Dental Mesenchymal Stem Cells in Inflamed Microenvironment: Potentials and Challenges for Regeneration

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412 Current Stem Cell Research & Therapy, 2015, 10, 412-421 Dental Mesenchymal Stem Cells in Inflamed Microenvironment: Potentials and Challenges for Regeneration

Yuan Zhou1,2, Liwei Zheng1,2, Xuedong Zhou1,2, Jiyao Li1,2 and Xin Xu1,2,*

1State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China; 2Department of Endodontics, West China Hospital of Stomatology, Sichuan Univer- sity, Chengdu, China

Abstract: Adult dental mesenchymal stem cells (DMSCs) are multi-potent stem cells that are involved in dental tissue repair and regeneration. DMSCs are able to differentiate into multiple lineages, including odontogenic, osteogenic, neurogenic, adipogenic, chondrogenic, hepatogenic lineages and insulin- producing cells. However, the DMSCs from functioning, impacted or exfoliated teeth may not be available when needed. Recently, DMSCs have been found in pulpal, periapical and periodontal tissue with inflammation from deciduous and immature/mature permanent teeth. DMSCs from inflamed tissue (iDMSCs) possess typical stem cell characteristics while they showing varied properties. Whether iDMSCs are comparable to healthy DMSCs and can be used for regeneration are not clear. Studies on the impact of infection/inflammation in the local microenvironment on DMSCs are widely conducted to investigate the specific influences and underlying mechanisms in vitro and in vivo. In this review, we introduced the discovery of iDMSCs from different sources, and also discussed the influence of dental inflammation and associated immune responses, particularly the effect from lipopolysaccharide (LPS) stimulation, on local DMSCs. In addition, the effects of dental procedures and materials on DMSCs are discussed. Keywords: Dental mesenchymal stem cells, inflammation, lipopolysaccharide, regeneration.

1. INTRODUCTION 2. STEMNESS AND IMMUNOLOGICAL PROPER- TIES OF DMSC Dental stem cells derived from different dental soft tissues are multi-potent mesenchymal stem cells (MSCs) which Five types of DMSCs have been isolated and character- are capable of self-renewal and multi-lineage differentiation ized from soft tissue of deciduous or permanent teeth so far, [1]. Dental pulp-composing cells are generally considered as including DPSCs, stem cells from exfoliated deciduous teeth neural crest ectomesenchyme-derived cells [2]. A recent (SHEDs), stem cells from apical papilla (SCAPs), periodon- study has demonstrated that a large population of dental tal ligament stem cells (PDLSCs), and dental follicle pro- mesenchymal stem cells (DMSCs) that produce pulp cells genitor cells (DFPCs) [1, 4, 7, 18, 19]. and odontoblasts, are derived from peripheral nerve- associated glia in a mouse model [3]. DMSCs can differenti- 2.1. DPSC ate into multiple lineages, including odontogenic, osteogenic, DPSCs were the first identified population of dental tis- neurogenic, adipogenic, chondrogenic, hepatogenic, and sue-derived mesenchymal stem cells, which were isolated insulin-producing cells [1, 2, 4-12]. In endodontic treatment, from adult third molar pulp tissue [1]. DPSCs demonstrate pulpectomy and root canal therapy are normally performed to clean pulpal infection and inflammation. The inflamed typical fibroblast-like morphology and are characterized by colony forming unit-fibroblast (CFU-F) assay. The niche of pulp is usually discarded as medical waste. Recent studies DPSCs is found at perivascular and perineural sheath regions have shown success in isolating DMSCs from inflamed pulp, [20]. Similar to the bone marrow-derived mesenchymal stem indicating that inflamed pulp could be a potential source of cells (BMMSCs), human DPSCs express putative stem cell allogenic or autogenic MSCs for regenerative medicine [13- surface markers such as STRO-1, CD44, CD73, CD90, 17]. Here we will briefly review the characteristics of DMSCs, and DMSCs isolated from inflamed dental soft tis- CD106, vascular markers or antigens such as -smooth muscle actin and MUC-18 (CD146), and embryonic stem cell sues. The influence of inflammation on DMSCs, particularly (ESC) markers such as Oct4, Nanog and Sox-2 [21, 22]. dental pulp stem cells (DPSCs), and potential challenges and Most of the DPSCs also express pericyte marker 3G5, perspectives for tissue regeneration will be discussed. whereas only minor populations of BMMSCs express 3G5 [20]. As neural crest originated cells, DPSCs express neu- *Address correspondence to this author at the Sichuan University State Key ronal and glial cell markers [23, 24]. However, hema- Lab Oral Dis, West China Hosp Stomatol, Chengdu 610041, Sichuan, Peoples topoietic lineage markers CD14, CD34 and CD45, odon- Republic of China; Tel: +86-028-85501439; E-mail: [email protected] toblast-specific markers dentin sialoprotein (DSP) and dentin

