F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.21058.1 Systematic Review Articles Dental stem cells in tooth repair: A systematic review

[version 1; peer review: 2 approved with reservations]

 Tawfik Tadros Mary Sabry Conceptualization Data Curation Funding Acquisition Methodology Resources Software Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-8423-1142 a 1 2 El-Baz Maha Abd-El Salam Supervision Writing – Review & Editing b 1 Khairy Mohamed Adel Ezzat Khairy Supervision Writing – Review & Editing c 1 Conservative Dentistry Department, Faculty of Dentistry, Cairo University,, Cairo, Egypt Helwan General Hospital, Ministry of Health, Cairo, Egypt mora.dentist@gmail.com mahaaelbaz@yahoo.com madelkhairy@yahoo.com

No competing interests were disclosed.

 22 11 2019 2019 8 1955 18 11 2019 Copyright: © 2019 Tawfik Tadros MS et al. 2019 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

 tooth repair dental stem cell pulp regeneration SCAPs SHEDs The author(s) declared that no grants were involved in supporting this work. 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, ScienceDirect, 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:

Population: Both in vitro and in vivo studies that investigate the endodontic regeneration following treatment with dental pulp stem cells.

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.

Outcomes: pulpal regeneration or repair.

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.

 Results 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.

PRISMA flow diagram of article selection in this systematic review. Summary of included studies.

This table was adapted from a previous systematic review with written permission from the authors and under a Creative Commons Attribution 4.0 International License 12 .

