Regenerative Capacity of Dental Pulp Stem Cells: A Systematic Review.

OBJECTIVES: The dental pulp contains undifferentiated mesenchymal cells, blood vessels and so on, which are responsible for routine functions of a tooth. The determination of stemness and regenerative properties using biomarkers and further application in routine practice may unravel its potential.
MATERIALS AND METHODS: Inclusion criteria-original research articles published in English, from 2000 to 2019, were collected both manually and by electronic search from databases of Cochrane, Medline, Embase, and PubMed. Exclusion criteria-articles other than English and review manuscripts were omitted. The shortlisted articles were reviewed for specific biomarkers, to assess the regenerative potential, stemness, and lineage of dental pulp stem cells.
RESULTS: Of 512 articles, 64 were selected and reviewed to determine the mesenchymal, neurogenic, vasculogenic, hematopoietic, and stem cell potential. On the basis of the search analysis, a panel of markers was proposed.
CONCLUSION: The application of proposed markers, on a pulpectomized tissue derived from human teeth, may be helpful to determine the regenerative potential and the usefulness in regenerative medicine and tissue engineering. Copyright:

INTRODUCTION

Dental pulp is a type of unique connective tissue that has an anatomical architecture closely restricted by its location within a rigid chamber of tooth. The dental pulp consists of cellular, noncellular components, collagen, and fibrillin fibers. The nonfibrous components include substances that are derived from the extracellular matrix, mainly glycosaminoglycan, proteoglycans, and other adhesion molecules. This matrix plays a pivotal role in the development, migration, division, shape, and function of the tissue. The presence of blood vessels and nerves plays an important role in the physiological functions of the tooth. It has varied functions ranging from detecting stimuli and also initiating and participating in response against insult.[1] Their regenerative capacity is explained on the basis of presence of various cellular constituents of the dental pulp, which includes odontoblasts, fibroblasts, defense, and undifferentiated cells.[2] The undifferentiated group of cells present in pulp tissue comes under the category of postnatal stem cells. Stem cells are seen in the pulp tissue of both adults and children, within the superficial cell-rich zone, underneath the Hoehl’s cell layer. They are believed to originate from the neural crest cells and segregate into different cell types.[34]

Dental pulp stem cells (DPSCs) are desirable for their unique properties to differentiate into various cell types, which include dentin-producing odontoblasts, neural predecessor cells, chondroblasts, endothelium formative cells, lipocytes, myoblasts, and osteoblasts.[3] DPSCs are mesenchymal cells that constitute one of the most broadly researched cells. Earlier studies have proved that DPSCs can form tissues such as dentin, pulp, and periodontal ligament fibers. These are proved to be a potential stem cell source for orthopedic and orofacial restoration, and it is postulated that these cells may contribute beyond the stomatognathic system.[4]

Regenerative potentials of the dental pulp tissue have been established in various fields such as, cure of neuro-deficit disorders,[5] cardiac-related disorders,[6] muscular disorders (muscular dystrophy),[7] genetic and lifestyle disorders,[8] liver diseases, ophthalmic-related defects,[9] immune diseases, diseases related to the orofacial, bone defects,[10] and infertility treatments. Hence based on the aforementioned facts, systematic review in DPSC was undertaken, which includes various biomarkers having regenerative potential and lineage, and with probable clinical applications were identified.

MATERIALS AND METHODS

This review was carried out based on the standard guidelines for making of a systematic review (Prisma Guidelines 2015) [Figure 1].[11]

Figure 1

PRISMA diagram

Eligibility criteria

Inclusion criteria: Original research articles published in English in the year from 2000 to 2019 and related to the title were selected for the review.

Exclusion criteria: Articles other than English language and review manuscripts were excluded. The stem cell markers and regenerative markers were the main method of determination of the potentiality of the dental pulp tissue.

Sources, search strategy, and study selection

Various standardized search engines were used, such as PubMed, Cochrane Library, Embase, and Medline. In addition, a manual search was performed on the personalized collection of journals.

Two oral pathologists reviewed the articles and an experienced reviewer specialized in stem cell gave final decision.