2212-3946/15 $58.00+.00 © 2015 Bentham Science Publishers Dental Mesenchymal Stem Cells in Inflamed Microenvironment Current Stem Cell Research & Therapy, 2015, Vol. 10, No. 5 413 sialophosphoprotein (DSPP), osteoblast-specific markers 3. DENTIN-PULP COMPLEX IS VULNERABLE TO alkaline phosphatase (ALP), osteocalcin (OCN), and osteo- INFECTION AND INFLAMMATION pontin (OPN) are absent in DPSCs [1, 21, 24]. Cultured Dental pulp residing in the pulp cavity contains connec- DPSCs demonstrate the ability to differentiate into odon- tive tissue and odontoblasts. Dentin surrounding the pulp toblasts and form mineral nodules. They are also able to form dentin/pulp-like complex when transplanted into im- contains not only dentinal tubules, but also the cytoplasmic extensions of odontoblasts. Both dentin and pulp share simi- munocompromised mice [21]. Moreover, DPSCs are able to lar embryonic origins. Given their interdependent functions differentiate into other cells, including osteogenic, chondro- and their close association, they are often collectively known genic, neurogenic, adipogenic and myogenic lineages [25, as the dentin-pulp complex. Because of the special tubular 26]. Treatment with transforming growth factor 1 (TGF- conjunction between dentin and pulp, extrinsic irritants can 1), alone or combined with fibroblast growth factor 2 (FGF- 2), and exposure to dentin matrix protein 1 (DMP1) induce easily injure the pulp through exposed dentinal tubules via both coronal and radicular routes [38, 39]. differentiation of DPSCs into odontoblasts in vitro [27]. Similar to other MSCs, DPSCs are immunosuppressive [28, Dental caries is the major cause of dental pulp inflamma- 29]. To modulate the local immune response, DPSCs could tion. In addition, dental anomalies, tooth fracture, marginal produce TGF-, which activates the p38 mitogen-activated micro-leakage at the restoration-tooth interface, periodontal protein kinase (MAPK) signaling pathway, and subsequently diseases, dental procedures, chemical irritation, aging, and suppresses the proliferation of peripheral blood mononuclear systemic diseases are also common causes of pulp inflamma- cells (PBMCs) in vitro [28]. tion. When caries progresses to dentin, bacteria with toxic metabolic by-products and degraded dentin matrix can dif- 2.2. Other DMSC fuse into the pulp through dentinal tubules. At the early stage of inflammation, odontoblasts, at the defending frontline SCAPs are precursors of dental pulp, which can be iso- against extrinsic irritants, will recognize invading pathogenic lated from apical papilla at the apices of developing perma- molecules and release low levels of interleukin-8 (IL-8), nent teeth [7, 30]. SCAPs also express stem cell markers chemokines (CC chemokine ligand 2 (CCL2), CCL26, CXC such as STRO-1, CD146 and CD24, among which CD24 can motif chemokine 2 (CXCL2), CXCL4, CXCL12, CXCL14) be used as a specific marker for this population. SCAPs can and chemokine receptors (CXC chemokine receptor type form odontoblast-like cells and produce dentin-pulp complex 2(CXCR2), CC chemokine receptor-like 1 (CCRL1), in the minipig model [30]. More importantly, SCAPs may CCRL2) [40-43]. Odontoblasts can recruit immature den- survive during the process of pulp necrosis, possibly due to dritic cells (DCs) by CCL2, CXCL2 and CXCL14 to initiate the adjacency to the periapical tissue with a rich blood sup- the innate immune response. As the disease progresses, ply [31]. Similarly, SCAPs can suppress the proliferation of odontoblasts as well as Hehl’s cells and stem/progenitor T cells in vitro [7]. SHEDs were identified from the remain- cells will be all involved as sensor cells to detect and respond ing pulp of exfoliated deciduous teeth, expressing a series of to the invaders [44]. Pattern recognition receptors (PRR) on stem cell markers [4]. Compared to DPSCs, SHEDs have a the immune sensor cell membrane are needed to detect vari- higher proliferation rate and longer population doubling ous bacterial components. PRR expressed by odontoblasts time. Transplanted SHEDs could induce bone-/dentin-like are mostly TLR1-6 and 9 [40], whose ligand binding leads to tissue formation in immunocompromised mouse model [32]. the activation of the nuclear factor B (NF-B) and p38 However, SHEDs are not able to regenerate complete den- MAPK intracellular signaling cascades [45-47]. In vitro tin/pulp-like complexes in vitro [4]. SHEDs also possess treatment of odontoblasts with lipoteichoic acid (LTA), a cell immunomodulatory characteristics by inhibiting T helper 17 wall component of Gram-positive bacteria, resulted in (Th17) cells and elevating the ratio of regulatory T cells upregulation of TLR-2, 3, 5 and 9, and release of CCL2, (Tregs) to Th17. Hence, SHEDs were able to reverse the CXCL10 and vascular endothelial growth factor (VEGF) systemic lupus erythematosus (SLE)-like symptom in mice which is a potent inducer of angiogenesis and endothelial permeability [40, 48]. TLR-2 and TLR-4 are also upregu- [32]. DFPCs are DMSCs derived from the dental follicle, lated when odontoblasts are challenged with lipopolysaccha- which can be found surrounding the impacted teeth in adults ride (LPS), a type of cell wall components of Gram-negative [18]. DFPCs are able to differentiate into osteoblasts, cemen- bacteria [49, 50]. toblasts, chondrocytes