Cell type Animal model Dose & dosage Route of administration Co-administrative factors TERM approach Time point Main results Reference
DPSC Rat Not declared Allogenic transplantation Into renal capsule NA AGS 2 weeks Inflamed DPSC has more tendency to osteogenesis rather than dentinogenesis Wang et al. (2013) 15
Mice 5 × 10 6 cells/ml Transplantation into subrenal capsule NA PLLA/(HA,TCP & CDHA) 5 weeks PLLA/TCP superiority for tooth tissue regeneration Zheng et al. (2011) 16
Mice 1 × 10 6 cells Xenograft subcutaneous tooth slice NA collagen TE 4 weeks Similar regeneration of MDPSCs from young and aged donors Horibe et al. (2014) 27
Mice 256,000/190 μL Xenograft subcutaneous tooth slice Nephronblastoma overexpressed 3D micro tissue Spheroids 4 weeks Vascular and pulp like tissue regeneration Dissanayaka et al. (2014) 38
Mice 1 × 10 6 cells Xenograft subcutaneous Transplantation NA Porous PLGA 4 weeks Promotion of dentinogenesis and odontoblastic differentiation Wang et al. (2014) 40
Mice 5 × 10 3 cells/well Xenograft subcutaneous Transplantation Plasmid vectors encoding BMP-7 NA 8 weeks Maintained MSC characteristics after implantation (DPSC > PDLSC) Lei et al. (2014) 41
Mice 1.0 × 10 6 cells/mL Xenograft subcutaneous Implantation NA Chitosan/collagen 4 weeks Odontoblast-like phenotype Differentiation Yang et al. (2012) 42
Mice 2.0 × 10 6 cells Xenograft subcutaneous Implantation NA Fibrin gel CBB 8 weeks Capability of mineralization (SHEDs > DPSCs) and CT formation (SHEDs < DPSCs) Wang et al. (2012) 43
Mice 3 × 10 6 cells/mL Xenogenic subcutaneous Transplantation NA HA/TCP Cell sheets/ powdery 12 weeks PL enhances the and layer of odontoblast-like cell formation Chen et al. (2012) 44
Mice 5 × 10 6 cells Xenogenic subcutaneous transplantation NA CBB particles 6 weeks Regular dentin–pulp complex and columnar odontoblast-like cells generation Wang et al. (2011) 45
Mice 10 6 cells Xenograft subcutaneous Transplantation NA NF-PLLA 8 weeks Enhanced odontogenic differentiation of human DPSCs and mineralization in NF-PLLA Wang et al.(2011) 17
Mice 1 × 10 7 cells Xenograft subcutaneous Transplantation pre ameloblast-CM HA/TCP ceramic powder 6–12 weeks Dentin deposition with palisaded odontoblast-like cells formation Oh et al. (2015) 18
Mice 1 × 10 6 cells/100μL Xenograft subcutaneous in dentin cylinder transplantation VEGF, TGFb1, FGF2 Laden peptide Hydrogel 6 weeks Generation of odontoblasts-like phenotypes, vascularization Galler et al. (2011) 19
Mice 4 × 10 6 cells Xenograft subcutaneous Transplantation NA HA and TCP Powder 8 weeks DPSCs from inflamed pulp formed pulp/dentin complexes in lesser extent than DPSCs–NPs Alongi et al. (2010) 20
Mice 1 × 10 6 cells Xenograft subcutaneous Transplantation BMP-7 + dexamethasone NF-PLLA 8 weeks More organized odontoblast like cells formation Wang et al. (2010) 21
Pig 2 × 10 6 Cells Allogeneic direct implantation into socket Vitamin C PDLSC sheet + HA/TCP/ DPSC 24 weeks Generation of bio-root with normal pulp and dentin-like matrix and natural biomechanical structure in low rate. Gao et al. (2016) 22
Pig 3 × 10 6 cells Autologous root fragment transplantation into jawbone NA Collagen PLGA 6–10 weeks Formation of continuous polarized & non-polarized cell along the canal wall Kodonas et al. (2012) 23
DPSC Rabbit 5 × 10 6 cells/ml Autologous transplantation into the extracted socket BMP-2 Collagen gel 12 weeks Similar tooth structure by different stem cells close to a normal living tooth Hung et al. (2011) 24
Rat 8 × 10 6 cells Xenograft intracanal Transplantation Hypoxic treatment PLLA Nanofibrous spongy microsphere 4 weeks Enhanced vascularization Kuang et al. (2016) 25
Dog 1 × 10 4 cell/cm 2 Autologous interacanal Transplantation G-CSF Atelocollagen 13–26 weeks Normal pulp-like tissue and apical secondary dentin formation Iohara e t al. (2018) 26
Dog 2 × 10 4 cells/100 mL Autologous interacanal Transplantation G-CSF Drug approved collagen 2, 4, 9, 26 weeks Regeneration of vascularized pulp tissue, dentin deposition along dentin wall and dense nerve plexus Nakashima and Iohara (2014) 28
Dog 1 × 10 6 cells/ml Autologous interacanal Transplantation G-CSF Atelocollagen 2–17 weeks Pulp-like tissue regeneration 60% apically, dentin & nerve formation Iohara et al. (2014) 29
Dog 1 × 10 6 cell per ml Autologous intracanal Transplantation G-CSF Clinical-graded atelocollagen 2, 4, 9, 26 weeks Over 90% pulp-like tissue regeneration, dentin & dense nerve plexus Formation Iohara et al. (2013) 30
Beagles 2.0 × 10 7 cells Autologous transplantation into the pulp canal NA Gel foam 24 Week Generation of pulp-like tissues Wang et al. (2013) 15
Mice 5 ×10 5 cells Allogenic subcutaneous implantation Costal chondrocytes/ FGF9 protein Collagen/PGA 4–8 weeks Enhance chondrogenesis and partially inhibit ossification in engineered cartilage Dai et al. (2012) 31
Mice 2×10 5 cells Subcutaneous implantation in mouse of human cells ND/NA TCP 8 weeks DPSC regulates dentin development and regeneration Zhou et al. (2015) 32
Mice 1×10 6 cells/mL Subcutaneous implantation in mouse of treated human tooth root PuraMatrix/MTA 3D microtissue Spheroids 4 weeks DPSC exhibited vascularized pulp-like tissue with patches of osteo-dentin after transplantation Dissanayaka et al (2015) 33
Mice NA Subcutaneous implantation in mouse of human tooth root and REP Growth factor Alginate 6 weeks Enhance chondrogenesis Schmalz et al. (2016) 37
Mice 1 × 10 6 cells in each Xenograft subcutaneous Implantation TDM Collagen TE 4 weeks Angiogenesis, neurogenesis and pulp regeneration induction Ishizaka et al. (2013) 34
Dog 1 × 10 6 cells in each Autologous intracanal Transplantation SDF-1 Collagen TE 2, 4, 13 week Full length pulp-like tissue formation, odontoblastic lining & tubular dentin along dental wall Iohara et al. (2011) 35
Rat NA Femur and autologous implantation of rat tooth chambers FGF2/VEGF/PDGF Collagen/gelatin 4,8 weeks Successful revascularization and tissue regeneration with direct vascular supply Srisuwan et al. (2013) 36
Mice 1 × 10 5 cells Subcutaneous implantation in mouse of human tooth root GCSF Collagen 6 weeks MDPSCs accelerated vasculo- genesis in an ischemic hindlimb model and augmented regenerated pulp tissue Murakami et al (2013) 39