DISCUSSION

The dental pulp is an intricate tissue that has got multiple potentials and functions to protect the pulp against challenges such as caries or dental trauma. Michael Goldberg[12] had earlier proposed that the knowledge of the inbuilt defense mechanisms employed by the dental pulp has given similar ideas to induce pulp regeneration therapeutically. Ingle’s has observed that multiple growth factors act as important controllers in the instigation of each of the phases of tooth development.[13] This supports the idea of regenerating an entire dentine–pulp complex from the dental pulp tissue itself.

Though earlier studies have been conducted regarding its regenerative potential, a proper categorization of the stem cells derived has not been made.[14] Hence review has been formulated on the following three criteria:

To determine the maximum number of immunohistochemical markers that were used in determining the potency of the pulpal tissue

To categorize the markers based on the respective properties such as mesenchymal, multi-lineage potency, vasculogenic, neurogenic, osteogenic, musculogenic, dentinogenic, and hematopoietic, thereby confirming the pluripotent nature of the pulpal tissue

To derive a standardized protocol of panel of markers.

Markers used by various authors and its application in the pulp are systematically analyzed in this review [Table 1]. On the basis of the results, the multi-lineage potency of the dental pulp tissue predicted by various markers has been tabulated [Table 2].

Table 1

The type of markers used by various authors and the interpretations derived[15161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778]