and adipocytes, also forming periodontal-ligament-like tissue after being transplanted into im- In irreversible pulpitis, TGF- expression increases in munocompromised mice [18, 33, 34]. DFPCs carry immuno- odontoblasts [43, 51, 52]. In healthy pulp, TGF- is impor- suppressive properties which are toll-like receptors (TLRs) tant for dentinogenesis and dentin repair by promoting ma- dependent [28]. PDLSCs can be isolated from periodontal trix metalloproteinase (MMP) secretion and dentin minerali- ligament [19]. PDLSCs can give rise to osteogenic, chondro- zation [53, 54]. In the early stages of pulp inflammation, genic, adipogenic and neurogenic cells. More importantly, TGF- acts as a pro-inflammatory factor and is involved in they can differentiate into periodontium-like cells in vitro recruiting immune cells [54]. At later stages, TGF- instead [19, 22, 35, 36]. PDLSCs can produce indoleamine 2, 3- displays anti-inflammatory effects, repressing lymphocyte dioxygenase (IDO), TGF-1 and hepatocyte growth factor proliferation, DC and macrophage activation, and TLR sig- (HGF) to further support immunosuppression by suppressing naling [55]. In inflamed pulp, DCs are the major antigen T cell proliferation [28, 37]. The secretion of these factors presenting cells that migrate and accumulate quickly at the odontoblast layer near the lesion region with foreign antigen. can be mediated by interferon gamma (IFN-) produced by After maturation, DCs release pro-inflammatory mediators activated PBMC during inflammation [28, 37]. such as tumor necrosis factor- (TNF-), IL-1, prostaglandin 414 Current Stem Cell Research & Therapy, 2015, Vol. 10, No. 5 Zhou et al.

E2 (PGE2) and vasoactive amines, and recruit more DCs and a broad range of bioactive molecules to trigger multiple sig- T cells to trigger further defense mechanisms [43, 56]. With naling cascades. The ignited effects include, but are not lim- lesion progression, neutrophils, monocytes, macrophages ited to: enhancement of cell proliferation and tissue vascu- and lymphocytes (including B cells, T cells and NK cells) larization; modulation of immune responses and reduction of gradually migrate and accumulate in the pulp and release cell aging and apoptosis. The typical mechanisms of immu- anti-inflammatory factors, for instance IFN- and stromal nosuppression by MSC include negative regulatory pathways cell-derived factor-1 (SDF-1), to combat the invading irri- associated with TNF- stimulated gene/protein 6 (TSG-6) tants and further promote tissue repair [44, 56]. Meanwhile, and PGE2 [59]. the interplay and anti-inflammatory effect of those cells can cause collateral tissue damage [57]. Short-term and mild 4.2. DMSC Isolated from Inflamed Tissue irritations usually cause acute pulp inflammation, which is self-retained and is possible to recover from. Dentinogenesis As a subset of MSCs, DMSCs have potential for use in of odontoblasts will be upregulated and generate reactionary various clinical settings, such as dental tissue destruction, dentin to protect pulp from caries invasion. In contrast, long- bone resorption, neurodegenerative diseases, heart failure term and severe stimuli will cause death of odontoblasts and and diabetes. However, adequate numbers of DMSCs from induce chronic and irreversible pulp inflammation [44]. healthy pulp are not always available as needed for regenerative treatment [13]. In this case, DMSCs from other sources, When infectious components contact with pulp tissue, the such as inflamed pulp, may serve as an alternative supply for odontoblast layer is mostly destroyed, however dentinogene- stem cells. sis may also occur [44, 58]. The progenitor/stem cells migrate to the dental pulp, and subsequently proliferate and Common pulp infection can be generally divided into differentiate into odontoblast-like cells or osteoblast-like three stages: reversible pulpitis, irreversible pulpitis and pulp cells to form reparative dentin or osteodentin [44]. If the necrosis. In the clinic, when irreversible pulpitis is diag- inflammation continues, pulp necrosis will occur [39, 55, nosed, the pulp is removed by pulpectomy, even though part 59]. The dental pulp may also respond to irritation with de- of the pulp may still be viable. Recently, dental generative changes such as fibrosis and calcification [39, stem/progenitor cells have been identified in dental pulp tis- 55]. The dynamics of periapical inflammation is similar to sue with various pathologies, including irreversible pulpitis, that of inflammation in the pulp. Moreover, alveolar bone necrosis, polyps/chronic hyperplastic pulpitis, deep caries, destruction and resorption develop in apical periodontitis, traumatically pulpal exposure, aggressive periodontitis and and associated signaling starts even earlier than apical in- other inflamed conditions [13-17]; from various tooth flammation. This is supported by the observation that peri- sources including deciduous, immature and mature perma- apical bone resorption signaling such as receptor activator of nent teeth [63-65]. Furthermore, periapical tissue with apical NF-B ligand (RANKL) occurs earlier than the inflamma- periodontitis or periapical cyst, and inflamed periodontal tory cell recruitment signaling such as interleukin-8 (IL-8) in tissue have also been reported to harbor stem/progenitor cells the development of apical periodontitis [60]. [14, 66-68]. These dental mesenchymal stem cells from inflamed tissue (iDMSCs) share similar characteristics with 4. DMSC IN INFLAMED DENTAL TISSUE DMSCs, while possessing distinct features gained from an inflamed microenvironment. 