SCAP Rat 1 × 10 6 cells Xenograft Transplantation Into renal capsules NA SCAP pellets /root segment 8 weeks MTA regulates dentinogenesis of SCAPs Yan et al. (2014) 49
Rat 1 × 10 6 cells Xenograft root fragment transplantation into renal capsule Different concentration of KH2PO4 (M2 > M1) Root segments containing SCAP pellets/AGS 2 weeks More mineralized tissues generation &, higher osteo /odontoblast differentiation in supplemented khpo4 medium Wang et al. (2013) 15
Mice 2 × 10 4 cells/well Subcutaneous VEGF Poly dioxanone Fiber 3 weeks Blood vessel formation Negligible inflammation Yadlapati et al. (2017) 50
Mice 1 × 10 7 cell/100 mg Powder Xenograft Subcutaneous Transplantation rhPAI-1 HA/TCP ceramic powder fibrin gel 12 weeks Dentin formation odontoblast presses inserted to dental tubules Jin and Choung (2016) 51
SCAP Mice 3 × 10 6 cells Xenograft Subcutaneous injection BMP-2 PLLA, NF-MS + PLGA, MS 4, 8 weeks Mineralized tissue with embedded cells resembling osteodentin excellent microenvironment for SCAP to regenerate dentin tissue Wang et al. (2016) 47
Mice 2x10 4 cells/ well Subcutaneous implantation Nuclear Factor I-C (NFIC) WNT3A/BMP9 5 weeks SCAPs may promote osteo/ odontoblastic differentiation Zhang et al (2015) 48
Mice 10 7 cells/mL Xenograft Subcutaneous root fragment transplantation NA PLG 21–28 weeks Fulfilling vascularization, continuous dentin-like tissue deposition Huang et al. (2010) 46
Mice NR Xenograft Subcutaneous transplantation NA HA/TCP hydrogel 8, 12 weeks Function of vascularized pulp- like tissue Hilkens et al. (2017) 52
SHED Mice 4 × 10 6 cells Xenograft subcutaneous transplantation NA HA/TCP 8 weeks Mineralization & DPC generation equally in SHED Fresh and SHED-Cryo Ma et al. (2012) 58
Mice 2.0 × 10 6 cells Xenograft subcutaneous Transplantation NA Fibrin gel CBB 8 weeks Capability of mineralization SHEDs > DPSCs CT formation SHEDs < DPSCs Wang et al. (2012) 43
Mice 3 × 10 6 cells Xenograft subcutaneous transplantation NA macroporous biphasic calcium phosphate 9 weeks Hard tissue formation (o-SHED > e-SHED) quality of hard tissue (o-SHED = e-SHED) Jeon et al. (2014) 59
Mice 5 × 10 4 cells Subcutaneous implantation in mouse of treated human tooth slice ND/NA PLLA NA SHED express markers of odontoblastic differentiation (DSPP, DMP-1, MEPE) Casagrande et al. (2010) 60
BMSC Mice 1 × 10 6 cells Allograft Transplantation Into renal capsules NA Lyophilized hydrogel 2 week Local mineralization production of dentin-like tissue Hashmi et al. (2017) 61
Rat 5 × 10 6 cells Allogenic Dentin slice Transplantation into renal capsule Dentin slice Dentin slices 6 weeks Polarized cells penetrating into dentin wall Lei et al. (2013) 62
Mice 1 × 10 7 cells/ml Xenograft subcutaneous cell-transplantation SDF-1 Collagen 3 weeks Participation of systemic BMSC in intracanal dental-pulp-like tissue regeneration Zhang et al. (2015) 63
Dog 5 × 10 5 cells Autologous interacanal Transplantation G-CSF Atelocollagen 2 weeks Potential pulp regeneration in MADSC & MBMSC but in less volume Murakami et al. (2015) 64
DFSC Pig 1 × 10 6 cells Direct implantation into socket APES/TDM/DPEM APES/TDM/DPEM 12 weeks Generation of uniform pulp-like tissue, predentin matrix formation Chen et al. (2015) 53
Mice 1 × 10 7 cells/mL Allograft subcutaneous Transplantation TDM TDM 6 weeks Similar dentin-like tissue Formation Chen et al (2015) 54
Mice 5 × 10 4 Cells/ scaffold Xenograft subcutaneous Transplantation TDM TDM 8 Weeks The structure of dentin tissues generated by DFCs was more complete Tian et al. (2015) 55
DFSC Mice 1 × 10 4 cells Xenograft subcutaneous Transplantation dentin matrix Human TDM and CDM 8 Weeks More mechanical properties dentinogenic protein release by CDM Jiao et al. (2014) 56
Mice 5 × 10 4 cells in each Xenograft subcutaneous Implantation TDM TDM 8 Weeks Formation of pulpdentin/ cementum/periodontium-like tissues Guo et al. (2013) 57
Mice 1 × 10 6 cells/mL Xenograft subcutaneous Implantation TDM TDM 8 Weeks New dentin-pulp like tissues and cementum-periodontal complexes Yang et al. (2012) 42
PDLSC Mice 3 × 10 4 cells/dish Xenograft subcutaneous Transplantat NA NA 8 Weeks Maintained MSC characteristics after implantation (DPSC > PDLSC) Lei et al. (2014) 41
Mice 5 × 10 4 Cells/ scaffold Xenograft subcutaneous Transplantat TDM TDM 8 Weeks The structure of dentin tissues generated by DFCs was more complete Tian et al. (2015) 55
Pig 2 × 10 5 Cells Allogeneic direct implantation into socket Vitamin C PDLSC sheet + HA/TCP/ DPSC 24 weeks Generation of bio-root with normal pulp and dentin-like matrix and natural biomechanical structure in low rate. Gao et al. (2016) 22
ADSC Rabbit 5 × 10 6 cells/ml Autologous transplantation into the extracted socket BMP-2 Collagen gel 12 weeks Similar tooth structure by different stem cells close to a Normal living tooth Hung et al. (2011) 24
RPSC Rat 10 6 cells each Allogeneic transplantation into the renalcapsules NA AGS 2 weeks Typical dentinogenesis by iRPSC, bone-like tissues by mRPSC Lei et al. (2011) 66
Mesenchymal Mice 2 × 10 5 cells Rat to mice Transplantation intorenal capsule hBMP4 hBMP7 PLGA 8 weeks Enamel and dentin-like tissues generation in two integrated layers with amelogenin expression and amelo blastin Jiang et al. (2014) 67
UCMSC Mice 5 × 10 4 cells/well Xenograft subcutaneous Transplantat hTDM TDM 8 Weeks Formation of layers of cells and calcifications Chen et al. (2015) 68
Human DP Progenitors Mice 10 6 cells/50 μl Xenograft subcutaneous Implantation Stem cell factor (SCF) Collagen sponge 4 weeks Induction of cell homing, angiogenesis, and tissue remodeling Pan et al. (2013) 69
Dermal multi potent cells Mice 2.0 × 10 6 cells Xenograft subcutaneous transplantation Embryonic and neonatal TGC-CM Fibrin gel 4 weeks Bone like structure formation from embryonic TGC-CM Huo et al. (2010) 70
DBCs Porcine 1 × 10 7 cells Autotransplantation into swine’s original alveolar socket GCHT Gelatin- chrondroitinhyaluronan- tricopolymer 36 weeks Successful rate of tooth regeneration from DBCs/GCHT scaffolds’ was about 33.3% Kuo et al. (2007) 71
NA Beagles NA Cell homing SDF-1α Silk/Fibroin 12 weeks Pulp tissue generation andmineralization along dentinal wall Yang et al. (2015) 72

