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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Isolation of DPSCs
  5. Characterization of DPSCs
  6. Regenerative potential
  7. Future perspectives
  8. References

Dental pulp-derived stem cells (DPSCs) are considered to be of great promise for use in tissue repair and regenerative medicine. DPSCs can easily be collected from discarded teeth with little ethical concerns and harvested in a minimally invasive and safe manner. However, unfractionated clonogenic DPSCs are heterogenous and have variations in their phenotype. In this review paper, we summarize further isolation methods of DPSC subpopulations including immunoselection methods and a granulocyte colony-stimulating factor (G-CSF) gradient mobilization method for therapeutic clinical applications. The fractionated DPSC subpopulations exhibit stem cell properties in vitro: (i) high expression of pluripotency markers, Oct3/4, Nanog, and Sox2; (ii) high stability in long-term expansion; (iii) multi-lineage differentiation capacity; (iv) high migratory activity; and (v) high expression of trophic factors to enhance proliferation, migration, and anti-apoptotic and immunomodulatory effects as well as angiogenesis and neurite extension. DPSC subpopulations have higher angiogenic, neurogenic, and regenerative potential compared with bone marrow stem cells and adipose stem cells, presenting an alternate versatile stem cell source for cellular therapies. Preclinical efficacy of DPSC subpopulations has also been investigated in various tissue/organ disease models including pulpitis, and currently a few clinical trials are underway to determine their safety and efficacy. Therefore, the major aim of this review is to highlight the recent progress in DPSC biology, trends in preclinical regenerative studies, and future perspectives.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Isolation of DPSCs
  5. Characterization of DPSCs
  6. Regenerative potential
  7. Future perspectives
  8. References

Stem cell-based therapies are rapidly emerging as a potential strategy for tissue regeneration in many diseases. The goal of cellular therapy in regenerative medicine is to replace, repair, or enhance the biological functions of the damaged tissues or organs. It is hoped that mesenchymal stem/progenitor cells (MSCs) will provide an inexhaustible source of therapeutic products in the cell therapy. MSCs are derived from mesoderm and are present in many postnatal tissues and organs. MSCs have been isolated from different tissue sources including bone marrow, adipose tissue, bone, periosteum, synovium of the joints, skeletal muscle, skin, pericytes of blood vessels, peripheral blood, periodontal ligament, umbilical cord, and dental pulp of permanent and deciduous teeth [1]. The autologous MSCs present no serious ethical issues in their utility. It is relatively easy to obtain large pools of autologous cells that possess self-renewal capability and multi-lineage differentiation potential. Of special note, MSCs from dental pulp tissue (dental pulp stem cells, DPSCs) can easily be collected from discarded permanent teeth and harvested in a minimally invasive and safe manner. DPSCs have higher angiogenic, neurogenic, and regenerative potential compared with bone marrow stem cells and adipose stem cells [2], presenting an alternate versatile stem cell source for cellular therapies.

There are numerous publications that have demonstrated the underlying biology and cellular properties of DPSCs. The preclinical efficacy of DPSCs has also been investigated in various tissue/organ disease models including pulpitis, and currently a few clinical trials are underway to determine their safety and efficacy. Therefore, the aim of this article is to highlight the recent progress in DPSC biology, trends in preclinical regenerative studies, and future perspectives.

Isolation of DPSCs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Isolation of DPSCs
  5. Characterization of DPSCs
  6. Regenerative potential
  7. Future perspectives
  8. References