4.1. MSC in Inflamed Microenvironment 4.2.1. DPSC from Inflamed Pulp For a tooth with pulpitis, what happens to the pulp progenitor/stem cells and how they react to pulp inflammation Mesenchymal stem cells isolated from inflamed pulp and the local immune response are crucial for pulp repair and (iDPSCs) of permanent tooth show CFU-F similar to DPSCs regeneration. Prior to looking into the DMSCs in inflamed isolated from normal pulp [13, 15]. iDPSCs also harbor self- pulp, it is necessary to take into account the responses and renewing and expanding abilities, with similar to higher pro- modulation properties of MSC in general infection/inflamed liferative rates compared to normal DPSCs [16, 69, 70]. microenvironment. Since being successfully isolated from However, iDPSCs have significantly lower total population bone marrow 30 years ago, human MSCs have thereafter doubling (PD) than normal DPSCs during long-term obser- been found in various tissues, such as bone, cartilage, dentin, vation, suggesting compromised cell division potential of muscle, fat and other connective tissues under certain condi- iDPSCs under inflammatory conditions [13]. The immuno- tions [61]. Under normal conditions, MSCs are pericytes in phenotype of iDPSCs is similar to normal DPSCs: express- the latent/dormant state. As vascular density decreases with ing stem cell surface markers such as STRO-1, CD73, CD90, age, the local availability of MSCs also decreases substan- CD105, CD146, while being positive for embryonic marker tially [62]. During local infection and injury, MSCs can pro- stage-specific embryonic antigen-4 (SSEA4) and negative liferate and differentiate. One of the most important features for hematopoietic markers CD14, CD34 and CD45 [13, 15, of MSCs is to sense and respond to local irritants and regu- 69]. Although some studies show no significant difference in late associated immune responses in the inflamed microenvi- cell morphology, proliferation and differentiation between ronment. MSCs activated during inflammation can partici- normal DPSCs and iDPSCs [69], accumulating data suggest pate in tissue repair, immunomodulation and have anti- that the multi-lineage differentiation capacity of iDPSCs inflammatary propoerties. At the onset of inflammation, lo- differs from that of normal DPSCs. cal sensor cells like DCs detect the irritant factors through In stimulated culture media, iDPSCs successfully differ- PRR, secreting inflammatory mediators such as TNF-, IFN- entiated into odontogenic, osteogenic, adipogenic and chon- , IL-1, IL-6, CCL2, CXCL8. Upon activation, MSCs secrete drogenic lineages, but the expression levels of odonto- Dental Mesenchymal Stem Cells in Inflamed Microenvironment Current Stem Cell Research & Therapy, 2015, Vol. 10, No. 5 415

/osteogenic markers as well as the mineral deposition ture and immature permanent teeth, stem cells can be iso- amount of iDPSCs were sometimes lower than normal lated from periapical lesions caused by endodontic infection, DPSCs [13, 16, 70, 71]. Studies have also shown that such as periapical cyst [66-68, 74, 76]. Cells isolated from iDPSCs may have impaired odontogenic but enhanced os- inflamed periapical tissue by apicoectomy contain fibroblast- teogenic differentiation potential relative to normal DPSCs like cells, which are able to form colonies, and express MSC [17]. iDPSCs from traumatically exposed pulp of rat had markers such as STRO-1, CD13, CD29, CD44, CD73, higher level of ALP, Runt-related transcription factor 2 CD90, CD105, and CD146. These cells show osteogenic and (Runx2) and Ocn and lower Dspp in vitro, while forming adipogenic differentiation potential in vitro, and form miner- Ocn-positive osteodentin-like tissue after transplantation in alized tissue in an immunocompromised mouse model [66, rat model [71]. iDPSCs from deep caries pulp also showed 67]. Similar to other DMSCs, periapical lesion stem cells increased ALP and OCN [70]. When transplanted in im- (PLSCs) have shown low immunogenicity, which is sup- munocompromised mice, iDPSCs were able to form den- ported by the absence of major histocompatibility complex tin/pulp-like complex, in which mineralized dentin-like tis- (MHC) class II expression on PLSCs [68]. Clinical cases sue was wrapped in connective tissue with blood vessels. showed that the SCAP from inflamed immature teeth (iS- However, the amount of dentin-pulp complex formed by CAP) may migrate and play a part in the formation of a sepa- iDPSCs compared to that of normal DPSCs varies between rate new tip from the main root after endodontical removal different studies [13, 15]. Elevated cytoplasmic phosphory- of