1.Introduction: Dental pulp is a soft connective tissue found inside the “pulp chamber” of a human tooth. Developmentally, the pulp organ is derived from a complex interplay between the ectodermal and the mesenchymal germ layers and thereby it is termed to be an ecto-mesenchymal organ. The dental pulp is comprised of a heterogeneous population of cells forming blood vessels, nerves, fibres and ground substances in addition to fibroblasts, odontoblasts and mesenchymal stem cells. Immunostaining experiments reveal the presence of stem cell niches in the “cell rich zone” and around the perivascular region . Biologically, the stem cell population functions as a reserve source of odontoblast which inturn produces the dentin required to maintain the dentin layer of the teeth [2–4].
The dental pulp stem cells were first isolated and reported as stem cells by Dr. Songato Shi in the year 2000 . This gave rise to an avalanche of studies targeted to de-mystify this interesting population of stem cells, which is evident by the steep increase in research publications since 2005 in this area (Fig. 1). The DPSCs are shown to express many mesenchymal specific cell surface markers like CD29, CD44, Stro1, CD 73 and CD45 [5,6], Nestin (neuronal marker) and Oct 4 (pluripotency marker) . In this review, we have attempted to consolidate the studies quoting recent advances and novel applications of DPSCs.
2.Novel biomaterials used for enhanced differentiation / regeneration
A usual trend in scaffolding studies is to combine a structural component such as hydrogel, carboxymethyl cellulose etc., with a functional component such as calcium, phosphate, zinc, hyaluronic acid etc. Many studies were also focused on using nanopatterning and surface enhancement of novel and commonly used biomaterials as a method for differentiation control and cell fate determination of human mesenchymal stem cells(hMSC)(Niehage, Karbanov, Steenblock, Corbeil, & Hoflack, 2016). Zinc-modification of titanium, which is a very commonly used material in the field of dentistry and oral maxillofacial surgery, such as bone fixation devices and dental implants was observed to increase the osteogenic differentiation of dental pulp stem cells as is evident by the overexpression of collagen (Col I), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteopontin (OPN), and vascular endothelial growth factor A (VEGF A) . Human dental pulp stem cells (hDPSCs) grown on polycaprolactone (PCL) scaffolds enhanced with hyaluronic acid and b-TCP (Tri-calcium phosphate) was also shown to increase osteogenic differentiation. An interesting study conducted by Jensen et al., demonstrated generation of a human dental matrix derived by demineralization of human teeth using 17% EDTA which is further enhanced by zinc and magnesium doped bio-ceramic scaffold and its ability to target hDPSCs towards dentin regeneration . Human DPSCs grown on a nano-patterned PEG-GelMA-HA showed a significant upregulation of the chondrogenic gene markers (Sox9, Alkaline phosphatase, Aggrecan, Procollagen type II, and Procollagen type X), while downregulating the pluripotent stem cell genes and epithelial–mesenchymal genes . Similarly, hDPSCs grown on five novel scaffolds; demineralized dentin matrix (DDM), ceramic bovine bone (CBB), small intestinal submucosa (SIS), poly-L-lactate-co-glycolate, and collagen-chondroitin sulfate-hyaluronic acid, actualised that the naturally mineralized scaffolds (DDM, CBB, SIS) showed increased levels of alkaline phosphatase (ALP) activity and mRNA expression of bone sialoprotein, osteocalcin, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) as compared to the artificial scaffolds .