DPSCs can be acquired from discarded permanent teeth including third molars, supernumerary teeth, displaced teeth or orthodontically unnecessary teeth. DPSCs can be isolated by the explant outgrowth technique or the enzymatic digestion technique. In the former technique, the tissues are cut into 2-mm3 pieces and are directly cultured in tissue culture dishes; cells migrating out from the tissues are collected 14 to 21 days later [3-5]. In the enzymatic cell isolation technique, after enzymatic digestion, the pulp single-cell suspensions are plated at a cell concentration of 0.005 to 0.5 × 105/mL to isolate a clonogenic population [6]. The DPSCs both from the explant outgrowth technique and from the colony formation technique exhibit highly proliferative, self-renewal, multi-lineage differentiation potential. However, these DPSCs are heterogenous and contain more than one stem cell population. Therefore, a more defined clonal subset of stem/progenitor cells has been isolated using immunoselection of some cell surface antigen markers by flow cytometry or magnetic-activated cell sorting. These antigen markers include STRO-1 [7-10], CD34 [11], c-kit/CD117 [8, 12], low-affinity nerve growth factor receptor (LANGFR/p75/CD271) [13], SSEA-4 [14], CXCR4 [15], and CD105/endoglin [16, 17]. Isolation of the “true” or “mother” adult stem cells which exhibited a lower level of the DNA binding fluorescent dye, Hoechst 33342, is an alternative method [2, 18-20]. So far, at least two different stem cell populations have been identified: one originating from the neural crest (ectomesenchyme) and the other arising from mesenchymal origin. Nevertheless, for clinical application of DPSCs, one major challenge in manufacturing clinical grade human DPSCs is the requirement for good manufacturing practice (GMP) grade cell isolation and processing. The safety of DPSCs isolated by flow cytometery has not yet been established. However, another isolation method using immuno-magnetic beads of CD34, such as the Isolex 300i Magnetic Cell Selection System device (Baxter) presently intended for clinical use [21, 22], is not suitable for human DPSCs because a limited volume of pulp tissue or a limited number of the primary pulp cells is available and CD34 is not a validated cell surface marker for pulp stem cell isolation. The costs would be prohibitive if other specific antibodies such as CD105 combined with magnetic beads are specially made to order for DPSC isolation.

Therefore, we have recently developed a granulocyte colony-stimulating factor (G-CSF) gradient mobilization method for the isolation of DPSC subpopulations that provides safe, efficient, and cost-effective isolation from a small amount of pulp tissue. The device consists of an upper chamber of a transwell membrane that was treated chemically to avoid cell attachment and a lower chamber to employ an optimized G-CSF gradient. G-CSF has the ability to mobilize mesenchymal stem cells from bone marrow [23, 24]. When unfractionated pulp cell suspensions are added in the upper chamber, DPSC subpopulations with high migration and regenerative potential are mobilized from the upper chamber to the lower chamber, supplemented with G-CSF in 48 h, and colonized in 8 days [25, 26]. We have named this DPSC subpopulation “mobilized DPSCs isolated by the G-CSF gradient” (MDPSCs). MDPSCs have similar stem cell properties and regenerative potential to pulp CD105+ cells and much higher regenerative potential, with higher expression of trophic factors than unfractionated DPSCs. The much higher stability of MDPSCs in long-term in vitro expansion has also been demonstrated compared with unfractionated DPSCs [25].

Thus, standardization of the isolation and culture procedures is needed for optimal reproducibility, quality, and safety of clinical grade DPSC subpopulations for regeneration of a variety of tissues in oral medicine and dentistry.

Characterization of DPSCs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Isolation of DPSCs
  5. Characterization of DPSCs
  6. Regenerative potential
  7. Future perspectives
  8. References

Self-renewal

The self-renewal potential of DPSCs is generally assessed by implantation in an ectopic site to unambiguously determine de novo renewal and tissue formation. Stromal-like cells, representing 85% human origin, were re-isolated 3 months after subcutaneous transplantation into immunocompromised mice. Re-transplantation of the human re-established cells generated a dentin–pulp like tissue, demonstrating their self-renewal capability [27]. Wnt signaling pathways play a critical role in stem cell self-renewal [28], and canonical Wnt/β-catenin signaling negatively regulates the odontoblast-like differentiation of DPSCs [29]. The hDPCs can sustain self-renewal in a medium containing bFGF, showing a higher ratio in the S-phase compared with medium without bFGF [30]. The activation of Notch signaling by Jagged-1 inhibits the odontoblastic differentiation of DPSCs in vitro and in vivo [31]. The deficient Notch signaling inhibits the self-renewal capacity of DPSCs [32]. These results indicate that Notch signaling plays a crucial role in regulating self-renewal and differentiation in DPSCs.