inflamed pulp [77]. Hence, teeth with advanced pulpitis or lated IB/P65 and nuclear P65 can be detected in parallel to even periapical inflammation can still serve as a source of the enhanced osteogenic and reduced odontogenic differen- stem cells for regenerative therapy. tiation associated markers in iDPSCs [17], suggesting that the NF-B pathway is involved in the differentiation regula- 4.2.3. PDLSC from Inflamed Periodontal Tissue tion of iDPSCs [16, 17, 71]. Similarly, iDPSCs isolated from When periodontal or combined periodontal-endodontic teeth with different degrees of aggressive periodontitis also inflammation develops, the PDLSCs are also affected. showed lower CFU-F, proliferation rate and odontogenic PDLSCs isolated from inflamed sites of periodontitis pa- differentiation, both in vitro and in transplantation mouse tients (iPDLSCs) show higher proliferation rates but reduced models, relative to normal DPSCs [14]. However, the osteogenic differentiation. In addition, iPDLSCs display im- changes in mineralization related markers and in vitro min- paired immunosuppressive properties, and are not able to eral deposition are not sufficient to reach a conclusion. More efficiently suppress T cell proliferation, induce Tregs, pro- in vivo evidence is needed to further validate the change in mote Th17 differentiation or IL-10, IL-17 and IFN- secre- odonto-/osteogenic differentiation of iDMSCs. tion, compared with normal PDLSCs [78]. The mechanisms Apart from permanent tooth, iDPSCs can also be isolated of defective osteogenic differentiation of iPDLSCs are pos- from the inflamed pulp of deciduous tooth [29, 63, 72, 73]. sibly associated with complex interplay among Wnt/- iDPSCs from deciduous tooth (idDPSCs) are positive for catenin, p38-MAPK, NF-B and also noncanonical Wnt/Ca2+ stem cell specific cell surface markers, including CD90, pathways [79-81]. Wnt/-catenin signaling is reported to CD105 and CD146, and show similar proliferation rates have both osteoinhibitory and osteoinductive roles to regu- compared to normal DPSCs from deciduous tooth (dDPSCs) late the balance of MSC differentiation depending on the [29]. idDPSCs can differentiate into osteogenic, adipogenic signaling extent [82-84]. When induced under osteogenic and chondrogenic lineages under certain stimulation in vitro. conditions, normal PDLSCs have elevated glycogen synthase However, lower expression levels of associated markers such kinase 3 (GSK3) and lower -catenin in the nucleus, as as Runx2 and Ocn were also observed [29, 63]. Importantly, well as increased noncanonical Wnt/Ca2+ signals (CaMKII, idDPSCs have impaired immunogenicity and immunomodu- NLK) and p38, resulting in upregulation of osteogenic dif- latory properties compared to dDPSCs, as indicated by lower ferentiation [79]. However, reports show that iPDLSCs have levels of human leukocyte antigen (HLA)-ABC/-G and lower levels of GSK3 and higher active -catenin and weaker inhibition of T cell proliferation in vitro [29]. phosphorylated p65/NF-B expression compared to normal idDPSCs also secrete higher levels of TNF-, TNF- and IL- PDLSCs [79, 81]. After osteogenic induction, the responses 2, but lower anti-inflammatory IL-10 relative to dDPSCs in of iPDLSCs in the aforementioned pathways are weaker than vitro [29]. Interestingly, treatment with FGF-2 induced odon- normal PDLSCs, which leads to impaired osteogenic differ- togenic differentiation of idDPSCs in vitro [73], supporting entiation [80, 81]. In vitro treatment using the pro- the combinatory use of various growth factors in expanding inflammatory cytokine TNF- on PDLSCs results in similar the potential of iDPSCs in clinical settings. effects. TNF- induces phosphorylation and inactivation of GSK3, further promoting the nuclear translocation of - 4.2.2. DMSC from Inflamed Periapical Tissue catenin, increasing -catenin/lef1 and subsequently decreas- ing Runx2-associated osteogenesis. The negative modulation If left untreated, pulpitis will eventually progress to pulp of NF-B pathway is also associated with the reduction of necrosis, and apical periodontitis may consequently occur. In osteogenic differentiation of iPDLSCs [79-81, 85]. this situation, the local infection and inflammation are so severe that barely any vital pulp remains. Surgical exposure of rat immature pulp induces pulpitis and further apical pe- 5. INTERACTIONS OF DMSC WITH THE INFLAM- MATORY MILIEU riodontitis, however, vital tissue can still be found in apical papilla and periapical lesions after a period of time [74]. In Since the inflammatory microenvironment affects the addition, osteogenic cells can be found in periapical granula- biological behavior of iDMSCs, interactions between tion lesions [75]. Recently it has been found that in both ma- DMSCs and the inflammatory milieu are worth investigat- 416 Current Stem Cell Research & Therapy, 2015, Vol. 10, No. 5 Zhou et al. ing. Several models have been employed to investigate the entiation of osteo-progenitor cells in bone tissue, which may interaction between DMSCs and the inflammatory milieu, be involved in the elevation of mineral formation of