BC, blood clot; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BMSC, bone marrow mesenchyme stem cell; CC, costal chondrocyte; CDHA, calcium carbonate hydroxyapatite; CNCC, cranial neural crest cell; CXCL14, chemokine (CXC motif) ligand-14; DFSC, dental follicle stem cell; DPSC, dental pulp stem cell; FGF, fibroblast growth factor; GCSF, granulocyte colony-stimulating factor; GF, growth factors; HA, hydroxyapatite; iPS, induced pluripotent stem cell; MCP1, monocyte chemoattractant protein-1; MSC, mesenchymal stem cell; MTA, mineral trioxide aggregate; NA, not exogenously added; ND, not determined; NGF, nerve growth factor; PCL, polycaprolactone; PGA, polyglycolic acid; PGDF, platelet-derived growth factor; PLGA, polylactic-co-glycolic acid; PLLA, poly-L-lactic acid; PRF, platelet-rich fibrin; PRP, platelet-rich plasma; REP, regenerative endodontic procedure; SCAP, stem cell of the apical papilla; SHED, stem cell from human exfoliated deciduous teeth; TCP, tricalcium phosphate; TDM, treated dentin matrix; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; DBCs, dental bud cells

 Variations of extracted data from reviewed articles.
Variables Number of studies (%) *
Cell type DPSCs 31 (46)
SCAP 8 (12)
DFSC 6 (9)
BMSC 4 (6)
SHED 4 (6)
PDLSC 3 (4.4)
ADSC 1 (1.5)
Other 7 (10.4)
Scaffold Collagen 15 (22.3)
TDM 9 (13.4)
HA/TCP 10 (15)
PLLA 6 (9)
PLGA 4 (6)
Atelocollagen 4 (6)
Fibrin gel 8 (12)
CBB 3 (4.4)
Silk fibroin 1 (1.5)
Other 3 (4.4)
Growth factors TDM 9 (13.4)
BMP 5 (7.4)
G-CSF 5 (7.4)
SDF-1 3 (4.4)
VEGF 3 (4.4)
b-FGF 3 (4.4)
Other 35 (52.2)
Transplantation site Subcutaneous 40 (59.7)
Inter canal 4 (6)
Renal capsule 8 (12)
Into socket 3 (4.4)
Other 8 (12)
Animals Mice 44 (65.6)
Rat 7 (10.4)
Pig 4 (6)
Dog 7 (10.4)
Rabbit 2 (2.9)
Beagles 2 (2.9)