Multi-lineage differentiation

DPSCs give rise to a variety of cell types in vitro, such as osteoblasts, chondrocytes, adipocytes, myoblasts, endotheliocytes, and melanocytes, as well as neurons and glia [9, 11, 12, 27, 33-41]. In vivo, DPSCs can differentiate into odontoblasts and induce host cells to participate in regeneration by generating a dentin/pulp-like complex after subcutaneous transplantation in conjunction with hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds into immunocompromised mice [6, 27, 42]. Differentiation into osteoblasts [8, 12] that secrete abundant extracellular matrix and that can build a woven bone has been demonstrated in vitro [43]. DPSCs can also differentiate into both osteoblasts and endothelial cells to produce adult bone-like tissue with an integral blood supply in vivo [36]. DPSCs can differentiate into mature oligodendrocytes or functionally active neurons after transplantation into the embryonic mesencephalon [37]. Smooth and skeletal muscle cells are induced from DPSCs in vivo [33, 44]. Preincubated DPSCs toward odontogenic, adipogenic, and myogenic lineages differentiate along distinct pathways after transplantation into immunocompromised mice [33, 39]. These multi-lineage differentiation potentials of DPSCs suggest their utility in various fields of regenerative medicine.

Migration

MSCs appear to both rely upon and generate a network that facilitates constant communication between normal and damaged cells in the body [45] and migrate to the site of insult or injury in response to signals of cellular damage, known as homing signals [46, 47]. The potential of MSCs to regenerate damaged tissue is attributed to the presence of chemokine receptors on the surface of MSCs, enabling these cells to migrate toward gradients of homing signals, chemokines, or growth factors secreted by injured tissues [48, 49]. DPSCs are known to respond to tooth injury by proliferating, migrating, and differentiating to replace lost odontoblasts, leading to the synthesis and secretion of tertiary dentin or reparative dentin [50]. The specific chemotactants for DPSCs leading to the response to tooth injury have not been rigorously identified. DPSCs show high migratory activity in vitro with many cytokines or growth factors, including stromal cell-derived factor-1α (SDF-1α), G-CSF, GM-CSF, and FGF-2 (our unpublished data). The migratory activity of pulp CD31 SP cells is significantly higher compared with bone marrow and adipose CD31 SP cells in the same canine or porcine subjects [2, 20], suggesting the higher migration potential of DPSCs. The involvement of SDF-1α as a migratory cue in the trafficking of C-X-C chemokine receptor type 4 (CXCR4)-positive DPSCs has been demonstrated in vivo [51]. Extracellular matrix proteins (especially laminin) and specific chemotactants (especially sphingosine-1-phosphate [S1P], transforming growth factor β1 [TGF-β1], and fibroblast growth factor-2) are suggested to be important promoters of DPSC migration [52, 53]. EphB/ephrin-B molecules play a role in restricting DPSC attachment and migration to maintain DPSCs within their stem cell niche under steady-state conditions [54]. These results indicate that enhancement of the migrating capacity of DPSCs can be achieved by modulating the response of DPSCs to a variety of cytokines and growth factors, thereby improving their regenerative potential.

Stem cell marker expression

Immunostaining and flow cytometry are the most common methods used to characterize MSCs based on their stem cell surface marker profile: positive for CD29, CD44, CD73, CD90, CD105, CD166, and STRO-1; negative for CD11b, CD19, CD34, CD45, and HLA-DR. The markers seem to be expanding with CD200 added as a marker with immunomodulatory properties [55]. Unfractionated clonogenic DPSCs are heterogenous and consist of mixed subpopulations, and their marker expression profiles are different among reports [56] (for further details, see the review by Kawashima, 2012 [56]). There is no distinct specific marker for characterizing DPSCs from other MSCs. The expression rate of CXCR4, GCSFR, and CD105 in MDPSCs or fractionated DPSCs such as CD31 SP cells and CD105+ cells is significantly higher compared with unfractionated clonogenic DPSCs [2, 20, 25, 26, 57]. Although the expression of Sox2 and CXCR4 mRNA in human MDPSCs and pulp CD105+ cells is lower compared with iPS cells, other stem cell marker expression (including Oct3/4, Nanog, Rex1, GDF3, LIN28, and Stat3) is at similar levels in both MDPSCs and CD105+ cells and the iPS cells [25]. These results suggest higher stemness of MDPSCs and fractionated DPSCs compared with unfractionated clonogenic DPSCs.