iDMSCs such as LPS treatment, inflammatory cytokine treatment, [97]. experimental inflammation using in vivo models and treat- Furthermore, repeated LPS stimulation induces the se- ment with endodontic medicine or appliances. nescence of DPSCs through TLR4-p16INK4A signaling [98]. Reactive oxygen species (ROS) levels rise after TLR4 acti- 5.1. LPS stimulates DMSC vation, causing DNA injury of local cells and inducing cellu- The LPS from the outer membrane of Gram negative lar aging [98]. During chronic pulpal inflammation, repeated bacteria is one of the major bacterial virulence factors in irritation has more adverse effects on DPSCs than acute in- pulpal infection [86]. LPS is a well-documented factor that flammation. This aspect needs to be considered during clini- initiates infection-stem cell interaction, and has been investi- cal application of iDPSCs. In addition to DPSCs, SCAP, gated widely. LPS stimulates local pulp cells such as odon- PDLSCs and dental follicle cells have been found to respond toblasts and fibroblasts, inducing the secretion of pro- similarly to LPS [96, 99, 100]. inflammatory cytokines and chemokines which further initi- LPS promotes the expression of multiple inflammatory ate a variety of immune responses and repair processes [47, mediators and changes the properties of DMSCs by binding 87-90]. to PRR TLRs. Other pathogen ligands including LTA, flag- LPS has a negative effect on DPSCs proliferation. The ellin, peptidoglycan and nucleic acids can also bind to TLRs LPS from Escherichia coli inhibits proliferation of DPSCs in [101, 102]. While most studies show that LPS from many vitro, and blockade of TLR4 can rescue this inhibitory effect bacterial strains recognize and bind to TLR4 on stem cell [91, 92]. LPS promotes the adhesion and migration of membranes, LPS from P. gingivalis has been shown to inter- DPSCs in vitro [91, 93]. After a moderate dose of E. coli act with both TLR4 and TLR2 [49, 103-105]. Intracellular LPS stimulation, the adhesion-related genes of DPSCs, in- signaling pathways associated with the pathological effects cluding fibronectin (FN), intercellular adhesion molecule 1 of LPS are the NF-B, MAPK and Wnt pathways [45, 50, (ICAM-1), 1 integrin (Integrin-1) and VEGF are upregu- 85, 92, 100, 106]. After activation of LPS-TLR4, phosphorylated [93]. Meanwhile, the levels of chemotactic factors such lation of NF-B p65, IB, extracellular signal-regulated as SDF-1, CXCR4, monocyte chemoattractant protein 1 kinases (ERK), c-Jun N-terminal kinase (JNK), p38-MAPK (MCP-1), laminin 5 (LAMA 5), FGF-2, macrophage in- can be detected, whereas blockade of NF-B and JNK has no flammatory protein-1 (MIP-1) and TGF-1, increase in effect on the reaction of DPSCs to LPS, indicating that the DPSCs after LPS treatment [93]. The increase in chemokine changes in odonto-/osteogenic differentiation triggered by production in inflamed pulp is believed to contribute to the LPS is more likely to be mediated by p38-MAPK rather than recruitment of DPSCs during tissue repair and regeneration NF-B pathway [13, 45, 107]. By activating signaling net- [94]. Other bacterial components such as the extract of works, LPS can further promote the secretion of pro- Streptococcus mutans can also promote the migration of inflammatory cytokines and chemokines, such as TNF-, IL- DPSCs [91]. In inflamed dental pulp cells, the mRNA ex- 1, IL-6, IL-8, and also enhance angiogenesis by upregulat- pression of high mobility group box 1 protein (HMGB1) and ing VEGF [108]. In addition, both NF-B and p38-MAPK its receptor for advanced glycation end products (RAGE) pathways are involved in the impact of LPS on stem cell increases. Similarly, when stimulated by E. coli LPS, the proliferation, adhesion, migration [91-93]. The Wnt signal- HMGB1 and RAGE are significantly upregulated in dental ing pathway has a key role in bone development and os- pulp cells. As a chemotactic factor, a higher level of teogenic differentiation [109, 110]. Wnt/-catenin pathway HMGB1 may aid in the recruitment of DPSCs during pulpal activated by LPS can mediate the inhibition of mineral nod- inflammation [95]. Other than enhancing recruitment, LPS ule formation of DPSCs, while upregulating the level of further interferes with the differentiation and secretion of OPN and type I collagen in vitro [100, 106]. Moreover, DPSCs. Wnt5a from the non-canonical Wnt pathway is also upregulated by LPS stimulation in DPSCs, possibly through the After treatment with LPS in vitro, paradoxical changes in TLR4/MyD88/PI3 kinase (PI3K)/AKT pathway [85]. The odonto-/osteogenic differentiation of DPSCs have been re- role of Wnt5a in LPS regulation on DMSCs is not clear yet. ported in different studies [16, 49, 92, 96]. Under E. coli LPS An additional level of complexity is borne by the fact that, stimulation, the mineral nodule formation of DPSCs during there exists a certain level of crosstalk between the NF-B, in vitro induction and expression of odonto-/osteogenic MAPK and Wnt signaling pathways in DMSCs. Further markers were both reported to be elevated [92]. In contrast, compounding the problem is the fact