*The number of included studies is 57 studies; however, the number of trials is 67 trials. Therefore, each study may include one or more types of cells.

BC, blood clot; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BMSC, bone marrow mesenchyme stem cell; CC, costal chondrocyte; CDHA, calcium carbonate hydroxyapatite; CNCC, cranial neural crest cell; CXCL14, chemokine (CXC motif) ligand-14; DFSC, dental follicle stem cell; DPSC, dental pulp stem cell; FGF, fibroblast growth factor; GCSF, granulocyte colony-stimulating factor; GF, growth factors; HA, hydroxyapatite; iPS, induced pluripotent stem cell; MCP1, monocyte chemoattractant protein-1; MSC, mesenchymal stem cell; MTA, mineral trioxide aggregate; NA, not exogenously added; ND, not determined; NGF, nerve growth factor; PCL, polycaprolactone; PGA, polyglycolic acid; PGDF, platelet-derived growth factor; PLGA, polylactic-co-glycolic acid; PLLA, poly-L-lactic acid; PRF, platelet-rich fibrin; PRP, platelet-rich plasma; REP, regenerative endodontic procedure; SCAP, stem cell of the apical papilla; SHED, stem cell from human exfoliated deciduous teeth; TCP, tricalcium phosphate; TDM, treated dentin matrix; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

 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), adipose-derived stem cell (ADSC), DPSC, SCAP, and SHED. The majority of the studies (n=31; 46%) used DPSCs for regeneration of dentine-pulp complexes 15– 45 . Two out of eight studies, in which stem cells were transplanted into the renal capsule, used DPSCs 15, 16 . Moreover, DPSCs were used by 19 out of 40 studies on subcutaneous transplantation 17– 21, 27, 31– 34, 37– 45 , three out of four studies on intra-canal transplantation 25, 30, 35 , and two studies about allogeneic direct implantation into socket 22, 24 .