Stability

The stability of the phenotype of stem cell lineages into multi-lineage cell differentiation is at the crux of regenerative medicine in general. Unfractionated human DPSCs undergo a change in morphology, lose their differentiation ability, and increase WNT16 expression during long-term passage [58]. Subculture-induced replicative senescence of the DPSCs leads to reduced expression of Bmi-1, which determines the self-renewal capacity of the stem cell and is required for transcriptional repression of its target genes through chromatin remodeling [59]. STRO-1+ DPSCs decrease their proliferation ability and restrict their differentiation capacity to osteoblast lineage in vivo at the 9th passage [41]. The expression of reprogramming markers, Oct-4, Sox2, and c-Myc peaked at the second passage of explant cultured DPSCs and decreased along the passages afterwards [60]. However, unfractionated clonogenic human DPSCs from young donors in media with 2% FCS supplemented with PDGF, EGF, and dexamethasone can be expanded over 60 population doublings and remain cytogenetically stable [61]. Human DPSCs cultured under 3% O2 show a significantly higher proliferation rate than those under 21% O2 for a long time period, increasing the possibility of obtaining DPSCs with differentiation capability from damaged and/or aged tissue [62]. MDPSCs at the 30th passage express senescence associated β-gal at a lower rate and cellular senescence markers, IL-1β, p16, IL-6, and IL-8 mRNA expression is significantly lower compared to unfractionated clonogenic DPSCs both at the 6th and the 30th passages, indicating the stability of MDPSCs in long-term in vitro expansion [25]. Furthermore, MDPSCs are more stable in proliferative and regenerative potential than unfractionated clonogenic DPSCs even from aged patients (our unpublished data). These results suggest that the stability of DPSCs after prolonged ex vivo culture is dependent on the isolation and culture method as well as the original pulp tissue condition.

Trophic effects

There is a growing and important realization of the potential for secretion of trophic factors from stem cells/progenitors. It has been noticed that transplanted MSCs are able to secrete a broad spectrum of cytokines and growth factors that affect neighboring cells (Fig. 1). The success in repairing/regenerating damaged tissues after transplantation is attributed, at least in part, to their resistance to stress and through the paracrine effect that they impart on host tissues [63]. The terminal differentiation capacity of transplanted MSCs is not the major determinant of their regenerative potential. The paracrine effects can stimulate the recruitment of host progenitor cells, enhance angiogenesis/neurogenesis, and possibly modulate the host immune response. The trophic/paracrine factors known to be highly expressed in MSCs are as follows: VEGF-A, ANG-1, TPO, HGF, LIF, IGF, IGF-binding proteins-1, -2, -3, -4, FGF-4, -6, -7, -9, and a number of other molecules such as leptin, fractalkine, neutrophil activating peptide-2 (NAP-2), macrophage inflammatory protein-1β (MIP-1β), and MIP-3α, as well as a small number of cytokines (IL-6, -7, -8, -10) and growth factors (G-CSF, M-CSF, GM-CSF, SDF-1, and SCF) [64] (see the review by Doorn et al., 2012 [64]). Conditioned medium (CM) alone exerts similar responses, underlining the particular importance of immunomodulatory and trophic mediators [65]. Our previous studies have shown that CM of pulp CD31 SP cells, CD105+ cells, and MDPSCs stimulate proliferation and migration and inhibit apoptosis in MSCs, human umbilical vein endothelial cells (HUVECs), and SH-SY5Y human neuroblastoma cell lines in vitro [25, 26, 57, 59]. CM from these cells have also demonstrated high immunomodulatory activity, and promotion of angiogenesis in NIH3T3 cells and neurite extension in TGW human neuroblastoma cell lines [20, 26]. A similar result has been reported on the promotion of neurite extension by CM of unfractionated DPSCs in cerebral granular neurons [66]. These trophic effects of CM of pulp CD31 SP cells are superior to CM of bone marrow and adipose CD31 SP cells [20], leading to higher angiogenic, neurogenic, and regenerative potential of pulp CD31 SP cells compared with bone marrow and adipose CD31 SP cells in vivo [20].

figure

Figure 1. Trophic and immunomodulatory effects of DPSCs. Factors secreted by DPSCs exhibit either trophic or immunomodulatory effects. Trophic factors secreted by DPSCs increase mobilization and proliferation of stem/progenitor cells, inhibit apoptosis, and induce angiogenesis/neurogenesis. Immunoregulatory factors exert anti-proliferative effects on T-cells, and increase anti-inflammatory profile and upregulate T cell regulatory (Tregs) stimulating immune tolerance.