3. Differentiation into new / specialized functional cell types:
Differentiation of hDPSCs into mesoderm derived lineages such as osteoblasts and adipocytes has been well reported in numerous studies. But, recent years saw an increase in research focusing on differentiating these stem cells into more functionally specialised lineages. Differentiation of hDPSCs into dopaminergic neuron like stem cells in vitro has been achieved using various inductive techniques such as knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) , exposure to midbrain cues (sonic hedgehog, fibroblast growth factor 8 and basic fibroblast growth factor)  and motor neuronal inductive media . A study conducted in University of Pittsburgh succeeded in differentiating hDPSCs to keratocytes (cells of the corneal stoma) using in vitro pellet culture in keratocyte differentiation medium containing advanced Dulbecco’s Modified Eagle’s Medium with 1 mM ascorbate-2-phosphate, 10 ng/ml fibroblast growth factor 2, and 0.1 ng/ml transforming growth factor-β3. Hepatic differentiation of hDPSCs was achieved by supplementing the media with hepatic growth factor, insulin-transferrin-selenium-x, dexamethasone, and oncostatin M. These cells expressed hepatic markers such as alpha fetoprotein, albumin, hepatic nuclear factor-4 alpha, insulin-like growth factor-1, and carbamoyl phosphate synthetase. hDPSCs were also differentiated into pancreatic cell lineage resembling islet-like cell aggregates (ICAs) using a media cocktail containing Dulbecco’s Modified Eagle’s Medium Knock Out (DMEM-KO), 1% BSA, 1x insulin-transferrin-selenium (ITS), 4 nM activin A, 1 mM sodium butyrate, and 50 µM 2-mercaptoethanol. The identity of these ICAs was confirmed as islets by dithiozone-positive staining, as well as by expression of C-peptide, Pdx-1(Pancreatic And Duodenal Homeobox 1), Pax4, Pax6, Neurogenin 3, and Isl-1.
4. Secretome, Proteome studies for therapeutic applications or characterization:
The anti-inflammatory, anti-microbial effects of trophic factors secreted by mesenchymal stem cells have been widely studied. An iconic study conducted in the Stanford University which involved surgical transplant of bone marrow derived mesenchymal stem cells in stroke patients showed preclinical evidence for the improvement in outcome. The study speculated that this effect may be associated with the secretion of trophic factors secreted by the mesenchymal cells , which stirred a new-found interest in the secretome of the MSCs and their therapeutic applications. Secreted paracrine factors from hDPSCs had neuroprotective and neuritogenic effect on retinal ganglion cells (RGC). Conditioned media, collected from cultured hDPSC, were assayed using ELISA and found to have growth factors such as NGF (Nerve Growth Factor), BDNF (Brain Derived Neurotrophic factors), NT-3 (Neurotrophin – 3), VEGF (Vascular Endothelial Growth Factor A), GDNF (Glial Cell Derived Neurotrophic Factor), PDGF (Platelet Derived growth Factor) -AA and PDGF-AB/BB . Secretome derived from dental pulp stem cells (DPSCs) reduced cytotoxicity and apoptosis caused by amyloid beta (Aβ) peptide in vitro, one of the main misfolded proteins in Alzheimer’s disease. DPSC secretome contains higher concentrations of VEGF, Fractalkine, RANTES, MCP-1(Monocyte Chemoattractant Protein -1), and GM-CSF (Granulocyte Macrophage – Colony Stimulating Factor), it also stimulated the endogenous survival factor Bcl-2 and decreased the apoptotic regulator Bax. A novel proteome study of cell surface markers of hDPSCs using label-free mass spectrometry identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. In rat models IGF1R+ hDPSCs cultured in low percentage of human umblical cord serum promoted neuroplasticity, therefore improving neurological function through increasing glucose metabolic activity, enhancing angiogenesis and anti-inflammatiory effects through a bidirectional cross-talk between IGF1R/IGF1 and CXCR4/SDF-1α signaling pathways .
The research on dental pulp stem cells has seen an exponential rise in the past decade. The dental pulp stem cells were observed to be very versatile in their application. The trends in studies were focused on regeneration using a functionalized scaffold material, direct cell replacement therapies and harnessing their tropic/secreted factors (Fig. 2). From the volume of studies conducted and through the myriad applications and abilities possessed by the DPSCs, we conclude that the therapeutic usage of DPSCs in regenerative and translational medicine seems to be very imminent.