SCAP was reported in eight studies 15, 46– 52 , six studies with subcutaneous implantation 46– 48, 50– 52 and two studies on renal capsule transplantation 15, 49 . There are no experiments utilizing transplanted SCAP into root canal. DFSC is reported in six studies 42, 53– 57 ; five subcutaneous implantation studies 42, 54– 57 and one into-socket transplantation 53 . Across four experiments that were all on subcutaneous transplants used SHEDs 43, 58– 60 . Four experiments used BMSCs; two were transplanted to the renal capsule 61, 62 , one to the subcutaneous 63 , and the other to the root canal 64 . Three trials tried to regenerate periodontal ligament (PDL) tissue using PDLSCs; two subcutaneously transplanted 41, 55 and one transplanted into a socket for extraction 22 .

 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– 21, 27, 31– 34, 37– 45 , three in rat models 15, 25, 36 , four in dogs 26, 28– 30 , and three in pigs 15, 22, 23 . It was observed that DPSC transplantation was associated with regeneration of pulp-like tissue 22, 29, 30, 32 , successful revascularization 36, 38 , enhanced chondrogenesis 31, 37 , and tissue regeneration with direct vascular supply. 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.

 Bone marrow derived mesenchymal stem cells

Murakami et al. 64 showed that BMSCs generated a potential pulp, but with less volume. Zhang et al. 63 proposed applying endogenous BM-MSC to a subcutaneous root canal tooth to a regenerative tissue after the systemic homing in the root canal, driven by the use of the stromal cell-derived factor-1 (SDF-1). The use of dentine-matrix-scaffolding cells was associated to the differentiation of the stem cells in a dentinal tubule in polarized odontoblast-like cells 62 .

 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, 56, 58, 61, 66, 70, 72 . Most of the included research assessed the 4–8 week dentine-pulp regeneration following transplant 57, 67, 69, 70 . These studies used the ectopic models of dentine pulp-complex. A few studies used long-term evaluation of up to 36 weeks after surgery 71 . However, there are some studies that had multiple time points for evaluation.

Like other tissues, three primary components are required to regenerate a necrotic pulp: 1) vital root canal cells, which can distinguish into normal pulp cells, 2) morphogenic and growth factors to activate and encourage cell distinction, and 3) a matrix that ensures an environment that maintains their vitality and growth and supports cells in a mechanical way 58, 59, 61, 62 .

Growth factors, drugs, bioactive products, glycosaminoglycans and other small molecules and motifs of peptide are considered promotive healing factors that can be used for stem cells and matrix to improve the effectiveness of stem cell therapy for dentine-pulp regeneration and biodegradation. Growth factors have a short half-life; therefore, degradable materials are required to control their release 47, 48, 73 .

Recent studies for dentine pulp regeneration have been done in various types of stem cells from various sources in body. DPSCs are the preferred cells in the majority of these studies and have demonstrated their capacity to regenerate the complex dentine 15, 22, 25, 46, 65 . Although the great tendency for dentine-pulp complex regeneration, SCAP and SHED were rarely administered 44, 45 . DPSCs and SHEDs were evaluated with adequate or partly successful histological outcomes in various REP research. DPSC, collagen or polylactic/glycol and scarce factors with or without growth factors are optimized when compared to REPs with growth factors but without amplified stem cells 19, 20 . 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.

 Reporting guidelines

Figshare: PRISMA checklist for ‘Dental stem cells in tooth repair: A systematic review’, https://doi.org/10.6084/m9.figshare.10315835.v1 74 .