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Immunomodulation

A major characteristic of MSCs has been the immunomodulatory properties of the cells. MSCs possess potent immunosuppressive effects by inhibiting the activity of both innate and adaptive immune cells [67-70]. This immunosuppression is mediated by cell contact-dependent and cell contact-independent mechanisms through the release of soluble factors. The immunomodulatory role of MSCs is due to activated T cell apoptosis via the FAS ligand/FAS-mediated death pathway by way of cell–cell contact, leading to the upregulation of T regulatory cells (Tregs) that results in immune tolerance [71]. Natural killer (NK) cells are important effector cells of innate immunity and play a key role via their cytotoxic potential and secretion of pro-inflammatory cytokines including TNF-α and IFN-γ. MSCs also inhibit NK cells from proliferation, cytokine production, and cytotoxic activity [72, 73]. While the suppressive effects of MSCs on T lymphocytes are known, the effects of MSCs on B-cells remain controversial: most studies have demonstrated that MSCs inhibit B-cell proliferation, differentiation, and antibody secretion in in vitro co-culture assays and in vivo multiple sclerosis models, whereas other in vitro studies have demonstrated a stimulatory role of MSCs on B-cell proliferation and antibody secretion. These results suggest that MSC-mediated regulation of B-cells may depend on the developmental stage of the B-cells and the local microenvironment [71]. The immunosuppressive and immunomodulatory roles of DPSCs are known [20, 74-79]. The candidate immunomodulators secreted by DPSCs include Prostaglandin E2, transforming growth factor-β, hepatic growth factors (HGF), interleukins-6 and -10, human leukocyte antigen-G5, matrix metalloproteinases, indoleamine-2, 3-dioxygenase, heme oxygenase-1 (HO-1), nitric oxide, and Fas ligand (FasL or CD95L) [76, 78, 80]. The potent immunomodulatory functions of DPSCs due to their soluble factors and cytokines via a paracrine mechanism may have a profound effect on clinical cell therapy by T-lymphocyte function inhibition and upregulation of T cell regulatory (Tregs) stimulating immune tolerance.

Regenerative potential

  1. Top of page
  2. Abstract
  3. Introduction
  4. Isolation of DPSCs
  5. Characterization of DPSCs
  6. Regenerative potential
  7. Future perspectives
  8. References

DPSCs offer regenerative potential of various damaged or lost tissues and organs including dentin, pulp, periodontal tissue, bone, neuronal tissue, blood vessels, muscle, cartilage, hair follicle, and cornea. We provide an overview of the work performed (Fig. 2).

figure

Figure 2. Regenerative potential of DPSCs in blood vessels, neuronal tissue, cartilage, hair follicle, pulp, dentin, bone, and periodontal tissue. (A) Angiogenesis after transplantation in a mouse ischemic hindlimb model. Confocal laser microscopic analysis after perfusion labeling with FITC-dextran in the muscles of the ischemic hindlimb. (B) Neurogenesis after transplantation in a sciatic nerve injury model. Electronmicrogram showing regenerated neurons surrounded by the schwann cells in the injury site. (C) Cartilage regeneration after ectopic transplantation of DPSCs in SCID mice. Alcian blue staining. (D) Regeneration of hair follicle after ectopic transplantation in SCID mice. (E) Regeneration of pulp tissue 14 days after orthotopic transplantation of DPSCs with G-CSF in the emptied root canal after pulpectomy in dogs. (F) Regeneration of tubular dentin 90 days after orthotopic transplantation of DPSCs with G-CSF in the emptied root canal after pulpectomy in dogs. (G) Bone regeneration surrounding tooth root after ectopic transplantation of tooth filled with DPSCs with collagen scaffold in SCID mice. (H) Regeneration of periodontal ligament and cementum surrounding tooth root after ectopic transplantation of tooth filled with DPSCs with collagen scaffold in SCID mice. (C–H) H-E staining.

