable tool to assess the quality of included studies and risk of bias.
In conclusion, the current evidence suggests that the DPSCs, SHEDs, and SCAPs are capable of providing a sufficient pulp regeneration and vascularization. Nevertheless, the efficacy of stem cell transplantation in therapy locations and their cost may be obstacles to their clinical use. Scaffolds and biomaterials provide a useful strategy for stronger incorporation of stem cells and development factors together with monitored regeneration rates. Hence, we suggest future studies to concentrate on offering definite guidance on appropriate and preferable biomaterial characteristics for use in regenerative endodontics.

Data availability
Underlying data All data underlying the results are available as part of the article and no additional source data are required.