S no.	Author name, year	Biomarkers used	Interpretation
1	Gronthos et al., 2000	DPSCs (Differentiation Potential)	Adipogenic, odontogenic, neurogenic, osteogenic, myoblasts formation, endothelial potency chondrogenic, cardiogenic potential
		Collagen type 1,2,3
		MyoD
		Alpha SMA
		Neurofilamin
		MUC-1 (CD 146)
		Osteocalcin
		Osteonectin
		Bone sialoprotein
		Osteopontin
		AlkPhos
		PPAR-gamma
		FGF-2
		CD 44, CD 45, CD 34, CD14
		VCAM-1 (calcium adhesion molecule)
		Integrin beta-1
2	Karöaz et al., 2010	Collagen type 2, SOX-9,collagen type 1, osteopontin, osteonectin, osteocalcin, beta III tubulin, NF, nestin, MAP proteins, alpha SMA, myosin IIa, myogenin, desmin	Regeneration of various structures
		Adipogenic markers— adipophillin, leptin
3	Karoaz et al., 2012	STRO-1	Positivity confirmed regenerative potential. Also neural crest origin of DPSC promoted neurogenic potential
4	Karoaz et al.,2011	Cytokeratin 18 and 19	Odontoblast differentiation and dentine repair.
5	Beatriz et al.	CD3, CD4, CD 5, CD 7, CD 8, CD 10, CD 11b, CD18, CD14, CD 15, CD 29, CD 33, CD 44, CD 45, CD71, CD 73, CD 90, CD106, CD 117, CD 123, CD 138, CD 146, CD 166 and HLA antigens.	Regenerative potential to repair neurogenic, cardiac, hepatic, ophthalmic, bony, and myogenic deficits
		CD 9, CD10, CD13, CD29, CD44, CD56, CD59, CD71, CD73, CD90, CD105, CD106, CD117, CD146, CD166, CD 127, CD 11b, CD14, CD19, CD31, CD34, CD43, CD45, CD 150, OCT ¾ SOX2 NANOG, c-myc KLF-4, LIN-28, STRO-1, SSEA-3, SSEA-4, TRA-1-60
6	Atari et al., 2012, Abou-Asi et al., 2015	HNF3beta+, SSEA-4+, Oct4+, Nanog+, FLK-1+, Sox2+, Lin28+, Nestin+, c- Myc+, CD13+, CD105+, CD34−, CD45−, CD90low, CD29+, CD73low, STRO-1low, and CD146	Potency to regenerate from DPSC’s demonstrated osteogenic induction
7	Ferro et al., 2012a	SSEA4, OCT3/4, NANOG, SOX2, LIN28, CD13, CD105, CD34, CD45, CD90, CD29, CD73, STRO1, and CD146	Demonstrated osteogenic induction
8	Ferro et al., 2012b	CD10, CD29, CD44, CD49a, CD49d, CD59, CD73, CD90, CD105 and CD133, CD117, CD 34,CD45, Oct4, Sox-2, and Nanog	Osteoblast differentiation, myocyte, hepatocyte, neural differentiation potential was highlighted. Hepatocytic differentiation potential
9	Ishkitiev et al., 2012	Presence of OCT4, CD 117 and various other hepatocytic growth factors	Osteogenic and hepatocytic potential
10	Miura et al., 2003	Stro-1, CD 146, GFAP, nestin, neurofilament, beta-3 tubulin	Adipogenic, neurogenic, odontogenic, osteogenic, and myoblastic potential, endothelial potency, hepatocytes formation
11	Kerkis et al., 2006	Nanog, Oct4, Nucleostemin, Slain-1, Jmjd1, Jmjd2, and Cyclin D1	Ability to regenerate myogenic (skeletal) tissues
12	Wang et al., 2010	STRO-1,CD29, CD90, CD146, CD34, vimentin, nestin, and TH, dentin sialoprotein, and βIII-tubulin	Differ differentiation into neurogenic, odontogenic cells, and lipocytic structures
13	Wang et al., 2012	STRO-1, CD 146, CD29,CD 105	Cell proliferative indices and osteogenic and adipogenic potential were elicited
14	Akpinar et al., 2014	CD3, CD4, CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD106, CD117, CD146, CD166, HLA-DR, and HLA-ABC	Ability to derive from all stem cell lines.
15	Trivanoic et al., 2015	Pluripotency markers (Nanog, Oct-4, SOX-2, and SSEA-4, CD90, CD44, CD73, and hematopoietic cells markers CD34 and CD45)	Higher proliferative indices
16	Nagako et al., 2012	Alpha SMA, STRO-1, nestin	Regeneration in wound healing.
17	Feng Juan etal., 2014	STRO-1 and CD 271	Cardiovascular repair. LowTrilineage differentiation
		SSEA-4, CD 146, CD 49f, 3G5, STRO-4	Tripotency, trilineage potency facilitates hematopoiesis, pericyte marker
18	Pereira et al.,	STRO-1	Proliferative capacity of MSCsisolated from normal and inflamed dental pulp
19	Alongi et al.,	STRO-1, CD 90, CD 105, CD 146	Inflamed dental pulps expressed higher levels of these markers
20	Evandro et al., 2017	CD 73, CD 90, CD 105, CD 45	Produced angiogenic proteins like endothelin, IGF, binding protein 3 (IL-3), pentraxin-3, serpin E (SE1), serpin F1 (SF1)
21	Paloma dias TELLES et al., 2010	CD 31, VE-Cadherin, VEGFR-2- Endothelial markers	Expression of these markers, and presence of VEGF, helped organize capillary-sprouts
22	Akihiro et al., 2015	STRO-1, ABCG2, CD90, alpha-smooth muscle actin, Bmi1, CD 31, CD90, CD 31/ CD 146	Positivity of cells in the perivascular region
23	Shi and Gronthos et al., 2003	STRO-1, CD 146 and Alpha smooth muscle actin	Niche of stem cells in the dental pulp
24	Shi et al., 2005	CD14, CD34, CD44, CD45, CD106 CD146,3GS,STRO-1, a- SM actin Collagen Type-I,	Found hematopoietic stem cells in dental pulp
25	Sloan and smith et al., 2007	Osteonectin, scleraxis, alkaline phosphatase, osteocalcin, osteopontin, collagen type-III, bone sialoprotein, dentin sialophosphoprotein	DPSCs have osteogenic potential
26	Struys et al., 2010	CD 29, CD44, CD105, CD 146, and STRO-1	Multi-lineage differentiation potential
27	W. Martens et al., 2012	CD 29, CD44, CD105,CD177, CD146, and STRO-1	Undifferentiated h DPSC’S- regenerative potential
28	Safford et al., 2002;	Neural markers like nestin, beta- III tubulin, neurofilament, S100, synaptophysin, Vimentin	Capable of deriving neural structures