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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CNS Neurol Disord Drug Targets. 2019;18(4):317–325. 30868968 10.2174/1871527318666190314101314 Tadros MST : PRISMA checklist for ‘Dental stem cells in tooth repair A systematic review’.pdf. figshare. Online resource. http://www.doi.org/10.6084/m9.figshare.10315835.v1 10.5256/f1000research.23175.r58864 Reviewer response for version 1 Tian Weidong 1 Referee State Key Laboratory of Oral Disease, West China Hospital of Stomatology, Sichuan University, Chengdu, China

Competing interests: No competing interests were disclosed.

 23 6 2020 Copyright: © 2020 Tian W 2020 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

In this review, the authors summarized recent studies of dental pulp regeneration using stem cells. In general, it is a timely and detailed review of stem cells used in dental regeneration. But this manuscript needs to be carefully modified. 

Comments:

In the abstract and Table 2, the authors described “TDM (treated dentin matrix)” as a growth factor. In fact, TDM is a matrix containing growth factors. “Growth factors derived from TDM” is more suitable. 

The language of this manuscript should be checked and polished by a native English speaker.

According to the methods, the authors searched the databases from January 2000 to June 2019: Since numbers of studies of dental pulp regeneration have published after June 2019, it is recommended for the authors to update their data to June 2020. 

Are the rationale for, and objectives of, the Systematic Review clearly stated?

Yes

Is the statistical analysis and its interpretation appropriate?

Not applicable

Are sufficient details of the methods and analysis provided to allow replication by others?

Partly

Are the conclusions drawn adequately supported by the results presented in the review?

Yes

Reviewer Expertise:

Dental regeneration

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

 10.5256/f1000research.23175.r56928 Reviewer response for version 1 Ruohola-Baker Hannele 2 Referee https://orcid.org/0000-0002-5588-4531 Hanson-Drury, D.D.S Sesha 1 Co-referee University of Washington, Seattle, USA Institute for Stem Cell and Regenerative Medicine, School of Medicine, University of Washington, Seattle, WA, 98195, USA

Competing interests: No competing interests were disclosed.

 21 1 2020 Copyright: © 2020 Hanson-Drury, D.D.S S and Ruohola-Baker H 2020 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The manuscript “Dental stem cells in tooth repair: A systematic review” is a review by Tadros et al. on a very timely topic, tooth regeneration. However, this paper does not read like a review but rather as a catalogue of papers. This review requires conceptual work towards synthesizing the great findings in the field. For example, many notable, recent papers are excluded from this review. In addition, the following comments need to be addressed.

Provide citations for every statement (e.g. “Loss of tooth pulp stops the development of permanent root teeth that can weaken the periodontal connection and lead to teeth loss” Page 3, Paragraph 2.)

Spelling and grammar review. Multiple incidences of unclear sentences (e.g. “Although the great tendency for dentine-pulp complex regeneration, SCAP and SHED were rarely administered.” Page 10, Paragraph 5) or incorrect word used (“SHEDs, which rare derived from extracted deciduous teeth” Page 10, Paragraph 1).

Develop appropriate tool to assess the quality of included studies and risk of bias such as that described in Cochrane Handbook for Systematic Reviews of Interventions.

How titles and abstracts were “deemed relevant” to the research question.

Why were studies excluded/removed?

What databases/sources were used?

What search terms were used?

Studies included:

Reviews are not listed though there are reviews included.

Humans are not listed in populations studied though human studies are cited (e.g. “All included experiments utilizing DPSCs were isolated from human healthy pulp tissues…” Page 4, Paragraph 3).

Why there was insufficient data for meta-analysis.

Meaning of “usually orthodontics” Page 4, Paragraph 3. Does this mean teeth were extracted due to orthodontic treatment plans?

“Fresh bio-root” and “success” when discussing PDLSCs and “potential pulp” on Page 4, Paragraph 4 and 5.

TERM approach.

“Scarce factors” and “excellent outcomes” discussed on Page 10, Paragraph 5.

Are the rationale for, and objectives of, the Systematic Review clearly stated?

No

Is the statistical analysis and its interpretation appropriate?

Not applicable

Are sufficient details of the methods and analysis provided to allow replication by others?

No

Are the conclusions drawn adequately supported by the results presented in the review?

No

Reviewer Expertise:

stem cells and regeneration

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.