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Dentin/pulp

DPSCs have dentin/pulp repair/regenerative potential. The dentin–pulp complex is induced on the exposed/amputated pulp in the cavity after transplantation of DPSCs alone or together with BMP2 [18, 81, 82]. Dentin regeneration has also been demonstrated in a simulated perforation repair model using DPSCs with dentin matrix protein 1 (DMP1) [83]. In ectopic tooth transplantation models in immunocompromised mice, the dentin–pulp complex with well-established vascularity is regenerated de novo in the emptied root canal space of tooth slices or tooth roots by DPSCs or MDPSCs [25, 84-87]. Regenerative dentin matrix formation with a continuous odontoblast-like cell layer has been shown on the canal walls after transplantation of tooth roots with swine DPSCs in the jaw bone of minipigs [88]. We have also used an orthotropic pulpectomized tooth model with complete apical closure in adult dogs as a simulated regenerative therapy in endodontics. Pulp tissue with well vasculature and innervation is completely regenerated after autologous transplantation of DPSCs with stromal cell-derived factor-1 (SDF-1) [2, 17, 20, 26, 89] or MDPSCs with G-CSF [26] in this model. Protein profiles, mRNA expression patterns, laser Doppler analysis of pulpal blood flow, and assessment of pulp vitality have demonstrated that the regenerated pulp tissue is true functional normal pulp [17, 26]. Dentin is also induced in the coronal and the apical parts of the regenerated pulp tissue as well as along the dentinal wall to prevent microleakage by day 180. Transplantation of MDPSCs with G-CSF yields a significantly larger amount of regenerated tissue compared with MDPSCs alone or G-CSF alone. A reduction in the number of inflammatory cells and apoptotic cells and significant increase in neurite outgrowth has been shown in the transplantation of MDPSCs with G-CSF compared with those alone. The transplanted stem cells highly express multiple angiogenic/neurotrophic factors. G-CSF together with conditioned medium of MDPSCs stimulates cell migration and neurite outgrowth, prevents cell death, and promotes immunosuppression in vitro compared with those alone. Thus, the combinatory effect of MDPSCs and G-CSF on accelerated pulp regeneration has been suggested to stimulate migration and proliferation of endogenous stem/progenitor cells from adjacent tissues and vessels, inhibit apoptosis and inflammation, and enhance angiogenesis and re-innervation [26]. Fractionated DPSCs such as CD105+ cells and CD31 SP cells or MDPSCs are more advantageous than unfractionated DPSCs to pulp regeneration, resulting in significantly less inflammation and apoptosis, significantly larger volumes of regenerated pulp tissue, higher density of angiogenesis and re-innervation, and much less mineralization in the root canal [17, 26].

Periodontal tissue

DPSCs transplanted into various periodontal defects result in inconsistencies among different research groups. Regeneration of cementum, bone, and periodontal ligament has been shown 8 weeks after autologous transplantation of DPSCs with Bio-oss in mesial three-walled periodontal defects with ligature-induced periodontitis in dogs [90]. Cementum, bone, and periodontal ligament are induced 3 weeks after ectopic transplantation of tooth root with injection of DPSCs (CD31 SP cells) or MDPSCs with collagen scaffold (our unpublished data). However, the periodontal defects that received DPSCs are very similar to those of the negative control, indicating no promotion of periodontal tissue regeneration, although periodontal ligament stem cells (PDLSCs) show the regenerating capacity of periodontal tissue as well as peripheral nerve and blood vessels [91].

Bone

DPSCs are capable of differentiating into osteoblasts [43, 92] that secrete abundant extracellular matrix and that can build a woven bone in vitro [8]. DPSCs transplanted with a variety of scaffold including HA/TCP [93-95], PLGA [96], collagen [97, 98], nano-fiber hydrogel [99], HA nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA) [100], fibroin [101], and platelet-rich plasma (PRP) [102] exhibit bone-like structure. Pretreatment of DPSCs with BMP2 promotes osteogenesis [100]. Critical size bone defects are repaired by DPSCs [95, 101]. DPSCs show higher osteogenic potential than bone marrow stem cells and periosteal cells and are a useful cell source for tissue-engineered bone around dental implants [103]. Thus, DPSCs can be used for therapeutic purposes such as the repair/regeneration of craniofacial bone and alveolar bone [98].