that various studies Porphyromonas gingivalis LPS suppressed the mineral ma- have used different parameters such as: the source of trix formation of DPSCs in vitro [49]. Thus, although LPS is DMSCs, the extent of stimulation and choice of defining a type of common stimulants used to treat DMSCs in studies, marker(s), resulting in sometimes paradoxical findings. More the variation in dose and source may result in slightly varied studies are needed to gain further clarification. reactions. E. coli LPS induced dental follicle cells usually show more potent reactions, such as increased secretion of 5.2. Other Factors that Regulate iDMSC pro-inflammatory factors and more elevated Runx and ALP activities compared to P. gingivalis LPS-treated cells [45, 49, Inflamed pulp or periapical lesions represent extremely 96]. Porphyromonas endodontalis LPS, which is commonly complicated and varied environments, in which iDMSCs found in endodontic infection, shows similar modulation of face highly dynamic modulation. In addition to bacterial the NF-B pathway compared with P. gingivalis LPS [45]. components, bacterial by-products are also involved in local The local elevation of TGF- signaling enhances the differ- inflammation. H2S, a representative volatile sulfur com- Dental Mesenchymal Stem Cells in Inflamed Microenvironment Current Stem Cell Research & Therapy, 2015, Vol. 10, No. 5 417 pound produced by a series of oral bacteria, can induce apop- showing weaker immunogenicity and immunosuppression. tosis of DPSCs [111]. As one of the initial steps of inflam- In addition, iDMSCs are more readily available than normal mation and immune responses, the chemotaxis of DMSCs DMSCs. All the above make iDMSCs more promising than can be induced by multiple factors, including chemoattrac- normal DMSCs as a cell source for regeneration. However, tants (e.g. sphingosine 1-phosphate (S1P), FGF and TGF-1) the knowledge of the physiological phenotypes and behavior and extracellular matrix proteins (EMPs) (e.g. laminin and of iDMSCs is still limited, and there are still multiple chal- FN) [112]. The levels of these chemotactic factors increase lenges to be overcome before translational application. in inflamed pulp or after LPS treatment, such as HMGBI and So far, iDMSCs have mostly been studied in vitro and its receptor RAGE [95]. About, I. and colleagues showed paradoxical results exist. For example, as mentioned above, that the complement system was activated during deep caries some studies have indicated that iDPSCs have similar cell and pulp infection, and that STRO-1 sorted pulp cells were morphology, proliferation and differentiation characteristics able to migrate along a C5a gradient [113]. compared with normal DPSCs [69]. Other studies have Other cells in inflamed pulp involved in inflammation shown that iDPSCs may form more, or less dentin-pulp may also influence the activities of DPSCs. For example, the complex in vitro compared to normal DPSCs, depending on nemotic pulp fibroblasts release PGE2 and IL-8, and further the study [13, 15]. Furthermore, the extent to which LPS promote the migration of DPSCs [114]. In the inflamed mi- affects the odonto-/osteogenic differentiation of DMSCs is croenvironment, in addition to regulation by extrinsic and inconsistent across studies [16, 49, 92, 96]. As differences local cells and signaling molecules, DPSCs may also modu- remain in tissue resource selection, local inflammation con- late repair process by autocrine/paracrine mechanisms. The dition and experiment design, the isolated iDMSCs may conditioned medium of DPSCs under stress conditions (e.g. show variation from study to study. With accumulating expe- hypoxia) can improve cell viability by impairing the cyto- rience and increasing interest towards iDMSCs investigation, toxic effects of triethylene glycol dimethacrylate (TEG- more standardized observations in vitro and in vivo are DMA), a common dentin bonding agent [115]. Since the needed to achieve consistent results and solid conclusions. inflammation may cause local tissue swelling and decrease blood supply, iDMSCs often encounter hypoxia. Further- The application of DMSCs in regenerative medicine is more, stem cells may experience short-term hypoxia imme- mainly in tissue engineering. From stem cells to regenera- diately after transplantation [6]. Vanacker et al. found hy- tion, there are multiple challenges that need to be overcome, poxia did not affect the proliferation of SCAP, but upregu- including the identification of cell populations, the physio- lated osteogenic and neurogenic differentiation as well as logical characterization of certain cells, the recruitment and angiogenesis related genes [6]. induction of proliferation and differentiation, the selection of scaffold from different biomaterials, and the lack of studies In the clinic, dental infection/inflammation is often in animal models and in the clinical setting [119-121]. Once treated with multiple appliances and materials. During con- the physiological characteristics of iDMSCs are elucidated, sideration of iDMSCs as a candidate for regeneration, one establishing