 S. Shi, S. Gronthos, J. Bone Miner. Res. 18 (2003) 696–704.
 a Almushayt, K. Narayanan, a E. Zaki, A. George, Gene Ther. 13 (2006) 611–20.
 S. Gronthos, M. Mankani, J. Brahim, P.G. Robey, S. Shi, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13625–30.
 A.J. Sloan, R.J. Waddington, Int. J. Paediatr. Dent. 19 (2009) 61–70.
 Y. Isobe, N. Koyama, K. Nakao, K. Osawa, M. Ikeno, S. Yamanaka, Y. Okubo, K. Fujimura, K. Bessho, Int. J. Oral Maxillofac. Surg. 45 (2016) 124–131.
 D. Ponnaiyan, V. Jegadeesan, Eur J Dent 8 (2014) 307–13.
 G. Ibarretxe, O. Crende, M. Aurrekoetxea, V. Garc, J. Etxaniz, F. Unda, Stem Cells Int. 2012 (2012).
 C. Niehage, J. Karbanov??, C. Steenblock, D. Corbeil, B. Hoflack, PLoS One 11 (2016) 1–25.
 K. Yusa, O. Yamamoto, H. Takano, M. Fukuda, M. Iino, Sci. Rep. (2016) 1–11.
 J. Jensen, D.C.E. Kraft, H. Lysdahl, C.B. Foldager, M. Chen, A.A. Kristiansen, J.H.D. Rölfing, C.E. Bünger, Tissue Eng. Part A 21 (2015) 729–39.
 C.L. Nemeth, K. Janebodin, A.E. Yuan, J.E. Dennis, M. Reyes, D.-H. Kim, Tissue Eng. Part A 20 (2014) 2817–29.
 H. Zhang, S. Liu, Y. Zhou, J. Tan, H. Che, F. Ning, X. Zhang, W. Xun, N. Huo, L. Tang, Z. Deng, Y. Jin, Tissue Eng. Part A 18 (2012) 677–691.
 S.Y. Chun, S. Soker, Y.-J. Jang, T.G. Kwon, E.S. Yoo, J. Korean Med. Sci. 31 (2016) 171–7.
 M. Kanafi, D. Majumdar, R. Bhonde, P. Gupta, I. Datta, J. Cell. Physiol. 229 (2014) 1369–1377.
 C.C. Chang, K.C. Chang, S.J. Tsai, H.H. Chang, C.P. Lin, J. Formos. Med. Assoc. 113 (2014) 956–965.
 F.N. Syed-Picard, Y. Du, K.L. Lathrop, M.M. Mann, M.L. Funderburgh, J.L. Funderburgh, Stem Cells Transl. Med. 4 (2015) 276–85.
 N. Ishkitiev, K. Yaegaki, T. Imai, T. Tanaka, T. Nakahara, H. Ishikawa, V. Mitev, M. Haapasalo, J. Endod. 38 (2012) 475–480.
 V. Govindasamy, V.S. Ronald, a N. Abdullah, K.R.G. Nathan, Z. a C. Ab Aziz, M. Abdullah, S. Musa, N.H.A. Kasim, R.R. Bhonde, J. Dent. Res. 90 (2011) 646–652.
 G.K. Steinberg, D. Kondziolka, L.R. Wechsler, L.D. Lunsford, M.L. Coburn, J.B. Billigen, A.S. Kim, J.N. Johnson, D. Bates, B. King, C. Case, M. McGrogan, E.W. Yankee, N.E. Schwartz, Stroke 47 (2016) 1817–1824.
 B. Mead, A. Logan, M. Berry, W. Leadbeater, B.A. Scheven, PLoS One 9 (2014).
 H.-T. Lee, H.-T. Chang, S. Lee, C.-H. Lin, J.-R. Fan, S.-Z. Lin, C.Y. Hsu, C.-H. Hsieh, W.-C. Shyu, Sci. Rep. 6 (2016) 32595.
Dr. Dannie Macrin
Saveetha Institute of Medical and Technical Sciences (SIMATS), India