29	Tropel et al., 2006	Neural markers	Success of differentiation to neurogenic structures
30	Alexanian et al., 2008	STRO-1	Cells with positivity showed fibroblastic behavior.
31	Arthur et al., 2008	Neuronal-specific markers	Neurogenic potential of DPSC. DPSCs provide a accessible source of precursor stem cells
32	Kiraly et al., 2009	Neural differentiation markers vimentin, nestin, N-tubulin, neurogenin-2 and neurofilament-M	Neurogenic potential
33	Karaöz et al., 2010	Differentiation Markers-CD13, CD44, CD90, CD146 and CD166, CD3, CD8, CD11b, CD14,CD15, CD19, CD33,CD34, CD45, CD117, and HLA-DR	Adipogenic, osteogenic, chondrogenic, myogenic, and neurogenic potential
34	Nosrat et al., 2001,	PGP9.5, Protein 43, synaptophysin	Production and secretion of neurotrophic factors
35	Nosrat et al., 2004;	Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and GDNF	Huge potential to treat neurological disease.
36	Arthur et al., 2008	Neurotrophic factors, like brain- derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell line–derived neurotrophic factor (GDNF) beta III Tubulin	Differentiation to neural structures
37	Apel et al., 2009	Neurotrophic factors NGF, GDNF, BDNF, and BMP2	Neurogenic potential
38	Shi and Gronthos et al., 2003	Endothelial cell marker and pericytic markers	Niches of cells, in the perivascular region, show positivity
39	Tecles et al., 2005	Anti-BrdU antibody	Positivity surrounding the perivascular area. Has odontoblastic capacity and helps in repair during odontoblastic injury
40	Sloan and Smith 2007	STRO-1, CD 146, alpha smooth muscle actin and the pericyte-associated antigen 3G5, collagen XVIII a1, IGF-2cyclin-dependent kinase 6	Positivity confirmed pluripotency of DPSC’s vasculogenic and myofibroblastic potential
41	Amera Alkasi et al., 2013	CD105, CD166	DPSCs show mesenchymal stem cell properties
42	Afshin Khorsand et al., 2013	CD 90, CD 44, CD 146, SSEA-4, and anti-macrophage marker	Capable of differentiating to bone, cartilage, and adipose tissues
43	Bressan et al., 2012	Collagen type 1 expressing osteopontin, RUNX, v WF VEGF, osteonectin, osteocalcin, CD 31,VEGF mRNAs	Osteogenic cells capable of producing an extracellular matrix is located
44	Chunwei Zhang et al., 2018	CD 71, CK 14, integrin alpha-6 and PCNA	Transplanted DPSCs are inducted to form esophageal stem cells in vivo, to cure esophageal problems
45	Tomoatsu Kaneka et al., 2013	CD146, CD 105, CD 166	Density of stem cell associated marker higher in coronal pulp, suggests that coronal pulp harbors more stem cells
46	Huang et al., 2010	Dentin sialophosphoprotein, bone sialoprotein, alkaline phosphatase, and CD105	Multipotency is demonstrated by its of osteogenic, adipogenic, and chondrogenic capacity
47	Ivanovski et al., 2006	CD 146	The ability to generate and regenerate vascular and muscular components
48	Huang et al., 2009	Oct4, Nanog, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81	Multipotentiality
49	Demarco et al., 2011	SHED express STRO-1 and CD146. Using different transcription factors (Oct4, Sox2, Klf4, Myc)	Positivity toward these markers helped in regenerative potential
50	Maurin et al., 2009	MAP1B	Generate neural components
51	Montzka et al., 2009	MAP1B, CD146,STRO‐1	Neurogenic potency
52	Askari et al., 2015	Olig2 and GFAP (glial fibrillary acidic protein)—markers for neuronal precursors and astrocytes	DPSC-derived OPCs can differentiate into more mature oligodendrocytes
53	Kerkis et al., 2007	Nanog, Oct4, nucleostemin, Slain-1, Jmjd1, Jmjd2, and Cyclin D1	Ability to regenerate myogenic skeletal tissues
54	Ebrahimi et al., 2011	Nanog, oct4, nucleostemin, slain-1, jmjd1a, jmjd2c, and cyclin D1	Neurogenic potential
55	Tatullo et al.,2014	STRO‐1, CD29, CD44,CD73, CD90, CD105,CD146, CD166, and CD271	Odontoblastic, osteoblastic, melanocytic, neurogenic, chondrocytic, and lipocytic potential of DPSCs derived
56	Kawashima et al., 2012	STRO-1, CD29, CD44, CD73, CD90, CD105, CD146, CD166, and CD271.	Dentinogenic, osteogenic, myogenic, chondrogenic potency. Cornea, neural, and hair follicles can be regenerated
57	Yan et al. 2010b	Lin28, Nanog, Oct4, and Sox2, or c-Myc, Klf4, Oct4, and Sox2	Forms induced pluripotent stem cells
58	Oda et al. 2010	Sox2, Oct3/4, and Klf4	iPSC generation using mesenchymal stem cells by retroviral transduction of Oct ¾, SOX-2, and Klf-4 without Myc
59	Tamaoki et al. 2010	NANOG, SSEA-3, Tra-1-81	iPS cell Banks are aided by this boon
60	Yoo et al., 2013	CD 34	Precursor/progenitor cells are identified (endothelial progenitor cells)
61	Nosrat et al., 2004	Neurotrophic factors, including nerve growth factor (NGF), BDNF, and GDNF	Capable of neurogenic curative and regenerative properties
62	Gronthos et al., 2000	CD 14,CD44, CD 34, CD45, Integrin beta-1, MyoD, VCAM-1, alpha-SM Actin, MUC-18, neurofilamin, collagen-1, collagen-3, collagen-2, osteocalcin, BSP, osteonectin, osteopontin, PPAR gamma AlkPhos, FGF-2	Increased clonogenicity and proliferative and regenerative capacity
63	Talaat et al., 2015	Markers dentin sialoprotein and bone sialoprotein	Lead to pulp regeneration and dentin pulp complex formation
64	Ferro et al., 2012	Markers dentin sialoprotein CD 14, CD44, CD 34, CD45	Proliferative and capable of regenerating a tissue