proper cell inclusion criteria will be very impor- has to factor in whether the tooth has undergone prior dental tant. In addition to the experience already gained from treatment, which may introduce further variables. During DMSCs studies, the evaluation and selection of originating treatment, medicine or materials that are commonly used in tissue for iDMSCs are essential yet difficult [122]. The dental practice may also affect the activities of iDMSCs. cause, extent and course of inflammation are variables that However, investigation of the influence of dental treatment on iDMSCs has rarely been reported. Recent studies have may affect the quality and quantity of iDMSCs [123]. focused on treating normal DMSCs with certain medical Whether cryopreserved iDMSCs show compatible character- compounds or materials. Mineral trioxide aggregate (MTA) istics as DMSCs also needs to be investigated [124]. can promote the migration of DPSCs and enhance odonto- On the other hand, to preserve and enhance the biological genic and osteogenic differentiation via the NF-B pathway behaviors of iDMSCs, various strategies of pre-treatment [71, 116]. However MTA may also cause apoptosis of the may need to be adopted before transplantation. Treatment dental pulp cells under long-term contact [117]. Camphor- with FGF-2 during expansion of iDPSCs in vitro enhances quinone (CQ) increases the secretion of pro-inflammatory cellular proliferation and migration, as well as reducing cytokines, further reducing the proliferation, odontogenic odontogenic differentiation; FGF-2 treatment during in vivo differentiation and mineral nodule formation of dental pulp transplantation instead results inincreased mineral material at cells [118]. In addition, it has been shown that TEGDMA the site of transplantation [73]. Induction of differentiation has a cytotoxic effect on DPSCs [115]. To reveal the impact by hypoxia may be one potential supplemental treatment for of endodontic and restoration treatment on iDMSCs, more the regenerative application of iSCAP [6, 125]. Non- specific studies are in need. ionizing, low-power laser (LPL) treatment of DPSCs can enhance the odontogenic differentiation in vitro and in vivo, 6. CHALLENGES IN IDMSC STUDY AND FUTURE by inducing ROS and then activating latent TGF-1 [126]. In CLINICAL APPLICATION particular, involvement of growth factors and immune me- In recent years, detailed investigation of the different in- diators to treat iDMSCs can also be considered after more flammation associated signals and conditions, as well as the information is gained from further study of signaling path- physiological response and alterations that iDMSCs display, way [127]. The osteogenic differentiation of human indicate the potential of iDMSCs in regenerative medicine. BMMSCs by nanostructured calcium phosphate biomaterials In brief, iDMSCs show the ability to proliferate, maintain is enhanced [128]. Whether similar effects exists or can aid stemness, and differentiate into multiple lineages, while the properties of iDMSCs is of interest for future utilization. 418 Current Stem Cell Research & Therapy, 2015, Vol. 10, No. 5 Zhou et al.

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Investigation of dental pulp stem sic irritants, local cells or their components, immune cells cells isolated from discarded human teeth extracted due to aggres- and multiple soluble regulatory molecules. The inflammatory sive periodontitis. Biomaterials 2014; 35(35): 9459-72. response and the adaptive immune system represent two [15] Attar A, Eslaminejad MB, Tavangar MS, et al. Dental pulp polyps highly regulated and dynamic networks, acting to impair, or contain stem cells comparable to the normal dental pulps. J Clin Exp Dent 2014; 6(1): e53-9. augment iDMSC characteristics. Compared to normal [16] Ma D, Cui L, Gao J, et al. Proteomic analysis of mesenchymal DMSCs, iDMSCs are more accessible, and can thus be con- stem cells from normal and deep carious dental pulp. PLoS One sidered as potential sources of cells for allo-/auto-graft; as 2014; 9(5): e97026. long as clinical standards for the inclusion of subjects and [17] Wang Y, Yan M, Wang Z, et al. Dental pulp stem cells from trau- the isolation of iDMSCs are clearly established. To further matically exposed pulps exhibited an enhanced osteogenic potential validate and explore the long-term effect and utilization po- and weakened odontogenic capacity. Arch Oral Biol 2013; 58(11): 1709-17. tential of iDMSCs, more in vivo models are needed to ad- [18] Morsczeck C, Gotz W, Schierholz J, et al. Isolation of precursor dress the aforementioned challenges. cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol 2005; 24(2): 155-65. CONFLICT OF INTEREST [19] Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet The authors confirm that this article content has no con- 2004; 364(9429): 149-55. flict of interest. [20] Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner ACKNOWLEDGEMENTS Res 2003; 18(4): 696-704. 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Received: January 03, 2015 Revised: February 19, 2015 Accepted: March 02, 2015