Neuronal tissue

DPSCs transplanted into the mesencephalon of embryonic day-2 chicken embryos acquire a neuronal morphology and function, suggesting that exposure to the appropriate environmental cues induces differentiation of DPSCs into active neurons [37]. DPSCs coordinate axon guidance via CXCR-4 and the stromal cell-derived factor-1 (SDF-1)/CXCL12 axis, inducing neuroplasticity within a receptive host nervous system [104]. Transplantation of DPSCs into the hippocampus of immune-suppressed mice promotes proliferation, cell recruitment, and maturation of endogenous neural cells by releasing neurotrophic factors. The results suggest the valuable therapeutic potential of DPSCs as a stimulator/modulator of the local response in the central nervous system [105]. Furthermore, in cerebral ischemia and spinal cord injury models, transplantation of DPSCs results in recovery from neurological dysfunction, suggesting the neural regenerative potential of DPSCs in the central nervous system [19, 66, 106, 107]. Its functional improvement is unlikely to be due to neural replacement, and is more likely to be mediated through DPSC-dependent paracrine effects [19, 108]. In a peripheral nerve injury model, artificial nerve conduits containing DPSCs promote nerve regeneration with myelinated fibers [109, 110]. Our recent study has shown various trophic effects of MDPSCs including migration, proliferation, anti-apoptosis, and induced differentiation on schwann cells in vitro and in vivo (our unpublished data).

Blood vessels

DPSCs accelerate blood flow and vasculogenesis/angiogenesis in an ischemic hindlimb model and a peripheral nerve injury model [16, 57, 109]. DPSCs differentiate into osteoblasts and endotheliocytes synergically after transplantation into immunocompromised rats and lead to generation of adult bone structure with an integral blood supply, suggesting that angiogenesis may be regulated by distinct mechanisms [36].

Muscle

Upon infusion into cardio toxin-induced muscle defects, cloned DPSCs that are sustainably Oct4+, Nanog+, and Stro1+ engraft and colonize host muscle, as well as express dystrophin and myosin heavy chain more efficaciously than their parent heterogenous cells, suggesting the therapeutic potential of cloned DPSCs in muscle regeneration [111]. In a myocardial infarction model in rats, DPSCs injected intramyocardially improve cardiac function, reduce infarct size, and increase angiogenesis without differentiating into endothelial cells, smooth muscle cells, or cardiac muscle cells. The result suggests that DPSCs can provide a novel alternative cell population for cardiac repair [112].

Cartilage

STRO-1+ DPSC cell pellets develop into dentin, bone, and cartilage 14 days after in vivo transplantation into the renal capsules in rats at the 1st passage and generate only osteoblast lineage at the 9th passage [41]. Similar results are obtained after subcutaneous transplantation of unfractionated DPSCs cultured with bFGF in immunocompromised mice [30].

Hair follicles

Human unfractionated DPSCs transplanted into surgically inactivated hair follicles interact with follicle epithelium to regenerate new end bulbs and create multiple differentiated hair fibers [113].

Cornea

Corneal transparency of rabbit eyes which had corneal defects is improved with the reconstruction of corneal epithelium after transplantation of a tissue-engineered human DPSC sheet onto the corneal bed and covered with de-epithelialized human amniotic membrane [114].

Future perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Isolation of DPSCs
  5. Characterization of DPSCs
  6. Regenerative potential
  7. Future perspectives
  8. References

MSCs are multi-potent, non-hematopoietic stem/progenitor cells that are being explored as a promising new treatment for tissue regeneration. DPSCs have been widely investigated in preclinical cell-based therapy studies as an alternative to bone marrow and adipose-derived MSCs. Although their immunomodulatory functions are not yet completely elucidated, their low immunogenic potential along with their immunomodulatory and immunosuppressive roles make them potent therapeutic tools in several human diseases, including pulpitis, periapical disease, ischemic diseases, and neurological disorders. A biocomplex composed of DPSCs and a collagen sponge scaffold have already been used in a clinical trial as a treatment for oro-maxillofacial bone defects, resulting in optimal bone repair and complete bone regeneration [98]. Another clinical trial using MDPSCs is ongoing by us for dentin/pulp regeneration in the treatment of pulpitis.

In conclusion, DPSCs and MDPSCs with high regenerative potential could be of great potential in treating and curing a multitude of human diseases and tissue injuries. The regenerative potential of DPSCs and MDPSCs can be experimentally tested and applied in clinical dentistry including endodontic surgery. Optimized manufacturing protocols, safety parameters, and cell transportation/manipulation protocols are a prerequisite for guaranteed utility, including the high quality and safety of the cells for the design and conduct of well-controlled clinical trials.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Isolation of DPSCs
  5. Characterization of DPSCs
  6. Regenerative potential
  7. Future perspectives
  8. References
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