Table 2

Pluripotency, markers, and number of articles

	Mesenchymal markers	Cell surface proteins	Embryonic stem cells markers	Hematopoietic markers	Dentinogenic markers	Neurogenic markers	Muscle and tendon markers
Markers	STRO-1	CD 90	Oct-4	CD 105	Bone sialoprotein	Nestin	Tenomodulin
	CD 29	CD 44	SOX-2	CD 34	Dentin sialoprotein	Neurofilamin	Tenascin
	CD 146	CD 14	C-myc	CD 45	DMP-1	CD 90	Collagen6
	Vimentin	CD 117	Klf	CD 31		CD 14	Eye absent homologue
	CD 73		Nanog			Tenascin- C
	CD 90		REX-1			Beta tubulin III
	CD 27		FOXO3			S 100
	CD 166		Nestin			Synaptophysin
	CD 217		Lin-28			P 75
			Stat-3
			CXCR-4
Number of articles	32	30	30	25	14	11	5

A panel of markers is proposed based on the markers used frequently by different authors, which shows the multi-lineage capacity and to support our aim of this review [Table 3].

Table 3

Proposed markers as investigation protocol for stemness and regeneration

Commonly used biomarkers	Application and uses
CD 146	This marker has the capacity to indicate cells that can be used for regeneration of structures from the three germ layers
CD 73	Positivity toward this marker indicates chondrogenic (cartilage forming), adipogenic, and osteogenic (bone and hard tissue forming) potential
CD 105	The positivity toward these markers elicit vasculogenic and angiogenic properties
STRO-1	STRO-1 positive cells DPSCs show highly potential bone forming, dentin forming and cartilage forming cells. Also positivity to this marker demonstrates capability of deriving structures from the endoderm, mesoderm and ectoderm (trilineage potency)
SOX-2	The stemness of the particular tissue derived can be proved, with this marker. Increase in S phase cells, that is positivity to this marker, shows the ability to produce increased number of cell lines and hence is implemented in regenerative research field

CONCLUSION

DPSC has a multi-lineage capacity, proved by various studies. The pulp tissue, which is considered as biological waste following extraction and endodontic treatment, can be harvested for the study. DPSC being mesenchymal and neuroectodermal origin has great potency for various therapeutic and regenerative purposes. It is imperative to study about this tiny tissue and its potential. On the basis of our review, we suggest that preservation of dental pulp tissue and subjecting them to the panel of biomarkers such as CD146, CD 73, CD 105, STRO-1, and SOX-2 will unravel its regenerative potential and lineage. The limitation of this analysis is that the panel of markers proposed is yet to apply practically in a pulp tissue to confirm its viability.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Databases	Keywords term and text word search
PubMed	Dental pulp AND stem cells
Medline	Immunohistochemistry AND dental pulp
Cochrane	Immunohistochemistry AND stem cells
Embase	Regeneration AND dental pulp
	Stem cells AND regenerative medicine
	In vitro studies AND dental pulp
	In vivo studies AND dental pulp
	Methods AND stem cell regeneration
	Biomarkers AND stem cell